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Biomedical subjects

G Chelvanayagam

Publications and source records attributed to G Chelvanayagam.

At least 37 records · Page 2Linked to original sources
PubMed1998

Anti-MUC1 antibodies react directly with MUC1 peptides presented by class I H2 and HLA molecules.

Peptides bound in the groove of MHC class I molecules and detected by CTLs are not normally accessible to Ab. We now report that MUC1 peptides that are bound within the groove of MHC class I molecules (H2 and HLA) and that can be detected by CTLs can also be detected by anti-MUC1 Abs. mAbs to the middle and C-terminal regions of the class I-associated peptides but not to the N terminus were able to react with MUC1 peptides bound to H2Kb and HLA-A*0201, and only to the mid-region for H2Db, by flow cytometry and also to block CTL activity. Molecular modeling showed that the N terminus is buried (and not accessible), whereas the midpeptide residues form a loop and the C terminus is free, making these two regions accessible to Ab. The findings demonstrate for the first time that peptides associated with class I molecules can be detected by anti-peptide Abs.

Amino Acid Sequence↗
PubMed1998

A viral peptide with limited homology to a self peptide can induce clinical signs of experimental autoimmune encephalomyelitis.

Molecular mimicry has been suggested as a mode of autoreactive T cell stimulation in autoimmune diseases. Myelin basic protein (MBP) peptide 1-11 induces experimental autoimmune encephalomyelitis (EAE) in susceptible strains of mice. Here we show that a herpesvirus Saimiri (HVS) peptide, AAQRRPSRPFA, with a limited homology to MBP1-11 peptide, ASQKRPSQRHG (underlined letters showing homology), can stimulate a panel of MBP-11-specific T cell hybridomas and more importantly cause EAE in mice. We demonstrate that this is due to cross-recognition of these two peptides by TCRs. Results presented in this communication are the first demonstration that a viral peptide with homology at just 5 amino acids with a self peptide can induce clinical signs of EAE in mice. These findings have important implications in understanding the breakdown of T cell tolerance to self Ags in autoimmune diseases by means of cross-reactivity with unrelated peptides.

Animals↗
PubMed1998

Human theta class glutathione transferase: the crystal structure reveals a sulfate-binding pocket within a buried active site.

BACKGROUND: Glutathione S-transferases (GSTs) comprise a multifunctional group of enzymes that play a critical role in the cellular detoxification process. These enzymes reduce the reactivity of toxic compounds by catalyzing their conjugation with glutathione. As a result of their role in detoxification, GSTs have been implicated in the development of cellular resistance to antibiotics, herbicides and clinical drugs and their study is therefore of much interest. In mammals, the cytosolic GSTs can be divided into five distinct classes termed alpha, mu, pi, sigma and theta. The human theta class GST, hGST T2-2, possesses several distinctive features compared to GSTs of other classes, including a long C-terminal extension and a specific sulfatase activity. It was hoped that the determination of the structure of hGST T2-2 may help us to understand more about this unusual class of enzymes. RESULTS: Here we present the crystal structures of hGST T2-2 in the apo form and in complex with the substrates glutathione and 1-menaphthyl sulfate. The enzyme adopts the canonical GST fold with a 40-residue C-terminal extension comprising two helices connected by a long loop. The extension completely buries the substrate-binding pocket and occludes most of the glutathione-binding site. The enzyme has a purpose-built novel sulfate-binding site. The crystals were shown to be catalytically active: soaks with 1-menaphthyl sulfate result in the production of the glutathione conjugate and cleavage of the sulfate group. CONCLUSIONS: hGST T2-2 shares less than 15% sequence identity with other GST classes, yet adopts a similar three-dimensional fold. The C-terminal extension that blocks the active site is not disordered in either the apo or complexed forms of the enzyme, but nevertheless catalysis occurs in the crystalline state. A narrow tunnel leading from the active site to the surface may provide a pathway for the entry of substrates and the release of products. The results suggest a molecular basis for the unique sulfatase activity of this GST.

Binding Sites↗
PubMed1998

Homology models for the PERB11 multigene family.

