Structure and expression of genes involved in T lymphocyte recognition and activation.
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Amino Acid Sequence↗
Biomedical subjects
M J Crumpton
Publications and source records attributed to M J Crumpton.
The regulation of T-cell proliferation: a role for protein kinase C.
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Enzyme Activation↗
The gene coding for the human T-lymphocyte CD2 antigen is located on chromosome 1p.
A cDNA clone encoding the human T lymphocyte sheep erythrocyte receptor [the CD2 (T11) antigen] was used as a probe to define the chromosomal location of the gene. The signal, revealed by hybridisation to Southern blots of genomic DNA from somatic cell hybrids, showed a high degree of concordance for human chromosome 1. In particular, the hybrid F4Sc13C19 which contained the short arm only of human chromosome 1 was positive. The location of the CD2 gene to 1p13 was confirmed by in situ hybridisation.
Animals↗
Chromosomal locations of the gene coding for the CD3 (T3) gamma subunit of the human and mouse CD3/T-cell antigen receptor complexes.
The gene coding for the Mr 26000 gamma chain of the human CD3 (T3) antigen/T-cell antigen receptor complex was mapped to chromosome band 11q23 by using a cDNA clone (pJ6T3 gamma-2), by in situ hybridization to metaphase chromosomes and by Southern blot analysis of a panel of human-rodent somatic cell hybrids. The mouse homolog, here termed Cdg-3, was mapped to chromosome 9 using the mouse gamma cDNA clone pB10.AT3 gamma-1 and a panel of mouse-hamster somatic cell hybrids. Similar locations for the CD3 delta genes have been described previously. Thus, the corporate results indicate that the CD3 gamma and delta genes have remained together since they duplicated about 200 million years ago.
Animals↗
Purification and identification by amino acid sequence analysis of the subunits of the CD3 antigen of human tonsil T-lymphocytes.
The CD3 antigen was purified from human tonsils by immunoaffinity chromatography and preparative SDS-PAGE; the overall yield was 10%. Amino acid sequence analysis of the separated gamma, delta and epsilon polypeptides revealed that the gamma and epsilon polypeptides were blocked at the N-terminus, whereas the partial N-terminal amino acid sequence for the delta chain was identical to that described by Borst et al. [Nature 312, 455-485 (1984)]. The gamma and epsilon chains were cleaved with formic acid and cyanogen bromide respectively in order to obtain amino acid sequence data. The sequences obtained corresponded exactly to the amino acid sequences deduced from the nucleotide sequences of putative gamma and epsilon cDNA clones.
Amino Acid Sequence↗
The role of class I and II antigens in T cell recognition.
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HLA Antigens↗
Homology between the subunits of S100 and a 10kDa polypeptide associated with p36 of pig lymphocytes.
The polypeptide of about 10,000-Mr which is associated with the p36 polypeptide of pig mesenteric lymph node lymphocytes has a N-terminal 51 amino acid sequence that is 56 and 42% homologous with the N-terminii of the alpha and beta subunits respectively of S100.
Amino Acids↗
The importance of conformation and of equilibria in the interaction of globular proteins and their fragments with antibodies.
Oligopeptide fragments of globular proteins often inhibit the interaction of antibodies with native protein. Although such fragments appear to possess in aqueous solution an unfolded conformation it is apparent, in those cases where the protein's three-dimensional structure is known, that the antibody-bound fragment possesses a folded conformation mimicking that of the corresponding portion of the whole protein. The probable explanation of this dichotomy is that the fragment has various conformations in equilibrium including a small proportion of molecules whose shape is recognized and stabilized by the antibody. A related situation can exist in the interaction of antibody(ies) with the whole protein. Thus, antibodies against an altered form of the protein can induce the native antigen to adopt the conformation of the altered form. In this case, it appears that localized regions of the protein's surface are flexible, adopting various conformations in equilibrium, one of which is stabilized (selected) by interaction with the appropriate antibody. In both instances interaction with antibody perturbs an equilibrium, leading to the selection of a particular conformation. Such dynamic effects have profound implications on the choice of peptides as synthetic vaccines.
