Biosynthesis Of Membrane Glycoproteins Research Articles

Loss of Specific Chaperones Involved in Membrane Glycoprotein Biosynthesis during the Maturation of Human Erythroid Progenitor Cells

The production of erythrocytes requires the massive synthesis of red cell-specific proteins including hemoglobin, cytoskeletal proteins, as well as membrane glycoproteins glycophorin A (GPA) and anion exchanger 1 (AE1). We found that during the terminal differentiation of human CD34(+) erythroid progenitor cells in culture, key components of the endoplasmic reticulum (ER) protein translocation (Sec61alpha), glycosylation (OST48), and protein folding machinery, chaperones BiP, calreticulin (CRT), and Hsp90 were maintained to allow efficient red cell glycoprotein biosynthesis. Unexpected was the loss of calnexin (CNX), an ER glycoprotein chaperone, and ERp57, a protein-disulfide isomerase, as well as a major decrease of the cytosolic chaperones, Hsc70 and Hsp70, components normally involved in membrane glycoprotein folding and quality control. AE1 can traffic to the cell surface in mouse embryonic fibroblasts completely deficient in CNX or CRT, whereas disruption of the CNX/CRT-glycoprotein interactions in human K562 cells using castanospermine did not affect the cell-surface levels of endogenous GPA or expressed AE1. These results demonstrate that CNX and ERp57 are not required for major glycoprotein biosynthesis during red cell development, in contrast to their role in glycoprotein folding and quality control in other cells.

Morphological and functional changes in the enterocyte induced by fructose

In the presence of 10-50 mM-fructose, enterocytes of organ-cultured pig intestinal-mucosal explants fail to glycosylate correctly their newly synthesized microvillar enzymes, and instead degrade them [Danielsen (1989) J. Biol. Chem. 264, 13726-13729]. In the present work, this degradation was shown to occur extremely rapidly as the microvillar enzyme aminopeptidase N (EC 3.4.11.2) was hardly detectable after a 10 min pulse with [35S]methionine. The abnormal biosynthesis of membrane glycoproteins affected both the morphology and the function of the Golgi complex as well as the microvillar membrane. Thus the stack of Golgi cisternae was condensed and devoid of dilated rims, and the secretion of a non-glycosylated protein, apolipoprotein A-1, was almost completely blocked in the presence of fructose, showing that transport through the secretory pathway is disturbed even for proteins unaffected by the defective glycosylation. The microvilli of the brush-border membrane were markedly shortened (by about 40%) in the presence of fructose, and incorporation of newly made actin into the microvillar cytoskeleton was similarly decreased. By affecting membrane glycoprotein synthesis, the common dietary sugar fructose thus profoundly perturbs the exocytic membrane traffic in the enterocyte.

Morphological analysis of surface immunoglobulin biosynthesis in mouse B lymphoid cells using immunoelectron microscopy

Immunoelectron microscopic techniques were employed to investigate the biosynthesis of surface immunoglobulin (sIg) molecules in murine splenic B lymphocytes. In addition, cryoultramicrotomy was used due to its unique ability to retain antigenicity and provide good accessibility of external immunolabeling reagents to the cellular interior. In order to induce the biosynthesis of sIg, preexisting surface molecules were first removed completely by treatment with antibody and chymotrypsin. And the cells were then allowed to regenerate the expression of sIg molecules in culture. Our results clearly indicate that cytoplasmic immunoglobulin (cIg) exists in a close association with endoplasmic reticulum, Golgi, and vesicle structures within the cytoplasm as early as 15–30 min after antibody/enzyme treatment. After approximately 60 min, intracellular cIg molecules were observed to be incorporated into the plasma membrane through the fusion of cIg containing vesicles with the surface membrane. Reexpression of sIg reaches about 50% of its original level at 6 hr and 90% of its original level at 20 hr of incubation. Both the intracellular appearance of Ig-positive structures and reexpression of sIg on the cell surface are found to be inhibited by both puromycin and tunicamycin which suggest that both protein synthesis and glycosylation are required for sIg synthesis and reexpression. To our knowledge these studies have provided the first morphological analysis of the biosynthesis of membrane glycoproteins with intact whole cells.