BACKGROUND: PERB11 is a multicopy polymorphic gene family found in association with HLA Class I genes within the major histocompatibility complex (MHC). Although its function is unknown, PERB11 has sequence similarities to HLA Class I and other related proteins. To explore the possible functional roles for PERB11, homology models have been constructed using both HLA Class I and Class I-like protein structures as templates. RESULTS: The models show that PERB11.1 appears to have an unusual distribution of charged residues that potentially give the molecule a distinct polarity. Furthermore, a cluster of negatively charged residues in the traditional P2 site may form a novel binding site for a positively charged ligand such as a metal ion or complex. Other charged residues line the floor and walls of the cleft and are able to form salt bridges, reminiscent of the closed cleft of the Class I-like mouse neonatal Fc receptor structure. The closely related PERB11.2 family has a different arrangement of charged residues in the cleft, but these residues are still able to form salt bridges. Unlike HLA Class I, the majority of polymorphic positions in the PERB11 family occur outside the cleft and on the surface of the molecule. CONCLUSIONS: Homology models for PERB11 suggest that the structure is capable of associating with beta2 microglobulin or a similar molecule. Furthermore, not all of the potential glycosylation sites suggested by the PERB11 sequences appear viable. Importantly, the models suggest that the molecule has a less accessible cleft than HLA Class I and is not, therefore, able to bind peptides. Other small ligands, including metal ions, might be bound, however.

Amino Acid Sequence↗
PubMed1998

A combinatorial distance-constraint approach to predicting protein tertiary models from known secondary structure.

BACKGROUND: Distance geometry methods allow protein structures to be constructed using a large number of distance constraints, which can be elucidated by experimental techniques such as NMR. New methods for gleaning tertiary structural information from multiple sequence alignments make it possible for distance constraints to be predicted from sequence information alone. The basic distance geometry method can thus be applied using these empirically derived distance constraints. Such an approach, which incorporates a novel combinatoric procedure, is reported here. RESULTS: Given the correct sheet topology and disulfide formations, the fully automated procedure is generally able to construct native-like Calpha models for eight small beta-protein structures. When the sheet topology was unknown but disulfide connectivities were included, all sheet topologies were explored by the combinatorial procedure. Using a simple geometric evaluation scheme, models with the correct sheet topology were ranked first in four of the eight example cases, second in three examples and third in one example. If neither the sheet topology nor the disulfide connectivities were given a priori, all combinations of sheet topologies and disulfides were explored by the combinatorial procedure. The evaluation scheme ranked the correct topology within the top five folds for half the example cases. CONCLUSIONS: The combinatorial procedure is a useful technique for identifying a limited number of low-resolution candidate folds for small, disulfide-rich, beta-protein structures. Better results are obtained, however, if correct disulfide connectivities are known in advance. Combinatorial distance constraints can be applied whenever there are a sufficiently small number of finite connectivities.

Amino Acid Sequence↗
PubMed1998

Peptide mimics of a tumor antigen induce functional cytotoxic T cells.

The ability to mimic peptide/peptide and/or peptide/carbohydrate structures may be important in generating cross-reactive antibodies for autoimmune and other diseases. We show that the peptide sequence DAHWESWL can mimic the conformation of the unrelated MUC1 peptide SAPDTRPAP(G). Mice immunized with mannan-MUC1-peptides make cytotoxic T lymphocytes (CTLs) and are protected from MUC1+ tumors. We show that the same specific anti-MUC1 responses can be produced by immunizing with the DAHWESWL peptide; furthermore, specific tumor protection is obtained in a manner similar to that with MUC1 immunization. The DAHWESWL peptide immunization leads to CTLs that recognize H2Dd and H2Ld but not H2b or human leukocyte antigens-group A (HLA-A) *0201 presented MUC1 peptides. However, mutation of the DAHWESWL peptide to a more HLA-A*0201-compatible structure with appropriate anchors (DLHWASWV), leads to the production of CTLs in HLA-A*0201 mice.

Amino Acid Sequence↗
PubMed1998

Correlating sequence variation with HLA-A allelic families: implications for T cell receptor binding specificities.

Six families of HLA-A alleles have been previously proposed on the basis of nucleotide sequence and phylogenetic analysis. Here, sequence polymorphism has been examined at both the protein and DNA levels in a family specific manner and new minimal signatures for each of the families have been delineated. The DNA and protein sites that constitute these signatures are distributed throughout the length of the sequence and generally do not appear to act to promote structural or functional features of the molecules. This is explained by the fact that traditional signatures suffer biases where, for example, recombination products of low frequency can obscure one family's trend by introducing 'impurities' intrinsic to another family. In the absence of complete frequency data, a closer approximation of family signatures can be defined by sites that show strong correlation with the family groups. Using this description, the amino acid positions 62, 97 and 114, localized in the antigen-binding cleft are, in combination, sufficient to discriminate between the six families. Thus, while the composition of the whole cleft defines the details of antigen specificity, these sites in particular, play a key role in modulating supertype peptide specificity and T cell recognition.

