Glycosylated Membrane Protein Research Articles

Freeze-fracture cytochemistry: localization of wheat-germ agglutinin and concanavalin A binding sites on freeze-fractured pancreatic cells.

The combined application of thin-section and critical-point-drying "fracture-label" is used to determine the pattern of distribution and partition of wheat-germ agglutinin and concanavalin A binding sites on the membrane faces of freeze-fractured exocrine and endocrine rat pancreatic cells. Whereas the exoplasmic face of plasma membrane is preferentially labeled by both lectins, the endoplasmic reticulum and nuclear envelope are strongly and uniformly labeled by concanavalin A but not by wheat-germ agglutinin. The results support current views in the glycosylation of membrane proteins and do not support the backflow of sialidated glycoproteins to the endoplasmic reticulum.

Nonenzymatic glycosylation of erythrocyte membrane proteins. Relevance to diabetes.

Nonenzymatic glycosylation of proteins of the erythrocyte membrane was determined by incubating erythrocyte ghosts with [3H]borohydride. The incorporation of tritium into protein provides a reliable assay of ketoamine linkages. The membrane proteins from 18 patients with diabetes incorporated twice as much radioactivity as membrane proteins from normal erythrocytes. After acid hydrolysis, amino acid analysis showed that the majority of radioactivity was localized to glucosyllysine. Autoradiograms showed that all of the major proteins of the erythrocyte membrane, separated by electrophoresis on sodium dodecyl sulfate gels, contained ketoamine linkages. No protein bands in either normal or diabetic erythrocytes showed significant preferential labeling. Erythrocyte membranes from three patients with hemolytic anemia showed reduced incorporation of tritium from [3H]-borohydride, indicating decreased nonenzymatic glycosylation. Two patients with diabetes and hemolytic anemia had incorporation of radioactivity similar to that of normal individuals. In these groups of patients the incorporation of tritium into erythrocyte membrane proteins correlated with levels of hemoglobin AIc. Thus the modification of membrane proteins like that of hemoglobin depends on blood glucose levels as well as erythrocyte age. These studies show that the enhanced nonenzymatic glycosylation of proteins in diabetics extends beyond hemoglobin to the proteins of the erythrocyte membrane and probably affects other proteins that have slow turnover and are exposed to high concentrations of glucose.

Membrane proteins specified by herpes simplex viruses. V. Identification of an Fc-binding glycoprotein

A glycoprotein with affinity for the Fc region of immunoglobulin was isolated from extracts of cultured cells infected with herpes simplex virus type 1, and experiments were done to characterize its properties and to investigate whether it could account for the Fc-binding activity previously demonstrated on the surfaces of intact herpes simplex virus-infected cells. The technique of affinity chromatography was used to identify and isolate the Fc-binding glycoprotein and to demonstrate the specificity of its interaction with immunoglobulin G-Fc. Although three electrophoretically distinguishable Fc-binding polypeptides were identified by affinity chromatography, these three species appear to be different forms of the same translation product, based on comparisons of proteolytic digestion products and on the kinetics of appearance of each form after a brief pulse with radioactive amino acids. The results suggest that one polypeptide, designated pE, is processed to yield gE1, which is in turn processed to yield gE2. Both gE1 and gE2 are glycosylated membrane proteins and both can be labeled by the lactoperoxidase-catalyzed radioiodination of intact infected cells, indicating the presence of these proteins in surface membranes of the cells. Increases in the amounts of gE1 and gE2 at the cell surface were found to parallel the increase in Fc-binding activity of intact infected cells.

A signal sequence for the insertion of a transmembrane glycoprotein. Similarities to the signals of secretory proteins in primary structure and function.

The biosynthesis of a secretory protein and a transmembrane viral glycoprotein are compared by two different experimental approaches. (a) NH2-terminal sequence analysis has been performed on various forms of the transmembrane glycoprotein of vesicular stomatitis virus synthesized in cell-free systems. The sequence data presented demonstrate that the nascent precursor of the glycoprotein contains a "signal sequence" of 16 amino acids at the NH2 terminus, whose sequence is Met-Lys-Cys-Leu-Leu-Tyr-Leu-Ala-Phe-Leu-Phe-Ile-(His-Val-Asn)-Cys. This signal sequence is proteolytically cleaved during the process of insertion into microsomal membranes prior to chain completion. The new NH2 terminus of the inserted, cleaved, and glycosylated membrane protein is located within the lumen of the microsomal vesicles and is identical to that of the authentic glycoprotein from virions. (b) Nascent chain competition experiments were performed between this glycoprotein, bovine pituitary prolactin (a secretory protein), and rabbit globin (a cytosolic protein). It was found that the nascent membrane glycoprotein, but not nascent globin, competed with nascent prolactin for membrane sites involved in the early biosynthetic event of transfer across membranes. These data suggest that an initially common pathway is involved in the biogenesis of secretory proteins and at least one class of integral membrane proteins.

Protein and lipid components of the pigeon erythrocyte membrane.

