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J G Cyster

Publications and source records attributed to J G Cyster.

48 records · Page 3Linked to original sources
PubMed1995

Protein tyrosine phosphatase 1C negatively regulates antigen receptor signaling in B lymphocytes and determines thresholds for negative selection.

Motheaten viable (mev) mice are deficient in the cytosolic protein tyrosine phosphatase, PTP1C, and exhibit severe B cell immunodeficiency and autoantibody production. The role of PTP1C in B cell selection and function was analyzed by breeding immunoglobulin transgenes specific for a defined antigen, hen egg lysozyme, into mev mice. Antigen triggered a greater and more rapid elevation of intracellular calcium in PTP1C-deficient B cells, indicating that this phosphatase negatively regulates immunoglobulin signaling. Elimination of self-reactive B cells carrying this signal-enhancing mutation was triggered during their development by binding a lower valency form of self-antigen than is normally required. These findings establish that activation of distinct repertoire-censoring mechanisms depends on quantitative differences in antigen receptor signaling, whose thresholds are determined by negative regulation through PTP1C.

Animals↗
PubMed1994

Competition for follicular niches excludes self-reactive cells from the recirculating B-cell repertoire.

Two different approaches to follow clones of B lymphocytes in a diverse preimmune repertoire reveal a new process for eliminating self-reactive cells in the periphery which depends on competition between cells with different specificities. A key feature of this censoring mechanism is the selective exclusion of self-antigen-binding B cells from the normal migration route into lymphoid follicles, resulting in their premature death. This is a striking example of homeostasis by cellular competition for limiting niches and may explain the paradoxical association between immunodeficiency and autoimmunity.

Animals↗
PubMed1994

Antigenic determinants encoded by alternatively spliced exons of CD45 are determined by the polypeptide but influenced by glycosylation.

Antibodies recognising the products of alternatively spliced exons near the N-terminus of the leukocyte common antigen, CD45, have been widely used to distinguish populations of lymphocytes with different functional properties. These alternatively spliced regions contain a high content of serine and threonine residues (average 35%) and are heavily O-glycosylated. Despite evidence that the O-glycosylation contributes significantly to the antigenic character of this region of CD45, work with leukosialin and mucin glycoproteins leads to the prediction that the majority of epitopes in the N-terminal exons should be linear protein determinants. In this study the exons of CD45 were expressed in Escherichia coli as non-glycosylated proteins fused to glutathione S-transferase (GST). Fourteen out of 17 mAbs specific for human CD45R reacted with a fusion protein containing exons 4, 5 and 6 (ABC) of human CD45, and four out of six mAbs specific for rat CD45R reacted with an equivalent rat protein. mAbs recognising the product of rat exon B are reported for the first time. Kinetic analysis of MRC OX22 antibody binding to spleen CD45 and to GST fusion proteins showed that the carbohydrate affected the kinetics of binding of antibodies to the protein backbone. In conclusion, heterogeneity in the glycosylation of heavily O-glycosylated cell surface proteins can affect interactions of these proteins both directly through the carbohydrate and indirectly through effects on the protein backbone.

Alternative Splicing↗
PubMed1993

Mutational analysis of the CD2/CD58 interaction: the binding site for CD58 lies on one face of the first domain of human CD2.

The adhesion interaction between the immunoglobulin superfamily molecules CD2 and CD58 (lymphocyte function-associated antigen 3) plays an important role in T cell and natural killer cell interaction with various antigen-presenting and target cells. Determination of the solution structure of rat CD2 domain 1 has allowed a model of human CD2 domain 1 to be generated, and a series of mutants based on this model have been made. Residues of domain 1 of human CD2 predicted to be solvent exposed were substituted with the equivalent residues present in the rat CD2 molecule. The ability of these mutants to mediate rosetting with human and sheep erythrocytes was studied. Results show that the binding site of CD2 for both human and sheep CD58 maps to the beta sheet containing beta strands CC'C"F and G. Residues K34 and E36 in beta strand C, R48 and K49 in beta strand C', and K91 and N92 in the loop connecting beta strands F and G are shown to be critical in the interaction. The data support the proposition that the interaction between CD2 and CD58 involves the major beta sheet face of CD2.