Animals↗
Primary structure of the T3 gamma subunit of the T3/T cell antigen receptor complex deduced from cDNA sequences: evolution of the T3 gamma and delta subunits.
cDNA clones, whose fusion proteins were recognised by an anti-(T3 gamma chain) serum, were isolated from a lambda gt11 expression library prepared from the human T leukaemia cell line J6. The clones encoded a unique sequence related to that of the T3 delta chain, and hybridised to two mRNA transcripts of 0.8 and 3.5 kb in size, whose expression was restricted to T lymphocytes. The 182 amino acid sequence deduced from the cDNA revealed a typical signal peptide, a predominantly hydrophilic 89 amino residue domain with two N-glycosylation sites, a hydrophobic domain with a centrally located glutamic acid residue and a 44-residue domain with at least one potential serine phosphorylation site for protein kinase C. Given this arrangement the T3 gamma polypeptide most probably has a transmembrane orientation with the N-terminal domain exposed on the cell surface. The amino acid and nucleotide sequences showed marked homology with those of the T3 delta chain, suggesting that the respective genes arose by duplication about 200 million years ago. The intracellular and membrane-proximal half of the extracellular domains were especially well conserved.
Amino Acid Sequence↗
Molecular cloning of the human T-lymphocyte surface CD2 (T11) antigen.
CD2 (T11, sheep erythrocyte receptor) is a surface antigen of the human T-lymphocyte lineage. cDNA clones encoding CD2 have been isolated by using the purified, denatured CD2 to raise a rat antiserum. Positive clones were recognized in a phage lambda gt11 expression library prepared from the human leukemia T-cell line J6. The DNA sequence contained an open reading frame encoding 360 amino acids. The N-terminal 24 amino acids were characteristic of a signal peptide and were followed by a region that matched all 25 residues of the CD2 N terminus previously determined by amino acid sequencing. The predicted amino acid sequence is consistent with that of a transmembrane glycoprotein containing three potential N-glycosylation sites on the N-terminal side of a 26-amino acid hydrophobic segment. There is a large cytoplasmic domain of 125 amino acids that is rich in proline and in basic residues. RNA blot-hybridization analysis demonstrated hybridization only in those T cells that were positive for surface CD2 antigen. There are limited regions of sequence similarity to members of the immunoglobulin supergene family.
Amino Acid Sequence↗
Identification of calcium-binding proteins associated with the lymphocyte plasma membrane.
The Nonidet P40 insoluble fraction of lymphocyte plasma membrane contains three polypeptides of about 68,000-, 33,000- and 28,000-Mr which are solubilised by Ca2+-chelators. As judged by various criteria the 33,000-Mr polypeptide is homologous to the 36,000-Mr pp60src kinase substrate of chicken fibroblasts and the 68,000-Mr polypeptide is related to the 67,000-Mr "calelectrin" of bovine liver. The 28,000-Mr polypeptide may also be related to calelectrin.
Animals↗
HLA-D region antigen-associated invariant polypeptides as revealed by two-dimensional gel analysis. Glycosylation and structural inter-relationships.
Two-dimensional polyacrylamide gel analyses of immunoprecipitates of HLA-D region antigens prepared from [35S]methionine-labeled B lymphoblastoid cells revealed a number of invariant polypeptides (Ii and theta) that co-precipitate with the alpha and beta polypeptides of the class II (Ia) antigens. The invariant polypeptides comprised at least three Ii spots of Mr = 31,000 (Ii1-Ii3) and a series of six theta spots of Mr = 34,000 (theta 1-theta 6). The structural inter-relationships of these polypeptides have been investigated. Tryptic peptide fingerprints showed that Ii and theta have closely related amino acid sequences. In contrast, the fingerprints of the HLA-DR alpha and beta polypeptides clearly differed from those of theta and Ii as well as from each other. Analyses of immunoprecipitates prepared from cells cultured in the presence of tunicamycin revealed the presence of two N-linked oligosaccharides on each invariant polypeptide and suggested that the more acidic theta polypeptides (theta 1 and theta 2) differed from the other invariant polypeptides by the presence of sialic acid on one or both N-linked oligosaccharides. Removal of sialic acid by neuraminidase simplified the pattern of theta spots into three distinct Ii-related polypeptides. Endo-beta-N-acetylglycosaminidase H digestion indicated that the individual theta polypeptides represent stages in carbohydrate processing whereby Ii with two N-linked immature oligosaccharides are converted initially to theta 6-theta 3 with one immature and one complex, but nonsialylated, oligosaccharide and finally to theta 2-theta 1 with two complex oligosaccharides. Digestion of the theta polypeptides with N-acetylgalactosamine oligosaccharidase indicated that the theta spots are also derived by O-glycosylation from the Ii polypeptides. This assignment is supported by results obtained using monensin to block glycosylation within the Golgi. At least three spots persisted after complete removal of the N- and O-linked oligosaccharides, suggesting the presence of a family of invariant polypeptides differing in amino acid sequence.
