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PubMed2003

Cellular binding of hepatitis C virus envelope glycoprotein E2 requires cell surface heparan sulfate.

The conservation of positively charged residues in the N terminus of the hepatitis C virus (HCV) envelope glycoprotein E2 suggests an interaction of the viral envelope with cell surface glycosaminoglycans. Using recombinant envelope glycoprotein E2 and virus-like particles as ligands for cellular binding, we demonstrate that cell surface heparan sulfate proteoglycans (HSPG) play an important role in mediating HCV envelope-target cell interaction. Heparin and liver-derived highly sulfated heparan sulfate but not other soluble glycosaminoglycans inhibited cellular binding and entry of virus-like particles in a dose-dependent manner. Degradation of cell surface heparan sulfate by pretreatment with heparinases resulted in a marked reduction of viral envelope protein binding. Surface plasmon resonance analysis demonstrated a high affinity interaction (KD 5.2 x 10-9 m) of E2 with heparin, a structural homologue of highly sulfated heparan sulfate. Deletion of E2 hypervariable region-1 reduced E2-heparin interaction suggesting that positively charged residues in the N-terminal E2 region play an important role in mediating E2-HSPG binding. In conclusion, our results demonstrate for the first time that cellular binding of HCV envelope requires E2-HSPG interaction. Docking of E2 to cellular HSPG may be the initial step in the interaction between HCV and the cell surface resulting in receptor-mediated entry and initiation of infection.

Cell Line↗
PubMed2005

Mechanism of chain selection in the assembly of collagen IV: a prominent role for the alpha2 chain.

Collagens comprise a large superfamily of extracellular matrix proteins that play diverse roles in tissue function. The mechanism by which newly synthesized collagen chains recognize each other and assemble into specific triple-helical molecules is a fundamental question that remains unanswered. Emerging evidence suggests a role for the non-collagenous domain (NC1) located at the C-terminal end of each chain. In this study, we have investigated the molecular mechanism underlying chain selection in the assembly of collagen IV. Using surface plasmon resonance, we have determined the kinetics of interaction and assembly of the alpha1(IV) and alpha2(IV) NC1 domains. We show that the differential affinity of alpha2(IV) NC1 domain for dimer formation underlies the driving force in the mechanism of chain discrimination. Given its characteristic domain recognition and affinity for the alpha1(IV) NC1 domain, we conclude that the alpha2(IV) chain plays a regulatory role in directing chain composition in the assembly of (alpha1)(2)alpha2 triple-helical molecule. Detailed crystal structure analysis of the [(alpha1)(2)alpha2](2) NC1 hexamer and sequence alignments of the NC1 domains of all six alpha-chains from mammalian species revealed the residues involved in the molecular recognition of NC1 domains. We further identified a hypervariable region of 15 residues and a beta-hairpin structural motif of 13 residues as two prominent regions that mediate chain selection in the assembly of collagen IV. To our knowledge, this report is the first to combine kinetics and structural data to describe molecular basis for chain selection in the assembly of a collagen molecule.

Amino Acid Sequence↗
PubMed2005

Structural and functional insights into the interaction of echoviruses and decay-accelerating factor.

Many enteroviruses bind to the complement control protein decay-accelerating factor (DAF) to facilitate cell entry. We present here a structure for echovirus (EV) type 12 bound to DAF using cryo-negative stain transmission electron microscopy and three-dimensional image reconstruction to 16-A resolution, which we interpreted using the atomic structures of EV11 and DAF. DAF binds to a hypervariable region of the capsid close to the 2-fold symmetry axes in an interaction that involves mostly the short consensus repeat 3 domain of DAF and the capsid protein VP2. A bulge in the density for the short consensus repeat 3 domain suggests that a loop at residues 174-180 rearranges to prevent steric collision between closely packed molecules at the 2-fold symmetry axes. Detailed analysis of receptor interactions between a variety of echoviruses and DAF using surface plasmon resonance and comparison of this structure (and our previous work; Bhella, D., Goodfellow, I. G., Roversi, P., Pettigrew, D., Chaudhry, Y., Evans, D. J., and Lea, S. M. (2004) J. Biol. Chem. 279, 8325-8332) with reconstructions published for EV7 bound to DAF support major differences in receptor recognition among these viruses. However, comparison of the electron density for the two virus.receptor complexes (rather than comparisons of the pseudo-atomic models derived from fitting the coordinates into these densities) suggests that the dramatic differences in interaction affinities/specificities may arise from relatively subtle structural differences rather than from large-scale repositioning of the receptor with respect to the virus surface.

