Search

Light-chain deposition disease (LCDD) of the kidney is defined as deposition of monotypic light chains (LC) within glomerular (GBM) and tubular (TBM) basement membranes. The morphologic features of pure renal LCDD have been presented only in case reports or small series. The aim of this study was to perform a comprehensive evaluation of the light (LM), immunofluorescence (IF), and electron microscopic (EM) features of pure renal LCDD in a large series of biopsies. Out of 46 cases assembled, 42 had multiple myeloma, 2 had monoclonal gammopathy of unknown significance, and in 2 patients no lymphoproliferative disease was identified. The most common LM lesion of LCDD, nodular glomerulosclerosis, was present in only 14 (30%) cases. GBM and/or TBM thickening was found in 3 (6%), mild to moderate mesangial matrix increase in 12 (23%), and unremarkable glomeruli and tubules were seen in 15 (32%) cases. Forty-two had IF and 40 (92%) showed characteristic linear LC immunoreactivity (24 kappa, 16 lambda) along GBM and/or TBM. Among 39 cases in which IF and EM was available, 25 (64%) were positive by both. Two (6%) were negative by IF, but had deposits by EM. In 12 (30%) with immunoreactivity to LC (4 kappa, 8 lambda), no deposits were identified ultrastructurally. This study shows heterogeneous LM lesions in pure LCDD cases. LM alone may be suspicious but not diagnostic of LCDD. Immunofluorescence is more sensitive than EM for detection of LC for the definitive diagnosis of LCDD. This study supports the importance of utilizing kappa and lambda stains in the routine IF panel for diagnosis of LCDD. [ABSTRACT FROM AUTHOR]

Abstract: Characterization of multiprotein complexes (MPCs) is an important step toward an integrative view of protein interaction networks and prerequisite for a molecular understanding of how a certain MPC functions. Here, we present a technique utilizing monoclonal subunit-specific antibodies for an electrophoretic immunoshift assay in Blue Native-gels (NAMOS-assay), which allows the determination of the stoichiometry of MPCs. First, we use the B cell antigen receptor as a model MPC whose stoichiometry is known, confirming the HC2LC2Igα/β1 stoichiometry. Second, we demonstrate that the digitonin-extracted T cell antigen receptor (TCR) extracted from T cells has a stoichiometry of αβε2γδζ2. We then show that the NAMOS-assay does not require purified MPCs, since it can determine the stoichiometry of an MPC in cell lysates. The NAMOS-assay is also compatible with use of epitope tags appended to the protein of interest, as e.g. the widely used HA-tag, and anti-epitope antibodies for the assay. Given its general applicability, this method has a wide potential for MPC research. [Copyright &y& Elsevier]

Aims: Bone marrow sampling is a key investigation in the work-up of amyloid light chain (AL) amyloidosis, but the relationship between bone marrow findings and the varied phenotype and clinical outcome of AL amyloidosis is unclear. The aim was to determine if bone marrow pathological parameters at diagnosis were related to clinical behaviour in AL amyloidosis patients. Methods and results: Bone marrow findings, clinical features and outcome of 80 patients referred with a diagnosis of systemic AL amyloidosis were evaluated; six patients were subsequently excluded due to re-categorization as other forms of amyloidosis. At latest follow-up (median 66 months), 11 of the 18 patients with no identifiable bone marrow neoplastic cells (61%) versus only seven of the 56 patients with neoplastic plasma cells or non-Hodgkin's lymphoma (13%) were alive ( P = 0.0046). However, neither the quantity of the neoplastic cells nor the serum light chain levels were correlated with amyloid burden or patient survival. Conclusions: Identification of a neoplastic population in the bone marrow of AL amyloidosis patients by histology and immunohistochemistry correlates with poor outcome; however, the neoplastic cell burden is not prognostically significant, suggesting that additional factors are important in determining disease behaviour in AL amyloidosis. [ABSTRACT FROM AUTHOR]

Abstract: The antibody heavy chain is generally more important than the light chain for the interaction with the antigen, although many reports demonstrate the influence of the light chain in the antibody binding properties. The heavy chains of anti-N-glycolyl-ganglioside P3 mAb and anti-idiotypic 1E10 mAb display complementary charged residues in their H-CDRs, particularly in H-CDR3. A basic residue in P3 mAb H-CDR1 was shown to be crucial for the interaction with the antigen and 1E10 mAb. The immunogenetic features of three other P3 mAb anti-idiotypic mAbs are now analyzed. One of them bears the same heavy chain as 1E10 mAb and a different light chain, but differs in its binding to P3 mAb mutants where H-CDR basic residues were replaced and in the binding to 1E10-specific phagotopes. Chimeric hybrid antibodies with P3 and 1E10 mAb heavy chains and unrelated light chains were obtained to further determine the importance of heavy chains in P3 and 1E10 mAb binding properties. One of the P3 heavy chain hybrid antibodies retained the specificity of P3 mAb with slight affinity differences. The heavy chains appear to play the main role in these mAb interactions, with the light chains modulating the affinity to their ligands. [Copyright &y& Elsevier]

