Michael J. Crumpton

Monoclonal Antibodies for Analysis of the HLA System

III Antibodies A Monoclonal antibodies against determinants of the HLA-A,-B,-C glycoproteins and pjmicroglobulin 1. Antibodies against species specific, non-polymorphic determinants 2. Antibodies against polymorphic HLA-A,-B,-C determinants B Monoclonal antibodies against HLA-DRw determinants 1. Antibody against a species specific HLA-DRw determinants 2. Antibodies against polymorphic HLA-DRw determinants

Isolation of glycoproteins from pig lymphocyte plasma membrane using Lens culinaris phytohemagglutinin.

Abstract Lymphocyte plasma membrane glycoprotiens solubilized in 1% sodium deoxycholate were selectively purified by adsorption to LcH ∗ , covalently attached to Sepharose 4B, and by subsequent elution with methyl-a-D-mannopyranoside. The yield of glycoprotein was more than twice that obtained using Concanavalin A-Sepharose. The purified glycoproteins inhibited the transformation of lymphocytes by LcH and PHA. The method is probably of general applicability to the purification of membrane glycoproteins.

Expression of HLA system antigens on placenta.

The levels of HLA-A and -B antigens expressed by placenta have been assessed relative to parental and other A and B antigen types that were not shared by the foetus. A purified preparation of placenta plasma membrane was used to estimate the antigen activities. The results indicate that maternally and paternally inherited A and B antigen activities and β2-microglobulin are expressed to similar extents but at much lower levels than in spleen lymphocytes (less than 5%). The possibility that the amounts detected were caused by contamination with blood or maternal tissue was ruled out. The low levels of A and B antigens may account for the lack of a cellular immune response to the other polymorphic cell surface antigens of the trophoblast. No evidence was obtained for the expression of a significant level of Ia antigens.

Orientation of cell-surface antigens in the lipid bilayer of lymphocyte plasma membrane.

Human HLA-A, B, C and Ia antigens were labelled by lactoperoxidase-catalysed iodination of the inner surface of lymphocyte plasma membrane and thus are transmembrane proteins. In contrast, membrane-bound human IgM and mouse IgM, IgD and Thy-1 antigen were not labelled on the inner membrane surface.

The structure of the human CD2 gene and its expression in transgenic mice.

We report the genomic organization of the human CD2 gene and its expression in transgenic mice. A 28.5 kb segment of DNA consisting of 4.5 kb 5′ flanking sequences, 15 kb containing the genes five exons and 9 kb of 3′ flanking sequences can direct the expression of the CD2 gene only on thymocytes, circulating T cells and megakaryocytes of the transgenic mice. The expression of each copy of the human CD2 transgene appears to be as high as the endogenous mouse CD2 gene and as high as the expression on the surface of human T lymphocytes, independent of the site of integration and dependent on the copy number of genes that have integrated.

Primary structure of the human, membrane-associated Ca2+-binding protein p68: a novel member of a protein family

cDNA clones encoding human ‘p68’, a membrane‐associated Ca2+‐binding protein, were isolated from a lambda gt11 expression library of the human T‐leukaemia cell line J6, by using a rabbit antiserum against denatured purified lymphocyte p68, and from a liver cDNA library by using 32P‐labelled p68 cDNA fragments. The amino acid sequence of p68, deduced from the sequences of overlapping cDNA clones, is described. The results show that p68 is closely related to a family of proteins which includes intracellular substrates of the EGF receptor and pp60src tyrosine kinases. The p68 amino acid sequence is internally repetitive, being constructed from eight repeats of varying lengths, each of which contains a highly conserved sequence. Multiple copies of the latter sequence are also present in the related proteins p36, lipocortin I and protein II. We discuss how the common structural features of these proteins may reflect common functions and, furthermore, how the eight repeat structure of p68 may have evolved. The sequences of independent cDNAs suggest that alternatively‐spliced mRNAs could encode different p68 protein species. This suggestion is consistent with the observation that purified p68 migrates as a closely‐spaced doublet when analysed by SDS‐PAGE.

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.