CD55 Antigens↗
PubMed1998

HLA-DR is strongly associated with Vogt-Koyanagi-Harada disease in Mexican Mestizo patients.

PURPOSE: To analyze the genetic background of human leukocyte antigens (HLA) of Vogt-Koyanagi-Harada (VKH) disease in Mexican Mestizo patients in order to establish whether the pathogenesis is related to the same genes or sequences described in other populations. PATIENTS AND METHODS: In 48 VKH patients, we performed HLA class I and class II typing using the standard microlymphocytotoxicity tests; a group of 100 nonrelated healthy subjects were analyzed for comparison. Antigen and gene frequencies were calculated for every antigen tested in patients and in controls. RESULTS: The frequency of HLA-DR4 was significantly increased in VKH Mexican patients (x2Y = 19.95; p = 0.00001; pc = 0.0002; RR = 5.3; EF = 0.52); a discrete increase in DR1 was also found (p = 0.02). HLA-DQ8 also showed a significant association with the disease with a lower RR (3.2) and EF (0.41) than DR4. CONCLUSION: The strong association found with HLA-DR4 and the slight DR1 increase shown in Mexican patients with VKH suggest that a common shared sequence present in the third hypervariable region of DRB1 genes is relevant for the expression of the disease. The stronger association with DR4 than the one with DQ8 suggests that the DR locus carries the primary susceptibility genes involved in the pathogenesis of VKH.

Adolescent↗
PubMed2003

Characterization of infectious bursal disease virus isolates from Indonesia indicates the existence of very virulent strains with unique genetic changes.

Sequencing of the hypervariable region of viral protein VP2 of infectious bursal disease virus (IBDV) isolates obtained from non-vaccinated chickens in Indonesia showed that the majority (16/17) were closely related to published very virulent (vv)IBDV strains. Four isolates contained identical amino acid sequences to Asian and European vvIBDVs, sharing vv-specific amino acid residues 222(Ala), 256(Ile), and 294(Ile). Eight isolates differed by one amino acid at position 222(Ala-->Ser); however, this change did not alter the pathogenicity or antigenicity of these strains. Two isolates, with amino acid substitutions at positions 272(Ile-->Thr) and 279(Asp-->Asn), did not cause clinical disease or mortality, and were therefore considered to be naturally occurring, attenuated mutants of vvIBDV. The results illustrate variability that might occur among vvIBDV strains.

Amino Acid Sequence↗
PubMed1977

Frequency of occurrence of idiotypes associated with anti-p-azophenylarsonate antibodies arising in mice immunologically suppressed with respect to a cross-reactive idiotype.

Inoculation of rabbit anti-idiotypic (anti-id) antibodies suppresses the subsequent appearance of a cross-reactive idiotype (CRI) associated with the anti-p-azophenylarsonate (anti-Ar) antibodies of A/J mice. Such suppressed mice produce normal concentrations of anti-Ar antibodies which lack the CRI, but against which anti-id antisera can be prepared. The anti-Ar antibodies of an individual, suppressed mouse do not in general share idiotype with anti-Ar antibodies of other A/J mice, either suppressed or nonsuppressed. The present experiments were undertaken to quantitate several "private idiotypes" in a large number of hyperimmunized A/J mice. Anti-Ar antibodies of three mice, suppressed for the CRI, were labeled with 125I and subjected to isoelectric focusing. Four single peaks, that were over 90% reactive with autologous antiid, were randomly selected for use as ligands in a radioimmunoassay, and ascitic fluids containing anti-Ar antibodies from 181 A/J mice were tested as inhibitors. Two of the four idiotypes could not be detected in any mouse other than the donor. The concentration of the idiotype was less than 1 part in 1,250 to less than 1 part in 25,000 of the anti-Ar antibody population; these are minimum values. A third idiotype was detected in 3 of the 181 mice, but at very low concentrations. The fourth idiotype was present in 28% of the mice, again at a low concentration. The data support the existence of a very large repertoire of anti-Ar antibodies in the A/J strain and are consistent with a process of random somatic mutation for generating diversity in hypervariable regions. It is proposed that the cross-reactive idiotype may be controlled by a germ line gene or a gene related to a germ line gene through a small number of somatic mutations; and that the idiotypes that were not detectable in other mice were the products of genes that had undergone extensive mutations, with a low probability of recurrence in other mice.