Acetylglucosaminidase↗
The mitogenic lectin from Phaseolus vulgaris does not recognize the T3 antigen of human T lymphocytes.
Human peripheral blood T lymphocytes are stimulated to grow and divide by lectins such as concanavalin A (Con A) and Phaseolus vulgaris phytohemagglutinin (PHA), as well as a few anti-T cell monoclonal antibodies. The latter antibodies recognize the T3 antigen. It has been suggested previously that PHA and Con A mediate T cell growth by interacting with T3. However, as reported in this study, affinity chromatography on immobilized lectins, and immunoprecipitation by lectin plus anti-lectin antibodies showed that T3 binds Con A but not PHA. Fab fragments of a monoclonal antibody against T3 (namely Leu-4) inhibited T lymphocyte proliferation induced by T3 antibodies and Con A, but not by PHA. Nevertheless, co-capping experiments performed with fluorescein-labeled lectins and rhodamine-labeled T3 antibodies showed that T3 co-caps with Con A and PHA receptors, although the co-capping with PHA was incomplete. Since the T cell receptor for antigen (Ti) has been shown to co-cap with T3 on the cell surface, we reasoned that PHA induced capping of the T3 antigen by interacting with Ti. A disulfide-linked heterodimer comprising subunits of about 49 000 and 41 000 mol. wt. that resembled the Ti molecule was detected in PHA-anti-PHA immunoprecipitates of various surface- and biosynthetically-labeled T cells, by two-dimensional (nonreduced vs. reduced) sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis. The results suggest that PHA triggers T lymphocytes by interacting with the carbohydrate moieties of Ti and imply that T lymphocytes can be stimulated by mitogens via at least two different cell surface molecules (Ti and T3).
Antibodies, Monoclonal↗
Characterization of the HLA-A2.2 subtype: T cell evidence for further heterogeneity.
Five blood donors were identified whose HLA-A2 is different from the common HLA-A2. Their A2 molecule (A2.2) had a more basic isoelectric point than normal A2 (A2.1). Cytotoxic T lymphocytes (CTL) restricted by HLA-A2.1, specific for influenza A and Epstein-Barr viruses, failed to lyse virus-infected target cells with HLA-A2.2. Identical patterns were obtained with both viruses. CTL from four of the A2.2-positive donors recognized target cells prepared from others in the group that shared only the HLA-A2.2 antigen. The A2.2 antigen from one donor seemed to be different in that target cells were not recognized by CTL from donors with the normal A2.1 nor with basic A2.2. There seems, therefore, to be heterogeneity within the HLA-A2.2 subtype.
Cytotoxicity, Immunologic↗
Activators of protein kinase C down-regulate and phosphorylate the T3/T-cell antigen receptor complex of human T lymphocytes.
As judged by indirect immunofluorescence, phorbol 12,13-dibutyrate and 1-oleoyl-2-acetylglycerol induced a rapid, concentration-dependent decrease of about 50% in the surface expression of the T3 antigen on human T lymphoblasts, and of T3 and the T-cell antigen receptor on HPB-ALL cells. Direct binding experiments using 125I-labeled antibody indicated that the reduction in T3 expression corresponded to a decrease in the number of antigen molecules rather than a change in their affinity. Biochemical analyses revealed that phorbol dibutyrate induced a rapid, prominent phosphorylation of the T3 Mr 26,000 gamma chain and to a lesser extent of the Mr 21,000 delta chain. No phosphorylation of the T3 epsilon chain or of the alpha and beta subunits of the T-cell antigen receptor was detected. The data suggest that protein kinase C induces a phosphorylation of the T3 gamma and delta chains that may lead to the down-regulation of the T3/T-cell antigen receptor complex.