Amino Acid Sequence↗
PubMed1993

The NH2-terminal domain of rat CD2 binds rat CD48 with a low affinity and binding does not require glycosylation of CD2.

CD2, CD48 and CD58 are structurally similar cell adhesion-molecules forming a subset of the immunoglobulin superfamily (IgSF). In humans CD58 is a ligand for CD2 while in mice CD2 binds CD48. We constructed a soluble chimeric molecule comprising the extracellular portion of rat CD48 and domains 3 and 4 of rat CD4 (sCD48-CD4) and used it to examine whether CD2 is a ligand for CD48 in rats. sCD48-CD4-coated polystyrene Dynabeads formed rosettes on rat CD2-transfected COS-7 cells, and this rosetting was blocked by anti-CD2 (OX34) and anti-CD48 (OX45) monoclonal antibodies. We used sucrose-gradient ultracentrifugation to show that sCD48-CD4 binds, in solution, to soluble forms of rat CD2 including the single NH2-terminal IgSF domain of rat CD2 expressed in bacteria. The upper limit of the affinity of the rat CD48-CD2 interaction is 4 x 10(5) M-1, lower than the published affinity of human CD2 for CD58. These results show that rat CD48 binds CD2 on its NH2-terminal IgSF domain with a low affinity and that binding is independent of glycosylation.

Animals↗
PubMed1993

Structural analysis of the CD2 T lymphocyte antigen by site-directed mutagenesis to introduce a disulphide bond into domain 1.

Many proteins have been predicted to contain domains with immunoglobulin-like folds and hence to be members of the immunoglobulin superfamily (IgSF). However, several members lack the Cys residues capable of forming the disulphide bond that forms a characteristic bridge between the beta sheets in the Ig fold, e.g. domain 1 of the lymphocyte antigen CD2. The assignment of beta strands in CD2 by sequence analysis was tested by attempting to introduce a disulphide bridge between the beta sheets by mutating two residues in the relevant positions to Cys. Mutant, soluble forms of CD2 were expressed in Chinese hamster ovary cell lines and amino acid sequencing showed that a disulphide bond was formed as predicted, but not in the control where one Cys residue was misplaced by four residues. Evidence that both mutated molecules folded correctly is given by the indistinguishable binding of three monoclonal antibodies recognising different epitopes on CD2. The 3-D structure of rat CD2 domain 1 has been determined by NMR spectroscopy and X-ray crystallography, confirming the predictions from the sequence. Applications of this method of insertion of disulphide residues for probing protein structures are discussed, together with other structures of IgSF domains lacking the typical inter-sheet disulphide bond.

Animals↗
PubMed1992

The importance of cross-linking in the homotypic aggregation of lymphocytes induced by anti-leukosialin (CD43) antibodies.

Leukosialin (CD43) is a major glycoprotein of T lymphocytes which has an extracellular domain of 45 nm in length that is heavily O-glycosylated. Monoclonal antibodies (mAb) to the extracellular domain of human leukosialin induce aggregation of T lymphocytes, monocytes and some cell lines that express leukosialin. The aggregation was reported in one case to be inducible by Fab fragments. In the present study, nine mAb specific for rat leukosialin were tested as inducers of thymocyte aggregation and all were effective. The level of aggregation was reduced by metabolic and cytoskeletal inhibitors, by removal of divalent cations and by reducing the temperature from 37 degrees C to 4 degrees C. The aggregation produced by mAb specific for certain epitopes was less sensitive to these inhibitors than aggregation induced by mAb to other epitopes. To examine the requirement for cross-linking, Fab fragments of four of the antibodies were tested and found to be inactive except for those derived from the OX75 mAb. However, OX75 Fab showed a tendency to dimerize, and monomeric OX75 Fab obtained directly after gel filtration was unable to induce aggregation. Thus induction of rat thymocyte aggregation by anti-leukosialin antibodies requires bivalent cross-linking and maximal aggregation is dependent on energy and an intact cytoskeleton. Mechanisms of antibody-induced aggregation are considered and it is proposed that in the case of leukosialin the antibodies may cross-link cells to overcome inherent repulsion between them and that subsequently other adhesion molecules complete the clustering process.