Alleles↗
PubMed1997

Zeta, a novel class of glutathione transferases in a range of species from plants to humans.

Sequence alignment and phylogenetic analysis has identified a new subgroup of glutathione S-transferase (GST)-like proteins from a range of species extending from plants to humans. This group has been termed the Zeta class. An atomic model of the N-terminal domain suggests that the members of the Zeta class have a similar structure to that of other GSTs, binding glutathione in a similar orientation in the G site. Recombinant human GSTZ1-1 has been expressed in Escherichia coli and characterized. The protein is a dimer composed of 24.2 kDa subunits and has minimal glutathione-conjugating activity with ethacrynic acid and 7-chloro-4-nitrobenz-2-oxa-1, 3-diazole. Although low in comparison with other GSTs, GSTZ1-1 has glutathione peroxidase activity with t-butyl and cumene hydroperoxides. The members of the Zeta class have been conserved over a long evolutionary period, suggesting that they might have a role in the metabolism of a compound that is common in many living cells.

Amino Acid Sequence↗
PubMed1997

Homology model for the human GSTT2 Theta class glutathione transferase.

A tertiary model of the human GSTT2 Theta class glutathione transferase is presented based on the recently solved crystal structure of a related thetalike isoenzyme from Lucilia cuprina. Although the N-terminal domains are quite homologous, the C-terminal domains share less than about 20% identity. The model is used to consolidate the role of Ser 11 in the active site of the enzyme as well as to identify other residues and mechanisms of likely catalytic importance. The T2 subfamily of theta class enzymes have been shown to inactivate reactive sulfate esters arising from arylmethanols. A possible reaction pathway involving the conjugation of glutathione with one such sulfate ester, 1-menaphthyl-sulfate, is described. It is also proposed that the C-terminal region of the enzyme plays an important role in allowing substrate access to the active site.

Amino Acid Sequence↗
PubMed1997

A roadmap for HLA-DR peptide binding specificities.

Peptide residue positional environments have previously been defined for class I MHC allelic products. These environments provide a less restrictive description of the traditional peptide binding pockets of class I molecules. When combined with the peptide anchor motifs that have been identified for some class I molecules, predictions as to likely motifs for other MHC molecules, which share the same potential environment can be made. Here, the same approach is used to derive peptide residue positional environments for class II MHC molecules. The environments are used to make predictions as to likely binding motifs for HLA-DR allelic products. The predictions are presented in the form of a Table and shown to have concordance with experimental results.

Alleles↗
PubMed1997

Correlating patterns in alignments of polymorphic sequences with experimental assays.

A general algorithm is presented for identifying sets of positions in multiple sequence alignments that best characterize an a priori partitioning such as those determined by inhibition studies or other experimental techniques. The algorithm explores combinations of polymorphic columns in the alignment and evaluates how well these sites reflect the original input partition. Partitions across the polymorphic columns are derived using a tree building procedure with conventional amino acid substitution matrices. Elucidation of those amino acids which govern the biochemical behaviour of a protein with a given substrate or inhibitor can provide insights towards an understanding of the tertiary conformation of the protein. Since it is likely that such positions will be spatially clustered in the protein fold, these positions may give rise to useful distance constraints for substantiating model protein structures. The method is exemplified using data for a set of human mu class glutathione S-transferases. A novel aspect for predicting the behaviour of new polymorphic sequences is also discussed.

Algorithms↗
PubMed1997

An analysis of simultaneous variation in protein structures.

The simultaneous substitution of pairs of buried amino acid side chains during divergent evolution has been examined in a set of protein families with known crystal structures. A weak signal is found that shows that amino acid pairs near in space in the folded structure preferentially undergo substitution in a compensatory way. Three different physicochemical types of covariation 'signals' were then examined separately, with consideration given to the evolutionary distance at which different types of compensation occur. Where the compensatory covariation tends towards retaining the combined residue volumes, the signal is significant only at very low evolutionary distances. Where the covariation compensates for changes in the hydrogen bonding, the signal is strongest at intermediate evolutionary distances. Covariations that compensate for charge variations appeared with equal strength at all the evolutionary distances examined. A recipe is suggested for using the weak covariation signal to assemble the predicted secondary structural elements, where the evolutionary distance, covariation type and weighting are considered together with the tertiary structural context (interior or surface) of the residues being examined.

