Specific Surface Glycoprotein Research Articles

Trypanosoma cruzi invasion is associated with trogocytosis

Trogocytosis was originally thought to be restricted to the interaction of cells of the immune system with cancer cells. Such membrane exchanges are probably a general process in cell biology, and membrane exchange has been demonstrated to occur between non-immune cells within an organism. Herein, we report that membrane and protein exchange, consistent with trogocytosis, between Trypanosoma cruzi (both the Brazil and Tulahuen strains) and the mammalian cells it infects. Transfer of labeled membrane patches was monitored by labeling of either parasites or host cells, i.e. human foreskin fibroblasts and rat myoblasts. Trypomastigotes and amastigotes transferred specific surface glycoproteins to the host cells along with membranes. Exchange of membranes between the parasite and host cells occurred during successful invasion. Extracellular amastigotes did not transfer membrane patches and were did not transfer either membranes or proteins to the host cells. Membrane exchange was also found to occur between interacting epimastigotes in cell-free culture and may be important in parasite–parasite interactions as well. Further studies should provide new insights into pathogenesis and provide targets for therapeutic intervention.

Structure and function of the Salmonella Typhi chimaeric A(2)B(5) typhoid toxin.

Salmonella Typhi differs from most other salmonellae in that it causes a life-threatening systemic infection known as typhoid fever1. The molecular bases for its unique clinical presentation are unknown2. Here we found that in an animal model, the systemic administration of typhoid toxin, a unique virulence factor of S. Typhi, reproduces many of the acute symptoms of typhoid fever. We identified specific carbohydrate moieties on specific surface glycoproteins that serve as receptors for typhoid toxin, which explains its broad cell target specificity. We present the atomic structure of typhoid toxin, which shows an unprecedented A2B5 organization with two covalently-linked A subunits non-covalently-associated to a pentameric B subunit. The structure provides insight into the toxin’s receptor-binding specificity and delivery mechanisms and reveals how the activities of two powerful toxins have been coopted into a single, unique toxin that can induce many of the symptoms characteristic of typhoid fever. These findings may lead to the development of potentially life-saving therapeutics against typhoid fever.

Ex vivo enzymatic treatment of aged CD4 T cells restores antigen-driven CD69 expression and proliferation in mice

Declines in immune function have been associated with declines in the function of naïve CD4 T cells. In vitro studies of naïve CD4 T cells in TCR-specific transgenic AND mice have shown age-related defects in immunosynapse formation, activation, proliferation and cytokine production. Previous work has also documented age-related alteration in the glycosylation of surface proteins involved in TCR signaling, and shown that enzymatic treatments to remove specific surface glycoproteins can restore in vitro function in CD4 cells from aged mice. Here an adoptive transfer system shows that a large percentage of naïve CD4 T cells from old mice fail to express CD69 and expand in antigen-primed mice, but these declines in CD69 and expansion can be restored by ex vivo pretreatment of the T cells with the bacterial enzyme O-sialoglycoprotein endopeptidase (OSGE). OSGE treatment also repairs the age-dependent loss of CD69 expression after in vivo activation.

Patients with active relapsing‐remitting multiple sclerosis synthesize antibodies recognizing oligodendrocyte progenitor cell surface protein: Implications for remyelination

In multiple sclerosis (MS), remyelination of demyelinated lesions diminishes with disease progression for unknown reasons. Oligodendrocyte progenitor cells contribute to remyelination; however, antibodies specific for oligodendrocyte progenitor antigens could block remyelination by eliminating or impeding these cells. In myelinating cultures, cell lysis with antibody recognizing a progenitor cell–specific surface glycoprotein (AN2) suppressed the synthesis of myelin proteins. Cerebrospinal fluid from patients with relapsing-remitting active MS contains antibodies against AN2, whereas cerebrospinal fluid from patients with nonactive disease does not. This is the first report describing antibodies in MS against a progenitor cell–specific antigen that may contribute to the development and progression of chronically demyelinated lesions. Ann Neurol 2000;48:362–371

Effect of Hypoxia on Endothelial Cell Surface Glycoprotein Expression: Modulation of Glycoprotein IIIa and Other Specific Surface Glycoproteins