Abstract: Botulinum neurotoxin type A (BoNT/A) light chain (LC) is a zinc endopeptidase that causes neuroparalysis by blocking neurotransmitter release at the neuromuscular junctions. The X-ray crystal structure of the toxin reveals that His223 and His227 of the Zn2+ binding motif HEXXH directly coordinate the active site zinc. Two Glu residues (Glu224 and Glu262) are also part of the active site, with Glu224 coordinating the zinc via a water molecule whereas Glu262 coordinates the zinc directly as the fourth ligand. In the past we have investigated the topographical role of Glu224 by replacing it with Asp thus reducing the side chain length by 1.4 Å that reduced the endopeptidase activity dramatically [L. Li, T. Binz, H. Niemann, and B.R. Singh, Probing the role of glutamate residue in the zinc-binding motif of type A botulinum neurotoxin light chain, Biochemistry 39 (2000) 2399–2405]. In this study we have moved the Glu 224 laterally by a residue (HXEXH) to assess its positional influence on the endopeptidase activity, which was completely lost. The functional implication of Glu262 was investigated by replacing this residue with aspartate and glutamine using site-directed mutagenesis. Substitution of Glu262 with Asp resulted in a 3-fold decrease in catalytic efficiency. This mutation did not induce any significant structural alterations in the active site and did not interfere with substrate binding. Substitution of Glu262 with Gln however, dramatically impaired the enzymatic activity and this is accompanied by global alterations in the active site conformation in terms of topography of aromatic amino acid residues, zinc binding, and substrate binding, resulting from the weakened interaction between the active site zinc and Gln. These results suggest a pivotal role of the negatively charged carboxyl group of Glu262 which may play a critical role in enhancing the stability of the active site with strong interaction with zinc. The zinc may thus play structural role in addition to its catalytic role. [Copyright &y& Elsevier]

Abstract: To elucidate the subunit composition of axonemal inner-arm dynein, we examined a 38kDa protein (p38) co-purified with a Chlamydomonas inner arm subspecies, dynein d. We found it is a novel protein conserved among a variety of organisms with motile cilia and flagella. Immunoprecipitation using specific antibody verified its association with a heavy chain, actin and a previously identified light chain (p28). Unexpectedly, mutant axonemes lacking dynein d and other dyneins retained reduced amounts of p38. This finding suggests that p38 is involved in the docking of dynein d to specific loci. [Copyright &y& Elsevier]

Myeloma kidney injury is caused by the large amount of light chain (LC) of immunoglobulins produced by cancerous plasma cells through stimulation of proinflamatory cytokines like TNF-α and IL-6. PACAP-38 suppressed LC-stimulated cytokine production by tubular epithelial cells in vitro and in vivo, and prevented injury of these epithelial cells . The suppressive effect is comparable or greater than dexamethasone (dex). Although dex produces adverse side effects when it is given for a long time period, PACAP-38 is a natural and safe neuropeptide and no adverse effect has been reported when administered to produce significant biological effects. Furthermore, PACAP-38 suppressed growth of myeloma cells in culture and also suppressed production of their growth factor, IL-6, production from the bone marrow stromal cells that was stimulated by adhesion of myeloma cells. These findings render PACAP-38 worth evaluation as a safe and potent renoprotectant in myeloma kidney as well as a new antitumor agent for myeloma cells. [ABSTRACT FROM AUTHOR]

The question of whether prion diseases can be transmitted by body fluids has important epidemiological, environmental and economical implications. In this work, we set to investigate whether urine collected from scrapie-infected hamsters can transmit fatal or subclinical infectivity to normal hamsters. After prolonged incubation times ranging from 300 to 700 days, a small number of animals inoculated with scrapie urine succumbed to scrapie disease, and several asymptomatic hamsters presented low levels of PrPSc in their brains. In addition, most of the asymptomatic hamsters inoculated with scrapie urine, as opposed to those inoculated with normal urine, presented extensive gliosis as well as protease-resistant light chain IgG in their urine, a molecule shown by us and others to be a surrogate marker for prion infection. Our results suggest that urine from scrapie-infected hamsters can transmit a widespread subclinical disease that in some cases develops into fatal scrapie. Copyright © 2006 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: A full-length synthetic gene encoding the light chain of botulinum neurotoxin serotype B, approximately 50kDa (BoNT/B LC), has been cloned into a bacterial expression vector pET24a+. BoNT/B LC was expressed in Escherichia coli BL21.DE3.pLysS and isolated from the soluble fraction. The resultant protein was purified to homogeneity by cation chromatography and was determined to be >98% pure as assessed by SDS–polyacrylamide gel stained with SilverXpress and analyzed by densitometry. Mass spectroscopic analysis indicated the protein to be 50.8kDa, which equaled the theoretically expected mass. N-terminal sequencing of the purified protein showed the sequence corresponded to the known reported sequence. The recombinant BoNT/B light chain was found to be highly stable, catalytically active, and has been used to prepare antisera that neutralizes against BoNT/B challenge. Characterization of the protein including pH, temperature, and the stability of the protein in the presence or absence of zinc is described within. The influence of pH differences, buffer, and added zinc on secondary and tertiary structure of BoNT/B light chain was analyzed by circular dichroism and tryptophan fluorescence measurements. Optimal conditions for obtaining maximum metalloprotease activity and stabilizing the protein for long term storage were determined. We further analyzed the thermal denaturation of BoNT/B LC as a function of temperature to probe the pH and added zinc effects on light chain stability. The synthetic BoNT/B LC has been found to be highly active on its substrate (vesicle associated membrane protein-2) and, therefore, can serve as a useful reagent for BoNT/B research. [Copyright &y& Elsevier]

