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Monogenetic disorders that cause cerebellar ataxia are characterized by defects in gait and atrophy of the cerebellum; however, patients often suffer from a spectrum of disease, complicating treatment options. Spinocerebellar ataxia autosomal recessive 16 (SCAR16) is caused by coding mutations in STUB1, a gene that encodes the multifunctional enzyme CHIP (C terminus of HSC70-interacting protein). The disease spectrum of SCAR16 includes a varying age of disease onset, cognitive dysfunction, increased tendon reflex, and hypogonadism. Although SCAR16 mutations span the multiple functional domains of CHIP, it is unclear whether the location of the mutation and the change in the biochemical properties of CHIP contributes to the clinical spectrum of SCAR16. In this study, we examined relationships between the clinical phenotypes of SCAR16 patients and the changes in biophysical, biochemical, and functional properties of the corresponding mutated protein. We found that the severity of ataxia did not correlate with age of onset; however, cognitive dysfunction, increased tendon reflex, and ancestry were able to predict 54% of the variation in ataxia severity. We further identified domain-specific relationships between biochemical changes in CHIP and clinical phenotypes and specific biochemical activities that associate selectively with either increased tendon reflex or cognitive dysfunction, suggesting that specific changes to CHIP-HSC70 dynamics contribute to the clinical spectrum of SCAR16. Finally, linear models of SCAR16 as a function of the biochemical properties of CHIP support the concept that further inhibiting mutant CHIP activity lessens disease severity and may be useful in the design of patient-specific targeted approaches to treat SCAR16.

Oxidation of malate to oxaloacetate, catalyzed by either malate dehydrogenase (Mdh) or malate quinone oxidoreductase (Mqo), is a critical step of the tricarboxylic acid cycle. Both Mqo and Mdh are found in most bacterial genomes, but the level of functional redundancy between these enzymes remains unclear. A bioinformatic survey revealed that Mqo was not as widespread as Mdh in bacteria but that it was highly conserved in mycobacteria. We therefore used mycobacteria as a model genera to study the functional role(s) of Mqo and its redundancy with Mdh. We deleted mqo from the environmental saprophyte Mycobacterium smegmatis, which lacks Mdh, and found that Mqo was essential for growth on nonfermentable carbon sources. On fermentable carbon sources, the Δmqo mutant exhibited delayed growth and lowered oxygen consumption and secreted malate and fumarate as terminal end products. Furthermore, heterologous expression of Mdh from the pathogenic species Mycobacterium tuberculosis shortened the delayed growth on fermentable carbon sources and restored growth on nonfermentable carbon sources at a reduced growth rate. In M. tuberculosis, CRISPR interference of either mdh or mqo expression resulted in a slower growth rate compared to controls, which was further inhibited when both genes were knocked down simultaneously. These data reveal that exergonic Mqo activity powers mycobacterial growth under nonenergy limiting conditions and that endergonic Mdh activity complements Mqo activity, but at an energetic cost for mycobacterial growth. We propose Mdh is maintained in slow-growing mycobacterial pathogens for use under conditions such as hypoxia that require reductive tricarboxylic acid cycle activity.

© 2017 by The American Society for Biochemistry and Molecular Biology, Inc. The constitutive heparin+ (HP) mast cells (MCs) in mice express mouseMCprotease (mMCP)-5 and carboxypeptidaseA (mMC-CPA). The amino acid sequence ofmMCP-5is most similar to that of human chymase-1, as are the nucleotide sequences of their genes and transcripts. Using a homologous recombination approach, a C57BL/6 mouse line was created that possessed a disrupted mMCP-5 gene. The resulting mice were fertile and had no obvious developmental abnormality. Lack of mMCP-5 protein did not alter the granulation of the IL-3/IL-9-dependent mMCP-2+ MCs in the jejunal mucosa of Trichinella spiralisinfected mice. In contrast, the constitutive HP+ MCs in the tongues of mMCP-5-null mice were poorly granulated and lacked mMC-CPA protein. Bone marrow-derived MCs were readily developed from the transgenic mice using IL-3. Although these MCs contained high levels of mMC-CPA mRNA, they also lacked the latter exopeptidase. mMCP-5 protein is therefore needed to target translated mMC-CPA to the secretory granule along with HP-containing serglycin proteoglycans. Alternately, mMCP-5 is needed to protect mMC-CPA from autolysis in the cell's granules. Fibronectin was identified as a target of mMCP-5, and the exocytosis ofmMCP-5from theMCs in the mouse's peritoneal cavity resulted in the expression of metalloproteinase protease-9, which has been implicated in arthritis. In support of the latter finding, experimental arthritis was markedly reduced in mMCP-5-null mice relative to wildtype mice in two disease models.

Ca2+ entering a cell through a torn or disrupted plasma membrane rapidly triggers a combination of homotypic and exocytotic membrane fusion events. These events serve to erect a reparative membrane patch and then anneal it to the defect site. Annexin A1 is a cytosolic protein that, when activated by micromolar Ca2+, binds to membrane phospholipids, promoting membrane aggregation and fusion. We demonstrate here that an annexin A1 function-blocking antibody, a small peptide competitor, and a dominant-negative annexin A1 mutant protein incapable of Ca2+ binding all inhibit resealing. Moreover, we show that, coincident with a resealing event, annexin A1 becomes concentrated at disruption sites. We propose that Ca2+ entering through a disruption locally induces annexin A1 binding to membranes, initiating emergency fusion events whenever and wherever required.

