Matthew F. Mescher

Glycosylation of the surface glycoprotein of Halobacterium salinarium via a cyclic pathway of lipid-linked intermediates

Assembly of a complex polysaccharide via a cyclic pathway involving polyprenol lipid-linked intermediates was first demonstrated in the case of peptidoglycan biosynthesis [l] and there is now considerable evidence that at least some of the glycoproteins of eukaryotic cells are glycosylatedvia similar intermediates [2-71. Halobacterium salinarium has a high molecular weight glycoprotein as its major surface component [ 8,9] and has recently been shown to have enzymatic activities for formation of lipid-linked sugars of the type expected as intermediates if glycosylation of the glycoprotein occurs via a polyprenol lipid carrier [lo] . Despite the fact that Halobacteria lack a peptidoglycan layer their growth is inhibited by bacitracin [ 1 l] , an antibiotic which inhibits growth of normal bacteria by complexing with the Css isoprenyl pyrophosphate released upon transfer of the newly formed subunit to the growing peptidoglycan chain [ 12,131. Formation of this complex prevents the enzymatic dephosphorylation of the lipid which is necessary for it again to act as an acceptor and allow continued peptidoglycan synthesis. The effects of bacitracin on H. salinarium have been examined and this report describes experiments which provide further evidence that glycosylation of the surface glycoprotein occurs via a cyclic pathway involving lipid intermediates.

Glycoproteins as cell-surface structural components

Abstract The major cell-surface component of Halobacterium salinarium is a glycoprotein having structural and biosynthetic features in common with eukaryotic glycoproteins. The occurrence of glycoproteins in eukaryotes and archaebacteria suggests that they may have originated in primitive cells, which existed before the divergence of the three major lines of descent, possibly as structural components to stabilize the cell membrane.

Large-scale purification of murine I-Ak and I-Ek antigens and characterization of the purified proteins☆☆☆

Detailed analysis of the role of the structural characteristics of these molecules will require isolation of relatively large amounts of these antigens in serologically active form. We have purified murine Ia antigens on a large scale by affinity chromatography using monoclonal antibodies coupled to Sepharose 4B. Both I-Ak and I-Ek were isolated by sequential passage of cell lysate over columns prepared using specific monoclonal antibodies. Elution of the bound antigens required high pH (11-12) but, nonetheless, the purified material was 50-75% serologically active. Using LPS-stimulated spleen cells or B-lymphocyte tumor cells as starting material, 0.5 mg of each antigen can be readily purified. Based on antigen yields, it can be estimated that normal B-cells have about the same surface density of Class I and Class II MHC antigens. LPS blasts, in contrast, have normal levels of Class I antigen but 3-5 times higher levels of Class II antigens. We have now purified I-Ak and I-Ek from a number of different cell sources and have noted differences in both the mol. wts of the alpha- and beta-chains and in their apparent associations with cytoskeletal components. Proteins having the same apparent mol. wts as actin and myosin co-purify with both I-Ak and I-Ek antigens from various sources. These proteins do not co-purify with H-2K and D molecules obtained by similar methods, suggesting that Ia antigens may specifically interact with cytoskeletal elements.

The plasma membrane skeleton of tumor and lymphoid cells: a role in cell lysis?

A detailed understanding of the structure and dynamics of the surface membrane of cells is crucial to study of the mechanisms of cell mediated cytotoxicity. The dynamics of the membrane receptors, target cell antigens and accessory proteins almost certainly affect the efficiency of establishing and maintaining cell-cell contact and delivery of the transmembrane signal resulting in triggering of the lytic mechanisms. Furthermore, whether the primary event involved in target cell lysis is formation of membrane lesions (pore formation) or transfer of a lytic component (enzyme?) from the effector cell to the target, the molecular requirements for these events will certainly depend upon the structure and dynamics of the target cell plasma membrane.

