160 results on '"Volanakis JE"'
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2. The crystal structure of cobra venom factor, a cofactor for C3- and C5-convertase CVFBb.
- Author
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Krishnan V, Ponnuraj K, Xu Y, Macon K, Volanakis JE, and Narayana SV
- Subjects
- Animals, Binding Sites genetics, Complement C3 Convertase, Alternative Pathway genetics, Complement C3-C5 Convertases genetics, Complement C3b genetics, Crystallography, X-Ray, Elapid Venoms genetics, Elapid Venoms isolation & purification, Elapid Venoms metabolism, Models, Chemical, Models, Molecular, Protein Binding genetics, Protein Structure, Secondary, Protein Structure, Tertiary, Complement C3 Convertase, Alternative Pathway metabolism, Complement C3-C5 Convertases metabolism, Complement C3b metabolism, Elapid Venoms chemistry
- Abstract
Cobra venom factor (CVF) is a functional analog of human complement component C3b, the active fragment of C3. Similar to C3b, in human and mammalian serum, CVF binds factor B, which is then cleaved by factor D, giving rise to the CVFBb complex that targets the same scissile bond in C3 as the authentic complement convertases C4bC2a and C3bBb. Unlike the latter, CVFBb is a stable complex and an efficient C5 convertase. We solved the crystal structure of CVF, isolated from Naja naja kouthia venom, at 2.6 A resolution. The CVF crystal structure, an intermediate between C3b and C3c, lacks the TED domain and has the CUB domain in an identical position to that seen in C3b. The similarly positioned CUB and slightly displaced C345c domains of CVF could play a vital role in the formation of C3 convertases by providing important primary binding sites for factor B.
- Published
- 2009
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3. The structure of C2b, a fragment of complement component C2 produced during C3 convertase formation.
- Author
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Krishnan V, Xu Y, Macon K, Volanakis JE, and Narayana SV
- Subjects
- Amino Acid Sequence, Complement C1s metabolism, Complement C2a chemistry, Complement C2a metabolism, Complement C2b metabolism, Complement C3-C5 Convertases metabolism, Complement C4b metabolism, Complement Factor B chemistry, Complement Factor B metabolism, Crystallography, X-Ray, Humans, Models, Molecular, Molecular Sequence Data, Mutagenesis, Site-Directed, Point Mutation, Protein Binding, Protein Conformation, Protein Folding, Protein Structure, Tertiary, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins metabolism, Sequence Alignment, Sequence Homology, Amino Acid, Structure-Activity Relationship, Complement C2b chemistry
- Abstract
The second component of complement (C2) is a multi-domain serine protease that provides catalytic activity for the C3 and C5 convertases of the classical and lectin pathways of human complement. The formation of these convertases requires the Mg(2+)-dependent binding of C2 to C4b and the subsequent cleavage of C2 by C1s or MASP2, respectively. The crystal structure of full-length C2 is not yet available, although the structure of its C-terminal catalytic segment C2a has been determined. The crystal structure of the N-terminal segment C2b of C2 determined to 1.8 A resolution presented here reveals the arrangement of its three CCP domains. The domains are arranged differently compared with most other CCP-domain assemblies, but their arrangement is similar to that found in the Ba part of the full-length factor B structure. The crystal structures of C2a, C2b and full-length factor B are used to generate a model for C2 and a discussion of the domain association and possible interactions with C4b during formation of the C4b-C2 complex is presented. The results of this study also suggest that upon cleavage by C1s, C2a domains undergo conformational rotation while bound to C4b and the released C2b domains may remain folded together similar to as observed in the intact protein.
- Published
- 2009
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4. The crystal structure of C2a, the catalytic fragment of classical pathway C3 and C5 convertase of human complement.
- Author
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Krishnan V, Xu Y, Macon K, Volanakis JE, and Narayana SV
- Subjects
- Catalysis, Complement C3-C5 Convertases chemistry, Crystallography, Humans, Models, Molecular, Protein Conformation, Protein Structure, Tertiary, Complement C2a chemistry, Complement C3-C5 Convertases metabolism
- Abstract
The multi-domain serine protease C2 provides the catalytic activity for the C3 and C5- convertases of the classical and lectin pathways of complement activation. Formation of these convertases requires the Mg(2+)-dependent binding of C2 to C4b, and the subsequent cleavage of C2 by C1s or MASP2, respectively. The C-terminal fragment C2a consisting of a serine protease (SP) and a von Willebrand factor type A (vWFA) domain, remains attached to C4b, forming the C3 convertase, C4b2a. Here, we present the crystal structure of Mg(2+)-bound C2a to 1.9 A resolution in comparison to its homolog Bb, the catalytic subunit of the alternative pathway C3 convertase, C3bBb. Although the overall domain arrangement of C2a is similar to Bb, there are certain structural differences. Unexpectedly, the conformation of the metal ion-dependent adhesion site and the position of the alpha7 helix of the vWFA domain indicate a co-factor-bound or open conformation. The active site of the SP domain is in a zymogen-like inactive conformation. On the basis of these structural features, we suggest a model for the initial steps of C3 convertase assembly.
- Published
- 2007
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5. The structure of complement C3b provides insights into complement activation and regulation.
- Author
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Abdul Ajees A, Gunasekaran K, Volanakis JE, Narayana SV, Kotwal GJ, and Murthy HM
- Subjects
- Animals, Binding Sites, Crystallography, X-Ray, Humans, Models, Molecular, Protein Conformation, Rats, Structure-Activity Relationship, Complement Activation, Complement C3b chemistry, Complement C3b metabolism
- Abstract
The human complement system is an important component of innate immunity. Complement-derived products mediate functions contributing to pathogen killing and elimination. However, inappropriate activation of the system contributes to the pathogenesis of immunological and inflammatory diseases. Complement component 3 (C3) occupies a central position because of the manifold biological activities of its activation fragments, including the major fragment, C3b, which anchors the assembly of convertases effecting C3 and C5 activation. C3 is converted to C3b by proteolysis of its anaphylatoxin domain, by either of two C3 convertases. This activates a stable thioester bond, leading to the covalent attachment of C3b to cell-surface or protein-surface hydroxyl groups through transesterification. The cleavage and activation of C3 exposes binding sites for factors B, H and I, properdin, decay accelerating factor (DAF, CD55), membrane cofactor protein (MCP, CD46), complement receptor 1 (CR1, CD35) and viral molecules such as vaccinia virus complement-control protein. C3b associates with these molecules in different configurations and forms complexes mediating the activation, amplification and regulation of the complement response. Structures of C3 and C3c, a fragment derived from the proteolysis of C3b, have revealed a domain configuration, including six macroglobulin domains (MG1-MG6; nomenclature follows ref. 5) arranged in a ring, termed the beta-ring. However, because neither C3 nor C3c is active in complement activation and regulation, questions about function can be answered only through direct observations on C3b. Here we present a structure of C3b that reveals a marked loss of secondary structure in the CUB (for 'complement C1r/C1s, Uegf, Bmp1') domain, which together with the resulting translocation of the thioester domain provides a molecular basis for conformational changes accompanying the conversion of C3 to C3b. The total conformational changes make many proposed ligand-binding sites more accessible and create a cavity that shields target peptide bonds from access by factor I. A covalently bound N-acetyl-l-threonine residue demonstrates the geometry of C3b attachment to surface hydroxyl groups.
- Published
- 2006
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6. A novel human complement-related protein, C1r-like protease (C1r-LP), specifically cleaves pro-C1s.
- Author
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Ligoudistianou C, Xu Y, Garnier G, Circolo A, and Volanakis JE
- Subjects
- Amino Acid Sequence, Cloning, Molecular methods, Complement C1 metabolism, DNA, Complementary genetics, Humans, Liver chemistry, Molecular Sequence Data, Peptide Hydrolases metabolism, Recombinant Proteins metabolism, Serine Endopeptidases blood, Serine Endopeptidases metabolism, Complement C1s metabolism, Serine Endopeptidases genetics
- Abstract
The availability of the human genome sequence allowed us to identify a human complement-related, C1r-like protease gene (c1r-LP) located 2 kb centromeric of the C1r gene (c1r). Compared with c1r, c1r-LP carries a large deletion corresponding to exons 4-8 of c1r. The open reading frame of the C1r-LP cDNA predicts a 50 kDa modular protein displaying 52% amino acid residue identity with the corresponding regions of C1r and 75% identity with a previously described murine C1r-LP. The serine protease domain of C1r-LP, despite an overall similarity with the AGY group of complement serine proteases, has certain structural features characteristic of C2 and factor B, thus raising interesting evolutionary questions. Northern blotting demonstrated the expression of C1r-LP mRNA mainly in the liver and ELISA demonstrated the presence of the protein in human serum at a concentration of 5.5+/-0.9 microg/ml. Immunoprecipitation experiments failed to demonstrate an association of C1r-LP with the C1 complex in serum. Recombinant C1r-LP exhibits esterolytic activity against peptide thioesters with arginine at the P1 position, but its catalytic efficiency (kcat/K(m)) is lower than that of C1r and C1s. The enzymic activity of C1r-LP is inhibited by di-isopropyl fluorophosphate and also by C1 inhibitor, which forms stable complexes with the protease. Most importantly, C1r-LP also expresses proteolytic activity, cleaving pro-C1s into two fragments of sizes identical with those of the two chains of active C1s. Thus C1r-LP may provide a novel means for the formation of the classical pathway C3/C5 convertase.
- Published
- 2005
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7. Structural analysis of engineered Bb fragment of complement factor B: insights into the activation mechanism of the alternative pathway C3-convertase.
- Author
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Ponnuraj K, Xu Y, Macon K, Moore D, Volanakis JE, and Narayana SV
- Subjects
- Animals, Binding Sites, Complement C3 Convertase, Alternative Pathway, Complement C3b genetics, Complement Inactivator Proteins metabolism, Crystallography, X-Ray, Disulfides chemistry, Elapid Venoms metabolism, Enzyme Activation, Enzyme Inhibitors chemistry, Enzyme Inhibitors metabolism, Models, Molecular, Peptide Fragments genetics, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, von Willebrand Factor metabolism, Complement C3-C5 Convertases metabolism, Complement C3b chemistry, Complement C3b metabolism, Complement Pathway, Alternative physiology, Peptide Fragments chemistry, Peptide Fragments metabolism, Protein Conformation, von Willebrand Factor chemistry
- Abstract
The C-terminal fragment, Bb, of factor B combines with C3b to form the pivotal C3-convertase, C3bBb, of alternative complement pathway. Bb consists of a von Willebrand factor type A (vWFA) domain that is structurally similar to the I domains of integrins and a serine protease (SP) domain that is in inactive conformation. The structure of the C3bBb complex would be important in deciphering the activation mechanism of the SP domain. However, C3bBb is labile and not amenable to X-ray diffraction studies. We engineered a disulfide bond in the vWFA domain of Bb homologous to that shown to lock I domains in active conformation. The crystal structures of Bb(C428-C435) and its inhibitor complexes reveal that the adoption of the "active" conformation by the vWFA domain is not sufficient to activate the C3-convertase catalytic apparatus and also provide insights into the possible mode of C3-convertase activation.