Preparation and Properties of Lymphocyte Plasma Membrane

The cell surface (plasma) membrane plays a crucial role in many diverse aspects of the cell’s behavior. Thus, the membrane acts not only as a barrier regulating the influx and efflux of ions, nutrients, and metabolites, but also provides the location for a multiplicity of molecules (“receptors”) which monitor the external environment for “messengers” such as hormones, antigens, and the surface components of other cells. The surfaces of different cell types vary with respect to the nature of their receptors and their antigenic specificities. A well-documented example of these distinctions is provided by the lymphocyte. Lymphocytes comprise essentially two populations [thymus-derived (T) and bone-marrow-derived (B) cells; Roitt et al., 1969] which can be distinguished functionally, antigenically, and in terms of their receptor activities. Thus, the surface of T lymphocytes plays major roles in cellular immunity, in immunosuppression by antilymphocytic serum, and in providing “help” in a humoral immune response, whereas the B-lymphocyte surface is primarily responsible for mediating humoral immunity (Greaves et al., 1973). Also, T cells (mouse) possess an antigenic specificity (θ)which is not shared by B cells (Raff, 1971), whereas receptors for Fc (Basten et al.,1972a,b) and the activated C3 component of complement (Nussenzweig and Pincus, 1972) are unique for B cells. Apart from these differences, the surfaces of T and B cells share histocompatibility antigens, mediate antigen recognition although not necessarily via an identical receptor, and possess receptors for plant lectins (Greaves and Janossy, 1972), polypeptide hormones [insulin (Hadden et al., 1972; Gavin et al., 1973) and growth hormone (Lesniak et al., 1973)], and small pharmacologically active molecules such as histamine (Melmon et al., 1972; Shearer et al., 1972).

Crystallization of p68 on lipid monolayers and as three-dimensional single crystals

Two-dimensional crystals of p68, a Ca2+ -binding protein that has homology with members of the lipocortin/calpactin family, were obtained by interaction with a phospholipid monolayer. By measuring surface pressure at constant surface area, p68 was found to interact in a Ca2+ -dependent manner specifically with phosphatidylethanolamine, less so with phosphatidylserine and not at all with phosphatidylcholine. With dimyristoyl-phosphatidylethanolamine, two-dimensional crystalline arrays were formed. Image analysis of electron micrographs of these crystals, which diffracted to about 50 A, revealed p3 symmetry with a unit cell of about 178 A by 178 A; the protein densities showed a two-domain structure giving a cylindrical molecule of about 100 A by 35 A diameter packed as trimers. Three-dimensional microcrystals obtained without lipid or Ca2+ were suitable for electron microscopy and gave a tetragonal unit cell of about 256 A by 68 A. The implications of these observations on the structure and lipid specificity of p68 binding are discussed.

Biosynthesis and molecular nature of the T3 antigen of human T lymphocytes.

Immunoprecipitates of the T3 antigen prepared from HPB‐ALL cells by using the monoclonal antibody UCH‐T1 were analysed by SDS‐polyacrylamide gel electrophoresis. Cells which had been biosynthetically labelled for up to 4 h gave a major polypeptide of mol. wt. 19 000 plus two weaker, more diffuse bands of mol. wts. 21 000 and 23 000, whereas surface labelled cells gave a prominent band of mol. wt. 19 000, a major band of 21 000 and a weaker diffuse band of approximately 26 000. As judged from their sensitivity to proteinase‐K digestion, all the above polypeptides possess a transmembrane orientation. Digestion with endoglycosidases H and F (endo‐H and endo‐F), and tunicamycin treatment indicate that all the polypeptides, except that of 19 000 mol. wt. are N‐glycosylated. The 21 000 and 23 000 mol. wt. chains possess both immature and mature oligosaccharide units, whereas the 26 000 mol. wt. band apparently has mature units only. Pulse chase experiments combined with digestion by endo‐F and endo‐H suggest that the N‐glycosylated polypeptides are derived from two polypeptides of mol. wts. 14 000 and 16 000. It is concluded that the T3 antigen is derived from three different non‐glycosylated polypeptides two of which are subsequently N‐glycosylated to give the 21 000, 23 000 and 26 000 forms. The cell surface T3 antigen most probably comprises at least two distinct, non‐covalently associated polypeptides, but the number and types of polypeptides giving rise to the whole molecule and whether different complexes exist is at present unclear.