The Role of Phosphorylated Dolichols in Membrane Glycoprotein Biosynthesis: Relation to Cholesterol Biosynthesis

Publisher Summary The participation of certain phosphorylated long-chain polyisoprenols (dolichols) as saccharide carriers and donors in the biosynthesis of complex carbohydrates has been among the most actively pursued discoveries in the field of membrane biosynthesis and glycoprotein metabolism in the last decade. Dolichols are major long-chain polyisoprenols found in animal cells. They mediate the assembly of asparagine-linked oligosaccharide units of exported and intracellular glycoproteins. This chapter focuses on the animal cell, particularly the cultured vascular smooth muscle cell. The chapter reviews the aspects of glycoprotein structure, the participation of phosphorylated dolichols in glycoprotein biosynthesis, and the interdependent regulation of biosynthesis of phosphorylated dolichols and cholesterol. HMG-CoA reductase is an inducible enzyme that, under steady-state cultural conditions, is in the suppressed state and behaves as a classic nonequilibrium enzyme. It has a rapid turnover rate and its activity is barely detectable in vitro. In some tissues it undergoes covalent modification by phosphorylation/dephosphorylation as a means of modulating its activity. These characteristics of HMG-CoA contribute to its position as a rate-controlling enzyme in polyisoprenol biosynthesis. The increased flux of substrates through this enzyme is useful in discerning regulatory enzymatic steps secondary to the reductase and specific to the pathways of cholesterol or other polyisoprenols such as the dolichols.

Synthesis of membrane glycoproteins in rat small-intestinal villus cells. Redistribution of L-[1,5,6-3H]fucose-labelled membrane glycoproteins among Golgi, lateral basal and microvillus membranes in vivo.

The biogenesis of plasmalemma glycoproteins of rat small-intestinal villus cells was studied by following the incorporation of l-[1,5,6-(3)H]fucose, given intraperitoneally with and without chase, into Golgi, lateral basal and microvillus membranes. Each membrane fraction showed distinct kinetics of incorporation of labelled fucose and was differently affected by the chase, which produced a much greater decrease in incorporation of label into Golgi and microvillus than into lateral basal membranes. The kinetic data suggest a redistribution of newly synthesized glycoproteins from the site of fucosylation, the Golgi complex, directly into both lateral basal and microvillus membranes. The observed biphasic pattern of label incorporation into the microvillus membrane fraction may be evidence for a second indirect route of incorporation. The selective effect of the chase suggests the presence of two different pools of radioactive fucose in the Golgi complex that differ in (1) their accessibility to dilution with non-radioactive fucose, and (2) their utilization for the biosynthesis of membrane glycoproteins subsequently destined for either the microvillus or the lateral basal parts of the plasmalemma. The radioactively labelled glycoproteins of the different membrane fractions were separated by sodium dodecyl sulphate/polyacrylamide-slab-gel electrophoresis and identified by fluorography. The patterns of labelled glycoproteins in Golgi and lateral basal membranes were identical at all times. At least 14 bands could be identified shortly after radioactive-fucose injection. Most seemed to disappear at later times, although one of them, which was never observed in microvillus membranes, increased in relative intensity. All but two of the labelled glycoproteins present in the microvillus membrane corresponded to those observed in Golgi and lateral basal membranes shortly after fucose injection. The patterns of labelled glycoproteins in all membrane fractions were little affected by the chase. These data support a flow concept for the insertion of most surface-membrane glycoproteins of the intestinal villus cells.

Biosynthesis of membrane glycoproteins in rat hepatoma tissue culture cells.

