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PubMed1986

Molecular mapping of idiotopes of anti-arsonate antibodies.

As part of understanding molecular function in structural terms, we have been attempting to map the idiotypic topography of specific anti-arsonate (Ars) antibodies. A panel of anti-Ars hybridomas of which the complete primary sequences are known were used. These molecules show a varied reactivity profile with a panel of monoclonal anti-idiotypic antibodies. By judicious chain recombination experiments and chemical modifications that altered this reactivity profile, we were able to identify particular amino acid residues or discreet regions of anti-Ars antibodies as having crucial roles in the expression of idiotypic determinants. Idiotopes were mapped to the heavy chain second hypervariable region and D segment, and to the light chain first and third hypervariable regions.

Amino Acid Sequence↗
PubMed1991

The Staphylococcus aureus mec determinant comprises an unusual cluster of direct repeats and codes for a gene product similar to the Escherichia coli sn-glycerophosphoryl diester phosphodiesterase.

The DNA sequence located between mecA, the gene that codes for penicillin-binding protein PBP2', and insertion sequence-like element IS431mec has been termed hypervariable because of its length polymorphism among different staphylococcal isolates. We sequenced and characterized the hypervariable region of the methicillin resistance determinant (mec) isolated from Staphylococcus aureus BB270. Within the 2,040-bp hypervariable region, we identified an unusual accumulation of long direct repeats. Analysis of the DNA sequence revealed a minimal direct repeat unit (dru) of 40 bp which was repeated 10 times within 500 bp. The dru sequences are responsible for the length polymorphism of mec. Moreover, we identified an open reading frame that codes for 145 amino acids (ORF145), whose deduced amino acid sequence showed 57% amino acid sequence similarity to the N terminus of the glycerophosphoryl diester phosphodiesterase (UgpQ) of Escherichia coli.

Amino Acid Sequence↗
PubMed1989

Receptor specificity of the Escherichia coli T-even type phage Ox2. Mutational alterations in host range mutants.

The T-even type Escherichia coli phage Ox2 uses the outer membrane protein OmpA as a receptor. The protein is recognized with the ends of the virion's long tail fibers. The 266 residue protein 38 is located at this site and acts as an adhesin. Host-range mutants had previously been isolated from Ox2. Mutant Ox2h5 is able to infect cells possessing an altered OmpA protein, which renders the cell resistant to Ox2. Ox2h10 was selected from Ox2h5. This phage recognizes the OmpC protein in addition to the OmpA protein. Ox2h12, which stems from Ox2h10, binds to OmpC with high affinity, but has lost efficient binding to OmpA. The mutational alterations caused in genes 38 are: Asp231----Asn(h5) and His170----Arg(h10). The triple mutant Ox2h12 possesses an insertion of a Gly residue next to Gly121. The three mutants have additionally acquired mutations affecting their base plate, making them "trigger-happy". When protein 38 was compared with the same protein derived from other E. coli phages, it was found to contain two constant and one variable domains, the latter harboring four hypervariable regions flanked by a largely conserved glycine-rich sequence. The h5 and h10 mutations occurred within two hypervariable areas, while the additional Gly residue was present in one of the flanking conserved sequences. On the basis of these results, as well as those obtained from host-range mutants analyzed previously, a model for such adhesins is proposed. Receptor recognition is most likely performed via the hypervariable regions, which may form loops held together in close proximity by the oligoglycine sequences. The latter may achieve this by being part of highly compact omega loops.

Bacterial Outer Membrane Proteins↗
PubMed1977

Heavy-chain variable-region sequence from an inulin-binding myeloma protein.

The entire variable-region sequence of the heavy chain from ABE-47N, a BALB/c inulin-binding myeloma protein, has been determined. This protein is unusual in that the third complementarity region (H3) is extremely short, consisting of at the most three and probably only one amino acid. A comparison of the heavy-chain hypervariable regions from mouse, human, and rabbit proteins shows that the variability in length of H3 is greater than that seen in the first or second hypervariable regions. This variability in H3 length suggests a specialized function for this region.

Amino Acid Sequence↗
PubMed1977

Towards a chemical definition of idiotypy.