Abstract: Combinatorial diversity is highly restricted during formation of the pre-immune heavy chain repertoire of swine, raising the question of whether the same is true for the pre-immune light chain repertoire. Before addressing this question, we first used competitive PCR to show that kappa and lambda light chains in swine are equally expressed in mature B cells similar to the situation in humans but alike that in other studied Ungulates. This justified efforts to examine the repertoire of both light chain types. These studies also revealed that lambda is preferentially expressed at sites of B cells lymphogenesis, perhaps because of the use of a surrogate light chain containing λ5. Data are presented here on >100 VκJκ-containing transcripts and 180 genomic Vκ genes to show that >90% of the pre-immune repertoire is generated from three subfamilies of IGKV2 genes and one of five Jκ segments. The kappa locus contains ≥50 IGKV2 genes belonging to at least five subfamilies and an undetermined but perhaps equal number of IGKV1 genes. The porcine IGKV1 and IGKV2 genes share 87% sequence similarity with their human counterparts and Jκ1 through Jκ5 share sequence and organizational homology with those in sheep, horse, human and mouse. Swine have a single Cκ gene. These findings contrast with those from rodents and primates but are reminiscent of those on the pre-immune heavy chain repertoire of swine in that it is generated using a relatively restricted number of gene segments. These restricted pre-immune repertoires may reflect the minimal exposure of the fetus to maternal factors and environmental antigens. The significance for swine immunology of characterizing the pre-immune repertoire is discussed. [Copyright &y& Elsevier]

The clathrin triskelion is composed of three light chain (LC) and three heavy chain (HC) subunits. Cellular control of clathrin function is thought to be aimed at the LC subunit, mainly on the basis of structural information. To test this hypothesis in vivo, we used evanescent-wave photobleaching recovery to study clathrin exchange from single pits using LC (LCa and LCb) and HC enhanced green fluorescent protein fusion constructs. The recovery signal was corrected for cytosolic diffusional background, yielding the pure exchange reaction times. For LCa, we measured an unbinding time constant τLEa = 18.9 ± 1.0 seconds at room temperature, faster than previously published; for LCb, we found τLCb = 10.6 ± 1.9 seconds and for HC τHC = 15.9 ± 1.0 seconds. Sucrose treatment, ATP or Ca2+ depletion blocked exchange of LCa completely, but only partially of HC, lowering its time constant to τ = 10.0 ± 0.9 seconds, identical to the one for LCb exchange. The latter was also not blocked by Ca2+ depletion or sucrose. We conclude that HCs bound both to LCa and to LCb contribute side by side to pit formation in vivo, but the affinity of LCa-free HC in pits is reduced, and the Ca2+- and ATP-mediated control of clathrin function is lost. [ABSTRACT FROM AUTHOR]

Catalytic antibodies capable of digesting crucial proteins of pathogenic bacteria have long been sought for potential therapeutic use. Helicobacter pylori urease plays a crucial role for the survival of this bacterium in the highly acidic conditions of human stomach. The HpU-9 monoclonal antibody (mAb) raised against H. pylori urease recognized the α-subunit of the urease, but only slightly recognized the β-subunit. However, when isolated both the light and the heavy chains of this antibody were mostly bound to the β-subunit. The cleavage reaction catalyzed by HpU-9 light chain (HpU-9-L) followed the Michaelis-Menten equation with a Km of 1.6 × 10−5 m and a kcat of 0.11 min−1, suggesting that the cleavage reaction was enzymatic. In a cleavage test using H. pylori urease, HpU-9-L efficiently cleaved the β-subunit but not the α-subunit, indicating that the degradation by HpU-9-L had a specificity. The cleaved peptide bonds in the β-subunit were L121-A122, E124-G125, S229-A230, Y241-D242, and M262-A263. BSA was hardly cleaved by HpU-9-L, again indicating the digestion by HpU-9-L was specific. In summary, we succeeded in the preparation of a catalytic antibody light chain capable of specifically digesting the β-subunit of H. pylori urease. [ABSTRACT FROM AUTHOR]