Antibodies, Monoclonal↗
Phaseolus vulgaris phytohaemagglutinin (PHA) binds to the human T lymphocyte antigen receptor.
The interaction of phytohaemagglutinin (PHA) with the human T lymphocyte antigen receptor (Ti) was explored. Nonidet-P40 lysates of surface-labelled HPB-ALL cells were immunoprecipitated with PHA, using a rabbit anti-(PHA)-serum, as well as clonotypic monoclonal antibodies (H1-2D4 and T40/25) and a rabbit antiserum (R-43) against Ti. One- and two-dimensional SDS-polyacrylamide electrophoresis under reducing and non-reducing conditions showed that both the clonotypic antibodies and PHA precipitated a disulphide cross-linked heterodimer having a mol. wt. of approximately 79 000 (unreduced) and a comprising subunits of mol. wts. approximately 50 000 and 39 000 (reduced). Further evidence that PHA binds Ti was obtained by (i) cross-immunodepletion with H1-2D4 and PHA; (ii) immunoprecipitation with H1-2D4 of a glycoprotein fraction specifically eluted from a PHA immunoprecipitate; (iii) immunoprecipitation with PHA of a solubilised H1-2D4 immunoprecipitate; (iv) 2-D (non-equilibrium pH gradient electrophoresis/SDS) analyses of H1-2D4 and PHA immunoprecipitates, indicated that H1-2D4 and PHA recognise coincident beta polypeptides. PHA also binds a Ti-like disulphide cross-linked heterodimer on tonsil lymphocytes and two other T-cell leukaemias (HUT-78 and J6). The data further suggest that PHA and R-43 recognise a subpopulation of Ti molecules on HPB-ALL cells that are not bound by H1-2D4, suggesting that there may be at least two forms of Ti. Similar experiments indicate that Concanavalin A (Con A) and wheat germ agglutinin (WGA) also probably bind Ti, whereas Helix pomatia agglutinin (HPA) does not.
Antibodies, Monoclonal↗
Nonidet P-40 extraction of lymphocyte plasma membrane. Characterization of the insoluble residue.
Purified preparations of lymphocyte plasma membrane were extracted exhaustively with Nonidet P-40 in Dulbecco's phosphate-buffered saline medium. The insoluble fraction, as defined by sedimentation at 10(6) g-min, contained about 10% of the membrane protein as well as cholesterol and phospholipid. The lipid/protein ratio, cholesterol/phospholipid ratio and sphingomyelin content were increased in the residue. Density-gradient centrifugation suggested that the lipid and protein form a common entity. As judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the Nonidet P-40-insoluble fractions of the plasma membranes of human B lymphoblastoid cells and pig mesenteric lymph-node lymphocytes possessed similar qualitative polypeptide compositions but differed quantitatively. Both residues comprised major polypeptides of Mr 28 000, 33 000, 45 000 and 68 000, together with a prominent band of Mr 120 000 in the human and of Mr 200 000 in the pig. The polypeptides of Mr 28 000, 33 000, 68 000 and 120 000 were probably located exclusively in the Nonidet P-40-insoluble residue, which also possessed a 4-fold increase in 5'-nucleotidase specific activity. The results indicate that a reproducible fraction of lymphocyte plasma membrane is insoluble in non-ionic detergents and that this fraction possesses a unique polypeptide composition. By analogy with similar studies with erythrocyte ghosts, it appears likely that the polypeptides are located on the plasma membrane's cytoplasmic face.
Animals↗
Isolation and characterization of a novel 68,000-Mr Ca2+-binding protein of lymphocyte plasma membrane.
A 68 000-Mr protein is a major component of a Nonidet P-40-insoluble fraction of lymphocyte plasma membrane prepared from human B lymphoblastoid cells ( BRI 8) and pig mesenteric lymph nodes. The association of the protein with the detergent-insoluble complex depends on free Ca2+ concentrations of greater than 10 microM. The human and pig 68 000-Mr proteins were purified and appear to be homologous on the basis of amino acid composition and peptide mapping. The protein is monomeric, has pI 5.8 and a single high-affinity Ca2+-binding site (KD 1.2 microM). The results are discussed in terms of the possible role of the 68 000-Mr protein as an intracellular Ca2+ receptor in lymphocytes.
Amino Acids↗