The idiotypic determinants have been precisely located to the variable regions of immunoglobulin polypeptide chains. Both chains are generally required to express the idiotype. The major idiotypic determinants are the result of the amino acid sequence of the hypervaiable regions, although some idiotypic determinants reside outside the antibody combining site and these so called "framework idiotypes" are important markers. In my view the hypervariable regions are spatially disposed so as to present adequate antigenic stimulation, and they display enough structural heterogeneity to account for the uniqueness of the idiotype in the general population of immunoglobulin molecules. Hypervariable regions, the antibody combining site, and the idiotypic determinants thus amalgamate three formerly diverse concepts into a unified theoretical construct.

Binding Sites, Antibody↗
PubMed1994

Molecular basis for degenerate T-cell recognition of one peptide in the context of several DR molecules.

We report the study of one CD4+ T-cell clone that recognizes peptide HA306-320 in the context of autologous DR1101 molecules as well as of allogeneic DR1301, DR0402, DR1501, and DR1601 molecules. This degenerate T-cell recognition is mediated by a single T-cell receptor (TCR) as judged by both TCR-V beta sequencing and cold-target competition assays. Restriction analysis shows that substitutions of DR residues within the third hypervariable region result in a loss of T-cell reactivity, which is restored by additional substitutions in the first and/or second hypervariable regions. Thus, there is no correlation between antigen presentation abilities of the different allelic DR products and the degree of sequence homology between these products. DR residues whose substitution is compatible with T-cell recognition potentially interact with peptides rather than with TCRs by virtue of their location in the floor of the groove or as previously documented for residues of the alpha-helix. Furthermore, antigen presentation by allogeneic DR molecules occurs independently of their affinity for the peptide, as determined in cell surface-binding assays using biotinylated HA306-320. Altogether these data suggest that degenerate T-cell recognition mainly depends on an influence of polymorphic DR residues on the configuration adopted by the peptide in the DR groove so that the epitope is left intact.

Amino Acid Sequence↗
PubMed1971

Heavy-chain variable regions in normal and pathological immunolobulins.

Aminoacid sequence analysis of immunoglobulin G heavy chains from each of 15 normal individuals showed the presence of VHIII subgroup sequences in all individuals, to an extent of about 15-25% of the total. Therefore, different subgroups of the heavy-chain variable region are not products of alleles of a single gene.N-terminal aminoacid sequences were determined for ten heavy chains of myeloma proteins that belonged to the VHIII subgroup. Our results demonstrate the existence of a "hypervariable" region of sequence between positions 30 and 35, homologous to one of the three hypervariable regions of light chains. At several positions, variants of the basic sequence are repeated in different proteins. It is likely that such variants are derived from several recently evolved genes that constitute the VHIII subgroup.

Amino Acid Sequence↗
PubMed1995

Variable region light chain genes encoding human antibodies to HIV-1.

In previous work, it was found that the heavy chain variable gene (VH) repertoire of human antibodies to HIV is markedly skewed and that the gp120 molecule is a ligand for VH3 gene products. Here, we have analysed the light chain (L-chain) variable region genes (VL) expressed by a panel of human monoclonal antibodies derived from an immunized volunteer, an AIDS patient and seropositive asymptomatic donors, and specific for HIV-1 p25, gp41 and gp120 proteins. We found that, in contrast to VH gene-family use, the VL repertoire does not exhibit a family-bias. We noticed however, a tendency to the use of VL genes that map to the downstream portion of the kappa locus. The VL genes expressed have mutated at lower rates than the corresponding VH genes and show no clustering of the replacement mutations in the hypervariable regions. We also found that the third hypervariable regions (CDR3) of the L-chains have undergone a marked diversification, with addition of untemplated nucleotides, frequent truncation at the 3' end of the VLs and somatic mutation. These molecular events result in a length heterogeneity of the CDR3s and an apparently positive selection of specific highly reactive amino acids. We conclude that the specificity of, at least some of the anti-HIV antibodies, is dictated by the L-chain CDR3 regions which bear the imprints of antigenic selection.

Amino Acid Sequence↗
PubMed1981

Possible conformation of major histocompatibility antigens: implication of amino acid sequence data.

Possible conformation of major histocompatibility antigens was proposed on the basis of homology between domains of major histocompatibility antigen polypeptide chains and domains of immunoglobulins, characterized by a beta-pleated structure. In the hypothetical three-dimensional structure, domains alpha 3 and alpha 2 interact along the chain similarly to the CH2 and CH3 domains of IgG. beta 2-Microglobulin together with domain alpha 2 forms a structure analogous to the CH3--CH3 or CH1--CL dimers in IgG. Domains alpha 2 ans alpha 1 interact in such a manner that the beta-pleated sheet of domain alpha 2 is directly joined to the beta-pleated sheet of domain alpha 1. Hypervariable regions in domains alpha 1 and alpha 2, which most likely take part in the formation of the alloantigen determinants, represent the loops joining the regions of the beta-pleated structure. Domains alpha 1 and alpha 2 may be packed in such a way that their hypervariable regions would become adjacent and would be located on their surface turned aside from the surface of the cell membrane.