Exposure to hypoxia alters many aspects of endothelial cell metabolism and function; however, changes in surface glycoconjugates under these conditions have not been extensively evaluated. In the current studies, we examined surface glycoproteins of cultured bovine aortic (BAEC) and pulmonary arterial (BPAEC) endothelial cells under standard culture conditions (21% oxygen) and following exposure to hypoxia (0% oxygen) for varying time periods (30 min to 18 h) using a system of biotinylation, lectin binding (concanavalin A, Con A; Griffonia simplicifolia , GSA; Arachis hypogaea, PNA; Ricinus communis, RCA; or Triticum vulgaris, WGA), subsequent strep-avidin binding, and staining. Using these methods, we identified differences in lectin binding between the two cell types cultured in 21% oxygen with all lectins except PNA. With exposure to 0% oxygen, there was no change in lectin binding to most surface glycoproteins. Several surface glycoproteins, including glycoprotein IIIa on both cell types, demonstrated a time-dependent decrease in lectin binding; in addition, there was an increase in lectin binding to a few specific surface glycoproteins on each cell type within 30-60 min of exposure to 0% oxygen. These changes in specific surface glycoproteins were confirmed in both cell types by 125I labeling. Increased lectin binding was observed for Con A binding BAEC glycoproteins at molecular weight (MW) 116, 130, and 205 kDa, GSA binding BAEC glycoproteins at MW 120 and 205 kDa, and RCA binding BPAEC glycoproteins at MW 140 and 205 kDa. Increased binding of WGA or PNA was not observed during exposure to hypoxia. The specificity of lectin binding was further confirmed by competitive inhibition with the appropriate sugar. These studies demonstrate that there are baseline differences between BAEC and BPAEC cell surface glycoproteins and that exposure to hypoxia is associated with little change in lectin binding to most surface glycoproteins. There is, however, increased surface expression of a few glycoproteins that differ depending of the origin of the endothelial cell. Although the mechanism of this increase in lectin binding is not yet clear, subsequent studies suggested that it is due to increased availability of select carbohydrate moieties. The time course of these alterations suggests a possible role in the endothelial cell response to decreases in ambient oxygen tension.

Structure of the glycosylated adhesion domain of human T lymphocyte glycoprotein CD2.

CD2, a T-cell specific surface glycoprotein, is critically important for mediating adherence of T cells to antigen-presenting cells or target cells. Domain 1 of human CD2 is responsible for cell adhesion, binding to CD58 (LFA-3) expressed on the cell to which the T cell binds. Human CD2 domain 1 requires N-linked carbohydrate to maintain its native conformation and ability to bind CD58. In contrast, rat CD2 does not require N-linked carbohydrate, and binds to a different ligand, CD48. The three-dimensional structure of the glycosylated form of domain 1 of human CD2 has been determined by NMR spectroscopy. The overall structure resembles the typical beta-barrel of an immunoglobulin variable domain. Nuclear Overhauser enhancement contacts between the protein and the N-linked glycan have been tentatively identified. Based on our results, we propose a model showing how the N-linked glycan might be positioned in the human CD2 domain 1 structure. The model provides an explanation for the observed instability of deglycosylated human CD2, and allows residues that are important for CD58 binding to be differentiated from those affecting conformational stability via interactions with the glycan.

IFN-gamma and 1,25(OH)2D3 induce on THP-1 cells distinct patterns of cell surface antigen expression, cytokine production, and responsiveness to contact with activated T cells.

Differentiation and maturation of monocytes are accompanied by the expression of specific surface glycoproteins, the secretion of cytokines, and the capacity to respond to ligands. These changes may be influenced by interactions with hormones, soluble lymphocytic products, or direct contact with lymphocytes. We have studied two distinct pathways in the differentiation of a human monocytic cell line, THP-1: one being induced by IFN-gamma and the other by 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3). In THP-1 cells, IFN-gamma induces cell surface expression of HLA-DR and CD54 and production of IL-1 beta, TNF-alpha, and IL-6. In contrast, 1,25(OH)2D3 increases cell surface expression of CD11b and CD14, but fails to stimulate cytokine production. Direct contact of THP-1 with stimulated fixed T cells markedly induces IL-1 beta, TNF-alpha, and IL-6 production by THP-1. Production is higher when THP-1 have been previously exposed to 1,25(OH)2D3 as compared to prior exposure to IFN-gamma. mAb raised against certain relevant cell surface glycoproteins on THP-1 were tested for their ability to block the response of THP-1 to T cells. Antibodies to CD11a, CD11b, and CD11c, alone or in combination, only partially blocked IL-1 beta production by THP-1, whereas antibodies to CD54 and CD14 did not. Thus other unknown structures on the THP-1 cells may be involved in the induction of THP-1 cytokine production by T cell contact.

Characterization of three related murine interleukin-3 surface receptor proteins

Iodinated interleukin-3 (IL-3) can be covalently cross-linked to three specific surface glycoproteins with net molecular masses of 170, 140, and 65-70 kDa under conditions in which ligand internalization and degradation do not occur. These three proteins plus two additional non-ligand-binding proteins of 90 and 55 kDa can be purified by IL-3 affinity chromatography. Comparative two-dimensional analysis of the tryptic digests of these five proteins indicates that the ligand-binding proteins are highly related at the peptide level. Incubation of cells with 125I-IL-3 at 37 degrees C results in rapid time- and energy-dependent internalization and degradation of ligand. Under these conditions only the 140- and 65-70-kDa binding proteins, which can recycle to the surface after internalization, can be identified. The lability of the 170-kDa protein indicates that it may not recycle. Thus, an energy-dependent mechanism is responsible for internalization and may be necessary for any potential interconversion of the higher 170- or 140-kDa proteins to the lower 140- and/or 65-70-kDa binding proteins.