Isoxyl (ISO) and thiacetazone (TAC), two prodrugs once used in the clinical treatment of tuberculosis, have long been thought to abolish Mycobacterium tuberculosis (M. tuberculosis) growth through the inhibition of mycolic acid biosynthesis, but their respective targets in this pathway have remained elusive. Here we show that treating M. tuberculosis with ISO or TAC results in both cases in the accumulation of 3-hydroxy C(18), C(20), and C(22) fatty acids, suggestive of an inhibition of the dehydratase step of the fatty-acid synthase type II elongation cycle. Consistently, overexpression of the essential hadABC genes encoding the (3R)-hydroxyacyl-acyl carrier protein dehydratases resulted in more than a 16- and 80-fold increase in the resistance of M. tuberculosis to ISO and TAC, respectively. A missense mutation in the hadA gene of spontaneous ISO- and TAC-resistant mutants was sufficient to confer upon M. tuberculosis high level resistance to both drugs. Other mutations found in hypersusceptible or resistant M. tuberculosis and Mycobacterium kansasii isolates mapped to hadC. Mutations affecting the non-essential mycolic acid methyltransferases MmaA4 and MmaA2 were also found in M. tuberculosis spontaneous ISO- and TAC-resistant mutants. That MmaA4, at least, participates in the activation of the two prodrugs as proposed earlier is not supported by our biochemical evidence. Instead and in light of the known interactions of both MmaA4 and MmaA2 with HadAB and HadBC, we propose that mutations affecting these enzymes may impact the binding of ISO and TAC to the dehydratases.

Conserved uncharacterized genes account for ~30% of genes in both eukaryotic and bacterial genomes and are predicted to encode what are often termed "conserved hypothetical proteins." Many of these proteins have a wide phylogenetic distribution and might play important roles in conserved cellular pathways. Using the bacterium Serratia as a model system, we have investigated two conserved uncharacterized proteins, YgfY (a DUF339 protein, renamed SdhE; succinate dehydrogenase protein E) and YgfX (a DUF1434 protein). SdhE was required for growth on succinate as a sole carbon source and for the function, but not stability, of succinate dehydrogenase, an important component of the electron transport chain and the tricarboxylic acid cycle. SdhE interacted with the flavoprotein SdhA, directly bound the flavin adenine dinucleotide co-factor, and was required for the flavinylation of SdhA. This is the first demonstration of a protein required for FAD incorporation in bacteria. Furthermore, the loss of SdhE was highly pleiotropic, suggesting that SdhE might flavinylate other flavoproteins. Our findings are of wide importance to central metabolism because SdhE homologues are present in α-, β-, and γ-proteobacteria and multiple eukaryotes, including humans and yeast.

Receptor for advanced glycation end products (RAGE), an immunoglobin superfamily cell surface receptor, contributes to the vascular pathology associated with multiple disorders, including Alzheimer disease (AD), diabetic complications, and inflammatory conditions. However, the underlying mechanisms remain largely unclear. Here, using the human umbilical vein endothelial cell line (ECV-304) expressing human RAGE, we report that RAGE expression leads to an altered F-actin organization and impaired membrane resealing. To investigate the underlying mechanisms, we showed that RAGE expression increases β-catenin level, which decreases F-actin stress fibers and attenuates plasma membrane resealing. These results thus suggest a negative function for RAGE in endothelial cell membrane repair and reveal a new mechanism underlying RAGE regulation of F-actin remodeling and membrane resealing.

The cell wall of mycobacteria consists of an outer membrane, analogous to that of Gram-negative bacteria, attached to the peptidoglycan (PG) via a connecting polysaccharide arabinogalactan (AG). Although the primary structure of these components is fairly well deciphered, issues such as the coverage of the PG layer by covalently attached mycolates in the outer membrane and the spatial details of the mycolic acid attachment to the arabinan have remained unknown. It is also not understood how these components work together to lead to the classical acid-fast staining of mycobacteria. Because the majority of Mycobacterium tuberculosis bacteria in established experimental animal infections are acid-fast negative, clearly cell wall changes are occurring. To address both the spatial properties of mycobacterial cell walls and to begin to study the differences between bacteria grown in animals and cultures, the cell walls of Mycobacterium leprae grown in armadillos was characterized and compared with that of M. tuberculosis grown in culture. Most fundamentally, it was determined that the cell wall of M. leprae contained significantly more mycolic acids attached to PG than that of in vitro grown M. tuberculosis (mycolate:PG ratios of 21:10 versus 16:10, respectively). In keeping with this difference, more arabinogalactan (AG) molecules, linking the mycolic acids to PG, were found. Differences in the structures of the AG were also found; the AG of M. leprae is smaller than that of M. tuberculosis, although the same basic structural motifs are retained.

Human group IIA-secreted phospholipase A(2) (sPLA(2)-IIA) is an important regulator of cytokine-mediated inflammatory responses in both in vitro and in vivo models of rheumatoid arthritis (RA). However, treatment of RA patients with sPLA(2)-IIA inhibitors shows only transient benefit. Using an activity-impaired sPLA(2)-IIA mutant protein (H48Q), we show that up-regulation of TNF-dependent PGE(2) production and cyclooxygenase-2 (COX-2) induction by exogenous sPLA(2)-IIA in RA fibroblast-like synoviocytes (FLSs) is independent of its enzyme function. Selective cytosolic phospholipase A(2)-α (cPLA(2)-α) inhibitors abrogate TNF/sPLA(2)-IIA-mediated PGE(2) production without affecting COX-2 levels, indicating arachidonic acid (AA) flux to COX-2 occurs exclusively through TNF-mediated activation of cPLA(2)-α. Nonetheless, exogenous sPLA(2)-IIA, but not H48Q, stimulates both AA mobilization from FLSs and microparticle-derived AA release that is not used for COX-2-dependent PGE(2) production. sPLA(2)-IIA-mediated AA production is inhibited by pharmacological blockade of sPLA(2)-IIA but not cPLA(2)-α. Exogenous H48Q alone, like sPLA(2)-IIA, increases COX-2 protein levels without inducing PGE(2) production. Unlike TNF, sPLA(2)-IIA alone does not rapidly mobilize NF-κB or activate phosphorylation of p38 MAPK, two key regulators of COX-2 protein expression, but does activate the ERK1/2 pathway. Thus, sPLA(2)-IIA regulates AA flux through the cPLA(2)-α/COX-2 pathway in RA FLSs by up-regulating steady state levels of these biosynthetic enzymes through an indirect mechanism, rather than direct provision of substrate to the pathway. Inhibitors that have been optimized for their potency in enzyme activity inhibition alone may not adequately block the activity-independent function of sPLA(2)-IIA.