Animals↗
PubMed1991

IgE and IgG binding of peptides expressed from fragments of cDNA encoding the major house dust mite allergen Der p I.

Large peptides expressed from cDNA fragments of a clone encoding the mite allergen Der p I were able to bind IgE and IgG in sera from allergic individuals. The binding was found for peptides from sequences throughout the molecule, with at least five regions, comprising residues 1-56, 53-99, 98-140, 166-194, and 188-222. The only limitation was that more than 30 amino acid residues were required for consistent binding. Each of seven sera examined showed a different profile of antibody binding to the peptides. For the most part the pattern of IgE and IgG binding to the peptides for each serum was similar, demonstrating a concordant repertoire. In 5/7 sera, however, IgG bound to some peptides which had little or no IgE binding activity, thus showing more diverse specificities. It is suggested that some divergence of repertoire can develop during the maturation of the B cell response.

Allergens↗
PubMed1991

Structure of domain 1 of rat T lymphocyte CD2 antigen.

The CD2 antigen is largely restricted to cells of the T-lymphocyte lineage and has been established as an important adhesion molecule in interactions between human T lymphocytes and accessory cells. In the adhesion reaction, CD2 on T cells binds to LFA-3 on other cells, with binding through domain 1 of CD2. CD2 can also be a target for the delivery of mitogenic signals to T lymphocytes cultured with combinations of anti-CD2 antibodies. Two predictions that are contradictory have been made for the structure of CD2 domain 1. One suggests an immunoglobulin (Ig) fold, on the basis of sequence patterns conserved in the Ig-superfamily (IgSF), whilst the other proposes a pattern of alternating alpha-helices and beta-strands, on the basis of secondary structure predictions. Thus CD2 domain 1 is an important test case for the validity of IgSF assignments based on sequence patterns. We report here the expression of domain 1 of rat CD2 in an Escherichia coli expression system and have determined a low-resolution solution structure by NMR spectroscopy.

Amino Acid Sequence↗
PubMed1991

The dimensions of the T lymphocyte glycoprotein leukosialin and identification of linear protein epitopes that can be modified by glycosylation.

Leukosialin (CD43) is a major glycoprotein of T lymphocytes whose extracellular domain of 224 amino acids contains on average one O-linked carbohydrate unit per three amino acids. This suggests an unfolded structure for the extracellular domain which has now been established to extend to a length of 45 nm by transmission electron microscopy following low angle rotary shadowing. The antigenicity of rat leukosialin has been studied using nine monoclonal antibodies (MAbs) whose binding is differentially affected by the cell type on which leukosialin is expressed and by the removal of sialic acid. From these observations it appears that the epitopes are affected by glycosylation, yet seven of the nine MAbs reacted clearly with the extracellular domain of leukosialian expressed in an unglycosylated form in Escherichia coli. The MAbs showing this positive reaction included three of the four antibodies whose epitopes were affected by neuraminidase treatment of leukosialin. It thus appears that linear protein epitopes are recognized and that some of these can be modified in the native structure by glycosylation. The positions of the antigenic determinants have been mapped by expressing fusion proteins of different lengths and the identity of one epitope was proven by the binding of two MAbs to an octapeptide expressed as a fusion protein. For three MAbs, the location of epitopes in the native protein was confirmed by electron microscopy of shadowed leukosialin--Fab complexes. Overall it is concluded that leukosialin is a major component at the periphery of the T lymphocyte and that despite its high level of glycosylation, protein determinants are exposed that could be ligands in cell interactions.

Amino Acid Sequence↗