Animals↗
PubMed1977

Novel idiotypic and antigen-binding characteristics in two anti-dinitrophenyl monoclonal antibodies.

Monoclonal anti-dinitrophenyl antibodies generated in the splenic focus system from B cells of adult BALB/c mice were studied for the presence or absence of murine anti-T15 (M anti-T15) reactivity and for their ability to bind phosphorylcholine (PC). Two foci of the 680 clones analyzed bound PC, and one of these antibodies reacted with M anti-T15 and anti-Fab on a 1:1 weight basis. The discovery of a clonotype reactive with M anti-15 but not with rabbit anti-T15 (R anti-T15) serum, the converse of the R anti-T15+, M anti-T15- clonotype identified in the PC-specific repertoire, points to the novel idiotypic relationships which may be found among homogeneous antibodies binding diverse antigens. The R anti-T15-, M anti-T15+ clonotype may represent a distinct set of hypervariable region sequences inserted into the T15 framework or may be a somatic variant of the T15 germ-line sequence. In addition, the maximum frequency with which this clonotype occurs within the B-cell pool is estimated.

Animals↗
PubMed1980

Presence of highly conserved idiotypic determinants in a family of antibodies that constitute an intrastrain cross-reactive idiotype.

It has been shown that A/J anti-p-azophenylarsonate antibodies that share a major cross-reactive idiotype (CRI) comprise a family of closely related, but nonidentical, molecules. Our results demonstrate that 12 of 14 monoclonal hybridoma products that express the CRI have in common at least one highly conserved idiotypic determinant. It is proposed that this reflects conservation of a portion of the amino acid sequence, presumably in hypervariable regions. That the conserved determinant(s0 are located in the region of the hapten-binding site is indicated by the ability of haptens to inhibit idiotype-anti-idiotype interactions involving the conserved, or public determinants.

Animals↗
PubMed1984

A class II gene conversion event defines an antigen-specific Ir gene epitope.

To assess the role of Ia epitopes in conferring specificity for the immune response to nominal antigen, we compared the insulin response of mice with a defined mutation in the I-Ab beta gene, the B6.C-H-2bm12 (bm12), with that of wild-type H-2b C57BL/6 (B6) mice. We report that the bm 12 mutation resulted in a selective alteration of the specificity of insulin recognition, such that bm 12 mice responded upon immunization with sheep but not beef insulin, which differ by only one amino acid at position 9 of the insulin A chain. Thus, the bm12 mutation allows for the definition of the actual nucleotide sequence coding for an Ia epitope that is responsible for controlling the specificity of immune recognition of insulin. Furthermore, we show that the sheep insulin response of H-2k mice is controlled by the E molecule and that sheep insulin can be recognized by primed bm12 and H-2k T cells in the context of either bm12, B10.A, or B10.A(5R) antigen-presenting cells. Our data suggest that the mechanism for the bm12 mutation was the intergenic transfer of a hypervariable region in the first domain that is identical in the I-Abm12 beta, I-Eb beta, and I-Ek beta genes.

Animals↗
PubMed1985

Characterization of an epibody. An antiidiotype that reacts with both the idiotype of rheumatoid factors (RF) and the antigen recognized by RF.

Recently, an antiidiotype to human monoclonal IgM anti-IgG autoantibodies (rheumatoid factors) was found to react also with human IgG. This peculiar antiidiotype was called an 'epibody'. We describe the induction of a similar epibody by immunization with a synthetic peptide (corresponding to one hypervariable region of the IgM-RF Glo). The results confirm the existence of epibodies, and provide the possible molecular basis of the epibody phenomenon.

Animals↗
PubMed1988

The importance of the location of antibody binding on the M6 protein for opsonization and phagocytosis of group A M6 streptococci.