- Published
- 2004
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8. Delayed lupus onset in (NZB x NZW)F1 mice expressing a human C-reactive protein transgene.
- Author
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Szalai AJ, Weaver CT, McCrory MA, van Ginkel FW, Reiman RM, Kearney JF, Marion TN, and Volanakis JE
- Subjects
- Animals, Antibodies, Antinuclear analysis, C-Reactive Protein metabolism, Capillaries ultrastructure, Complement C3 metabolism, Disease Models, Animal, Enzyme-Linked Immunosorbent Assay, Genetic Predisposition to Disease, Humans, Immunoglobulin G metabolism, Immunoglobulin M metabolism, Kidney Glomerulus blood supply, Kidney Glomerulus metabolism, Kidney Glomerulus ultrastructure, Longevity, Lupus Erythematosus, Systemic metabolism, Lupus Erythematosus, Systemic pathology, Mice, Mice, Inbred C57BL, Mice, Inbred NZB, Transgenes, C-Reactive Protein genetics, Lupus Erythematosus, Systemic genetics, Mice, Transgenic
- Abstract
Objective: Human C-reactive protein (CRP) binds apoptotic cells and alters blood clearance of injected chromatin in mice. To test whether CRP participates in the pathogenesis of systemic lupus erythematosus (SLE), we examined disease development in lupus-prone (NZB x NZW)F(1) (NZB/NZW) mice expressing a human CRP transgene (hCRPtg/BW)., Methods: Mortality was monitored, proteinuria was determined by dipstick, and serum levels of human CRP and anti-double-stranded DNA (anti-dsDNA) were determined by enzyme-linked immunosorbent assay in NZB/NZW and hCRPtg/BW mice. Thin sections of kidneys were analyzed by immunofluorescence microscopy to compare deposition of IgG, IgM, C3, and human CRP, and electron microscopy was used to reveal differences in ultrastructure. In situ hybridization was performed to detect human CRP messenger RNA expression., Results: The hCRPtg/BW mice had less proteinuria and longer survival than NZB/NZW mice. They also had lower IgM and higher IgG anti-dsDNA titers than NZB/NZW mice, although the differences were transient and small. In hCRPtg/BW mice, accumulation of IgM and IgG in the renal glomeruli was delayed, reduced, and more mesangial than in NZB/NZW mice, while end-stage accumulation of IgG, IgM, and C3 in the renal cortex was prevented. There was less glomerular podocyte fusion, basement membrane thickening, mesangial cell proliferation, and occlusion of capillary lumens in hCRPtg/BW mice, but dense deposits in the mesangium were increased. With disease progression in hCRPtg/BW mice, there was little rise in the plasma CRP level, but CRP in the kidneys became increasingly apparent due to local, disease-independent, age-related expression of the transgene., Conclusion: In hCRPtg/BW mice, CRP protects against SLE by increasing blood and mesangial clearance of immune complexes and by preventing their accumulation in the renal cortex.
- Published
- 2003
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9. A novel murine complement-related gene encoding a C1r-like serum protein.
- Author
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Circolo A, Garnier G, and Volanakis JE
- Subjects
- Amino Acid Sequence, Animals, Blood Proteins biosynthesis, COS Cells, Chlorocebus aethiops, Chromosome Mapping, DNA, Complementary genetics, Exons genetics, Gene Duplication, Humans, Introns genetics, Lipopolysaccharides pharmacology, Liver metabolism, Mice, Molecular Sequence Data, Multigene Family, Open Reading Frames, Protein Structure, Tertiary, RNA, Messenger genetics, Sequence Alignment, Sequence Deletion, Sequence Homology, Amino Acid, Sequence Homology, Nucleic Acid, Serine Endopeptidases biosynthesis, Blood Proteins genetics, Complement C1r genetics, Genes, Serine Endopeptidases genetics
- Abstract
C1r and C1s are highly specific serine proteases that initiate the classical pathway of complement activation. We recently demonstrated that, in the mouse, the genes encoding these proteins are duplicated. Analysis of the 5'-flanking region of the murine C1rA gene, the homologue of human C1r, revealed the presence of a novel gene encoding a C1r-like protein (c1r-LP). Although this gene carries a large deletion, it shows an overall structure similar to that of c1rA, suggesting that it may have arisen from a duplication of the C1r gene. The c1r-LP gene is expressed primarily in the liver, and is not regulated by lipopolysaccharide. The open reading frame of full-length cDNA clones encodes a pre-protein with a calculated molecular mass of 50.6 kDa which, except for an internal deletion of several modules, has a modular organization similar to that of C1r and shows 51% overall amino acid identity to corresponding regions of C1rA. Western blot analysis demonstrates the presence of C1r-LP in mouse serum. The serine protease domain of C1r-LP displays 60% amino acid residue identity to that of C1rA, however, certain atypical features of the active center, and primarily the absence of the activation/cleavage site, suggest that C1r-LP is either an atypical enzyme, or it lacks proteolytic activity, perhaps serving a regulatory function in the classical pathway.
- Published
- 2003
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10. Complement C1r and C1s genes are duplicated in the mouse: differential expression generates alternative isomorphs in the liver and in the male reproductive system.
- Author
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Garnier G, Circolo A, Xu Y, and Volanakis JE
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Cloning, Molecular, DNA, Complementary genetics, Exons, Gene Duplication, Humans, Introns, Isoenzymes genetics, Male, Mice, Molecular Sequence Data, Recombinant Proteins biosynthesis, Sequence Alignment, Sequence Homology, Amino Acid, Complement C1r genetics, Complement C1s genetics, Gene Expression Regulation, Genitalia, Male enzymology, Liver enzymology
- Abstract
C1r and C1s are the serine proteases that form the catalytic unit of the C1 complex, the first component of complement. In the present study, we found that the genes encoding murine C1r and C1s are duplicated. One set of these genes, referred to as c1rA and c1sA, are primarily expressed in the liver and are therefore the homologues of the human C1r and C1s genes. The other two genes, termed c1rB and c1sB, are expressed exclusively in male reproductive tissues, specifically the coagulating gland and the prostate. The predicted C1rB and C1sB proteins share 96 and 93% amino acid identity with C1rA and C1sA respectively. Most of the substitutions are clustered in the serine protease domains, suggesting differences in catalytic efficiencies and/or substrate specificities or alternatively adaptation to different physiological environments. The high homology of C1rB and C1sB with C1rA and C1sA in the non-catalytic regions indicates that they are probably capable of assembling the C1 complex. The expression of alternative genes encoding isomorphs of activating components of complement in male reproductive tissues raises the possibility of new mechanisms of complement activation in the male genital tract or of novel functions for complement proteases in reproduction.
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- 2003
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11. Determining alleles of the C2 gene by Southern Blotting.
- Author
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Zhu ZB and Volanakis JE
- Subjects
- DNA Probes genetics, DNA Probes metabolism, Humans, Major Histocompatibility Complex genetics, Polymorphism, Restriction Fragment Length, Alleles, Blotting, Southern methods, Complement C2 genetics
- Published
- 2003
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12. Structure, function and molecular genetics of human and murine C1r.
- Author
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Arlaud GJ, Gaboriaud C, Garnier G, Circolo A, Thielens NM, Budayova-Spano M, Fontecilla-Camps JC, and Volanakis JE
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- Animals, Complement C1s chemistry, Complement C1s physiology, Enzyme Activation, Humans, Mice, Protein Isoforms, Protein Structure, Secondary, Structure-Activity Relationship, Complement C1r chemistry, Complement C1r genetics, Complement C1r physiology, Complement Pathway, Classical physiology
- Abstract
C1r, the enzyme responsible for intrinsic activation of the C1 complex of complement, is a modular serine protease featuring an overall structural organization homologous to those of C1s and the mannan-binding lectin-associated serine proteases (MASPs). This review will initially summarize current information on the structure and function of C1r, with particular emphasis on the three-dimensional structure of its catalytic domain, which provides new insights into the activation mechanism of C1. The second part of this review will focus on recent discoveries dealing with a truncated, C1r-related protein, and the occurrence in the mouse of two isoforms, C1rA and C1rB, exhibiting tissue-specific expression patterns.
- Published
- 2002
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13. The three-dimensional structure of calcium-depleted human C-reactive protein from perfectly twinned crystals.
- Author
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Ramadan MA, Shrive AK, Holden D, Myles DA, Volanakis JE, DeLucas LJ, and Greenhough TJ
- Subjects
- Binding Sites, Calcium chemistry, Calcium metabolism, Crystallization, Crystallography, X-Ray, Humans, Ligands, Models, Molecular, Polymers chemistry, Protein Conformation, C-Reactive Protein chemistry
- Abstract
C-reactive protein is a member of the pentraxin family of oligomeric serum proteins which has been conserved through evolution, homologues having been found in every species in which they have been sought. Human C-reactive protein (hCRP) is the classical acute-phase reactant produced in large amounts in response to tissue damage and inflammation and is used almost universally as a clinical indicator of infection and inflammation. The role of hCRP in host defence and the calcium-dependent ligand-binding specificity of hCRP for phosphocholine moieties have long been recognized. In order to clarify the structural rearrangements associated with calcium binding, the reported affinity of calcium-depleted hCRP for polycations and other ligands, and the role of calcium in protection against denaturation and proteolysis, the structure of calcium-depleted hCRP has been determined by X-ray crystallography. Crystals of calcium-depleted hCRP are invariably twinned and those suitable for analysis are merohedral type II twins of point group 4 single crystals. The structure has been solved by molecular replacement using the calcium-bound hCRP structure [Shrive et al. (1996), Nature Struct. Biol. 3, 346-354]. It reveals two independent pentamers which form a face-to-face decamer across a dyad near-parallel to the twinning twofold axis. Cycles of intensity deconvolution, density modification (tenfold NCS) and model building, eventually including refinement, give a final R factor of 0.19 (R(free) = 0.20). Despite poor definition in some areas arising from the limited resolution of the data and from the twinning and disorder, the structure reveals the probable mode of twinning and the conformational changes, localized in one of the calcium-binding loops, which accompany calcium binding.