Abstract: This contribution initially discusses the development of T-cells and the recognition of antigen; processing and presentation of antigen; and professional antigen-presenting cells. It then focuses on the activation and effector function of CD8+ and CD+ T-cells; and B-cells and the production of antibody. It finishes with a brief look at the concept of immunological memory. [Copyright &y& Elsevier]

Abstract: The necessity of a non-invasive in-vivo test for prion diseases has become more apparent since the transmission of vCJD from the blood of a healthy individual incubating the disease. Here we show that prion urine comprises an array of protease resistant peptides, among them light chain immunoglobulin (LC). This was observed by sequencing gel bands comprising hamster urine samples, as well as by immunoblotting of similar samples with anti mouse IgG reagents for hamster samples, or with anti human IgG reagents for human samples. Our result suggests that urine samples from CJD patients can be identified by the presence of protease resistant proteins such as LC. [Copyright &y& Elsevier]

We have recently shown that the anti-cardiolipin activity of human anti-phospholipid antibody UK4 (lambda) resides on its heavy chain. We now show that UK4 possesses strong reactivity to the plasma-protein β2-Glycoprotein I (β2-GPI) also. Utilizing chain shuffling experiments involving an unrelated anti-p185 antibody 4D5 (kappa) with no reactivity to β2-GPI, we now demonstrate that both the constructs possessing the auto-antibody-derived light chain exhibited significant binding to β2-GPI. However, the construct possessing UK4 heavy chain in association with 4D5 light chain, exhibited no anti-β2-GPI activity. Furthermore, there was a low increase (≅10%) in the binding of UK4 to cardiolipin in the presence of β2-GPI. The results demonstrate that anti-β2-GPI activity resides on UK4 light chain and, importantly, this activity could be transferred to a novel antibody construct via the light chain alone. Computer-generated models of the three-dimensional structures of UK4 and its hybrids, suggest predominant interaction of UK4 light chain with domain IV of β2-GPI. Molecular docking experiments highlight a number of potential sites on β2-GPI for interaction of UK4 and indicate as to how β2-GPI recognition may occur primarily via the autoantibody light chain. The study provides first demonstration of the occurrence of anti-phospholipid and anti-β2-GPI activities separately on heavy and light chains of an autoantibody. The possible mechanisms that such antibodies may employ to recognise their antigens, are discussed. [Copyright &y& Elsevier]

Case report of a 70-year-old woman with the diagnosis of multiple myeloma and acute renal failure due to rhabdomyolysis (RDM) that was caused from the deposition of κ light chain in muscle fibers. In addition, the deposition was found in the liver. [ABSTRACT FROM AUTHOR]

Somatic hypermutation, which diversifies the immunoglobulin repertoire by introducing mutations into rearranged IgV genes, is dependent on the expression of activation-induced cytidine deaminase (AID). It has been proposed that AID deaminates DNA directly, generating mutations at C bases. Mutations from C and G are linked, and it has been suggested that mutations from G arise either during repair of DNA following deamination of C or by deamination of C on both DNA strands. Studies demonstrating that AID deaminates ssDNA on the non-transcribed strand support the former hypothesis. However, analyses of microsequences surrounding mutations suggest that the G·C mutator acts on both DNA strands equivalently. Unusually, in human IgVλ genes, there is G·C strand bias favoring mutation from G. In IgVλ, 92% of mutations from G occur in GNW motifs. Hotspots for mutation from G IgVλ are often independent of C nucleotides in the context of local microsequence. This independence of G and C mutation, yet retained dependence on local microsequence suggests that mutations from G arise independent of C on the non-transcribed strand. We suggest that both DNA strands are deaminated and that the transcribed strand is preferentially deaminated in human IgVλ resulting in bias towards mutations from G. [Copyright &y& Elsevier]

Amyloid protein was isolated from formalin-fixed paraffin-embedded heart tissue sections from a patient with primary (AL) amyloidosis by extraction with 6 M guanidine HCl. SDS-PAGE analysis of extracted material showed a major band at 16 kDa and a minor band at 18 kDa. Edman degradation analysis before and after pyroglutamate aminopeptidase treatment showed that the amyloid protein contained N-terminal pyroglutamic acid and was derived from an immunoglobulin λ light chain. Analysis of tryptic peptides from the extract identified the amyloid protein as a λI. Of particular interest is that almost the entire amyloid protein amino acid sequence could be obtained from the cardiac sections. These results demonstrate that formalin-fixed paraffin-embedded tissue sections can be used for extensive biochemical characterization of amyloid proteins and will become a valuable source for isolation and extensive biochemical characterization of amyloid proteins as they are now a valuable source for isolation of DNA for genetic analysis. [ABSTRACT FROM AUTHOR]