Cytolytic T Lymphocyte Recognition of Subcellular Antigen

Specific antigen recognition is required to stimulate generation of a cytolytic T lymphocyte (CTL1) response and to allow the resulting effector CTLs to bind and lyse the target cells. Reliable assays allow in vitro assessment of stimulation of a primary and secondary response and of the effector-target interaction. The antigens required to trigger generation of a response are the same as those required to allow binding and lysis by the effector CTL.

Inhibition of normal lymphocyte responses by cell membranes.

Cell membranes bearing the appropriate antigen are known to stimulate a variety of cell-mediated immune responses. This report confirms that tumor cell membranes at doses of 2-5 micrograms protein/ml will stimulate in vitro generation of allogeneic cytotoxic T lymphocytes (CTL). However, higher doses (50-100 micrograms protein/ml) of the same membranes completely abrogate the generation of lytic activity. Responding lymphocytes are inhibited by membranes from either syngeneic or allogeneic cells. The inhibition appears to act at a proliferative or differentiation step in the generation of the CTL response, since membranes are known to have little direct effect on the lytic phase of CTL activity. Similar doses of membranes also inhibit LPS-induced B-cell proliferation. B-Cell proliferation is inhibited equally well by allogeneic and syngeneic membranes, and membranes from normal spleen cells are as inhibitory as tumor cell membranes. The inhibitory activity copurifies with the plasma membrane. The results raise important considerations regarding the use of subcellular forms of antigen in studies of lymphocyte recognition. In addition, these data suggest that cell-cell contacts might provide signals regulating the proliferation of lymphocytes.

Induction of allogeneic cytolytic T lymphocytes by partially purified membrane glycoproteins

Abstract It has been previously shown that P815 (H-2 d ) purified plasma membranes can induce cytolytic activity from primed C57BL/6 (H-2 b ) spleen cells. The secondary cytolytic T lymphocyte (CTL) inducing activity is retained when these P815 plasma membranes are solubilized in deoxycholate. Evidence is now presented that the cell surface antigens responsible for CTL induction can be partially purified in active form and these antigens can be incorporated into reconstituted membranes and phospholipid vesicles. The active antigens have the properties expected for H-2 molecules on lentil lectin chromatography and gel filtration.

Cytolytic T lymphocyte mediated chromium-51 release versus spontaneous release from blast and spleen cell targets at low temperatures

Abstract The rate of spontaneous 51Cr release from spleen cell and LPS blast target cells is strongly temperature dependent. Between 32 and 37 °C the rate of spontaneous release increases dramatically with temperature. Cytolytic T-lymphocyte-mediated lysis of these target cells is also temperature dependent, but lysis does not increase greatly above 32 °C. The ratio of specific 51Cr release to spontaneous release can be significantly improved by doing 51Cr-release assays at temperatures below 37 °C.

INDUCTION OF VIRUS-SPECIFIC H-2 RESTRICTED MURINE CTL BY LIPOSOMES

ABSTRACT Reconstituted membranes and liposomes have been used to study the requirements for the induction of in vitro secondary CTL responses to allogeneic and virus-infected, syngeneic cells. Evidence is presented that partially purified H-2 antigens are able to induce CTL activity from primed allogeneic spleen cells. These partially purified H-2 antigens when incorporated into reconstituted membranes or liposomes with Sendai virus antigen are able to induce virally primed syngeneic spleen cells to give a virus specific, H-2 restricted CTL response. Furthermore, when the hemagglutinin/neuraminadase (HN) protein of Sendai virus was isolated and incorporated into liposomes with partially purified H-2 antigens, virus specific CTL are induced.

Structural heterogeneity of murine IgD during ontogeny

We have studied the expression of the two membrane delta heavy chains (delta 1 and delta 2) and the two native IgD structures (IgDI and IgDII) in neonatal mice. Both delta-chains appear simultaneously during development and neonatal mice, like adults, express equal amounts of delta 1 and delta 2. IgDI and IgDII also appear simultaneously during ontogeny and in the same ratio as expressed by adult mice of the same strain. Thus, when IgD first appears during ontogeny it shows the same structural heterogeneity as observed in adult mice.