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- 2002
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14. Calnexin is associated with and induced by overexpressed human complement protein C2.
- Author
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Tsukamoto H, Tousson A, Circolo A, Marchase RB, and Volanakis JE
- Subjects
- Animals, CHO Cells metabolism, COS Cells metabolism, Calcium-Binding Proteins analysis, Calnexin, Chlorocebus aethiops, Complement Factor B metabolism, Cricetinae, Fluorescent Antibody Technique, Humans, Molecular Chaperones analysis, Transfection, Up-Regulation, Calcium-Binding Proteins metabolism, Complement C2 metabolism, Molecular Chaperones metabolism
- Abstract
C2 is a serum glycoprotein that is essential for activation of the classical and lectin pathways of the complement system. We reported previously that in transiently transfected COS cells, C2 accumulates in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC). Transfection with a cDNA corresponding to a variant C2 mRNA in which exon 17 is spliced out, C2Delta(17), resulted in retention of the mutant polypeptide in the ER. We now show that calnexin, a lectin-like chaperone, colocalizes with wild-type (wt) C2 and C2Delta(17). Biosynthetic labeling and sequential immunoprecipitation experiments indicated that colocalization is due to a physical association between calnexin and C2. Immunofluorescence analysis indicated that calnexin was upregulated in cells transfected with either C2 species. Upregulation of calnexin was not affected by castanospermine, which inhibits glucosidases I and II. However, castanospermine inhibited translocation of calnexin to the ERGIC in wt C2 transfected cells. Upregulation of calnexin was also observed in cells transfected with the complement protein factor B, a glycoprotein with extensive structural and functional similarities to C2, but not in cells transfected with complement proteins C3 or factor D, which have no structural similarity to C2, and low or no glycan content, respectively. Calnexin upregulation by transfection with C2 or factor B, but not factor D, was also demonstrated by quantitative analysis of calnexin immunoprecipitates from biosynthetically labeled cells. Increased calnexin expression by overexpressed C2 and factor B appears to be triggered either by the high glycan content of these proteins or, since it also occurs in the presence of castanospermine, by shared features of the structure of these two proteins., (Copyright 2002 Wiley-Liss, Inc.)
- Published
- 2002
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15. The role of complement in innate and adaptive immunity.
- Author
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Volanakis JE
- Subjects
- Animals, Antibody Formation immunology, Humans, Immunity, Active immunology, Immunity, Innate immunology, Complement System Proteins immunology
- Published
- 2002
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16. Complement activation in factor D-deficient mice.
- Author
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Xu Y, Ma M, Ippolito GC, Schroeder HW Jr, Carroll MC, and Volanakis JE
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- Animals, Complement C3 metabolism, Complement Factor B metabolism, Complement Factor D genetics, Elapid Venoms pharmacology, Erythrocytes immunology, Female, Kinetics, Magnesium pharmacology, Male, Mice, Mice, Knockout, Opsonin Proteins metabolism, Rabbits, Streptococcus pneumoniae immunology, Zymosan pharmacology, Complement Factor D deficiency, Complement Pathway, Alternative
- Abstract
To assess the contribution of the alternative pathway in complement activation and host defense and its possible role in the regulation of systemic energy balance in vivo, factor D-deficient mice were generated by gene targeting. The mutant mice have no apparent abnormality in development and their body weights are similar to those of factor D-sufficient littermates. Complement activation could not be initiated in the serum of deficient mice by the alternative pathway activators rabbit erythrocytes and zymosan. Surprisingly, injection of cobra venom factor (CVF) caused a profound and reproducible reduction in serum C3 levels, whereas, as expected, there was no C3 reduction in factor B-deficient mice treated similarly. Studies of C3 and factor B activation in vitro by CVF demonstrated that in factor D-deficient serum the alpha chain of C3 was cleaved gradually over a period of 60 min without detectable cleavage of factor B. CVF-dependent C3 cleavage in the deficient serum required the presence of Mg(2+), whereas in normal mouse serum the presence of divalent cations was not required. These results suggest that in mouse proteolytic cleavage of factor B by factor D is not an absolute requirement for the zymogen to active enzyme conformational transition of CVF-bound factor B. Kinetics of opsonization of Streptococcus pneumoniae by C3 fragments was much slower in factor D-deficient serum, suggesting a significant contribution of the alternative pathway to antibacterial host defense early after infection.
- Published
- 2001
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17. Human C-reactive protein: expression, structure, and function.
- Author
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Volanakis JE
- Subjects
- C-Reactive Protein chemistry, Complement Pathway, Classical, Gene Expression Regulation, Humans, Phagocytes, Protein Conformation, C-Reactive Protein physiology
- Abstract
C-reactive protein (CRP) is an acute-phase protein featuring a homopentameric structure and Ca-binding specificity for phosphocholine (PCh). Expression of CRP is regulated mainly at the transcriptional level with interleukin-6 being the principal inducer of the gene during the acute phase. The crystal structure of CRP has been determined and the topology and chemical composition of its ligand-binding site determined. The wide distribution of PCh in polysaccharides of pathogens and in cellular membranes allows CRP to recognize a range of pathogenic targets as well as membranes of damaged and necrotic host cells. CRP bound to a multivalent ligand can efficiently initiate the assembly of a C3 convertase through the classical pathway and thus decorate the surface of the ligand with opsonic complement fragments. However, the protein does not favor the formation of a C5 convertase and therefore, CRP-initiated complement activation does not mediate acute inflammatory reactions and membrane damage. CRP also interacts with Fc receptors on phagocytic cells and acts as an opsonin. Other CRP-initiated signals through interactions with neutrophil Fc receptors have an overall anti-inflammatory effect. Thus, the main biological function of CRP appears to be host defense against bacterial pathogens and clearance of apoptotic and necrotic cells. Protection from lethal bacterial infection, from complement-induced alveolitis, and from endotoxemia has been confirmed in vivo using transgenic mice. Additional functions, including participation in atherogenesis and pathogenesis of myocardial injury after myocardial infarction have been reported. However, the weight of the evidence is that CRP like other acute-phase proteins is a component first line of innate host defense.
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- 2001
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18. Structural biology of the alternative pathway convertase.
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Xu Y, Narayana SV, and Volanakis JE
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- Animals, Binding Sites, Catalytic Domain, Complement C3 Convertase, Alternative Pathway, Complement C3-C5 Convertases physiology, Complement C3b immunology, Complement C3b metabolism, Complement Factor B metabolism, Complement Factor D physiology, Enzyme Activation, Enzyme Precursors chemistry, Mice, Models, Molecular, Peptide Fragments metabolism, Protein Binding, Protein Conformation, Protein Structure, Tertiary, Serine Endopeptidases chemistry, Substrate Specificity, von Willebrand Factor chemistry, Complement C3-C5 Convertases biosynthesis, Complement C3-C5 Convertases chemistry, Complement Factor D chemistry, Complement Pathway, Alternative
- Abstract
Complement convertases are bimolecular complexes expressing protease activity only against C3 and C5. Their action is necessary for production of the biological activities of the complement system. Formation of these complexes proceeds through sequential protein-protein interactions and proteolytic cleavages of high specificity. Recent structural, mutational and functional data on factors D and B have significantly enhanced our understanding of the assembly, action, and regulation of the alternative pathway convertase. These processes were shown to depend critically on conformational changes, only some of which are reversible. The need for such changes is dictated by the zymogen-like configurations of the active centers of these unique serine proteases. The structural determinants of some of these changes have been defined from structural and mutational analyses of the two enzymes. Transition of factor D from the zymogen-like to the catalytically active conformation is completely reversible, while the active conformation of the catalytic center of the Bb fragment of factor B is irreversibly attenuated to a great extent on dissociation of the convertase complex. Both mechanisms contribute to the regulation of the proteolytic activity of these enzymes. Additional studies are necessary for a complete description of the elegant mechanisms mediating these processes.
- Published
- 2001
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19. Topology and structure of the C1q-binding site on C-reactive protein.
- Author
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Agrawal A, Shrive AK, Greenhough TJ, and Volanakis JE
- Subjects
- Amino Acid Substitution genetics, Amino Acid Substitution immunology, Asparagine genetics, Binding Sites genetics, C-Reactive Protein genetics, Complement Activation genetics, Glutamic Acid genetics, Histidine genetics, Humans, Models, Molecular, Mutagenesis, Site-Directed, Phosphorylcholine metabolism, Polysaccharides, Bacterial metabolism, Serum Albumin, Bovine metabolism, Streptococcus pneumoniae immunology, Threonine genetics, Tyrosine genetics, C-Reactive Protein chemistry, C-Reactive Protein metabolism, Complement C1q metabolism
- Abstract
The host defense functions of human C-reactive protein (CRP) depend to a great extent on its ability to activate the classical complement pathway. The aim of this study was to define the topology and structure of the CRP site that binds C1q, the recognition protein of the classical pathway. We have previously reported that residue Asp(112) of CRP plays a major role in the formation of the C1q-binding site, while the neighboring Lys(114) hinders C1q binding. The three-dimensional structure of CRP shows the presence of a deep, extended cleft in each protomer on the face of the pentamer opposite that containing the phosphocholine-binding sites. Asp(112) is part of this marked cleft that is deep at its origin but becomes wider and shallower close to the inner edge of the protomer and the central pore of the pentamer. The shallow end of the pocket is bounded by the 112-114 loop, residues 86-92 (the inner loop), the C terminus of the protomer, and the C terminus of the pentraxin alpha-helix 169-176, particularly Tyr(175). Mutational analysis of residues participating in the formation of this pocket demonstrates that Asp(112) and Tyr(175) are important contact residues for C1q binding, that Glu(88) influences the conformational change in C1q necessary for complement activation, and that Asn(158) and His(38) probably contribute to the correct geometry of the binding site. Thus, it appears that the pocket at the open end of the cleft is the C1q-binding site of CRP.
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- 2001
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20. Human C-reactive protein is protective against fatal Salmonella enterica serovar typhimurium infection in transgenic mice.