The catalytic activity of the highly potent botulinum neurotoxins are confined to their N-terminal light chains (∼50 kDa). A full-length light chain for the type E neurotoxin with a C-terminal 6× His-tag, BoNT/E-LC, has been cloned in a pET-9c vector and over-expressed in BL21 (DE3) cells. BoNT/E-LC was purified to homogeneity by affinity chromatography on Ni–NTA agarose followed by exclusion chromatography using a Superdex-75 sizing column. The purified protein has very good solubility and can be stored stably at −20 °C; however, it seems to undergo auto-proteolysis when stored at temperature ⩾4–10 °C. BoNT/E-LC is active on its natural substrate, the synaptosomal associated 25 kDa protein, SNAP-25, indicating that it retains a native-like conformation and therefore can be considered as a useful tool in studying the structure/function of the catalytic light chain. Recombinant BoNT/E-LC has been crystallized under five different conditions and at various pHs. Crystals diffract to better than 2.1 A˚. [Copyright &y& Elsevier]

Transmission ratio distortion is a dramatic example of non-Mendelian transmission. In mice, t-haplotype males produce dysfunctional +-sperm and normal t-sperm, leading to transmission in favor of t-sperm. Genetic studies have indicated that the t-complex responder locus, Tcr, rescues t-sperm but not +-sperm from defective products of t-complex distorter loci, Tcds. Light chain 1 (LC1) and LC3 from sea urchin sperm outer arm dynein have sequence similarities to Tctex2 and Tctex1, respectively, both of which are wild-type products of Tcds. We show here that LC1 and LC3 are able to make a 1:1 complex. Since Tcr is a member of the Smok (sperm motility kinase) family and LC1 is phosphorylated at the activation of sperm motility in a cAMP-dependent manner, this complex in a dynein motor molecule might be a direct target of Smok/Tcr kinase in a signal cascade that regulates sperm motility. Thus, we designate it as Smoac (sperm motility activating complex). [Copyright &y& Elsevier]

Immunoglobulin light chain cDNA sequences of a perciform fish, the mandarin fish Siniperca chuatsi were amplified from head kidney mRNA by reverse transcription (RT)-PCR and RACE methods using degenerated primer and gene specific ones. In cDNA sequences of the VL region, nucleotide exchanges were present mainly within CDRs, although a lesser degree of variability was also found in FRs. Moreover, the length of CDR1 and CDR3 in the mandarin fish is shorter than in most other fish species. In the middle of S. chuatsi CL region, a microsatellite sequence (AGC)6–8 was found, which is also present in another perciform species, the spotted wolffish (Anarhichas minor). The comparison of amino acid sequence of the mandarin fish CL domain with those of other vertebrates showed the highest degree of similarity of 94.5% to the spotted wolffish, while the similarity with rainbow trout (Oncorhynchus mykiss) Ig L1 (62.7%) and channel catfish (Ictalurus punctatus) Ig LG (55.9%) isotypes is also higher. However, there is only 50% identity in the VL regions between the mandarin fish and the wolffish. The sequence similarity of the mandarin fish CL domain with those of higher vertebrate did not readily allow it to be classified as κ or λ isotype. The phylogenetic analyses also demonstrated that the CL genes of the mandarin fish and most other teleost fish cluster as a separate branch out of the mammal κ and λ branches. [Copyright &y& Elsevier]

Clostridial neurotoxins are potent inhibitors of synaptic function, with the zinc-dependent proteolytic light chain (LC) portion of the toxin cleaving one of three neural SNARE proteins. In nature, the LC is expressed as a part of a much larger toxin and hemagglutinin complex, protecting it from environmental degradation and preserving its catalytic activity. We developed forms of the LC of type A botulinum neurotoxin (BoNT-A) with parts of the larger toxin gene, for use as reagents in high-throughput assays to screen for potential LC antagonists, to further elucidate the toxin''s mechanism of action, and to study immunological responses to the toxin. Three BoNT-A constructs were engineered and expressed: the LC, LC with translocation region (LC+Hn), and the LC with the belt portion of the translocation region (LC+Belt). Purification was optimized to a two-step process, with relatively high yields of all three constructs obtained. Activity assays showed all three constructs to be active, with the LC being the most active. Immunogenic protection against native BoNT-A toxin challenge was observed for all three constructs, with the best protection observed with the LC+Hn and LC+Belt proteins. [Copyright &y& Elsevier]

A probabilistic model of allelic exclusion fails to explain the status of receptor genes and the receptor phenotype of most B cells. A large proportion of B cells have incompletely rearranged H and/or L chain genes (e.g. κ0/κ+) and most B cells express only one receptor. These properties seem to require deterministic features of B cell development such as special mechanisms that stop rearrangement. However, receptor editing has revealed that rearrangement-stop is not stable and that multi-receptor lymphocytes make up a significant fraction of certain B and T cell populations. Consequently we have revived the purely probabilistic approach in a model that now includes receptor editing and allows for some multi-receptor B cells. We find that this model can explain the observed properties of B cells when the frequency of self-reactive B cells is high. Indeed, as we illustrate for anti-DNA, this is the case. Hence the probabilistic model has life and assiduous use of the model suggests unexpected but not unrealistic features of lymphocyte development. [Copyright &y& Elsevier]