The early steps in the biosynthesis of glycoproteins associated with the plasma membranes of rat hepatoma tissue culture cells has been analyzed. By measuring the effect of tunicamycin on the incorporation of [3H] mannose and [3H] fucose into cell glycoproteins, it was determined that an interval of about 1 h was required to transfer the glycoprotein from site of mannosylation to the site of fucosylation. This result was corroborated by an analysis of the time required for the appearance of either mannose or fucose-labeled glycoproteins at the cell surface. The separation of membrane glycoproteins by a two-dimensional gel system allowed the visualization of the modifications leading to both size and charge heterogeneity of these proteins. By following the changes in electrophoretic mobility introduced into membrane glycoproteins during a chase period after a pulse labeling, the time course of these molecular alterations could be estimated. Several glycoproteins have apparently higher rates of synthesis than the bulk of membrane-associated glycoproteins. Most of these glycoproteins were released within 2 h after biosynthesis from the intracellular membrane fraction and appear after 3 h in the medium. In addition to the glycoproteins that contain both mannose and fucose and that show a high degree of charge heterogeneity, there are other membrane-bound species that are not noticeably modified by the incorporation of fucose or sialic acids. These glycoproteins could represent constituents limited to the internal membrane system of the HTC cell.

Stimulation of the biosynthesis of membrane glycoproteins from zajdela ascites hepatoma cells by Robinia lectin

Membrane glycoprotein biosynthesis of ascites hepatoma cells is followed by [ 14C]glucosamine and [ 3H]leucine incorporation into cells in culture. The rate of incorporation is strongly increased by the addition of Robinia lectin in culture medium. Labeled glycoproteins are released from lectin stimulated and non-stimulated cells by trypsin digestion. Studies of labeled trypsinates on sodium dodecyl sulfate gel electrophoresis and Sephadex G-200 filtration exhibit two fractions both labeled with [ 14C]glucosamine and [ 3H]leucine and having different molecular weights, one over 200 000 and the other about 2000. Identical results are obtained when external membrane glycoproteins are solubilized by sodium deoxycholate. Comparison of surface glycoproteins isolated by trypsinization from control cells labeledwith [ 3H]-glucosamine and from lectin stimulated cells labeled with [ 14C]glucosamine displays no significant qualitative differences between glycoprotein fractions released from both cell groups.

Stimulation of the biosynthesis of membrane glycoproteins from zajdela ascites hepatoma cells by Robinia lectin

Membrane glycoprotein biosynthesis of ascites hepatoma cells is followed by [ 14C]glucosamine and [ 3H]leucine incorporation into cells in culture. The rate of incorporation is strongly increased by the addition of Robinia lectin in culture medium. Labeled glycoproteins are released from lectin stimulated and non-stimulated ceils by trypsin digestion. Studies of labeled trypsinates on sodium dodecyl sulfate gel electrophoresis and Sephadex G-200 filtration exhibit two fractions both labeled with [ 14C]glucosamine and [ 3H]leucine and having different molecular weights, one over 200 000 and the other about 2000. Identical results are obtained when external membrane glycoproteins are solubilized by sodium deoxycholate. Comparison of surface glycoproteins isolated by trypsinization from control cells labeled with [ 3H]glucosamine and from lectin stimulated cells labeled with [ 14C]glucosamine displays no significant qualitative differences between glycoprotein fractions released from both cell groups.

Biosynthesis of membrane glycoproteins in the rat small intestine

Biosynthesis of membrane glycoproteins in the rat small intestine

Permanent Mixed‐Field Polyagglutinability (PMFP)

Abstract. With the use of endless belt fluid electrophoresis, these studies confirm previous reports of two cell populations in PMFP having different mobilities. Our method shows two clearly separated bands, while with the microelectrophoresis method a distinctly bimodal distribution is reported. The abnormal (A) cells show a 33‐ to 42‐percent reduction in mobility and a 47‐ to 48‐percent reduction in sialic acid as compared with standard cells. The serologically normal (N) cell population, on mixture with standard cells and subjected to electrophoresis, exhibits no increase in streak width indicative of increased electrophoretic heterogeneity. This is consistent with the findings in the preceding report of no serological abnormalities in the (N) cells. Results of hematological studies, red cell isozyme determinations and assays of glycolytic enzymes activity were not significantly different between the two cell populations.These observations lead us to suggest that the basic abnormality in PMFP (A) cells (Tn transformation) represents a point mutation involving a single genetic step in erythrocyte membrane glycoprotein biosynthesis. A block in the transfer of terminal D‐galactose to N‐acetylgalactosamine in a substantial proportion of (A) cell heterosaccharides is postulated to be common to all of the serological and biophysical aberrations. This would be consistent with the observed reduction of T, NVG, NHmn (also MHmn), electrostatic charge, sialic acid, and increased ‘saline agglutinability’ in incomplete antisera. The defect in galactose linkage could also leave exposed underlying N‐acetylgalactosamines with the consequent manifestation of AHmn, ADB, AHP, NBV and Tn.