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Szalai AJ, VanCott JL, McGhee JR, Volanakis JE, and Benjamin WH Jr
- Subjects
- Animals, Antibodies, Bacterial blood, Bacteremia microbiology, C-Reactive Protein genetics, C-Reactive Protein metabolism, Humans, Liver microbiology, Male, Mice, Mice, Inbred C57BL, Mice, Inbred DBA, Mice, Transgenic, Salmonella Infections, Animal microbiology, Salmonella Infections, Animal mortality, Salmonella typhimurium isolation & purification, Spleen microbiology, Transgenes, C-Reactive Protein immunology, Salmonella Infections, Animal immunology, Salmonella typhimurium immunology
- Abstract
C-reactive protein (CRP) is an acute-phase protein with a well-known association with infection and other inflammatory conditions. We have shown that expression of human CRP by CRP transgenic (CRPtg) mice is protective against lethal infection by Streptococcus pneumoniae, an effect likely mediated by CRP's ability to bind to this gram-positive pathogen. In the present study we tested whether CRPtg mice are resistant to infection with Salmonella enterica serovar Typhimurium, a gram-negative pathogen that causes the murine equivalent of typhoid fever. CRPtg mice experimentally infected with a virulent Typhimurium strain lived longer and had significantly lower mortality than their non-tg littermates. The greater resistance of CRPtg mice could be attributed to significantly increased early (0 to 4 h) blood clearance of salmonellae and significantly decreased numbers of bacteria in the liver and spleen on day 7 postinfection. In addition, 14 days after infection with an avirulent Salmonella strain, the serum titer of anti-Salmonella immunoglobulin G antibodies was higher in CRPtg than non-tg mice. This study provides unequivocal evidence that CRP plays an important role in vivo in host defense against salmonellae during the early stages of infection. In addition, as the beneficial effect of CRP includes enhancement of the host's humoral immune response, CRP may also contribute indirectly to host defense during later stages of infection.
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- 2000
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21. Complement-dependent acute-phase expression of C-reactive protein and serum amyloid P-component.
- Author
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Szalai AJ, van Ginkel FW, Wang Y, McGhee JR, and Volanakis JE
- Subjects
- Acute-Phase Reaction genetics, Animals, C-Reactive Protein genetics, Complement Activation, Complement C5a administration & dosage, Complement Inactivator Proteins administration & dosage, Complement System Proteins deficiency, Dose-Response Relationship, Immunologic, Elapid Venoms administration & dosage, Gene Expression Regulation immunology, Humans, Injections, Intraperitoneal, Interleukin-1 physiology, Interleukin-6 physiology, Lipopolysaccharides administration & dosage, Male, Mice, Mice, Inbred C57BL, Mice, Inbred DBA, Mice, Knockout, Mice, Transgenic, Serum Amyloid P-Component genetics, Transgenes immunology, Up-Regulation genetics, Up-Regulation immunology, Acute-Phase Reaction immunology, C-Reactive Protein biosynthesis, Complement System Proteins physiology, Serum Amyloid P-Component biosynthesis
- Abstract
The acute-phase response (APR) is regulated by TNF-alpha, IL-1beta, and IL-6 acting alone, in combination, or in concert with hormones. The anaphylotoxin C5a, generated during complement activation, induces in vitro the synthesis of these cytokines by leukocytes and of acute-phase proteins by HepG2 cells. However, there is no clear evidence for a role of C5a or any other complement activation product in regulation of the APR in vivo. In this study, using human C-reactive protein (CRP) transgenic mice deficient in C3 or C5, we investigated whether complement activation contributes to induction of the acute-phase proteins CRP and serum amyloid P-component (SAP). Absence of C3 or C5 resulted in decreased LPS-induced up-regulation of the CRP transgene and the mouse SAP gene. Also, LPS induced both the IL-1beta and IL-6 genes in normocomplementemic mice, but in complement-deficient mice it significantly induced only IL-6. Like LPS injection, activation of complement by cobra venom factor led to significant elevation of serum CRP and SAP in normocomplementemic mice but not in complement-deficient mice. Injection of recombinant human C5a into human CRP transgenic mice induced the IL-1beta gene and caused significant elevation of both serum CRP and SAP. However, in human CRP transgenic IL-6-deficient mice, recombinant human C5a did not induce the CRP nor the SAP gene. Based on these data, we conclude that during the APR, C5a generated as a consequence of complement activation acts in concert with IL-6 and/or IL-1beta to promote up-regulation of the CRP and SAP genes.
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- 2000
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22. High prevalence of complement component C6 deficiency among African-Americans in the south-eastern USA.
- Author
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Zhu Z, Atkinson TP, Hovanky KT, Boppana SB, Dai YL, Densen P, Go RC, Jablecki JS, and Volanakis JE
- Subjects
- Amino Acid Sequence, Base Sequence, Gene Deletion, Humans, Incidence, Meningitis, Meningococcal epidemiology, Meningitis, Meningococcal genetics, Meningitis, Meningococcal immunology, Molecular Sequence Data, Neisseria meningitidis immunology, Point Mutation, Prevalence, Southeastern United States epidemiology, White People genetics, Black or African American, Black People genetics, Complement C6 deficiency, Complement C6 genetics
- Abstract
Complement component C6 is a part of the membrane attack complex that forms a pore-like structure in cell membranes following complement activation. Deficiency of terminal complement components including C6 predisposes individuals to infection with Neisseriae. Using polymerase chain reaction/single-strand conformation polymorphism analysis followed by DNA sequencing, we screened genomic DNA from 200 randomly chosen blacks and an equal number from whites for three loss-of-function C6 mutations. Ten blacks and two whites were found to be heterozygous for one of the mutations. Two of the mutations, 1195delC and 1936delG, were found exclusively in black individuals. A third previously undescribed mutation, 878delA, was found at equal frequency among the two groups. The difference between the two groups was significant (P = 0.027), indicating that C6 deficiency due to these three mutations is more common among blacks than whites in the local area, principally Jefferson County, Alabama. In addition, three previously undescribed point mutations, two of which result in amino acid substitutions, were identified within exon 6. A review of the county health department records over the past 6 years revealed a higher incidence of meningococcal meningitis in blacks due to serogroups Y and W-135 which paralleled the difference in the estimated prevalence of C6 deficiency. Among black residents of the county (n = 235 598) there were 15 cases of meningitis due to these two serogroups, compared with two cases in the white population (n = 422 604) (P = 0.002). We conclude that C6 deficiency is more common among blacks than whites in the south-eastern United States, with a frequency approaching 1 in 1600 black individuals.
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- 2000
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23. New structural motifs on the chymotrypsin fold and their potential roles in complement factor B.
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Jing H, Xu Y, Carson M, Moore D, Macon KJ, Volanakis JE, and Narayana SV
- Subjects
- Amino Acid Sequence, Binding Sites, Chymotrypsin metabolism, Chymotrypsinogen chemistry, Complement C2 chemistry, Complement Factor B metabolism, Conserved Sequence, Crystallography, X-Ray, Models, Molecular, Molecular Sequence Data, Mutagenesis, Protein Conformation, Protein Folding, Protein Structure, Secondary, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Sequence Alignment, Sequence Deletion, Sequence Homology, Amino Acid, Chymotrypsin chemistry, Complement Factor B chemistry, Serine Endopeptidases chemistry
- Abstract
Factor B and C2 are two central enzymes for complement activation. They are multidomain serine proteases and require cofactor binding for full expression of proteolytic activities. We present a 2.1 A crystal structure of the serine protease domain of factor B. It shows a number of structural motifs novel to the chymotrypsin fold, which by sequence homology are probably present in C2 as well. These motifs distribute characteristically on the protein surface. Six loops surround the active site, four of which shape substrate-binding pockets. Three loops next to the oxyanion hole, which typically mediate zymogen activation, are much shorter or absent. Three insertions including the linker to the preceding domain bulge from the side opposite to the active site. The catalytic triad and non-specific substrate-binding site display active conformations, but the oxyanion hole displays a zymogen-like conformation. The bottom of the S1 pocket has a negative charge at residue 226 instead of the typical 189 position. These unique structural features may play different roles in domain-domain interaction, cofactor binding and substrate binding.
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- 2000
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24. Mutational analysis of the primary substrate specificity pocket of complement factor B. Asp(226) is a major structural determinant for p(1)-Arg binding.
- Author
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Xu Y, Circolo A, Jing H, Wang Y, Narayana SV, and Volanakis JE
- Subjects
- Amino Acid Sequence, Asparagine, Aspartic Acid, Catalytic Domain, Complement C3-C5 Convertases metabolism, Complement Factor B genetics, Complement Factor D metabolism, Elapid Venoms metabolism, Hemolysis, Models, Molecular, Molecular Sequence Data, Mutation, Sequence Homology, Amino Acid, Substrate Specificity, Complement Factor B metabolism
- Abstract
Factor B is a serine protease, which despite its trypsin-like specificity has Asn instead of the typical Asp at the bottom of the S(1) pocket (position 189, chymotrypsinogen numbering). Asp residues are present at positions 187 and 226 and either one could conceivably provide the negative charge for binding the P(1)-Arg of the substrate. Determination of the crystal structure of the factor B serine protease domain has revealed that the side chain of Asp(226) is within the S(1) pocket, whereas Asp(187) is located outside the pocket. To investigate the possible role of these atypical structural features in substrate binding and catalysis, we constructed a panel of mutants of these residues. Replacement of Asp(187) caused moderate (50-60%) decrease in hemolytic activity, compared with wild type factor B, whereas replacement of Asn(189) resulted in more profound reductions (71-95%). Substitutions at these two positions did not significantly affect assembly of the alternative pathway C3 convertase. In contrast, elimination of the negative charge from Asp(226) completely abrogated hemolytic activity and also affected formation of the C3 convertase. Kinetic analyses of the hydrolysis of a P(1)-Arg containing thioester by selected mutants confirmed that residue Asp(226) is a primary structural determinant for P(1)-Arg binding and catalysis.
- Published
- 2000
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25. The Arthus reaction in rodents: species-specific requirement of complement.
- Author
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Szalai AJ, Digerness SB, Agrawal A, Kearney JF, Bucy RP, Niwas S, Kilpatrick JM, Babu YS, and Volanakis JE
- Subjects
- Animals, Arthus Reaction pathology, Arthus Reaction prevention & control, Complement Activation drug effects, Complement C1 Inactivator Proteins pharmacology, Complement Factor D antagonists & inhibitors, Complement System Proteins immunology, Complement System Proteins metabolism, Dose-Response Relationship, Immunologic, Guinea Pigs, Humans, Kinetics, Mice, Mice, Inbred C57BL, Rats, Rats, Sprague-Dawley, Serine Proteinase Inhibitors pharmacology, Species Specificity, Arthus Reaction immunology, Complement System Proteins physiology, Thiophenes pharmacology
- Abstract
We induced reverse passive Arthus (RPA) reactions in the skin of rodents and found that the contribution of complement to immune complex-mediated inflammation is species specific. Complement was found to be necessary in rats and guinea pigs but not in C57BL/6J mice. In rats, within 4 h after initiation of an RPA reaction, serum alternative pathway hemolytic titers decreased significantly below basal levels, whereas classical pathway titers were unchanged. Thus the dermal reaction proceeds coincident with systemic activation of complement. The serine protease inhibitor BCX 1470, which blocks the esterolytic and hemolytic activities of the complement enzymes Cls and factor D in vitro, also blocked development of RPA-induced edema in the rat. These data support the proposal that complement-mediated processes are of major importance in the Arthus reaction in rats and guinea pigs, and suggest that BCX 1470 will be useful as an anti-inflammatory agent in diseases where complement activation is known to be detrimental.