Highly purified recombinant zinc-endopeptidase light chain of the botulinum neurotoxin serotype A underwent autocatalytic proteolytic processing and fragmentation. In the absence of added zinc, initially 10-28 residues were cleaved from the C-terminal end of the 448-residue protein followed by the appearance of an SDS-stable dimer and finally fragmentation near the middle of the molecule. In the presence of added zinc, the rate of fragmentation was accelerated but the specificity of the cleavable bond changed, suggesting a structural role for zinc in the light chain. The C-terminal proteolytic processing was reduced, and fragmentation near the middle of the molecule was prevented by adding the metal chelator TPEN to the light chain. Similarly, adding a competitive peptide inhibitor (CRATKML) of the light-chain catalytic activity also greatly reduced the proteolysis. With these results, for the first time, we provide clear evidence that the loss of C-terminal peptides and fragmentation of the light chain are enzymatic and autocatalytic. By isolating both the large and small peptides, we sequenced them by Edman degradation and ESIMS-MS, and mapped the sites of proteolysis. We also found that proteolysis occurred at F266-G267, F419-T420, F423-E424, R432-G433, and C430-V431 bonds in addition to the previously reported Y250-Y251 and K438-T439 bonds. [ABSTRACT FROM AUTHOR]

We report here the structure of cDNA clones encoding axolotl light chains of the lambda type. A single IGLC gene and eight different potential IGLV genes belonging to four different families were detected. The axolotl Cλ domain has several residues or stretches of residues that are typically conserved in mammalian, avian, and Xenopus C λ, but the KATLVCL stretch, which is well conserved in the Cλ and T-cell receptor Cβ domains of many vertebrate species, is not well conserved. All axolotl Vλ sequences closely match several human and Xenopus Vλ-like sequences and, although the axolotl Cλ and Vλ sequences are very like their tetrapod homologues, they are not closely related to nontetrapod L chains. Southern blot experiments suggested the presence of a single IGLC gene and of a limited number of IGLV genes, and analysis of IGLV-J junctions clearly indicated that at least three of the IGLJ segments can associate with IGLV1, IGLV2, or IGLV3 subgroup genes. The overall diversity of the axolotl Vλ CDR3 junctions seems to be of the same order as that of mammalian Vλ chains. However, a single IGLV4 segment was found among the 45 cDNAs analyzed. This suggests that the axolotl IGL locus may have a canonical tandem structure, like the mammalian IGK or IGH loci. Immunofluorescence, immunoblotting, and microsequencing experiments strongly suggested that most, if not all L chains are of the λ type. This may explain in part the poor humoral response of the axolotl. [ABSTRACT FROM AUTHOR]

Epidermolysis bullosa acquisita and bullous systemic lupus erythematosus are blistering skin diseases characterized by IgG autoantibodies that predominantly target the noncollagenous domain 1 of type VII collagen, a skin basement membrane component. The basic immunologic events leading to the blistering processes in these diseases remains unclear. We defined the subclass and light chain compositions of the IgG autoantibodies in 15 patients, in order to gain insight into the blistering mechanism. Immunofluorescence correlated the patients' in vivo-bound and circulating antibasement membrane autoantibodies. Four eukaryotic recombinant proteins, including one full-length and three truncated noncollagenous domain 1 proteins generated by sequential deletion of C-terminal amino acids, were used to perform enzyme-linked immunosorbent assay to detect the patients' anti-type VII collagen autoantibodies. The majority of patients' autoantibodies contained both complement-activating and non-complement-activating IgG subclasses. The presence or absence of complement-activating IgG autoantibody subclasses did not correlate with the inflammatory or noninflammatory clinical phenotype. The majority of tested sera contained both κ and λ light chain autoantibodies. All sera that reacted to the full-length noncollagenous domain 1 also reacted to the smallest truncated protein containing the cartilage matrix protein and the first three fibronectinlike repeats. The patients' anti-type VII collagen autoantibodies, likely to be polyclonal in nature, may contribute to the pathogenesis of the blistering process by both complement-dependent inflammatory injury and complement-independent mechanical disruption of the anchoring function of type VII collagen. The N-terminal region of the noncollagenous domain 1 may contain an important antigenic epitope targeted by the IgG autoantibodies. [ABSTRACT FROM AUTHOR]

A syngeneic monoclonal idiotypic antibody was prepared by immunizing the sequence peptide of complementary determining region-1 (CDRL-1) of 41S-2-L which is an antibody light chain capable of catalytically decomposing the antigen peptide (gp41 peptide:original antigen) as well as the intact gp41 molecule of HIV-1 envelope. The obtained idiotypic antibody, i41SL1-2, showed a high specificity to the CDRL-1 peptide. The intact i41SL1-2 and its heavy and light chains displayed apparent affinity constants to the CDRL-1 peptide of 3.6 × 10, 2.7 × 10, 1.8 × 10/M, respectively. The i41SL1-2 recognized the artificial molecule CA2, which has a more complex steric conformation than the CDRL-1, while the i41SL1-2 showed very low affinity to the original monoclonal antibody 41S-2 and its light chain 41S-2-L. However, a homologous sequence, EGG-D, with the gp41 peptide was expressed in the complementary determining region-3 (CDRH-3) of the heavy chain of i41SL1-2. Furthermore, the consensus sequence EGG was located at the important position of the CDRH-3 loop of i41SL1-2. Although the sequence of CDRL-1 (16 mer) is quite shorter than that of whole light chain (112 mer), the CDRL-1 could induce the rearrangement of CDRH-3 gene of i41SL1-2 so as to express a homologous sequence with the original antigen. [ABSTRACT FROM AUTHOR]