Permanent Mixed-Field Polyagglutinability (PMFP)

With the use of endless belt fluid electrophoresis, these studies confirm previous reports of two cell populations in PMFP having different mobilities. Our method shows two clearly separated bands, while with the microelectrophoresis method a distinctly bimodal distribution is reported. The abnormal (A) cells show a 33- to 42-percent reduction in mobility and a 47- to 48-percent reduction in sialic acid as compared with standard cells. The serologically normal (N) cell population, on mixture with standard cells and subjected to electrophoresis, exhibits no increase in streak width indicative of increased electrophoretic heterogeneity. This is consistent with the findings in the preceding report of no serological abnormalities in the (N) cells. Results of hematological studies, red cell isozyme determinations and assays of glycolytic enzymes activity were not significantly different between the two cell populations. These observations lead us to suggest that the basic abnormality in PMFP (A) cells (Tn transformation) represents a point mutation involving a single genetic step in erythrocyte membrane glycoprotein biosynthesis. A block in the transfer of terminal D-galactose to N-acetylgalactosamine in a substantial proportion of (A) cell heterosaccharides is postulated to be common to all of the serological and biophysical aberrations. This would be consistent with the observed reduction of T, N(vg), N(Hmn) (also M(Hmn)), electrostatic charge, sialic acid, and increased ‘saline agglutinability’ in incomplete antisera. The defect in galactose linkage could also leave exposed underlying N-acetylgalactosamines with the consequent manifestation of A(Hmn), A(db), A(hp), N(bv) and Tn.

Cellular membranes: The biosynthesis of glycoprotein and glycolipid in HeLa cell membranes

The biosynthesis of glycoprotein and glycolipid in HeLa cells was studied by following the distribution of glucosamine- 14C, fucose- 14C, and leucine- 3H as a function of time. The HeLa cells were useful for studying the biosynthesis of membrane components; nearly all of the glycoprotein and glycolipid synthesized by these cells is membrane-bound or intracellular. The extracellular material constituted only 1–4%. Most of the glucosamine- 14C and fucose- 14C was incorporated into glycoprotein and glycolipid found in the smooth internal and plasma membranes; by contrast, the majority of the leucine- 3H was found in the microsomal fraction and soluble protein. It was concluded that glucosamine is not attached to protein at the ribosomal or microsomal level. From chase experiments it appeared that the soluble and smooth membrane glycoprotein and glycolipid was first labeled; the labeled material then migrates progressively to the plasma membrane. A hypothesis was proposed concerning the events involved in membrane glycoprotein biosynthesis and the localization of these events within the cell. The smooth internal membranes were specified as the subcellular site for assembly of membrane glycoprotein and glycolipid. The incorporation of glycoprotein and glycolipid membrane subunits, which are eventually integrated into the substructure of the plasma membrane, was discussed.

Membrane glycoprotein biosynthesis: changes in levels of glycosyl transferases in fibroblasts transformed by oncogenic viruses.

AbstractFibroblasts transformed by oncogenic viruses were found to have greater enzymic activities of four membrane glycoprotein:glycosyl transferases on a cell or protein basis then two non‐transformed fibroblastic lines. These enzymes are responsible for the synthesis of membrane glycoproteins; each of the four transferases studied, the polypeptide:N‐acetylgalactosaminyl, glycoprotein:galactosyl, fetuin:fucosyl and PSM:fucosyl transferases, was more than twice as active in the transformed cell lines using both endogenous and added receptor. The most pronounced differences occurred with the doubly (SV‐PY‐3T3) transformed fibroblasts in all cases; with the N‐acetylgalactosaminyl and galactosyl transferases the increase was 8–16 fold over the non‐transformed cells. It was demonstrated that these results do not arise from a changed level of glycosidase activities.