Amino Acid Sequence↗
PubMed1994

Characterization of the 5' noncoding and structural region of the hepatitis C virus genome from patients with non-A, non-B hepatitis responding differently to interferon treatment.

We examined 14 patients with hepatitis C caused by infection with the hepatitis C virus-II genotype to understand differences in responsiveness to interferon. The patients were classified into two groups according to their response to interferon: eight responding and six non-responding patients. The 5' noncoding and structural regions of the hepatitis C virus-II genome from each patient specimen were amplified by reverse transcription followed by the polymerase chain reaction. The nucleotide sequences of these amplified DNAs were then determined. By comparing the nucleotide sequences and the deduced amino acid sequences of samples from both groups, no group-specific sequence was observed in the analyzed regions despite the presence of considerable sequence diversity. However, additional cysteine residues were observed in half the responding group. The degree of micro-heterogeneity in hypervariable region 1 of the hepatitis C virus in relation to the sensitivity to interferon treatment was also examined; however, no significant correlation was observed. In addition, frequent alterations in the amino acid sequences were observed in hypervariable region 1 during the course of interferon treatment.

Adolescent↗
PubMed1992

Interaction of immune sera with synthetic peptides corresponding to the structural protein region of hepatitis C virus.

Comparison of the deduced amino acid sequence from the structural region of the Hutchinson strain of hepatitis C virus (HCV-H) with four other HCV isolates clearly divides the five isolates into two groups based on sequence homology. The first group includes HCV-H, HCV-1, and HC-J1, while the second includes HCV-J1 and HC-J4. Among the five isolates the first 190 residues (putative nucleocapsid) are highly conserved whereas residues 196-513 exhibit significant diversity and include a hypervariable region encompassing residues 386-404. A series of overlapping decapeptides were synthesized by solid-phase pin technology according to sequence from HCV-H (amino acids 1-513), HC-J4 (amino acids 181-513), and regions from the three other isolates which exhibited sequence variation. A modified ELISA was used to measure immunoreactivity of sera from clinical posttransfusion cases and experimentally infected chimpanzees. Comparison of pre- and postinfection samples revealed 16 clusters of immunoreactive peptides within the structural region, none of which was found in the hypervariable region. Only one cluster (amino acids 73-89) was recognized by all human and chimpanzee sera. Clear variation in the immune response was observed between individuals, although no obvious difference in reactivity between acute and chronic cases was observed. Within individual profiles, the reactivity to each peptide cluster and the total number of reactive clusters increased over time.

Amino Acid Sequence↗
PubMed1985

The VK gene expressed by BALB/c ABPC48 cross-reactive idiotypes induced by anti-idiotypic immunization is identical to that of BALB/c anti-oxazolone and A/J anti-arsonate antibodies.

Anti-idiotypic immunization triggers the production of antibodies that are structurally related to the idiotype. We have shown that the heavy chain variable regions of antibodies produced after anti-ABPC48 (A48) anti-idiotypic immunization of BALB/c mice are homologous to that of A48, except for the third hypervariable region. We present here partial light chain sequences of A48 and of antibodies induced by anti-idiotypic immunization. Nearly perfect homology is found, suggesting that these chains are the products of genes derived from a unique VK germ-line gene. These observations indicate that the H and L hypervariable regions contribute to define the structure of A48 idiotopes. Remarkably, the VK sequence we identify is the same as that described for anti-arsonate and anti-oxazolone antibodies. We discuss the relative importance of particular amino acids for idiotype expression and antigen-binding activity.

Animals↗
PubMed1976

Studies on the structural localization of rabbit H chain allotypic determinants controlled by the a locus. Purification and immunological properties of an immunopeptide bearing a3 allotypic determinants.