One of 19 mAbs against the native group A streptococcal M6 protein proved opsonic for type 6 organisms in a bactericidal assay. The opsonic and three nonopsonic antibodies were selected for isotype and complement fixation studies based on previous knowledge of their epitope site on the M6 molecule. While mAb 3B8 (IgG3), whose epitope is in the NH2-terminal hypervariable region of the molecule (distal from the cell), and mAbs 10B6 (IgG2a) and 10F5 (IgG2b), both located in the conserved central region of the molecule, all fix complement, 10A11 (IgG1) did not. Only mAb 3B8 was opsonic despite the fact that mAbs 10B6 and 10F5 both exhibited similar complement-fixing capacity, binding titer, and surface exposure of epitopes. Analysis of antibodies raised against synthetic peptides representing various regions of the M6 protein showed that only the amino-terminal peptide (residues 1-21) was capable of eliciting opsonic antibodies, despite the fact that peptides from other areas produced antibodies with high-binding titers to the native M6 protein and also with the ability to bind to intact streptococcal cells. These results not only support the observed type specificity of opsonic antibodies, but also clearly point to the importance of the location of antibody binding on the M molecule relative to the actual functional capacity of the antibody with respect to the opsonization and phagocytosis of M6 streptococci. These results may underscore the recently observed role of complement Factor H in the antiphagocytic activity of the M protein.

Amino Acid Sequence↗
PubMed1989

T cells sensitized to synthetic HLA-DR3 peptide give evidence of continuous presentation of denatured HLA-DR3 molecules by HLA-DP.

T cell clones raised against a synthetic peptide, identical to the third hypervariable region of the DR3 BI chain, were tested for secondary proliferative responses against a panel of PBLs. All seven DR3 DPw3+ stimulators could induce proliferation. DR3- DPw3+ PBLs were recognized when the synthetic peptide was added to the cultures. Inhibition studies with mAbs showed that in both cases the HLA-DP molecule is involved in the recognition of both types of stimulators. We conclude that the clones recognize the DR3 peptide presented by HLA-DPw3. This stimulus can be obtained in two different ways: (a) by addition of synthetic peptide to DPw3+ PBLs or (b) by using DR3 DPw3+ stimulator cells where DR3 peptides are present in the culture as a product of denaturation of the DR3 molecule. Because all DR3 DPw3+ PBLs tested could stimulate the line and clones, we assume that the presentation of the DR3 peptide by DP is a naturally and continuously occurring phenomenon.

Antibodies, Monoclonal↗
PubMed1989

Novel rearrangements at the immunoglobulin D locus. Inversions and fusions add to IgH somatic diversity.

IgH rearrangements (VH-D, D-JH) are central to the generation of antibody diversity. The majority of the diversity seen in the third hypervariable region is generated by the D segment and at the joints formed by both junctional and N segment variation during D-JH and VH-D rearrangements. The mechanisms that regulate rearrangement are thought to obey the 12/23 rule, wherein D-D or VH-JH rearrangements are precluded. Here, we present evidence that D-D fusions do in fact occur, either as direct or inverted rearrangements. The fused D segments so generated may be fully capable of proceeding in subsequent D-JH and VH-D rearrangements. The resultant VH-D-D-JH recombinations add another dimension to the potential repertoire of IgH V regions by increasing the level of combinatorial diversity and by providing additional sites for N region variation.

Animals↗
PubMed1990

An immunoglobulin light chain from a lupus-prone mouse induces autoantibodies in normal mice.

Autoantibodies against the 70-kD U1 RNP nucleoprotein autoantigen and DNA were elicited in normal BALB/c mice with a purified Ig light chain. This light chain, derived from a lupus-prone MRL-lpr/lpr mouse, has two distinctive properties: it contains an idiotypic marker recognized by a monoclonal MRL-lpr/lpr anti-snRNP autoantibody, and the amino acid sequence of its third hypervariable region (CDR3) is homologous to a sequence in an antigenic region of the 70-kD U1 RNP polypeptide. The results demonstrate that an Ig idiotype that mimics an autoantigen can induce autoimmunization.

Amino Acid Sequence↗
PubMed1993

Antigen recognition properties of mutant V beta 3+ T cell receptors are consistent with an immunoglobulin-like structure for the receptor.

We examined the effect of mutations in the V beta portion of a pigeon cytochrome c (cyto c)-specific V beta 3+/V alpha 11+ T cell receptor on its ability to recognize cyto c/IEk and various superantigens. The results were consistent with an immunoglobulin-like structure for the receptor V beta domain and with separate interaction sites on V beta for conventional antigen and superantigens. An amino acid predicted to lie in CDR1 was critical for cyto c/IEk but not superantigen recognition, while several amino acids predicted to lie in the hypervariable region 4 loop were critical for superantigen but not cyto c/IEk recognition.