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- 2000
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26. Genetic disruption of the murine complement C3 promoter region generates deficient mice with extrahepatic expression of C3 mRNA.
- Author
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Circolo A, Garnier G, Fukuda W, Wang X, Hidvegi T, Szalai AJ, Briles DE, Volanakis JE, Wetsel RA, and Colten HR
- Subjects
- Animals, Base Sequence, Blotting, Northern, Chloramphenicol O-Acetyltransferase biosynthesis, Chloramphenicol O-Acetyltransferase genetics, Complement C3 biosynthesis, Complement C3 immunology, DNA genetics, Exons, Female, Gene Deletion, Gene Expression, Gene Targeting, Immunity, Innate, In Situ Hybridization, Liver metabolism, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Molecular Sequence Data, Organ Specificity, Pneumococcal Infections immunology, RNA, Messenger biosynthesis, RNA, Messenger genetics, Recombinant Fusion Proteins biosynthesis, Recombinant Fusion Proteins genetics, Streptococcus pneumoniae, Complement C3 deficiency, Complement C3 genetics, Promoter Regions, Genetic genetics, RNA, Messenger metabolism
- Abstract
Genetic deficiencies of the complement protein C3 occur naturally in humans and animal models and have been induced in mice by targeted deletion of the C3 gene. The study of these deficiencies has provided evidence for C3 functions in immune responses. C3 deficient mice were generated by replacing the 5'-flanking region of the C3 gene with the neomycin-resistance (neo) gene. Serum from these mice had no detectable C3 protein or complement activity. Challenge with Streptococcus pneumoniae revealed approximately 2000-fold increase in bacteremia as compared to littermate controls. C3 mRNA was absent in the liver, but it was detected in the lung, kidney, fat tissue, heart and spleen. Metabolic labeling of the lung tissue and peritoneal macrophages showed synthesis of pro-C3, but no post-synthetic intracellular processing of the protein and no secretion of mature C3. cDNA analysis at the cap site indicated that extrahepatic transcription of the targeted gene was initiated in the neo cassette, close to the C3/neo junction and predicted a primary translation product lacking the leader peptide. The data indicate that these mice provide a good animal model for the study of complete C3 deficiencies and a potential probe for tissue-specific C3 gene regulatory elements.
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- 1999
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27. The evolution, structure, biology and pathophysiology of complement.
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Lambris JD, Reid KB, and Volanakis JE
- Subjects
- Anaphylatoxins chemistry, Anaphylatoxins genetics, Anaphylatoxins physiology, Animals, Antibody Formation, Complement Activation, Complement Membrane Attack Complex chemistry, Complement Membrane Attack Complex genetics, Complement Membrane Attack Complex physiology, Evolution, Molecular, Genetic Variation, Humans, Molecular Structure, Complement System Proteins chemistry, Complement System Proteins genetics, Complement System Proteins physiology
- Published
- 1999
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28. C-reactive protein: structural biology and host defense function.
- Author
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Szalai AJ, Agrawal A, Greenhough TJ, and Volanakis JE
- Subjects
- Animals, Binding Sites, C-Reactive Protein metabolism, Complement System Proteins metabolism, Complement System Proteins physiology, Humans, Ligands, Protein Conformation, C-Reactive Protein chemistry, C-Reactive Protein immunology
- Abstract
Human C-reactive protein is a Ca2+-binding acute phase-protein with binding specificity for phosphocholine. Recent crystallographic and mutagenesis studies have provided a solid understanding of the structural biology of the protein, while experiments using transgenic mice have confirmed its host-defense function. The protein consists of five identical protomers in cyclic symmetry. On one face of each protomer there is a binding site for phosphocholine consisting of two Ca2+ ions that ligate the phosphate group and a hydrophobic pocket that accommodates the methyl groups of phosphocholine. On the opposite face is a deep cleft formed by parts of the N and C termini and bordered by an alpha-helix. Mutational studies indicate that the C1q-binding site of the molecule is located at the open end of this cleft with Asp112 and Tyr175 representing contact residues. Using human C-reactive protein transgenic mice, we investigated the host defense functions of the protein. Transgenic mice infected with Streptococcus pneumoniae had increased lifespan and lowered mortality compared to wild-type mice. This was attributable to an up to 400-fold reduction in bacteremia mediated mainly by the interaction of C-reactive protein with complement. A complement-independent host protective effect was also demonstrated.
- Published
- 1999
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29. Structural basis of profactor D activation: from a highly flexible zymogen to a novel self-inhibited serine protease, complement factor D.
- Author
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Jing H, Macon KJ, Moore D, DeLucas LJ, Volanakis JE, and Narayana SV
- Subjects
- Amino Acid Sequence, Binding Sites, Crystallography, X-Ray, Enzyme Activation, Humans, Models, Molecular, Molecular Sequence Data, Protein Conformation, Protein Processing, Post-Translational, Protein Structure, Secondary, Recombinant Proteins chemistry, Sequence Homology, Amino Acid, Complement Factor D chemistry, Enzyme Precursors chemistry, Protein Precursors chemistry, Serine Endopeptidases chemistry
- Abstract
The crystal structure of profactor D, determined at 2.1 A resolution with an Rfree and an R-factor of 25.1 and 20.4%, respectively, displays highly flexible or disordered conformation for five regions: N-22, 71-76, 143-152, 187-193 and 215-223. A comparison with the structure of its mature serine protease, complement factor D, revealed major conformational changes in the similar regions. Comparisons with the zymogen-active enzyme pairs of chymotrypsinogen, trypsinogen and prethrombin-2 showed a similar distribution of the flexible regions. However, profactor D is the most flexible of the four, and its mature enzyme displays inactive, self-inhibited active site conformation. Examination of the surface properties of the N-terminus-binding pocket indicates that Ile16 may play the initial positioning role for the N-terminus, and Leu17 probably also helps in inducing the required conformational changes. This process, perhaps shared by most chymotrypsinogen-like zymogens, is followed by a factor D-unique step, the re-orientation of an external Arg218 to an internal position for salt-bridging with Asp189, leading to the generation of the self-inhibited factor D.
- Published
- 1999
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30. Structures of native and complexed complement factor D: implications of the atypical His57 conformation and self-inhibitory loop in the regulation of specific serine protease activity.
- Author
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Jing H, Babu YS, Moore D, Kilpatrick JM, Liu XY, Volanakis JE, and Narayana SV
- Subjects
- Binding Sites, Coumarins chemistry, Coumarins metabolism, Crystallography, X-Ray, Enzyme Inhibitors chemistry, Enzyme Inhibitors metabolism, Histidine, Isocoumarins, Models, Molecular, Oxazines metabolism, Protein Conformation, Serine Endopeptidases chemistry, Serine Endopeptidases metabolism, Serine Proteinase Inhibitors chemistry, Serine Proteinase Inhibitors metabolism, Water, Complement Factor D chemistry, Complement Factor D metabolism, Oxazines chemistry
- Abstract
Factor D is a serine protease essential for the activation of the alternative pathway of complement. The structures of native factor D and a complex formed with isatoic anhydride inhibitor were determined at resolution of 2.3 and 1.5 A, respectively, in an isomorphous monoclinic crystal form containing one molecule per asymmetric unit. The native structure was compared with structures determined previously in a triclinic cell containing two molecules with different active site conformations. The current structure shows greater similarity with molecule B in the triclinic cell, suggesting that this may be the dominant factor D conformation in solution. The major conformational differences with molecule A in the triclinic cell are located in four regions, three of which are close to the active site and include some of the residues shown to be critical for factor D catalytic activity. The conformational flexibility associated with these regions is proposed to provide a structural basis for the previously proposed substrate-induced reversible conformational changes in factor D. The high-resolution structure of the factor D/isatoic anhydride complex reveals the binding mode of the mechanism-based inhibitor. The higher specificity towards factor D over trypsin and thrombin is based on hydrophobic interactions between the inhibitor benzyl ring and the aliphatic side-chain of Arg218 that is salt bridged with Asp189 at the bottom of the primary specificity (S1) pocket. Comparison of factor D structural variants with other serine protease structures revealed the presence of a unique "self-inhibitory loop". This loop (214-218) dictates the resting-state conformation of factor D by (1) preventing His57 from adopting active tautomer conformation, (2) preventing the P1 to P3 residues of the substrate from forming anti-parallel beta-sheets with the non-specific substrate binding loop, and (3) blocking the accessibility of Asp189 to the positive1y charged P1 residue of the substrate. The conformational switch from resting-state to active-state can only be induced by the single macromolecular substrate, C3b-bound factor B. This self-inhibitory mechanism is highly correlated with the unique functional properties of factor D, which include high specificity toward factor B, low esterolytic activity toward synthetic substrates, and absence of regulation by zymogen and serpin-like or other natural inhibitors in blood., (Copyright 1998 Academic Press.)
- Published
- 1998
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31. A novel type II complement C2 deficiency allele in an African-American family.
- Author
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Zhu ZB, Atkinson TP, and Volanakis JE
- Subjects
- Amino Acid Substitution genetics, Black People genetics, Child, Complement C2 metabolism, Cysteine genetics, Exons, Glycine genetics, Humans, Male, Mutation, Pedigree, Polymorphism, Genetic, Protein Biosynthesis, Tyrosine genetics, Black or African American, Alleles, Complement C2 deficiency, Complement C2 genetics
- Abstract
A 9-yr-old African-American male presenting with severe recurrent pyogenic infections was found to have C2 deficiency (C2D). Analysis of his genomic DNA demonstrated that he carried one type I C2D allele associated with the HLA-A25, B18, DR15 haplotype. Screening all 18 exons of the C2 gene by exon-specific PCR/single-strand conformation polymorphism indicated abnormal bands in exons 3, 7, and 6, the latter apparently caused by the 28-bp deletion of the typical type I C2D allele. Nucleotide (nt) sequencing of the PCR-amplified exons 3 and 7 revealed a heterozygous G to A transition at nt 392, causing a C111Y mutation, and a heterozygous G to C transversion at nt 954, causing a E298D mutation and a polymorphic MaeII site. Cys111 is the invariable third half-cystine of the second complement control protein module of C2. Pulse-chase biosynthetic labeling experiments indicated that the C111Y mutant C2 was retained by transfected COS cells and secreted only in minimal amounts. Therefore, this mutation causes a type II C2D. In contrast, the E298D mutation affected neither the secretion of C2 from transfected cells nor its specific hemolytic activity. Analysis of genomic DNA from members of the patient's family indicated that 1) the proband as well as one of his sisters inherited the type I C2D allele from their father and the novel type II C2D allele from their mother; 2) the polymorphic MaeII site caused by the G954C transversion is associated with the type I C2D allele; and 3) the novel C111Y mutation is associated in this family with the haplotype HLA-A28, B58, DR12.