Trypanosoma brucei gambiense and T. b. rhodesiense: concanavalin A binding to the membrane and flagellar pocket of bloodstream and procyclic forms.

We have measured binding of fluorescein-conjugated succinyl-concanavalin A (Fl-s-Con A) to bloodstream and procyclic forms of Trypanosoma brucei gambiense and to bloodstream forms of T. b. rhodesiense by flow cytofluorimetry. Bloodstream forms bound an order of magnitude less lectin than procyclic forms. Trypsin-treating cells enhanced binding of Fl-s-Con A to bloodstream forms 3-16-fold depending on the strain and the length of trypsinization but had little effect on Fl-s-Con A binding by procyclics. The trypsinization protocol used did not remove major common glycoproteins detected on lectin blots of either life cycle form but removed greater than 95% of the variant specific glycoprotein and fragments derived from this protein of bloodstream forms. Microscopically detectable Fl-s-Con A binding to bloodstream forms was confined to the flagellar pocket. Trypsinized bloodstream forms and procyclics bound Fl-s-Con A in the flagellar pocket, on the flagellum, and on the cell surface. Lectin remained cell associated but appeared to redistribute towards the flagellum and pocket when cells that had bound lectin on ice were subsequently incubated at physiological temperatures. The Fl-s-Con A binding had specificity characteristic of the interaction between the lectin and oligosaccharides. These results are consistent with the hypothesis that the variant specific surface glycoprotein blocks binding of the lectin to surface glycoproteins of bloodstream forms and suggest that concanavalin A-binding glycoproteins are abundant in the flagellar pocket of both life cycle forms.

The anatomy and transcription of a telomeric expression site for variant-specific surface antigens in T. brucei

The variant specific surface glycoprotein (VSG) genes of T. brucei are expressed in telomeric expression sites. We have determined the structure of the active site in trypanosome variant 221a, which contains VSG gene 221, by analysis of cloned DNA segments that represent 65 kb of the 5′-flanking region of the VSG gene. In nuclear run-on experiments, 57 kb of adjacent sequences are cotranscribed with the VSG gene at approximately similar rates and in the α-amanitin-resistant manner characteristic of VSG genes. Besides the VSG mRNA, this expression site yields at least seven stable RNAs, suggesting that it is a multicistronic transcription unit. Our results also show that insertion of a transcriptional terminator is not the general mechanism of switching off expression sites.

Lectin interactions with the variant surface glycoprotein from Trypanosoma brucei brucei incorporated into liposomes

The variant specific surface glycoprotein from Trypanosoma brucei brucei is incorporated into lipid vesicles using 8M urea as an unfolding reagent. Pronase treatment of these proteoliposomes removes most of the protein, leaving a glycophospholipopeptide which is the membrane attachment site. We show here that lectins, specific for mannose and galactose are able to recognize oligosaccharide residues on these proteoliposomes, using a straightforward aggregation assay. The relevance of these results obtained with the liposome model system to the accessibility of the surface antigens in living trypanosomes is discussed.

Physical and immunological analysis of the two domains isolated from a variant surface glycoprotein of Trypanosoma brucei

A specific surface glycoprotein of a variant of Trypanosoma brucei was cleaved with trypsin and the two major domains of the molecule have been purified. We have studied the chemical composition of each domain and compared the data to published results of the specific cDNA sequence. Circular dichroïsm measurements show that the aminoterminal domain includes preferentially α-helical or β-sheet structure. The physicochemical analyses are supplemented by a prediction of secondary structure and a statistical pattern of hydrophilicity-hydrophobicity. The results are discussed in light of the internal limits that were described in the process of partial gene conversion occurring between the variant gene sequence and related members of the same gene family. Immunoblots with homologous antiserum indicate that the amino-terminal domain is implicated in antigenicity. In addition, immunoblotting with heterologous antiserum on native antigen, tryptic hydrolysates, or purified domains suggests a site of interaction supported by the two domains.

In vitro studies on the biosynthesis of the surface glycoprotein of Trypanosoma congolense.