An immunopeptide bearing a3 allotypic determinant(s) was isolated from the gamma chain of an a3 homozygous rabbit (G222-2) immunized with type III pneumococcal vaccine. Immunocogical properties of peptides were studied using a radioimmunoassay that involved inhibition by these peptides of a reaction between 125I-labeled anti-a3 antibody and Sepharose-bound a3 immunoglobulin G (IgG). The gamma chain was isolated from IgG of restricted heterogeneity and then citraconylated and digested with trypsin. The tryptic digest (TD1) was passed through an anti-a3 immunoabsorbent column either directly or after an intermediate step of Sephadex G-75 chromatography. The bound peptides (T1) were eluted with 0.1 M acetic acid and further digested with trypsin. The digest (TD2) was again run on the anti-a3 immunoabsorbent column to purify the bound immunopeptide T2. In the radioimmunossay this immunopeptide was found to have major a3 determinant(s). Its molecular weight was found to be approximately 6,000, which decreased to about 3,000 after reduction and alkylation. These data, together with NH2- and COOH-terminal analyses and cysteine peptide mapping, demonstrated that T2 is composed of two polypeptide chains linked by a disulfide bond, one from the cysteine 22 region having lysine at the COOH terminus and the other from the cysteine 92 region arginine at the COOH terminus. The lysine peptide was separated from the arginine peptide and its NH2-terminal sequence was found to be Gly-Asx-Glx-Ser-Thr-Cys. Since the cysteine is at position 22, the lysine peptide starts at position 17. It has approximately 22 residues. The framework sequence from 17 to 20 is different from those reported so far. In addition, the heavy chain used in these studies has some other unusual features including a histidine, probably in the first hypervariable region. The presence of histidine in the first hypervariable region of rabbit heavy chain has not been reported previously. The other peptide which is about 30 amino acids in length and ends with arginine 94, probably includes positions 67, 70, 71, 84, and 85 that are believed to have substitutions correlating with a allotypes. In a hypothetical three-deminsional model of the Fv portion of rabbit anti-SIII antibody BS-5, residues 17 to 33 of the lysine peptide and 67 to 79 and 84 to 85 which may be present in the arginine peptide are fully exposed on the surface and are far removed from the antibody combining site.

Amino Acids↗
PubMed1990

Sequence analysis of amphotropic and 10A1 murine leukemia viruses: close relationship to mink cell focus-inducing viruses.

Viral interference studies have demonstrated the existence of four distinct murine leukemia virus (MuLV) receptors on NIH 3T3 mouse cells. The four viral interference groups are ecotropic MuLV; mink cell focus inducing virus (MCF); amphotropic MuLV; and 10A1, a recombinant derivative of amphotropic MuLV that uses a unique receptor but also retains affinity for the amphotropic MuLV receptor. We report here that 10A1 infects rat and hamster cells, unlike its amphotropic parent. We isolated an infectious molecular clone of 10A1 and present here the sequences of the env genes and enhancer regions of amphotropic MuLV and 10A1. The deduced amino acid sequences of amphotropic MuLV and 10A1 gp70su are remarkably similar to those of MCF and xenotropic MuLV (for which mouse cells lack receptors), with 64% amino acids identical in the four groups. We generated a consensus from these comparisons. Further, the differences are largely localized to a few discrete regions: (i) amphotropic MuLV has two short insertions relative to MCF, at residues 87 to 92 and 163 to 169, and (ii) amphotropic MuLV and MCF are totally different in a hypervariable region, which is greater than 30% proline, at residues approximately 253 to 304. 10A1 closely resembles amphotropic MuLV in its N terminus but contains an MCF-type hypervariable region. These results suggest the possibility that receptor specificity is localized in these short variable regions and further that the unique receptor specificity of 10A1 is due to the novel combination of amphotropic MuLV and MCF sequences rather than to the presence of any novel sequences. The Env proteins of ecotropic MuLV are far more distantly related to those of the other four groups than the latter are to each other. We also found that the enhancer regions of amphotropic MuLV and 10A1 are nearly identical, although 10A1 is far more leukemogenic than amphotropic MuLV.

Amino Acid Sequence↗
PubMed1994

Sequence variability in the env-coding region of hepatitis C virus isolated from patients infected during a single source outbreak.