- Published
- 1998
32. Testosterone and IL-6 requirements for human C-reactive protein gene expression in transgenic mice.
- Author
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Szalai AJ, van Ginkel FW, Dalrymple SA, Murray R, McGhee JR, and Volanakis JE
- Subjects
- Age Factors, Animals, C-Reactive Protein biosynthesis, C-Reactive Protein metabolism, Drug Combinations, Female, Humans, Injections, Intraperitoneal, Interleukin-1 administration & dosage, Interleukin-6 administration & dosage, Lipopolysaccharides administration & dosage, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Mice, Transgenic, Serum Amyloid P-Component biosynthesis, Sex Factors, Testosterone administration & dosage, Transgenes immunology, C-Reactive Protein genetics, Gene Expression Regulation immunology, Interleukin-6 physiology, Testosterone physiology
- Abstract
In vitro, IL-6 is the main inducer of the human C-reactive protein (CRP) gene, and IL-1 and steroids can enhance this effect. However, in mice, IL-6 is necessary but not sufficient for induction of the human CRP transgene, and testosterone is required for its constitutive expression by males. To examine the relative contributions of testosterone and IL-6 in the regulation of CRP gene expression, we produced CRP-transgenic (CRPtg), IL-6-deficient (IL-6-/-) mice. Male CRPtg/IL-6-/- mice expressed CRP constitutively, but CRP levels were not increased after injection of LPS. However, acute-phase CRP levels were attained after injection of IL-6. In contrast, female CRPtg/IL-6-/- mice did not express CRP constitutively or after administration of LPS, IL-6, IL-1, or IL-6 plus IL-1. Like males, testosterone-treated CRPtg/IL-6-/- females expressed CRP constitutively, and their transgene responded to injection of IL-6. The endogenous acute-phase protein serum amyloid P (SAP) was expressed constitutively equally by male and female IL-6-/- mice, responded minimally to LPS, and did not respond to either IL-6 or IL-1 alone. Acute-phase levels of SAP were induced in IL-6-/- mice by injection of IL-6 together with IL-1 or LPS. We conclude that in vivo, both constitutive and IL-6-dependent acute-phase expression of the CRP transgene require testosterone. In contrast, testosterone is not required for expression of the SAP gene, which requires IL-1 plus IL-6 for acute-phase induction.
- Published
- 1998
33. Susceptibility locus for IgA deficiency and common variable immunodeficiency in the HLA-DR3, -B8, -A1 haplotypes.
- Author
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Schroeder HW Jr, Zhu ZB, March RE, Campbell RD, Berney SM, Nedospasov SA, Turetskaya RL, Atkinson TP, Go RC, Cooper MD, and Volanakis JE
- Subjects
- Adult, Disease Susceptibility, Female, Genetic Markers, Haplotypes genetics, Humans, Infant, Male, Pedigree, Common Variable Immunodeficiency genetics, HLA-A1 Antigen genetics, HLA-B8 Antigen genetics, HLA-DR3 Antigen genetics, IgA Deficiency genetics
- Abstract
Background: A common genetic basis for IgA deficiency (IgAD) and common variable immunodeficiency (CVID) is suggested by their occurrence in members of the same family and the similarity of the underlying B cell differentiation defects. An association between IgAD/CVID and HLA alleles DR3, B8, and A1 has also been documented. In a search for the gene(s) in the major histocompatibility complex (MHC) that predispose to IgAD/CVID, we analyzed the extended MHC haplotypes present in a large family with 8 affected members., Materials and Methods: We examined the CVID proband, 72 immediate relatives, and 21 spouses, and determined their serum immunoglobulin concentrations. The MHC haplotype analysis of individual family members employed 21 allelic DNA and protein markers, including seven newly available microsatellite markers., Results: Forty-one (56%) of the 73 relatives by common descent were heterozygous and nine (12%) were homozygous for a fragment or the entire extended MHC haplotype designated haplotype 1 that included HLA- DR3, -C4A-0, -B8, and -A1. The remarkable prevalence of haplotype 1 was due in part to marital introduction into the family of 11 different copies of the haplotype, eight sharing 20 identical genotype markers between HLA-DR3 and HLA-B8, and three that contained fragments of haplotype 1., Conclusion: Crossover events within the MHC indicated a susceptibility locus for IgAD/CVID between the class III markers D821/D823 and HLA-B8, a region populated by 21 genes that include tumor necrosis factor alpha and lymphotoxins alpha and beta. Inheritance of at least this fragment of haplotype 1 appears to be necessary for the development of IgAD/CVID in this family.
- Published
- 1998
34. Molecular defects leading to human complement component C6 deficiency in an African-American family.
- Author
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Zhu ZB, Totemchokchyakarn K, Atkinson TP, and Volanakis JE
- Subjects
- Adolescent, Adult, Alleles, Black People genetics, Child, Female, Humans, Male, Meningitis, Meningococcal immunology, Neisseria meningitidis immunology, Pedigree, Black or African American, Complement C6 deficiency, Complement C6 genetics, Meningitis, Meningococcal genetics, Mutation, Neisseria meningitidis isolation & purification
- Abstract
Complement component C6 deficiency (C6D) was diagnosed in a 16-year-old African-American male with meningococcal meningitis. The patient's father and two brothers also had C6D, but gave no history of meningitis or other neisserial infection. By using exon-specific polymerase chain reaction (PCR)/single-strand conformation polymorphism as a screening step and nucleotide sequencing of target exons, we determined that the proband was a compound heterozygote for two C6 gene mutations. The first, 1195delC located in exon 7, is a novel mutation, while the second, 1936delG in exon 12, has been described before to cause C6D in an unrelated African-American individual. Both mutations result in premature termination codons and C6 null alleles. Allele-specific PCR indicated that the proband's two brothers also inherited the 1195delC mutation from their heterozygous mother and the 1936delG mutation from their homozygous father.
- Published
- 1998
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35. Evaluation of IgA deficiency in Sardinians indicates a susceptibility gene is encoded within the HLA class III region.
- Author
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Cucca F, Zhu ZB, Khanna A, Cossu F, Congia M, Badiali M, Lampis R, Frau F, De Virgiliis S, Cao A, Arnone M, Piras P, Campbell RD, Cooper MD, Volanakis JE, and Powis SH
- Subjects
- Disease Susceptibility, HLA-DR3 Antigen immunology, Haplotypes, Humans, IgA Deficiency epidemiology, IgA Deficiency immunology, Italy epidemiology, HLA-DR3 Antigen genetics, IgA Deficiency genetics
- Abstract
IgA deficiency (IgA-D) has been associated with the HLA region, in particular with the North European haplotype HLA-A1, -B8, -DR3, but the exact location of the susceptibility gene(s) is unknown. Some reports suggest that a susceptibility gene is encoded in the class II region, while others implicate the class III region. We exploited differences between the common Sardinian and North European HLA-DR3 haplotypes to help localize the IgA-D susceptibility gene(s). With the knowledge that approximately 13% of HLA-DR3 homozygous individuals of North European origin are IgA-D, we examined 43 HLA-DR3 homozygous Sardinians to find that all had normal serum IgA, IgG and IgM levels. A detailed analysis of their MHC haplotypes indicated a common Sardinian HLA-DR3 haplotype TAP1A, TAP2A, HLA-DQB1*0201, -DQA1*0501, -DRB1*0301, LH1-(Z + 2), D3A-(Z + 2), C4B-0, C4A-L, G11-15, Bf-0-4, C2-a, HSP70-7.5, 9N3-(Z + 10), 82I-(Z - 2), TNFalpha-9, 62-(Z - 20), HLA-B18, -Cw5, -A30 which diverges from the common North European HLA-DR3 haplotype telomeric to the HLA-DR region. In parallel studies of five Sardinians with IgA-D, two of the 10 HLA haplotypes (20%) contained HLA-DR3, a frequency similar to that observed in the background population. One of these was the HLA-DR3- B8 North European haplotype, which occurs rarely in Sardinia. Our data favour the hypothesis that a class III region allele, present on the common North European but not on the Sardinian HLA-DR3 haplotype, confers susceptibility to IgA-D.
- Published
- 1998
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36. The atypical serine proteases of the complement system.
- Author
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Arlaud GJ, Volanakis JE, Thielens NM, Narayana SV, Rossi V, and Xu Y
- Subjects
- Amino Acid Sequence, Animals, Binding Sites, Complement System Proteins chemistry, Humans, Molecular Sequence Data, Protein Conformation, Serine Endopeptidases chemistry, Complement System Proteins physiology, Serine Endopeptidases physiology
- Published
- 1998
37. Contribution of the complement control protein modules of C2 in C4b binding assessed by analysis of C2/factor B chimeras.
- Author
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Xu Y and Volanakis JE
- Subjects
- Antibodies, Monoclonal, Base Sequence, Chimera, Complement C3b immunology, Complement Factor B immunology, Complement Pathway, Classical, Epitopes analysis, Humans, Molecular Sequence Data, Complement C2 analysis, Complement C2 physiology, Complement C4b immunology, Complement Factor B analysis
- Abstract
To identify the complement control protein (CCP) module(s) of C2 that are required for C4b recognition, we constructed a panel of C2/factor B chimeras by substituting intact or partial factor B CCP modules for the corresponding ones of C2. Epitope mapping indicated that the anti-C2b mAb 3A3.3, which inhibits binding of C2 to C4b, reacts with the second CCP of C2 and similarly the anti-Ba mAb HA4-1A, which inhibits binding of factor B to C3b, reacts with the second CCP of factor B. The hemolytic activity of the chimeras CP1, CP2, and CP3a containing CCP1, CCP2, and a fragment of CCP3 of factor B, respectively, was substantially decreased compared with that of wild-type C2. The CP3 and CP1-3 chimeras, in which CCP3 and all three CCP modules of factor B, respectively, were substituted, had no hemolytic activity. Loss of activity could be attributed to the resistance of these two chimeras to C1s cleavage, which was probably due to conformational changes of the cleavage site. The combined results indicate that all three CCP modules of C2 contribute structural elements to the C4b-binding site of C2b. This site has been shown previously to be necessary for the initial binding of C2 to C4b which leads to the formation of the classical pathway C3 convertase.