LP2086 is a family of outer membrane lipoproteins from Neisseria meningitidis, which elicits bactericidal antibodies and are currently undergoing human clinical trials in a bivalent formulation where each antigen represents one of the two known LP2086 subfamilies. Here we report the NMR structure of the recombinant LP2086 variant B01, a representative of the LP2086 subfamily B. The structure reveals a novel fold composed of two domains: a “taco-shaped” N-terminal β-sheet and a C-terminal β-barrel connected by a linker. The structure in micellar solution is consistent with a model of LP2086 anchored to the outer membrane bilayer through its lipidated N terminus. A long flexible chain connects the folded part of the protein to the lipid anchor and acts as spacer, making both domains accessible to the host immune system. Antibodies broadly reactive against members from both subfamilies have been mapped to the N terminus. A surface of subfamily-defining residues was identified on one face of the protein, offering an explanation for the induction of subfamily-specific bactericidal antibodies.

Current evidence supports a binding model in which the insulin molecule contains two binding surfaces, site 1 and site 2, which contact the two halves of the insulin receptor. The interaction of these two surfaces with the insulin receptor results in a high affinity cross-linking of the two receptor alpha subunits and leads to receptor activation. Evidence suggests that insulin-like growth factor-I (IGF-I) may activate the IGF-I receptor in a similar mode. So far IGF-I residues structurally corresponding to the residues of the insulin site 1 together with residues in the C-domain of IGF-I have been found to be important for binding of IGF-I to the IGF-I receptor (e.g. Phe(23), Tyr(24), Tyr(31), Arg(36), Arg(37), Val(44), Tyr(60), and Ala(62)). However, an IGF-I second binding surface similar to site 2 of insulin has not been identified yet. In this study, we have analyzed whether IGF-I residues corresponding to the six residues of the insulin site 2 have a role in high affinity binding of IGF-I to the IGF-I receptor. Six single-substituted IGF-I analogues were produced, each containing an alanine substitution in one of the following positions (corresponding insulin residues in parentheses): Glu(9) (His(B10)), Asp(12) (Glu(B13)), Phe(16) (Leu(B17)), Asp(53) (Ser(A12)), Leu(54) (Leu(A13)), and Glu(58) (Glu(A17)). In addition, two analogues with 2 and 3 combined alanine substitutions were also produced (E9A,D12A IGF-I and E9A,D12A,E58A IGF-I). The results show that introducing alanine in positions Glu(9), Asp(12), Phe(16), Leu(54), and Glu(58) results in a significant reduction in IGF-I receptor binding affinity, whereas alanine substitution at position 53 had no effect on IGF-I receptor binding. The multiple substitutions resulted in a 33-100-fold reduction in IGF-I receptor binding affinity. These data suggest that IGF-I, in addition to the C-domain, uses surfaces similar to those of insulin in contacting its cognate receptor, although the relative contribution of the side chains of homologous residues varies.

The complex cell wall of Mycobacterium tuberculosis is the hallmark of acid fast bacteria and is responsible for much of its physiological characteristics. Hence, much effort has been made to determine its primary structure. Such studies have been hampered by its extreme complexity. Also, its insolubility leads to difficulties determining the presence or absence of base labile groups. We have used an endogenous arabinase to solubilize the arabinan region of the cell wall and have shown using mass spectrometry and NMR that succinyl esters are present on O2 of the inner-branched 1,3,5-α-d-arabinofuranosyl residues. In addition, an inner arabinan region of 14 linear α-1,5 arabinofuranosyl residues has been identified. These and earlier results now allow the presentation of a model of the entire primary structure of the mycobacterial mycolyl arabinogalactan highlighted by three arabinan chains of 31 residues each.

Insulin and the insulin-like growth factors (IGFs) bind with high affinity to their cognate receptor and with lower affinity to the noncognate receptor. The major structural difference between insulin and the IGFs is that the IGFs are single chain polypeptides containing A-, B-, C-, and D-domains, whereas the insulin molecule contains separate A- and B-chains. The C-domain of IGF-I is critical for high affinity binding to the insulin-like growth factor I receptor, and lack of a C-domain largely explains the low affinity of insulin for the insulin-like growth factor I receptor. It is less clear why the IGFs have lower affinity for the insulin receptor. In this study, 24 insulin analogues and four IGF analogues were expressed and analyzed to explore the role of amino acid differences in the A- and B-domains between insulin and the IGFs in binding affinity for the insulin receptor. Using the information obtained from single substituted analogues, four multiple substituted analogues were produced. A "quadruple insulin" analogue ([Phe(A8), Ser(A10), Thr(B5), Gln(B16)]Ins) showed affinity as IGF-I for the insulin receptor, and a "sextuple insulin" analogue ([Phe(A8), Ser(A10), Thr(A18), Thr(B5), Thr(B14), Gln(B16)]Ins) showed an affinity close to that of IGF-II for the insulin receptor, whereas a "quadruple IGF-I" analogue ([His(4), Tyr(15), Thr(49), Ile(51)]IGF-I) and a "sextuple IGF-II" analogue ([His(7), Ala(16), Tyr(18), Thr(48), Ile(50), Asn(58)]IGF-II) showed affinities similar to that of insulin for the insulin receptor. The mitogenic potency of these analogues correlated well with the binding properties. Thus, a small number of A- and B-domain substitutions that map to the IGF surface equivalent to the classical binding surface of insulin weaken two hotspots that bind to the insulin receptor site 1.