The variability of the hepatitis C virus genome was investigated in a group of German patients who developed chronic hepatitis C after parenteral administration of contaminated immunoglobulin to prevent Rh sensitization after pregnancy. The nucleotide and deduced amino acid sequence alterations of the E1 and the first hypervariable region of the E2 gene of the hepatitis C virus (HCV) genome from sera of two randomly selected patients were studied by comparison of HCV sequences obtained from the original inoculum (anti Rh immunoglobulin) and from patient sera collected in 1979 and 1989. All isolates were classified as subtype 1b but showed nucleotide insertions of up to 12 nucleotides at the cleavage site of E1/E2. Microheterogeneity of HCV genomes was found in the immunoglobulin supporting the quasispecies model of HCV distribution. Remarkable nucleotide exchanges over the 10 year period in the E1 region (0.9-5.2 x 10(-3) base substitutions per genome site per year) and especially in the first hypervariable region of the E2 gene (about 1.5 x 10(-2)) occurred. The HCV genome undergoes a selection of variants, though it is not known if this derives from mutation or selection of pre-existing rare variants.

Amino Acid Sequence↗
PubMed1994

The affinity-selection of a minibody polypeptide inhibitor of human interleukin-6.

A major challenge in basic and applied biological research is the engineering of small proteins with pre-determined structures and novel functions. In a limited number of cases, this has been achieved by de novo design. An alternative combinatorial approach is based on the construction of large libraries of random peptides and on methods for the selection of the desired molecules. Here we describe a successful combination of both the rational design and the combinatorial approaches for developing proteins with useful biological functions, in this case the construction of a specific inhibitor of the cytokine human interleukin-6. In previous work, the 'minibody', a 61 residue polypeptide consisting of a beta-pleated framework and two hypervariable regions, was designed, synthesized and expressed on f1 phage surface. We report the construction of a repertoire of 50 million minibodies displayed on phage in which the hypervariable regions have been randomized. One polypeptide which binds tightly and specifically to human interleukin-6 was isolated from this collection of minibody mutants. This particular minibody is an effective inhibitor of the cytokine's biological activity. The approach described here could in principle be applied to other molecular targets.

Amino Acid Sequence↗
PubMed1992

An X chromosome inactivation assay based on differential methylation of a CpG island coupled to a VNTR polymorphism at the 5' end of the monoamine oxidase A gene.

A CpG island has been identified just upstream of the first exon of the human monoamine oxidase A (MAOA) gene, localized to Xp11.4-Xp11.23. Southern blotting following digestion with the methylation sensitive restriction endonucleases SmaI, HpaII and HhaI, indicated that CpG dinucleotides within the CpG island were unmethylated on the active X chromosome and extensively methylated on the inactive X chromosome. These sites of differential methylation were close to a polymorphic GT-dinucleotide/VNTR region, which is located 1 kb 3' of the first exon and has a heterozygosity value of 75%. PCR primers were designed for amplification of 1.2-1.3 kb DNA fragments, encompassing both the hypervariable region and a cluster of six HpaII sites within the CpG-rich region. Cleavage of HpaII sites was found to be restricted to active X chromosomes. Therefore, following HpaII digestion, DNA fragments were exclusively amplified from inactive X chromosomes. The resulting PCR products were digested with SacI, which reduced the size of the DNA fragments containing the hypervariable region to 230-330 bp, and were subsequently analyzed on denaturating polyacrylamide gels. Because amplified fragments were exclusively derived from the inactive X chromosome, the relative densities of the two allelic fragments should reflect the proportions of cells that have either of the two X chromosome inactivated. The results of this PCR-based X chromosome inactivation assay were fully concordant with Southern blotting methylation analyses at the PGK locus. It therefore provides a rapid and informative method in tumour clonality analysis and carrier detection in X-linked diseases.

Base Sequence↗
PubMedDate not supplied

The HLA association with rheumatoid arthritis.

Rheumatoid arthritis (RA) is primarily associated with HLA-DR4 in a wide range of ethnic groups. DNA analysis of HLA-DRB1 sequences shows that a limited set of alleles are positively associated with the disease. Third hypervariable region sequences QRRAA, QKRAA and RRRAA are found in up to 85% of RA patients and may constitute the basic unit of association. Risk estimates for alleles and hypervariable regions differ between ethnic groups and subsets of patients. Severe RA and Felty's syndrome are significantly associated with DR4 Dw4 and, to a lesser extent, with DR4 Dw14. In patients the latter allele is almost exclusively found in combination with Dw4, suggesting that complementation is occurring. Critical substitutions in the peptide binding groove correlate with the presence of RA, suggesting that the disease may be driven by the presentation of specifically bound peptide and/or may be influenced by differential selection of the T cell repertoire.

Arthritis, Rheumatoid↗