- Published
- 1997
38. Surface loops adjacent to the cation-binding site of the complement factor B von Willebrand factor type A module determine C3b binding specificity.
- Author
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Tuckwell DS, Xu Y, Newham P, Humphries MJ, and Volanakis JE
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Binding Sites, Cattle, Chickens, Complement C3b chemistry, Complement Factor B genetics, Complement Factor B metabolism, Elapid Venoms metabolism, Electrochemistry, Hemolysis, Humans, Mice, Molecular Sequence Data, Protein Structure, Secondary, Recombinant Fusion Proteins, Sequence Homology, von Willebrand Factor metabolism, Cations, Complement C3b metabolism, Complement Factor B chemistry, von Willebrand Factor chemistry
- Abstract
The interaction of factor B with C3b deposited on the surface of pathogens is the first step in the activation of the alternative complement pathway. The role of the von Willebrand factor type A (VWFA) module of factor B in this interaction has been investigated by generating three chimeras, Ch1-Ch3, in which surface loops of the VWFA module flanking the cation-binding residues were replaced by the corresponding sequences of C2, a factor B-like molecule which does not bind C3b. The location of the three loops was inferred from a homology model based on the structure of the integrin alphaM VWFA module [Ch1, betaA-alpha1 loop: Ch2, alpha3-alpha4 loop; and Ch3, betaD-alpha5 loop; Lee, J.-O., et al. (1995b) Cell 80, 631-638]. The function of the chimeras was studied by means of hemolytic assays and assays of the individual steps of the alternative complement pathway, i.e., binding to the C3b analogue cobra venom factor and factor D cleavage. These experiments showed that Ch1 and Ch3 define regions that are involved in C3b binding whereas Ch2 does not appear to be involved in binding specificity. The inability of Ch1 to register the enhancement of cobra venom factor binding normally seen after factor D cleavage suggested that the betaA-alpha1 loop mediates the conformational regulation of ligand binding affinity. Homology modeling of the chimeras has been used to visualize the surface structures which potentially define the C3b binding site.
- Published
- 1997
- Full Text
- View/download PDF
39. Age-related changes in serum immunoglobulins in patients with familial IgA deficiency and common variable immunodeficiency (CVID).
- Author
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Johnson ML, Keeton LG, Zhu ZB, Volanakis JE, Cooper MD, and Schroeder HW Jr
- Subjects
- Adolescent, Adult, Age Factors, Child, Child, Preschool, Female, Haplotypes, Humans, Major Histocompatibility Complex, Male, Common Variable Immunodeficiency immunology, IgA Deficiency immunology, Immunoglobulins blood
- Abstract
The concentration of serum immunoglobulins in individuals with IgA deficiency (IgAD) and CVID can vary with age to have practical implications for evaluation, therapy, and genetic analysis. Most IgAD and CVID patients in our clinic population in the Southeastern United States have inherited part or all of two extended MHC haplotypes, referred to as haplotype 1 (HLA-DQB1 0201, HLA-DR3, C4B-Sf, C4A-0, G1-15, Bf-0.4, C2-a, HSP-7.5, TNF alpha-5, HLA-B8, HLA-A1) and haplotype 2 (HLA-DQB1 0201, HLA-DR-7, C4B-S, C4A-L, G11-4.5, Bf-0.6, C2-b, HSP-9, TNF alpha-9, HLA-B44, HLA-A29). In the present study, the clinic records of 68 CVID patients and 73 IgAD patients were reviewed to determine whether patients with familial or MHC-associated IgAD or CVID experience changes in serum immunoglobulin concentrations. An increase in serum immunoglobulin to the normal range was associated with clinical improvement in one patient with CVID and haplotype 2, two patients with IgAD and haplotype 2, and one IgAD patient whose haplotype was not determined. Two patients with haplotype 1 and one with haplotype 2 had a significant decline in serum immunoglobulin: one progressed from normal to IgAD associated with IgG subclass deficiencies, and two progressed from IgAD to CVID. Five of the seven patients with notable changing serum immunoglobulin levels have a family member with either IgAD or CVID. The findings suggest that familial, MHC-associated IgAD and CVID may be either progressive or reversible disorders, and emphasize the value of monitoring immunoglobulin levels in affected individuals and their family members.
- Published
- 1997
- Full Text
- View/download PDF
40. Structure of diisopropyl fluorophosphate-inhibited factor D.
- Author
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Cole LB, Chu N, Kilpatrick JM, Volanakis JE, Narayana SV, and Babu YS
- Abstract
Factor D (D) is a serine protease, crucial for the activation of the alternative complement pathway. Only a limited number of general serine protease inhibitors are known to inhibit D, most of which covalently bind to the serine hydroxyl of the catalytic triad. The structure of the first enzyme:inhibitor covalent adduct of D with diisopropyl fluorophosphate (DIP:D) to a resolution of 2.4 A is described. The inhibited enzyme is similar in overall structure to the native enzyme and to trypsin, yet exhibits notable differences in the active site. One region of the active site is conserved between D and trypsin with respect to amino-acid sequence and to conformation. Another reflects the amino-acid substitutions and conformational flexibility between these enzymes. The active-site histidine residue is observed in the gauche+ conformation, not the normal gauche- orientation seen in the classic catalytic triad arrangement required for enzymatic activity in serine proteases. Comparisons of the active sites between native D, the DIP:D adduct, and DIP-inhibited trypsin have provided fundamental insights currently being employed in the design of novel small-molecule pharmaceutical agents capable of modulating the alternative complement pathway.
- Published
- 1997
- Full Text
- View/download PDF
41. Site-directed mutagenesis of the phosphocholine-binding site of human C-reactive protein: role of Thr76 and Trp67.
- Author
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Agrawal A, Lee S, Carson M, Narayana SV, Greenhough TJ, and Volanakis JE
- Subjects
- Humans, Models, Molecular, Protein Binding genetics, Protein Binding immunology, Serum Amyloid P-Component metabolism, Threonine analysis, Tryptophan analysis, C-Reactive Protein genetics, Mutagenesis, Site-Directed, Phosphorylcholine metabolism
- Abstract
We have reported previously that residues Lys57, Arg58, and Trp67 of human C-reactive protein (CRP) contribute to the structure of the phosphocholine (PCh)-binding site. In this study, based on the three-dimensional structures of human CRP and serum amyloid P, we constructed an additional mutant, T76Y, to probe the structural determinants of the PCh-binding site of CRP. Binding properties of four mutant CRPs, K57Q/R58G, W67K, K57Q/R58G/W67K, and T76Y were compared. Wild-type (wt) and all mutant CRPs were purified by affinity chromatography on PCh-, pneumococcal C-polysaccharide (PnC)-, or phosphoethanolamine-conjugated agarose columns. Purified mutant CRPs, K57Q/R58G/W67K and T76Y failed to bind to solid phase, PCh-substituted BSA. They did, however, bind to immobilized PnC, although with substantially decreased avidity compared with wt CRP. W67K, K57Q/R58G/W67K, and T76Y CRP required a 10-fold higher Ca2+ concentration than wt CRP to bind PnC and exhibited decreased avidity for mAb EA4.1, which recognizes a Ca2+-dependent epitope. We conclude that Thr76 is a determinant of the PCh-binding site, probably interacting with the choline group. This conclusion is supported by recent crystallographic data indicating that this residue participates in the formation of a hydrophobic pocket that constitutes the binding site for choline. Trp67, Lys57, and Arg58 do not directly contact PCh, but appear to be required for the proper conformation of the binding site.
- Published
- 1997
42. C-reactive protein: structural biology, gene expression, and host defense function.
- Author
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Szalai AJ, Agrawal A, Greenhough TJ, and Volanakis JE
- Subjects
- Acute-Phase Reaction genetics, Amino Acid Sequence, Animals, C-Reactive Protein immunology, Humans, Immunity, Innate, Mice, Acute-Phase Reaction immunology, C-Reactive Protein chemistry, C-Reactive Protein genetics, Gene Expression Regulation immunology, Models, Molecular
- Abstract
Over the past few years substantial insight was gained into the biology and biochemistry of human C-reactive protein (CRP). X-ray crystallography in conjunction with mutational analyses, generated the three-dimensional structure of the protein and indicated the topology and structure of ligand-binding sites. Using human CRP transgenic mice infected with Streptococcus pneumoniae, we obtained data that clearly established CRP as an important host defense molecule. Studies using the same mice revealed a previously unknown testosterone-dependence of constitutive expression of human CRP. In this article we provide a brief overview of these recent findings.
- Published
- 1997
- Full Text
- View/download PDF
43. Genetic bases of human complement C7 deficiency.
- Author
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Nishizaka H, Horiuchi T, Zhu ZB, Fukumori Y, and Volanakis JE
- Subjects
- Adult, Alleles, Amino Acid Sequence, Base Sequence, Complement C7 genetics, DNA Mutational Analysis, Exons genetics, Female, Humans, Male, Pedigree, Polymerase Chain Reaction, Polymorphism, Single-Stranded Conformational, Complement C7 deficiency, Point Mutation, Sequence Deletion
- Abstract
Complement C7 deficiency (C7D) is associated frequently with recurrent bacterial infections, especially meningitis caused by Neisseria meningitidis. We report in this work the molecular bases of C7D in two unrelated Japanese males. We used exon-specific PCR/single-strand conformation polymorphism analysis as a screening step for mutations. Subsequent direct sequencing of the target exons identified homozygous mutations in exon 16 of case 1 and in exon 15 of case 2. The mutation of case 1 was a homozygous T to A transversion at nucleotide 2250, the third nucleotide of the codon TGT for Cys728, leading to a stop codon TGA (C728X). In case 2, a homozygous 2-bp deletion (2137delTG/2138delGT/2139delTG) caused a frameshift, generating a premature termination codon 4 to 6 nucleotides downstream. Family study in case 1 confirmed the genetic nature of the defect. Moreover, we detected a novel polymorphism in intron 11 that presumably is linked to the mutation responsible for C7D in case 1. Our results indicate that the pathogenesis of C7D is heterogeneous like most of the other deficiencies of complement components.