Computer Simulation↗
PubMed1996

Mutagenesis of the active site of the human Theta-class glutathione transferase GSTT2-2: catalysis with different substrates involves different residues.

The role of serine-11 in the catalytic mechanism of recombinant human GSTT2-2 was examined by site-directed mutagenesis. Amino acid sequence comparison of the Theta-class isoenzymes has identified a conserved serine residue in the N-terminal domain [Wilce, Board, Feil and Parker (1995) EMBO J. 14, 2133-2143]. This conserved serine has been implicated in the activation of the enzyme-bound glutathione [Board, Coggan and Parker (1995) Biochem. J. 311, 247-250]. Mutating the equivalent serine (residue 11) of GSTT2-2 to Ala, Thr or Tyr abolished the catalytic properties of GSTT2-2 with cumene hydroperoxide and ethacrynic acid as second substrate. However, with l-menaphthyl sulphate (MSu) as the second substrate, the specific activity of the S11A mutant was doubled, while the S11T mutant retained half the wild-type activity and the S11Y mutant was inactive. The role of Ser-11 in catalysis seems to vary with different second substrates. In the substitution reaction with MSu, GSTT2-2 activity appears to depend on the size of the Ser-11 replacement rather than the presence of a side-chain hydroxy group. In addition, the reaction rate appears to be a function of pH, and there is no non-enzymic reaction even at high pH. We demonstrated that a reaction between MSu and an alternative thiol such as L-cysteine or 2-mercaptoethanol can take place in the presence of S-methylglutathione and GSTT2-2. We propose that the catalytic activity of GSTT2-2 with MSu is preceded by a conformational or charge modification to the enzyme upon the binding of glutathione or S-methylglutathione. This is followed by the binding of MSu and the subsequent removal of the sulphate group, giving rise to the carbonium ion of l-methylnaphthelene as the electrophile that reacts with the nucleophilic species. The reaction mechanism of GSTT2-2 with MSu may represent a novel function of GSTT2-2 as a glutathione-dependent sulphatase.

Benzene Derivatives↗
PubMed1996

A roadmap for HLA-A, HLA-B, and HLA-C peptide binding specificities.

The high level of polymorphism in major histocompatibility complex (MHC) molecules leads to many allele-specific peptide binding repertoires that can generally be characterized by sequence motifs. Such motifs have previously been elucidated experimentally for several MHC molecules and shown to bind in specificity pockets in the antigen binding cleft. Here, a new and less restrictive description of the traditional antigen binding pockets is derived. These regions are referred to as peptide binding environments and are defined as those residues in a fixed neighborhood of the peptide residues in known crystal structure complexes. By examining the antigen binding environments from MHC molecules with known motifs, we made predictions as to likely motifs for other MHC molecules which share the same environments. The predictions are presented in the form of Tables and are pertinent to class I HLA-A, HLA-B, and HLA-C MHC sequences, and are shown to correlate well with experiments.

Alleles↗
PubMed1996

Structural comparison of major histocompatibility complex class I molecules and homology modelling of five distinct human leukocyte antigen-A alleles.

The peptide complexes of 19 major histocompatibility complex class I alpha 1 and alpha 2 domains have been compared to identify similarities that can be interpreted as constraints necessary for the function or stability of the molecule. It was found that nearly half of the residues maintained their side-chain conformations (or had no side chain), with the remaining residues being highly solvent exposed and/or polymorphic. Seven hydrogen bonds between the molecule and peptide are conserved in all the structures and serve to orientate the ends of the peptide in the binding groove. Furthermore, the general orientations of most residue side chains in the peptide are similar. Based on these constraints, homology models for the distinct human leukocyte antigen-A alleles A*0302, A*2403, A*2603, A*3101 and A*8001 have been constructed and the implications for peptide binding discussed. The models provide a useful framework from which to engineer allele-specific peptides with a high binding affinity.

Alleles↗
PubMed1995

A predicted consensus structure for the protein kinase C2 homology (C2H) domain, the repeating unit of synaptotagmin.

A secondary structure has been predicted for the protein kinase C2 regulatory domain found in homologous form in synaptotagmin, some phospholipases, and some GTP activated proteins. The proposed structure is built from seven consecutive beta strands followed by a terminal alpha helix. Considerations of overall surface exposure of individual secondary structural elements suggest that these are packed into a 2-sheet beta sandwich structure, with one of only three of the many possible folds being preferred.

Amino Acid Sequence↗