The plasma membrane of the nucleated pigeon erythrocyte was isolated by a method that is simple, reproducible and minimally disruptive, the final preparation consisting of whole cell 'ghosts', recovered at over 40% yield. Alternative methods, which yield membrane fragments, were also tested and some of their possible disadvantages demonstrated. Analysis of the protein components of the isolated membranes by gel elctrophoresis in the presence of sodium dodecyl sulphate revealed that their composition is very similar to that of the proteins of human erythrocyte membranes. However, two major proteins are unique to the nucleated cell membrane; these have apparent mol.wts. of 97000 and 57000. Also, the bands designated 4.2 (74500 mol.wt.) and 6 (35000 mol wt.) by Steck [(1974) J. Cell Biol. 62, 1-19] for the human cell membrane are absent from pigon cell membrane. Glycosylated membrane proteins could not be detected in gels stained with the periodate-Schiff-base procedure. Analysis of membrane phospholipids revealed the same components known to be present in mammalian erythrocytes, though in different proportions. These findings are discussed in the light of known physiological and biochemical differences between avian and mature mammalian erythrocytes.

Immediate glycosylation of Sindbis virus membrane proteins

The mechanisms by which the membrane proteins of Sindbis virus are initially glycosylated during growth of the virus in chick cells was studied. The experiments suggest strongly that the two viral glycoproteins are glycosylated before release from the polysome, and that this glycosylation involves transfer of a large 1800 dalton oligosaccharide to the polypeptide chains. The donor of the oligosaccharide is most probably a lipid.

Evidence for the participation of saccharide-lipids in the synthesis of the oligosaccharide chain of ovalbumin.

To obtain information on the mechanism of glycosylation of ovalbumin, three types of experiments were performed with either hen oviduct membrane preparations or tissue slices and the antibiotic tunicamycin. First, experiments involving the addition of tunicamycin to oviduct membranes demonstrated that this antibiotic inhibited the synthesis of a N-acetylglucosaminyl-lipid with the properties of N-acetyl-glucosaminylpyrophosphorylpolyisoprenol. No inhibitory effects on the other steps in the synthesis of oligosaccharide-lipid were observed. Thus, tunicamycin inhibits the lipid-linked pathway for membrane protein glycosylation by blocking the first step in the synthesis of oligosaccharide-lipid, namely, the synthesis of N-acetylglucosaminylpyrophosphorylpolyisoprenol. Second, in experiments using membranes prepared from oviduct tissue slices preincubated with tunicamycin, it was found that mannosylphosphoryldolichol was the only saccharide-lipid synthesized. This result indicates that tunicamycin administered in vivo depletes endogenous pools of N-acetylglucosamine-lipid precursors to oligosaccharide-lipid in a manner consistent with its activity in vitro. Finally, it was found that tissue slices incubated in the presence of tunicamycin synthesized the polypeptide chain of ovalbumin at almost normal rates. However, the protein newly synthesized in vivo did not contain labeled N-acetylglucosamine or mannose and had the properties of unglycosylated ovalbumin. These results indicate that saccharide-lipids participate in the assembly of the core oligosaccharide of the secretory glycoprotein, ovalbumin.

Vesicular stomatitis virus envelope glycoprotein alterations induced by host cell transformation

Vesicular stomatitis virus (VSV) contains a single structural glycoprotein in which the sugar sequences are largely host specified. We have used VSV as a probe to study the changes in cell glycoprotein metabolism induced by virus transformation. Analysis of purified VSV grown in baby hamster kidney (BHK) or polyoma transformed BHK cells showed that the virus glycoproteins have identical apparent molecular weights. The glycopeptides derived from the glycoproteins by extensive pronase digestion have an identical molecular weight distribution. On the basis of labeling experiments with fucose, mannose, and glucosamine, the oligosaccharide moieties of the VSV glycoprotein were different in virus from the two cell lines. The VSV glycopeptides from transformed cells showed an increased resistance to cleavage by an endoglycosidase, indicating structural changes in the core region of the oligosaccharides. They also showed an increased ratio of sialic acid to N-acetylglucosamine. VSV grows in a wide variety of cell types, and the carbohydrate structures of its single glycoprotein are amenable to analysis with specific glycosidases. The virus thus provides an excellent tool with which to study alterations induced by cell transformation in the glycosylation of membrane proteins.

Proteins specified by herpes simplex virus, IV. Site of glycosylation and accumulation of viral membrane proteins.

The membrane glycoproteins specified by herpes simplex virus are synthesized concurrently with structural viral proteins and accumulate in the cytoplasm and in the membranes lining it. Analyses of free and membranebound polyribosomes, the cytoplasmic pool of soluble proteins, and purified smooth membranes showed that viral membrane proteins bind to membranes soon after synthesis and become glycosylated in situ.

Proteins specified by herpes simplex virus. 3. Viruses differing in their effects on the social behavior of infected cells specify different membrane glycoproteins.

We examined the electrophoretic properties of the proteins and glycoproteins in the smooth membranes and virions purified from cells infected with herpes simplex virus strains differing with respect to their effects on the interaction of cells among themselves. The data show the following: (1) The glycoproteins in virions and binding to membranes share common features but vary quantitatively and qualitatively depending on the virus strain. (2) The binding to smooth membranes is ordered and not random. (3) Differences in the glycosylation of membrane proteins in African green monkey (VERO) and human (HEp-2) cells indicate that glycosylation is at least in part determined by the host.

Erythrocyte life span in small animals; comparison of two methods employing radioiron.

Erythrocyte life span in small animals; comparison of two methods employing radioiron.