- Published
- 1996
44. Role of complement in C-reactive-protein-mediated protection of mice from Streptococcus pneumoniae.
- Author
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Szalai AJ, Briles DE, and Volanakis JE
- Subjects
- Animals, Bacteremia immunology, Bacteremia microbiology, C-Reactive Protein genetics, Colony Count, Microbial, Complement C3 analysis, Elapid Venoms pharmacology, Female, Humans, Male, Mice, Mice, Inbred C57BL, Mice, Transgenic, Pneumococcal Infections microbiology, Streptococcus pneumoniae growth & development, C-Reactive Protein immunology, Complement System Proteins immunology, Pneumococcal Infections immunology, Streptococcus pneumoniae immunology
- Abstract
Expression of a human C-reactive protein (CRP) transgene has been shown to protect mice from lethal Streptococcus pneumoniae infection. In the present study, we used cobra venom factor-induced decomplementation to investigate the role of complement in this CRP-mediated protection. An intact complement system significantly reduced pneumococcal bacteremia at 24 h postinfection and extended median survival time of both CRP-transgenic and nontransgenic mice. However, mortality was significantly lowered only for CRP transgenic mice. The transgene significantly reduced bacteremia for both normocomplementemic and decomplemented mice, but it resulted in a significantly longer median survival time and lower mortality only for normocomplementemic mice. These data suggest that in vivo complement and CRP amplify each other's protective capacity, particularly during the early course of infection.
- Published
- 1996
- Full Text
- View/download PDF
45. Down-regulation of secretion of human complement component C2 by the product of an alternatively spliced C2 messenger RNA.
- Author
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Tsukamoto H, Tousson A, Marchase RB, and Volanakis JE
- Subjects
- Alternative Splicing, Animals, Base Sequence, Brefeldin A, Cell Compartmentation drug effects, Cells, Cultured, Chlorocebus aethiops, Complement C2 genetics, Cyclopentanes pharmacology, Down-Regulation, Fluorescent Antibody Technique, Indirect, Gene Expression, Humans, Molecular Sequence Data, Oligodeoxyribonucleotides chemistry, RNA, Messenger genetics, Complement C2 metabolism
- Abstract
We have previously described alternatively spliced transcripts of the human C2 gene. Among those, C2delta(17), in which exon 17 has been spliced out, encodes a polypeptide that contains the C4b binding and the CIs cleavage sites of C2, but lacks the serine protease active center. To study the possible function of this variant, we constructed C2delta(17) cDNA by deletional mutagenesis and expressed it transiently in COS cells. Transfected COS cells secreted only trace amounts of the C2delta(17) polypeptide, which had no detectable hemolytic activity and could not be cleaved by CIs. Pulse-chase experiments using [35S]methionine demonstrated that the majority of the 88-kDa C2delta(17) remained intracellular. Control wild-type (wt) C2 in the intracellular compartment consisted of two bands of 93 and 99 kDa, the latter corresponding to mature secreted C2. Intracellular C2delta(17) and only the 93-kDa wt C2 were sensitive to endoglycosidase H, a marker for transport from the endoplasmic reticulum (ER) to the Golgi. Experiments using brefeldin A and double label immunofluorescence staining indicated that C2delta(17) exhibited a typical ER distribution pattern, while wt C2 accumulated in the ER-Golgi intermediate compartment. Secretion of C2 by COS cells cotransfected with wt C2 and C2delta(17) cDNA was significantly decreased compared with that by cells transfected with wt C2 alone. These combined results indicate that C2delta(17) is retained in the ER probably because it is incorrectly folded and that it could down-regulate the expression of the wt C2 gene.
- Published
- 1996
46. Complement factor D, a novel serine protease.
- Author
-
Volanakis JE and Narayana SV
- Subjects
- Amino Acid Sequence, Animals, Complement Factor D chemistry, Crystallography, X-Ray, Humans, Molecular Sequence Data, Protein Conformation, Serine Endopeptidases chemistry, Substrate Specificity, Complement Factor D physiology, Serine Endopeptidases physiology
- Abstract
Factor D is unique among serine proteases in that it requires neither enzymatic cleavage for expression of proteolytic activity nor inactivation by a serpin for its control. Regulation of factor D activity is instead attained by a novel mechanism that depends on reversible conformational changes for expression and control of catalytic activity. These conformational changes are believed to be induced by the single natural substrate, C3bB, and to result in realignment of the catalytic triad, the specificity pocket, and the nonspecific substrate binding site, all of which have atypical conformations. Mutational studies have defined structural determinants responsible for these unique structural features of factor D and for the resultant low reactivity with synthetic esters.
- Published
- 1996
- Full Text
- View/download PDF
47. Three dimensional structure of human C-reactive protein.
- Author
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Shrive AK, Cheetham GM, Holden D, Myles DA, Turnell WG, Volanakis JE, Pepys MB, Bloomer AC, and Greenhough TJ
- Subjects
- Amino Acid Sequence, C-Reactive Protein metabolism, Calcium metabolism, Crystallography, X-Ray, Humans, Models, Molecular, Molecular Sequence Data, Phosphorylcholine metabolism, Protein Binding, Protein Structure, Secondary, Sequence Homology, Amino Acid, Serum Amyloid P-Component chemistry, C-Reactive Protein chemistry, Protein Conformation
- Abstract
The structure of the classical acute phase reactant human C-reactive protein provides evidence that phosphocholine binding is mediated through calcium and a hydrophobic pocket centred on Phe 66. The residue Glu 81 is suitably positioned to interact with the choline group. A cleft on the pentameric face opposite to that containing the calcium site may have an important functional role. The structure provides insights into the molecular mechanisms by which this highly conserved plasma protein, for which no polymorphism or deficiency state is known, may exert its biological role.
- Published
- 1996
- Full Text
- View/download PDF
48. Molecular bases for inherited human complement component C6 deficiency in two unrelated individuals.
- Author
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Nishizaka H, Horiuchi T, Zhu ZB, Fukumori Y, Nagasawa K, Hayashi K, Krumdieck R, Cobbs CG, Higuchi M, Yasunaga S, Niho Y, and Volanakis JE
- Subjects
- Adult, Amino Acid Sequence, Asian People, Base Sequence, Black People genetics, Complement C6 genetics, Complement C6 isolation & purification, DNA isolation & purification, Exons immunology, Humans, Male, Middle Aged, Molecular Sequence Data, Mutation, Polymerase Chain Reaction, Polymorphism, Genetic immunology, Polymorphism, Single-Stranded Conformational, Black or African American, Complement C6 deficiency, Immunologic Deficiency Syndromes genetics
- Abstract
Deficiency of the sixth component of complement (C6D) is frequently associated with recurrent neisserial infections, especially meningitis caused by Neisseria meningitidis. We here report the molecular bases of C6D in two unrelated subjects, one African American (case 1) and the other Japanese (case 2). Screening all 17 exons of the C6 gene and their boundaries by exon-specific PCR/single strand conformation polymorphism demonstrated aberrant single stranded DNA fragments in exon 12 of case 1 and exon 2 of case 2. Nucleotide sequencing of the amplified DNA fragments revealed a homozygous single-base deletion (G1936) in exon 12 case 1 and a heterozygous single base deletion (C291/C292/C293/C294) in exon 2 of case 2. Both mutations resulted in frame shifts and premature termination of the C6 polypeptide. Sequence-specific oligonucleotide probe hybridization and direct sequencing of exon 12 amplified from genomic DNA further supported the homozygosity of the mutation in case 1. Case 2 is apparently compound heterozygote, but the putative mutation in the other allele of the C6 gene remains unknown. Both case 1 and case 2 were homozygous for the C6A allotype. These data indicate that at least three distinct mutational events can cause C6D, single nucleotide deletions in exons 2 and 12, and a mutation yet unidentified. Thus, similar to other complement protein deficiencies, the pathogenesis of C6D appears to be heterogeneous.
- Published
- 1996
49. Crystal structure of a complement factor D mutant expressing enhanced catalytic activity.
- Author
-
Kim S, Narayana SV, and Volanakis JE
- Subjects
- Amino Acid Sequence, Animals, Aspartic Acid, Base Sequence, Binding Sites, Catalysis, Cattle, Chymotrypsinogen chemistry, Histidine, Humans, Models, Molecular, Molecular Sequence Data, Mutagenesis, Site-Directed, Oligodeoxyribonucleotides, Recombinant Proteins biosynthesis, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Sequence Homology, Amino Acid, Serine, Serine Endopeptidases biosynthesis, Swine, Protein Conformation, Serine Endopeptidases chemistry, Serine Endopeptidases metabolism
- Abstract
Complement factor D is a serine protease regulated by a novel mechanism that depends on conformational changes rather than cleavage of a zymogen for expression of proteolytic activity. The conformational changes are presumed to be induced by the single natural substrate, C3bB, and to result in reversible reorientation of the catalytic center and of the substrate binding site of factor D, both of which have atypical conformations. Here we report that replacement of Ser94, Thr214, and Ser215 of factor D (chymotrypsinogen numbering has been used for comparison purposes) with the corresponding residues of trypsin, Tyr, Ser, and Trp, is sufficient to induce substantially higher catalytic activity associated with a typical serine protease alignment of the catalytic triad residues His57, Asp102, and Ser195. These results provide a partial structural explanation for the low reactivity of "resting-state" factor D toward synthetic substrates.
- Published
- 1995
- Full Text
- View/download PDF
50. Human C-reactive protein is protective against fatal Streptococcus pneumoniae infection in transgenic mice.
- Author
-
Szalai AJ, Briles DE, and Volanakis JE
- Subjects
- Animals, C-Reactive Protein biosynthesis, Female, Humans, Male, Mice, Mice, Transgenic, Pneumococcal Infections blood, Pneumococcal Infections mortality, Sex Factors, Testosterone pharmacology, C-Reactive Protein physiology, Pneumococcal Infections prevention & control
- Abstract
C-reactive protein (CRP) is an acute phase protein with a well known association with infection and other inflammatory conditions. Studies with use of purified CRP in in vitro assays provided early evidence that CRP has antibacterial activity. Subsequently it was shown that passively administered human CRP can protect mice from lethal infection with Streptococcus pneumoniae. In this study, we extend these observations to an in vivo model of host resistance by using human CRP transgenic mice. CRP transgenic mice experimentally infected with S. pneumoniae lived longer and had significantly lower mortality than their nontransgenic littermates. This increased resistance to infection was associated with q 10- to 400-fold reduction of bacteremia. Furthermore, male transgenics exhibited longer survival time than females, and this difference could be attributed to increased expression of CRP by males, which was mediated by testosterone. This study provides the first unequivocal evidence that CRP plays an important role in vivo in host defense against pneumococcal infections, and shows that sex hormones can affect expression of the human CRP transgene in mice.
- Published
- 1995
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