D-Arabinofurans, attached to either a galactofuran or a lipomannan, are the primary constituents of mycobacterial cell wall, forming the unique arabinogalactan (AG) and lipoarabinomannan (LAM), respectively. Emerging data indicate that the arabinans of AG and LAM are distinguished by virtue of the additional presence of linear termini in LAM, which entails some unknown feature of the EmbC protein for proper synthesis. In common with the two paralogous EmbA and EmbB proteins functionally implicated for the arabinosylation of AG, EmbC is predicted to carry 13 transmembrane spanning helices in an integral N-terminal domain followed by a hydrophilic extracytoplasmic C-terminal domain. To delineate the function of this C-terminal domain, the embC knock-out mutant of Mycobacterium smegmatis was complemented with plasmids expressing truncated embC genes. The expression level of serially truncated EmbC protein thus induced was examined by EmbC-specific peptide antibody, and their functional implications were inferred from ensuing detailed structural analysis of the truncated LAM variants synthesized. Apart from critically showing that the smaller arabinans are mostly devoid of the linear terminal motif, beta-D-Araf(1-->2)-alpha-D-Araf(1-->5)-alpha-D-Araf(1-->5)-alpha-D-Araf, our studies clearly implicate the C-terminal domain of EmbC in the chain extension of LAM. For the first time a full range of arabinan chains as large as 18-22 Araf residues and beyond could be released intact by the use of an endogenous endo-D-arabinanase from M. smegmatis, profiled, and sequenced directly by tandem mass spectrometry. In conjunction with NMR studies, our results unequivocally show that the LAM-specific linear termini are an extension on a well defined inner branched Ara-(18-22) core. This hitherto unrecognized feature not only allows a significant revision of the structural model of LAM-arabinan since its first description a decade ago but also furnishes a probable molecular basis of selectivity in biosynthesis, as conferred by the EmbC protein.

Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne disease in cattle and other ruminants, is proposed to be at least one of the causes of Crohn disease in humans. MAP and Mycobacterium avium subspecies avium, a closely related opportunistic environmental bacterium, share 95% of their genes and exhibit homologies of more than 99% between these genes. The identification of molecules specific for MAP is essential for understanding its pathogenicity and for development of useful diagnostic tools. The application of gas chromatography, mass spectrometry, and nuclear magnetic resonance led to the structural identification of a major cell wall lipopeptide of MAP, termed Para-LP-01, defined as C20 fatty acyl-d-Phe-N-Me-l-Val-l-Ile-l-Phe-l-Ala methyl ester. Variations of this lipopeptide with different fatty acyl moieties (C16 fatty acyl through C17, C18, C19, C21 to C22) were also identified. Besides the specificity of this lipopeptide for MAP, the presence of an N-Me-l-valine represents the first reported N-methylated amino acid within an immunogenic lipopeptide of mycobacteria. Sera from animals with Johne disease, but not sera from uninfected cattle, reacted with this lipopeptide, indicating potential biological importance.

Temperature-sensitive mutant 2-20/32 of Mycobacterium smegmatis mc(2)155 was isolated and genetically complemented with a Mycobacterium tuberculosis H37Rv DNA fragment that contained a single open reading frame. This open reading frame is designated Rv3265c in the M. tuberculosis H37Rv genome. Rv3265c shows homology to the Escherichia coli gene wbbL, which encodes a dTDP-Rha:alpha-D-GlcNAc-pyrophosphate polyprenol, alpha-3-L-rhamnosyltransferase. In E. coli this enzyme is involved in O-antigen synthesis, but in mycobacteria it is required for the rhamnosyl-containing linker unit responsible for the attachment of the cell wall polymer mycolyl-arabinogalactan to the peptidoglycan. The M. tuberculosis wbbL homologue, encoded by Rv3265c, was shown to be capable of restoring an E. coli K12 strain containing an insertionally inactivated wbbL to O-antigen positive. Likewise, the E. coli wbbL gene allowed 2-20/32 to grow at higher non-permissive temperatures. The rhamnosyltransferase activity of M. tuberculosis WbbL was demonstrated in 2-20/32 as was the loss of this transferase activity in 2-20/32 at elevated temperatures. The wbbL of the temperature-sensitive mutant contained a single-base change that converted what was a proline in mc(2)155 to a serine residue. Exposure of 2-20/32 to higher non-permissive temperatures resulted in bacteria that could not be recovered at the lower permissive temperatures.

The emb genes are conserved among different mycobacteria. In Mycobacterium smegmatis and Mycobacterium tuberculosis, they belong to an operon comprising three genes, embC, embA, and embB. The EmbB protein has been proposed to be the target of ethambutol, a drug which is known to inhibit the synthesis of the arabinan portion of the mycobacterial cell wall arabinogalactan (AG). To further define the role of EmbB protein in arabinan biosynthesis, embA, -B, and -C genes were inactivated individually by homologous recombination in M. smegmatis. All three mutants were viable, and among the three, the slowest growing embB(-) mutant encountered profound morphological changes and exhibited a higher sensitivity to hydrophobic drugs and detergents, presumably due to an increase in cell wall permeability. Furthermore, chemical analyses showed that there was a diminution in the arabinose content of arabinogalactan from the embA(-) and embB(-) mutants. Specifically, in comparison with the wild-type strain, the crucial terminal hexaarabinofuranosyl motif, which is a template for mycolylation, was altered in both embA(-) and embB(-) mutants. Detailed nuclear magnetic resonance studies coupled with enzyme digestion, chromatography, and mass spectrometry analyses revealed that the disaccharide beta-d-Ara(f)-(1-->2)-alpha-d-Ara(f) extension from the 3-position of the 3,5-linked alpha-d-Ara(f) residue is markedly diminished. As a consequence, a linear terminal beta-d-Ara(f)-(1-->2)-alpha-d-Ara(f)-(1-->5)-alpha-d-Ara(f)-(1-->5)-alpha-d-Ara(f) is formed, a motif which is a recognized, nonreducing terminal feature of lipoarabinomannan but not of normal AG. Upon complementation with the embB and embA wild-type genes, the phenotype of the mutants reverted to wild-type, in that normal AG was resynthesized. Our results clearly show that both EmbA and EmbB proteins are involved in the formation of the proper terminal hexaarabinofuranoside motif in AG, thus paving the way for future studies to identify the complete array of arabinosyltransferases involved in the synthesis of mycobacterial cell wall arabinan.