Botulinum neurotoxins, the most potent of all toxins, induce lethal neuromuscular paralysis by inhibiting exocytosis at the neuromuscular junction. The light chains (LC) of these dichain neurotoxins are a new class of zinc-endopeptidases that specifically cleave the synaptosomal proteins, SNAP-25, VAMP, or syntaxin at discrete sites. To facilitate the structural and functional characterization of these unique endopeptidases, we constructed a synthetic gene for the LC of the botulinum neurotoxin serotype A (BoNT/A), overexpressed it in Escherichia coli, and purified the gene product from inclusion bodies. Our procedure can provide 1.1 g of the LC from 1 L of culture. The LC product was stable in solution at 4°C for at least 6 months. This rBoNT/A LC was proteolytically active, specifically cleaving the Glu-Arg bond in a 17-residue synthetic peptide of SNAP-25, the reported cleavage site of BoNT/A. Its calculated catalytic efficiency kcat/ Km was higher than that reported for the native BoNT/A dichain. Treating the rBoNT/A LC with mercuric compounds completely abolished its activity, most probably by modifying the cysteine-164 residue located in the vicinity of the active site. About 70% activity of the LC was restored by adding Zn2+ to a Zn2+-free, apo-LC preparation. The LC was nontoxic to mice and failed to elicit neutralizing epitope(s) when the animals were vaccinated with this protein. In addition, injecting rBoNT/A LC into sea urchin eggs inhibited exocytosis-dependent plasma membrane resealing. For the first time, results of our study make available a large amount of the biologically active toxin fragment in a soluble and stable form. [ABSTRACT FROM AUTHOR]

Chemical catalysis, an effector mechanism utilized by fully assembled antibodies, can also be mediated by the isolated antibody subunits. Because trace amounts of free light chains (L chains) are present in IgG preparations, a detailed study was undertaken to identify the constituents responsible for the polyreactive proteolytic activity of IgG purified from human sera, determined as the extent of cleavage of the model peptide substrate Pro-Phe-Arg-methylcoumarinamide. Two proteolytic species with approximate mass of 50 kD and 150 kD were separated by repetitive gel filtration in a denaturing solvent (6 m guanidine hydrochloride). The activity of the renatured 50-kD fraction (in fluorescence units/μg protein) was more than 45-fold greater than of the 150-kD fraction. Both fractions lost the activity following immunoadsorption on immobilized anti-IgG antibody. Fab fragments prepared from the 150-kD IgG fraction retained the activity. Reducing and non-reducing SDS-electrophoresis suggested the 50-kD fraction isolated from the IgG preparations to be a mixture of heavy chain (H chain) monomers and disulphide bonded L chain dimers. Electrophoretically homogeneous monomers of 50-kD H chains and 25-kD L chains were prepared by gel filtration of reduced and alkylated IgG from seven human subjects. Each of the alkylated L chain preparations displayed the proteolytic activity. The activity in alkylated H chains was undetectable or only marginally greater than the background values. L chain dimers appear to be the major species responsible for the polyreactive proteolytic activity of serum IgG preparations, with a smaller contribution furnished by tetrameric IgG. [ABSTRACT FROM AUTHOR]

A portion of the mouse complement factor I (mCFI) gene encoding for the mCFI light chain was cloned from a mouse 129/SVJ1 bacterial artificial chromosome library. It contains five exons and four introns. The intron sizes are remarkably different from the human homolog. Several polymorphisms were found in exon 13. One polymorphism was in the coding region, which causes a threonine in the Balb/c mCFI to be replaced by an isoleucine in the 129/SVJ1 mCFI. The other two polymorphisms are located in the 3' untranslated region. The organization of the serine protease domain in mCFI is similar to that of trypsin but very different from that of the other complement serine proteases. [ABSTRACT FROM AUTHOR]