Tritiated leucine, glucosamine, mannose, and galactose were incorporated into the variant specific surface glycoprotein (VSG) of Trypanosoma congolense in vitro. The uptake of the precursors is shown by SDS-polyacrylamide electrophoresis and fluorography, by assay of the radioactivity in immunoprecipitates obtained with specific antisera, and by the isolation of the labeled antigens by affinity chromatography on concanavalin A-sepharose and isoelectric focusing. The in vitro labeled VSG exhibits the same degree of microheterogeneity as that observed in the VSG isolated from trypanosomes grown in animals. Analysis of the incorporated sugars after hydrolysis of the glycoprotein showed that glucosamine and mannose were utilized in biosynthesis of the carbohydrate moiety directly whereas galactose was converted possibly to other intermediates before being incorporated into the antigen. Tunicamycin completely prevented the incorporation of the radiolabeled sugars into the surface glycoprotein. The unglycosylated VSG with a molecular weight of 47 kDa had completely lost its size heterogeneity.

Isolation of plasma membrane domains from murine T lymphocytes.

Murine T-lymphoma cells have been homogenized in dense sucrose solution and centrifuged under isopycnic conditions for membrane components. Floating fractions banding between 10% and 22.5% sucrose ("light" membranes) and between 22.5% and 35% sucrose ("heavy" membranes) were shown to consist of smooth membrane vesicles. The amounts of cholesterol and phospholipids recovered after chloroform/methanol extraction were similar in both fractions, but heavy membranes contained two to three times more protein than light membranes. The most striking difference between the two membrane fractions was revealed by their labeled surface glycoprotein patterns on polyacrylamide gels, suggesting that (i) the smooth membrane vesicles originated from the plasma membrane and (ii) two distinct segments of the plasma membrane can be recovered in fractions characterized by specific surface glycoproteins. Light membranes were enriched in Thy-1 antigen, whereas Ly-5 antigen and a 170,000-dalton surface glycoprotein were recovered almost exclusively from heavy membranes, as were metabolically labeled protein spots comigrating with the H-2k/d antigen in two-dimensional electrophoresis. The patterns of the unlabeled proteins in light and heavy membranes appeared similar, except for polypeptides of 180,000 and 85,000 daltons that were found preferentially in heavy membranes. These results support the concept of plasma membrane domains by showing that two distinct populations of plasma membrane vesicles can be isolated and that these populations contain different sets of cell surface glycoproteins.

The polypeptide profiles of strains of the Trypanosoma subgenera Schizotrypanum and Trypanozoon: peptideme characterization

Examination, by SDS-PAGE, of the polypeptide profiles of 36 Brazilian strains of Trypanosoma (Schizotrypanum) cruzi has revealed five peptidemes (subpopulations having similar polypeptide profiles) recognizing differences not apparent from zymodeme analysis. T. cruzi zymodeme (Z) 1 has been subdivided into peptidemes 3, 4 and 5 (one example of Z2 was included in peptideme 5). Peptideme 1 consisted mostly of Z2 strains except for one example each of Z1 and Z3. One strain of Z2 (Tc 003) was sufficiently dissimilar to merit separation into peptideme 2. Peptideme characterization was not possible with the Trypanozoon trypanosomes since there were no significant differences in the polypeptide profiles of 56 strains from Africa and South America; their profiles could only be compared in the molecular weight ranges 94,000 to 70,000 and below 36,000 because of distortion resulting from the variant specific surface glycoproteins. It was possible to differentiate between eight VATs of T. (Trypanozoon) brucei (BruTat 2 to 9) by isoelectric focusing of their soluble proteins.

Disappearance of a major cell-type specific surface glycoprotein antigen (SF) after transformation of fibroblasts by Rous sarcoma virus.

AbstractNormal chick fibroblasts are known to contain a tissue‐specific surface component, a major glycoprotein (SF) antigen that is also present in chicken serum. This antigen was greatly reduced in amount or absent in fibroblasts transformed by five different Rous sarcoma virus strains. Fibroblasts infected with virus mutants temperature sensitive for transformation recovered the SF antigen when maintained at the non‐permissive temperature. Productive infection with a non‐transforming avian type‐C virus did not alter the level of SF antigen characteristic of normal fibroblasts. Polyacrylamide gel electrophoresis of total cell extracts indicated that proteins with apparent mol. wt. of 145,000 and mol. wt. 210,000 were greatly decreased following transformation of the fibroblasts. That the ability to synthetize the 145,000 mol. wt. polypeptide was lost upon transformation was implied by pulse labelling experiments, in which normal fibroblasts incorporate [35S] methionine into the above polypeptide whereas transformed cells lose this ability. This polypeptide, unique in its presence in normal but not in transformed cells, appears to have an exceptionally high rate of turnover. Indirect evidence suggests that the 145,000 and 210,000 polypeptides are molecular counterparts of the SF antigen. The transformation‐associated change in the quantity of the SF molecule supports our proposal that tissue‐specific surface glycoproteins, such as the SF antigen, express the stage of differentiation at the membrane of normal cells and may be involved in intercellular control of growth and movement.