The cell wall of Mycobacterium tuberculosis and related genera is unique among prokaryotes, consisting of a covalently bound complex of mycolic acids, D-arabinan and D-galactan, which is linked to peptidoglycan via a special linkage unit consisting of Rhap-(1-->3)-GlcNAc-P. Information concerning the biosynthesis of this entire polymer is now emerging with the promise of new drug targets against tuberculosis. Accordingly, we have developed a galactosyltransferase assay that utilizes the disaccharide neoglycolipid acceptors beta-d-Galf-(1-->5)-beta-D-Galf-O-C(10:1) and beta-D-Galf-(1-->6)-beta-D-Galf-O-C(10:1), with UDP-Gal in conjunction with isolated membranes. Chemical analysis of the subsequent reaction products established that the enzymatically synthesized products contained both beta-D-Galf linkages ((1-->5) and (1-->6)) found within the mycobacterial cell, as well as in an alternating (1-->5) and (1-->6) fashion consistent with the established structure of the cell wall. Furthermore, through a detailed examination of the M. tuberculosis genome, we have shown that the gene product of Rv3808c, now termed glfT, is a novel UDP-galactofuranosyltransferase. This enzyme possesses dual functionality in performing both (1-->5) and (1-->6) galactofuranosyltransferase reactions with the above neoglycolipid acceptors, using membranes isolated from the heterologous host Escherichia coli expressing Rv3808c. Thus, at a biochemical and genetic level, the polymerization of the galactan region of the mycolyl-arabinogalactan complex has been defined, allowing the possibility of further studies toward substrate recognition and catalysis and assay development. Ultimately, this may also lead to a more rational approach to drug design to be explored in the context of mycobacterial infections.

The structural core of the cell walls of Mycobacterium spp. consists of peptidoglycan bound by a linker unit (-alpha-L-Rhap-(1-->3)-D-GlcNAc-P-) to a galactofuran, which in turn is attached to arabinofuran and mycolic acids. The sequence of reactions leading to the biogenesis of this complex starts with the formation of the linker unit on a polyprenyl-P to produce polyprenyl-P-P-GlcNAc-Rha (Mikusova, K., Mikus, M., Besra, G. S., Hancock, I., and Brennan, P. J. (1996) J. Biol. Chem. 271, 7820-7828). We now establish that formation of the galactofuran takes place on this intermediate with UDP-Galf as the Galf donor presented in the form of UDP-Galp and UDP-Galp mutase (the glf gene product) and is catalyzed by galactofuranosyl transferases, one of which, the Mycobacterium tuberculosis H37Rv3808c gene product, has been identified. Evidence is also presented for the growth of the arabinofuran on this polyprenyl-P-P-linker unit-galactan intermediate catalyzed by unidentified arabinosyl transferases, with decaprenyl-P-Araf or 5-P-ribosyl-PP as the Araf donor. The product of these steps, the lipid-linked-LU-galactan-arabinan has been partially characterized in terms of its heterogeneity, size, and composition. Biosynthesis of the major components of mycobacterial cell walls is proving to be extremely complex. However, partial definition of arabinogalactan synthesis, the site of action of several major anti-tuberculosis drugs, facilitates the present day thrust for new drugs to counteract multiple drug-resistant tuberculosis.

We have investigated which region(s) of bovine insulin-like growth factor binding protein-2 (bIGFBP-2) interact with insulin-like growth factors (IGFs) using C-terminally truncated forms of bIGFBP-2. Initially to aid in mutant design, we defined the disulfide bonding pattern of bIGFBP-2 C-terminal region using enzymatic digestion. The pattern is Cys186-Cys220, Cys231-Cys242, and Cys244-Cys265. In addition, cyanogen bromide cleavage of bIGFBP-2 revealed that the N- and C-terminal cysteine-rich domains were not linked by disulfide bonds. Taking the disulfide bonding pattern into consideration, C-terminal truncation mutants were designed and expressed in COS-1 mammalian cells. Following IGF binding assays, a region between residues 222 and 236 was identified as important in IGF binding. Specifically, mutants truncated by 14, 36, and 48 residues from the C terminus bound IGFs to the same extent as wild type (WT) bIGFBP-2. Removal of 63 residues resulted in a greatly reduced (up to 80-fold) ability to bind IGF compared with WT bIGFBP-2. Interestingly this mutant lacked the IGF-II binding preference of WT bIGFBP-2. Residues 236-270 also appeared to play a role in determining IGF binding specificity as their removal resulted in mutants with higher IGF-II binding affinity.

Polyprenylphosphate-arabinose (in which the polyprenyl unit is found both as decaprenyl and octahydroheptaprenyl) is a donor of mycobacterial cell wall arabinosyl residues. Because of this important role, its biosynthetic pathway, and that of the related lipid, polyprenylphosphate-D-ribose, was investigated. Surprisingly, phosphoribose pyrophosphate was shown to be a key intermediate on the pathway to both polyprenylphosphate-D-pentoses. Thus, incubation of 5-phospho-D-[14C]ribose pyrophosphate with membranes prepared from Mycobacterium smegmatis resulted in the presence of organic-soluble radioactivity that was shown to be, in part, polyprenylphosphate-[14C]arabinose and polyprenylphosphate-[14C]ribose. Two additional intermediates, polyprenylphosphate-5-phospho[14C]ribose and polyprenylphosphate-5-phospho[14C]arabinose, were identified. Further experiments showed that the mature polyprenylphosphate-ribose is formed from phosphoribose pyrophosphate via a two-step pathway involving a transferase to form polyprenylphosphate-5-phosphoribose and then a phosphatase to form the final polyprenylphosphateribose. Polyprenylphosphate-arabinose is formed by a similar pathway with an additional step being the epimerization at C-2 of the ribosyl residue. This epimerization occurs at either the level of phosphoribose pyrophosphate or at the level of polyprenylphosphate-5-phosphoribose.