The autoimmune repertoire is well known from previous studies to be capable of producing catalytic antibodies directed to self-antigens. In the present study, we explored the ability of 26 monoclonal light chains (Lchains) from multiple myeloma patients to cleave radiolabeled gp 120, a foreign protein. One L chain with this activity was identified. 125I-gp120 and unlabeled gp 120 were cleaved at several sites by the L chain, as shown by SDS-polyacrylamide gel electrophoresis, autoradiography, and immunoblotting, respectively. The apparent dissociation constant of the L chain was 130–145 n M, indicating high-affinity gp 120 recognition. 125I-albumin was not cleaved by the L chain, and various proteins and peptides did not inhibit gp 120 cleavage by the L chain, suggesting that the activity is not a nonspecific phenomenon. The substrate recognition determinants may be conserved in different HIV-1 strains, because gp 120 isolated from strains SF2, MN, and IIIB was found to be cleaved by the L chain. Micromolar concentrations of a synthetic peptide corresponding to residues 23–30 of gp 120 inhibited the cleavage of 125I-gp 120, suggesting that these residues are components of the epitope recognized by the L chain. The toxic effect of gp120 in neuronal cultures was reduced by about 100-fold by pretreatment of the protein with the L chain. These observations open the possibility of utilizing gp120-cleaving antibodies in the treatment of AIDS. [ABSTRACT FROM AUTHOR]

Virus infection, inflammation and genetic factors are important factors in the pathogenesis of diabetes mellitus. The nuclear factor-kappa B (NF-κB) is a family of transcription factors that bind the enhancer of the κ light chain gene of B cell immunoglobulin. NF-κB plays an essential role in the activation and development of B cells, and the activation of NF-κB is critical in the inflammation and development of diabetes mellitus. Recently, immunoglobulin-free light chain (FLC) λ was found to be increased in the sera of patients with diabetes mellitus, and the FLC λ and κ/λ ratios are more specific and sensitive markers for the diagnosis of diabetes relative to glycated hemoglobin A1c. Thus, FLCs may be promising biomarkers of inflammation that could relate to the activation of NF-κB. We suggest that NF-κB could be a target for an anti-inflammatory strategy in preventing and treating diabetes when FLCs are modified. FLCs could be a surrogate endpoint in the management of diabetes. In this review, the role of inflammation in the pathogenesis of diabetes, as well as the novel inflammatory biomarkers of FLCs for the management of diabetes, are discussed.

Prominent inclusion bodies can develop in the endoplasmic reticulum (ER) when overexpressed antibodies possess intrinsically high condensation propensities. These observations suggest that antibodies deemed to show notable solubility problems may reveal such characteristics preemptively in the form of ER-associated inclusion bodies during antibody overexpression. To define the relationships between solubility problems and inclusion body phenotypes, we investigated the biosynthesis of a model human IgG2λ that shows severe opalescence in an acidic formulation buffer yet retains high solubility at physiological pH. Consistent with the pH-dependent solubility characteristics, the model antibody did not induce notable inclusion body in the physiological pH environment of the ER lumen. However, when individual subunit chains of the antibody were expressed separately, the light chain (LC) spontaneously induced notable crystal-like inclusion bodies in the ER. The LC crystallization event was readily reproducible in vitro by simply concentrating the purified LC protein at physiological pH. Two independent structural determinants for the LC crystallization were identified through rational mutagenesis approach by monitoring the effect of amino acid substitutions on intracellular LC crystallogenesis. The effect of mutations on crystallization was also recapitulated in vitro using purified LC proteins. Importantly, when introduced directly into the model antibody, a mutation that prevents the LC crystallization remediated the antibody's solubility problem without compromising the secretory output or antigen binding. These results illustrate that the ER can serve as a "physiological test tube" that not only reports secretory cargo's high condensation propensity at physiological pH, but also provides an orthogonal method that guides antibody engineering strategy. • The LC subunit of a poorly soluble model IgG2λ crystallized spontaneously in the ER. • LC crystallization was readily reconstituted using purified protein in vitro at physiological pH. • Structural determinants underlying the LC crystallization were elucidated. • Mutation to the determinant remediated the solubility problem when introduced directly to IgG2λ. • The ER can not only detect secretory cargo's high condensation propensity but also guide antibody engineering strategy. [ABSTRACT FROM AUTHOR]

Background: Monoclonal gammopathy is a biological reality encountered in approximately 1% of the general population. In the absence of clinical and biological signs, it is considered of undetermined significance; however, it can be a biological signature of a monoclonal lymphocytic or plasma-cell proliferation. Their localisation to the oral mucosa remains rare and difficult to diagnose, particularly in indolent forms that escape imaging techniques., Case Presentation: Here, we report the case of a 73-year-old woman with a history of IgM kappa gammopathy followed for 13 years. The patient did not have a chronic infection or an autoimmune disease, and all the biological investigations and radiological explorations were unremarkable during this period. The discovery of a submucosal nodule in the cheek led to the diagnosis of MALT lymphoma and regression of half of the IgM kappa level after resection. The review of the literature shows the dominance of clinical signs (i.e., a mass or swelling) in the diagnosis of primary MALT lymphomas of the oral cavity after surgical resection., Conclusions: Our case illustrates the role of examination of the oral cavity in the context of a monoclonal gammopathy. The absence of clinical and radiological evidence in favor of lymphoplasmacytic proliferation, does not exclude a primary indolent MALT lymphoma of the oral mucosa., (© 2021. The Author(s).)