Amino Acid Sequence↗
PubMed1993

Compartmentalization of T lymphocytes to the site of disease: intrahepatic CD4+ T cells specific for the protein NS4 of hepatitis C virus in patients with chronic hepatitis C.

The adult liver is an organ without constitutive lymphoid components. Therefore, any intrahepatic T cell found in chronic hepatitis should have migrated to the liver after infection and inflammation. Because of the little information available on the differences between intrahepatic and peripheral T cells, we used recombinant proteins of the hepatitis C virus (HCV) to establish specific T cell lines and clones from liver biopsies of patients with chronic hepatitis C and compared them with those present in peripheral blood mononuclear cells (PBMC). We found that the protein nonstructural 4 (NS4) was able to stimulate CD4+ T cells isolated from liver biopsies, whereas with all the other HCV proteins we consistently failed to establish liver-derived T cell lines from 16 biopsies. We then compared NS4-specific T cell clones obtained on the same day from PBMC and liver of the same patient. We found that the 22 PBMC-derived T cell clones represent, at least, six distinct clonal populations that differ in major histocompatibility complex restriction and response to superantigens, whereas the 27 liver-derived T cell clones appear all identical, as further confirmed by cloning and sequencing of the T cell receptor (TCR) variable and hypervariable regions. Remarkably, none of the PBMC-derived clones has a TCR identical to the liver-derived clone, and even with polymerase chain reaction oligotyping we did not find the liver-derived clonotypic TCR transcript in the PBMC, indicating a preferential intrahepatic localization of these T cells. Functionally, the liver-derived T cells provided help for polyclonal immunoglobulin (Ig)A production by B cells in vitro that is 10-fold more effective than that provided by the PBMC-derived clones, whereas there is no difference in the help provided for IgM and IgG production. Altogether these results demonstrate that the protein NS4 is highly immunogenic for intrahepatic CD4+ T cells primed by HCV in vivo, and that there can be compartmentalization of some NS4-specific CD4+ T cells to the liver of patients with chronic hepatitis C.

Adult↗
PubMed1993

Antigen-driven B cell differentiation in vivo.

The secretion of specific antibodies and the development of somatically mutated memory B cells in germinal centers are consequences of T cell-dependent challenge with the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP). Using six-parameter flow cytometry and single cell molecular analysis we can directly monitor the extent of somatic hypermutation in individual responsive (isotype switched) antigen-specific B cells. The current study provides a direct quantitative assessment of recruitment into the antibody-secreting compartment on the one hand, and the germinal center pathway to memory on the other. Cellular expansion in both compartments is exponential and independent during the first week after challenge. The first evidence of somatic mutation, towards the end of the first week, was restricted to the germinal center pathway. Furthermore, germinal center cells express a significantly shorter third hypervariable region (CDR3), even when unmutated, than their antibody-secreting counterparts, suggesting a secondary selection event may occur at the bifurcation of these two pathways in vivo. By the end of the second week, the majority of mutated clones express a shorter CDR3 and affinity-increasing mutations as evidence of further selection after somatic mutation. These data provide evidence for substantial proliferation within germinal centers before the initiation of somatic mutation and the subsequent selection of a significant frequency of mutated clonotypes into the memory compartment.

Amino Acid Sequence↗
PubMed1995

Self tolerance to T cell receptor V beta sequences.

T cell tolerance to self is achieved by deletion or inactivation of clones recognizing peptides of self proteins presented by major histocompatibility complex molecules. A considerable fraction of self proteins accessible to the immune system is contributed by the system itself, for example, the receptors used for antigen recognition (antibodies and T cell receptors [TCRs]). Thus far, it has remained unclear, whether antigen receptors are subject to self tolerance, or on contrary, engage into network interactions implying immunity rather than tolerance. In this study, we demonstrate self tolerance to synthetic peptides corresponding to the first hypervariable region of the V beta 8.1 and V beta 8.2 TCR proteins. We also show that the tolerogenic synthetic peptide corresponds to a fragment produced by processing of the V beta protein, and conversely, that a V beta peptide not produced by processing is also not subject to self tolerance. Thus, the rules of tolerance seem to apply to antigen receptors, at least to their germline-encoded portions, in a similar fashion as to other self proteins. This finding has important implications for studies of natural and artificially induced immune networks.

Amino Acid Sequence↗