The entire surface of the cercarial stage of the human blood fluke Schistosoma mansoni is covered by a 1-microns thick, highly immunogenic, fucose-rich glycocalyx (GCX). Using strategies based on enzymatic, chemical, and mass spectrometric analysis, we have defined the structures of the major glycans released by reductive elimination from GCX. They comprise a heterogeneous population of multifocosylated complex oligosaccharides with the following nonreducing terminal sequences: [formula: see text] Our structural data suggest that these tri- to pentafucosylated epitopes are carried on type 1, R--Gal beta-1--3GalNAc, and type 2, R--Gal beta 1--3(R--GlcNAc beta-1--6)GalNAc, core structures via repeat units of (3GalNAc beta 1--4(Fuc alpha 1--2Fuc alpha 1--2Fuc alpha 1--3)GlcNAc beta-1--3Gal alpha--)n, where n is mainly 0 and 1, and all sugars are in the pyranose form. The proposed structure represents the first instance where an alpha-galactosylated beta-GalNAc(1--4)-beta-GlcNAc sequence occurs as a repeating unit in a glycoprotein. It is also unique in being substituted with oligofucosyl appendages. The unusual oligosaccharide structures described here, particularly the potentially immunodominant oligofucosyl moieties, are most likely responsible for the known potency of GCX in modulating various immune responses including complement activation, B cell mitogenesis, and delayed type hypersensitivity in schistosomiasis.

Despite major advances in our understanding of the structure of mycobacterial cell walls, little is known of their biogenesis, and yet they are the site of action of many anti-tuberculosis drugs and implicated in much of the pathology of tuberculosis and leprosy. A family of monoglycosyl polyprenylphosphates was isolated from Mycobacterium smegmatis, containing arabinose, ribose, and mannose. The isoprenoid nature of the lipid components was established by 1H NMR, and fast atom bombardment mass spectroscopy (FAB-MS) demonstrated the presence of C50 decaprenyl-P derivatives and smaller amounts of the C35 octahydroheptaprenyl-P products. The configuration of the mycobacterial decaprenol was established as mono-trans, octa-cis, pointing to carriers of unusual structure. Combined gas chromatography (GC)/MS, FAB-MS/MS, and 1H NMR allowed characterization of one of the primary components as beta-D-arabinofuranosyl-1-monophosphodecaprenol. Pulse-chase metabolic labeling of cells with D-[14C]glucose indicated that the decaprenyl-P-arabinose is an active intermediate in the biosynthesis of the arabinan of cell wall arabinogalactan and arabinomannan. The identification of polyprenyl-P-ribose suggests the existence of ribose-containing polysaccharides in the cell walls of M. smegmatis or/and of a novel epimerase in the D-arabinose biosynthetic pathway. Ethambutol, a powerful anti-tuberculosis drug known to inhibit arabinogalactan and arabinomannan biosynthesis, results in the rapid accumulation of decaprenyl-P-arabinose, indicating that the drug interferes with either the transfer of arabinose from the donor or, alternatively, the synthesis of the arabinose acceptor itself.

The genes encoding both the cytosolic and mitochondrial serine hydroxymethyltransferases (SHM2 and SHM1, respectively) and a third unidentified gene of the yeast Saccharomyces cerevisiae have been isolated and their nucleotide sequences determined. Analysis of the predicted amino acid sequence of the amino-terminal regions, sequence comparison with other genes encoding SHMT enzymes, and subcellular fractionation studies all suggested that the SHM1 gene encodes the mitochondrial SHMT, while the SHM2 gene encodes the cytosolic enzyme. The SHM2 gene but not the SHM1 gene has putative GCN4 sites upstream of the putative TATA box, suggesting regulation of its transcription by the general amino acid control system. Yeast mutants with disruptions at each SHM gene and in both genes were constructed and all mutants had the same growth requirements as the parental strains. Mutagenesis of the double-disrupted, shm1 shm2 yeast yielded strains of a single complementation group that are auxotrophic for glycine. Complementation of the glycine auxotrophy using a yeast genomic library retrieved the SHM1 and SHM2 genes and a third gene designated GLY1. Gene disruption studies demonstrated that inactivation of SHM1, SHM2, and GLY1 is required to yield yeast that are completely auxotrophic for glycine.

Matrilysin, a member of the matrix metalloproteinase family, is structurally different from the other matrix metalloproteinases by virtue of the absence of a conserved COOH-terminal protein domain. In addition, matrilysin mRNA is regulated in a specific and distinct manner in normal and malignant tissues. Analysis of the genomic structure of the human matrilysin gene revealed that the organization of the first five exons is highly conserved among the different members of the matrix metalloproteinase family, but that matrilysin contains an atypical sixth exon. The promoter region of the matrilysin gene has several features that are conserved among several other matrix metalloproteinase family members, including the presence of TATA, AP-1, and PEA3 elements. Comparison of the expression of the human matrilysin promoter with rat stromelysin promoter/chloramphenicol acetyltransferase constructs in HeLa cells revealed that constructs containing AP-1 and PEA3 elements respond similarly to epidermal growth factor and tumor promoter (12-O-tetradecanoyl-phorbol-13-acetate) induction, but that the addition of upstream stromelysin sequences results in an increased transcriptional activity not observed with upstream matrilysin sequences. The similarities and differences observed between the promoters of matrilysin and the other metalloproteinases may provide insights into the molecular mechanisms that regulate the expression of this family of enzymes as a whole and the factors that distinguish the expression patterns of individual family members.

Toward studying the genetics, biosynthesis, and roles in the pathogenesis of the dominant surface glycopeptidolipid antigens of Mycobacterium avium, rough colony variants of M. avium serovar 2 were picked, cultured in quantity, and their lipid composition examined. Two of the rough (Rg) variants, Rg-3 and Rg-4, were devoid of glycopeptidolipids or any more elemental structures and thus were similar to those described previously. Two others, Rg-0 and Rg-1, each contained two novel lipopeptides, devoid of any of the carbohydrate substituents that confer serotypic activity on the glycopeptidolipids. The application of gas chromatography, fast atom bombardment-mass spectrometry and 1H NMR to lipopeptide I established the structure C32:2-beta-OH-fatty acyl-D-Phe-D-allo-Thr-D-Ala-L-alaninol. Lipopeptide II represented a minor variation of this structure: C32:1-beta-OH-fatty acyl-D-Phe-D-allo-Thr-D-Ala-L-alaninol. These newly discovered lipopeptides are identical to the fatty acyl-tripeptide-amino alcohol "core" component of the glycopeptidolipids of the M. avium complex, and thus the Rg-0 and Rg-1 variants represent a form of "deep rough" mutation in M. avium. Separately, we report that these rough variants of M. avium differ genetically from the smooth, virulent form by major deletions of portions of the genes responsible for glycopeptidolipid synthesis (Belisle, J. T., Klaczkiewicz, K., Brennan, P. J., Jacobs, W. R., Jr., and Inamine, J. M. (1993) J. Biol. Chem. 268, 10517-10523). The isolation of different sets of spontaneous mutants of M. avium differentially defective in the capacity to synthesize glycopeptidolipids provides the means to explore their biosynthesis and roles in opportunistic pathogenesis.

Although transcription of mast cell (MC) secretory granule neutral protease genes has been shown to distinguish MC subclasses in mucosal and serosal environments, the specific cytokines that regulate the expression of these genes have not been determined. To examine cytokine-mediated gene regulation, bone marrow-derived MC (BMMC) differentiated in vitro were obtained by culturing mouse bone marrow progenitor cells in the presence of WEHI-3 cell-conditioned medium, concanavalin A-stimulated splenocyte-conditioned medium (BMMCC), or recombinant (r) interleukin (IL)-3 (BMMCIL-3). All three populations of BMMC expressed the serosal MC-specific transcripts that encode mouse MC serine protease (MMCP)-5, MMCP-6, and MC carboxypeptidase A. However, only BMMCC contained MMCP-2 mRNA, a late expressed gene selectively transcribed by intestinal mucosal MC that proliferate during helminthic infestation in response to the T cell-derived cytokines IL-3, IL-4, and IL-10. When BMMCIL-3 were exposed to rIL-10 in the presence of either rIL-3 or rIL-4, they expressed MMCP-2 mRNA. Not only was the transcription of the MMCP-2 gene in BMMC dependent on continuous exposure of the cells to rIL-10, but the level of MMCP-2 mRNA in these cells could be down-regulated by rIL-3. These studies comparing the effects of two cytokines on the transcriptional regulation of secretory granule protease genes in MC demonstrate that rIL-10 induces BMMCIL-3 to express the mucosal MC protease MMCP-2, that rIL-3 attenuates the rIL-10-induced expression of this gene, and that transcription of the MMCP-2 gene is reversed in the absence of rIL-10.

The lipopolysaccharides of mycobacteria, lipoarabinomannan (LAM) and lipomannan (LM), of key importance in host-pathogen interaction, were recently shown to contain a phosphatidylinositol "anchoring domain." We now have established that LAM and LM are based on the phosphatidylinositol mannosides, the characteristic glycophospholipids of mycobacteria. Digestion of the arabinose-free LM with an endo-alpha 1----6-mannosidase yielded evidence for the presence of the 1-(sn-glycerol-3-phospho)-D-myo-inositol-2,6-bis-alpha-D-mannopyranoside unit, indistinguishable from that derived from phosphatidylinositol dimannoside. This same inositol substitution pattern was shown to be present in LAM by methylation analysis before and after dephosphorylation. Positions C-2 and C-6 of the inositol unit of LAM are occupied by mannosyl residues and C-1 by a phosphoryl group. Partial acid hydrolysis of per-O-methylated LAM and comparison by gas chromatography-mass spectrometry of the resulting derivatized oligosaccharides with like products from phosphatidylinositol hexamannoside demonstrated that the C-6 of inositol is the point of attachment of the mannan core of LAM, which consists of an alpha 1----6-linked backbone with considerable alpha-1----2 side chains. Thus, a structural and presumably biosynthetic relationship is established between some of the membranous mannosylphosphatidylinositols described some 25 years ago and the newly emerging, biologically active lipopolysaccharides of mycobacteria.

Previously we had demonstrated that the termini of the arabinan component of mycobacterial cell wall arabinogalactan, the site of mycolic acid location, consists mostly of clusters of a pentaarabinofuranoside, [beta-D-Araf-(1----2)-alpha-D-Araf-(1----]2----(3 and 5)-alpha-D-Araf. Subsequently, the same arrangement was shown to dominate the non-reducing ends of lipoarabinomannan (LAM), a key component in the interaction of mycobacteria with host cell. Accordingly, we had proposed that mycobacteria universally elaborate the same Araf-containing motifs in two settings for different pathophysiological purposes. However, we now report that the termini of LAM from the virulent, Erdman, strain of Mycobacterium tuberculosis, unlike those from the attenuated H37Ra strain, are extensively capped with mannosyl (Manp) residues, either a single alpha-D-Manp, a dimannoside (alpha-D-Manp-(1----2)-alpha-D-Manp), or a trimannoside (alpha-D-Manp-(1----2)-alpha-D-Manp-(1----2)-alpha-D-Manp ). The use of monoclonal antibodies demonstrates a clear difference in the antigenicity of the basic and mannose-capped LAM. The possibility that these structures are a factor in the virulence of some strains of M. tuberculosis and represent an example of carbohydrate mimicry in mycobacterial infections is discussed.

The question of the precise location of mycolic acids, the single most distinctive cell wall entity of members of the Mycobacterium genus, has now been addressed. The free hydroxyl functions of the arabinogalactan component of the mycobacterial cell wall were O-methylated under conditions in which the mycolyl esters were not cleaved. Subsequent replacement of the mycolyl functions with O-ethyl groups resulted in an acid- and base-stable differentially O-alkylated surrogate polysaccharide, more amenable to analysis. Complete hydrolysis, reduction, acetylation, and gas chromatography/mass spectrometry revealed the unexpected finding that the mycolyl substituents were selectively and equally distributed on the 5-hydroxyl functions of terminal- and 2-linked arabinofuranosyl (Araf) residues. Further analysis of the O-alkylated cell wall through partial acid hydrolysis, NaB[2H]4 reduction, pentadeuterioethylation, and gas chromatography/mass spectrometry demonstrated that the mycolyl units are clustered in groups of four on the previously recognized nonreducing terminal pentaarabinosyl unit [beta-Araf-(1----2)-alpha-Araf)2-3, 5-alpha-Araf. However, only about two-thirds of the available pentasaccharide units are so substituted. Thus, the antigenicity of the arabinan component of mycobacterial cell walls may be explained by the fact that about one-third of the pentaarabinosyl units are not mycolyated and are available for interaction with the immune system. On the other hand, the extreme hydrophobicity and impenetrability of the mycobacterial cell may be explained by the same motif also acting as the fulerum for massive esterified paraffin residues. New fundamental information on the structure of mycobacterial cell walls will aid in our comprehension of its impenetrability to antibiotics and role in immunopathogenesis and persistence of disease.

The recent availability of pure lipoarabinomannan (LAM) from Mycobacterium spp. has resulted in its implication in host-parasite interaction, which events may be mediated by the presence of a phosphatidylinositol unit at the reducing end of LAM. Herein we address the structure of the antigenic, nonreducing end of the molecule. Through the process of 13C NMR analysis of the whole molecule and gas chromatography/mass spectrometry of alditol acetates derived from the differential per-O-alkylated lipopolysaccharide, the majority of the arabinosyl residues were recognized as furanosides. Second, through analysis of per-O-alkylated oligoarabinosyl arabinitol fragments of partially hydrolyzed LAM, it was established that the internal segments of the arabinan component consists of branched 3,5-linked alpha-D-arabinofuranosyl (Araf) units with stretches of linear 5-linked alpha-D-Araf residues attached at both branch positions, whereas the nonreducing terminal segments of LAM consist of either of the two arrangements, beta-D-Araf-(1----2)-alpha-D-Araf-(1----5)- alpha-D-Araf---- or [beta-D-Araf-(1----2)-alpha-D-Araf-(1----]2---- (3 and 5)-alpha-D-Araf----. Since this latter arrangement also characterizes the terminal segments of the peptidoglycan-bound arabinogalactan of Mycobacterium spp., we propose that mycobacteria elaborate unique terminal arabinan motifs in two distinct settings. In the case of the bound arabinogalactan, these motifs provide the nucleus for the esterified mycolic acids, entities which dominate the physicochemical features of mycobacteria and their peculiar pathogenesis. In the case of LAM, these motifs, non-mycolylated, are the dominant B-cell antigens responsible for the majority of the copious antibody response evident in most mycobacterial infections.

The long-posed question of the nature of the link between the mycolylarabinogalactan and the underlying peptidoglycan of the cell walls of Mycobacterium sp. has been addressed. The insoluble cell wall matrix of Mycobacterium leprae, Mycobacterium tuberculosis, and Mycobacterium bovis was partially hydrolyzed with acid either before or after per-O-methylation and the resulting oligosaccharides further derivatized and analyzed by gas chromatography/mass spectrometry. The structures of fragments arising from the reducing end of arabinogalactan demonstrated the existence of the terminal sequence—-5)-D-Galf-(1—-4)-L-Rhap-(1—3)-D-GlcNAc. Other analyses confirmed the presence of muramyl-6-P within the peptidoglycan of these mycobacteria. Based on the acid lability of the 3-linked GlcNAc unit, the presence of about equimolar amounts of Rhap-(1—-3)-D-GlcNAc and muramyl-6-P in an isolated cell wall fragment, and 31P NMR analysis, it was concluded that the GlcNAc residue of the terminal triglycosyl unit of arabinogalactan is joined by 1-O-phosphoryl linkage to the 6-position of some muramyl residues within the peptidoglycan. Thus, it is reasoned that the massive mycolylarabinogalactan of mycobacteria, responsible for aspects of disease pathogenesis and much of the antibody response in infections, is attached to the peptidoglycan framework by the actinomycete-specific diglycosylphosphoryl bridge, L-Rhap-(1—-3)-D-GlcNAc-(1—-P, perhaps thereby providing a unique target for site-directed chemotherapy of mycobacterial infections.

The peptidoglycan-bound arabinogalactan of a virulent strain of Mycobacterium tuberculosis was per-O-methylated, partially hydrolyzed with acid, and the resulting oligosaccharides reduced and O-pentadeute-rioethylated. The per-O-alkylated oligoglycosyl alditol fragments were separated by high pressure liquid chromatography and the structures of 43 of these constituents determined by 1H NMR and gas chromatography/mass spectrometry. The arabinogalactan was shown to consist of a galactan containing alternating 5-linked beta-D-galactofuranosyl (Galf) and 6-linked beta-D-Galf residues. The arabinan chains are attached to C-5 of some of the 6-linked Galf residues. The arabinan is comprised of at least three major structural domains. One is composed of linear 5-linked alpha-D-arabinofuranosyl (Araf) residues; a second consists of branched 3,5-linked alpha-D-Araf units substituted with 5-linked alpha-D-Araf residues at both branched positions. The non-reducing terminal region of the arabinan was characterized by a 3,5-linked alpha-D-Araf residue substituted at both branched positions with the disaccharide beta-D-Araf-(1----2)-alpha-D-Araf. 13C NMR of intact soluble arabinogalactan established the presence of both alpha- and beta-Araf residues in this domain. This non-reducing terminal motif apparently provides the structural basis of the dominant immunogenicity of arabinogalactan within mycobacteria. A rhamnosyl residue occupies the reducing terminus of the galactan core and may link the arabinogalactan to the peptidoglycan. Evidence is also presented for the presence of minor structural features involving terminal mannopyranosyl units. Models for most of the heteropolysaccharide are proposed which should increase our understanding of a molecule responsible for much of the immunogenicity, pathogenicity, and peculiar physical properties of the mycobacterial cell.