Biosynthesis of the human asialoglycoprotein receptor

The asialoglycoprotein receptor (ASGP-R) isolated from human liver is a single polypeptide of Mr = 46,000. Monospecific polyclonal anti-human ASGP-R antibodies as well as anti-rat ASGP-R antibodies specifically inhibit binding of 125I-asialoorosomucoid to human hepatoma Hep G2 ASGP-R. These anti-ASGP-R antibodies specifically immunoprecipitate the 46,000-Da polypeptide from hepatoma cells labeled biosynthetically with 35S-amino acid. The receptor is initially synthesized as a 40,000-Da precursor which is converted to the mature 46,000-Da species with a t1/2 of approximately 45 min. The precursor species is sensitive to endo-beta-N-acetylglucosaminidase H and becomes resistant coincident with the appearance of the mature 46,000-Da receptor. In addition, the receptor synthesized in the presence of tunicamycin is approximately 34,000 Da. The newly synthesized ASGP-R reaches the cell surface after 45-60 min, where only the mature 46,000-Da species is present. In Hep G2 cells, the ASGP-R has a mean lifetime of approximately 30 h, a value which is unaltered during maximal rates of receptor-mediated endocytosis of ASGP.     

Biosynthesis and glycosylation of the human complement regulatory protein decay-accelerating factor

The biosynthesis and oligosaccharide structure of the human complement regulatory glycoprotein decay-accelerating factor (DAF) were studied in erythrocytes and cell lines. Initial information relative to carbohydrate moieties of DAF was obtained by enzymatic digestions. The 74,000 Mr erythrocyte DAF was lowered 3000 by endoglycosidase F, whereas endoglycosidase H had no effect, indicating one N-linked complex-type unit. Treatment with endo-alpha-N-acetylgalactosaminidase to remove O-linked oligosaccharides resulted in a 48,000 Mr molecule (67% of the Mr shift being due to sialic acid), which decreased to 45,000 Mr after sequential endoglycosidase F treatment. To additionally define the oligosaccharide structure and identify precursors in biosynthetic pathways, DAF was studied in the HL-60 cell line differentiated by vitamin D toward monocytes. Pulse-chase experiments with [35S]methionine revealed a precursor species of 43,000 Mr that underwent an early post-translational modification to a 46,000 Mr intermediate, and subsequently was chased into a mature species of 80,000 Mr that aligned with 125I surface-labeled DAF from these cells. All three forms of DAF were approximately 3000 lower in Mr in the presence of tunicamycin. The two lower Mr DAF species were sensitive to endoglycosidases F and H but not to neuraminidase or endo-alpha-N-acetylgalactosaminidase. In summary, DAF is synthesized as a 43,000 Mr precursor species containing one N-linked high-mannose unit. Before entering the central region of the Golgi, it is converted to a 46,000 Mr species by an as yet unknown post-translational modification. The 46,000 Mr form is converted to the 74,000 Mr (erythrocyte) or 80,000 Mr (leukocyte) membrane form of DAF by the addition of multiple, sialylated O-linked oligosaccharide chains (responsible for the large electrophoretic mobility shift) and conversion of the single N-linked high-mannose unit to a complex-type structure. The cell-specific Mr variation between red and white blood cells arises during this post-translational modification from the 46,000 Mr biosynthetic intermediate to the mature DAF species expressed on the cell surface.     

Glycosylation of the epidermal growth factor receptor in A-431 cells. The contribution of carbohydrate to receptor function

A-431 cells were treated with inhibitors of either N-linked glycosylation (tunicamycin or glucosamine) or of N-linked oligosaccharide processing (swainsonine or monensin) to examine the glycosylation of epidermal growth factor (EGF) receptors and to determine the effect of glycosylation modification on receptor function. The receptor was found to be an Mr = 130,000 polypeptide to which a relatively large amount of carbohydrate is added co-translationally in the form of N-linked oligosaccharides. Processing of these oligosaccharides accounts for the 10,000-dalton difference in electrophoretic migration between the Mr = 160,000 precursor and Mr = 170,000 mature forms of the receptor. No evidence was found for O-linked oligosaccharides on the receptor. Mr = 160,000 receptors resulting from swainsonine or monensin treatment were present on the cell surface and retained full function, as judged by 125I-EGF binding to intact cells and detergent-solubilized extracts and by in vitro phosphorylation in the absence or presence of EGF. On the other hand, when cells were treated with tunicamycin or glucosamine, ligand binding was reduced by more than 50% in either intact cells or solubilized cell extracts. The Mr = 130,000 receptors synthesized in the presence of these inhibitors were not found on the cell surface. In addition, no Mr = 130,000 phosphoprotein was detected in the in vitro phosphorylation of tunicamycin or glucosamine-treated cells. It appears, therefore, that although terminal processing of N-linked oligosaccharides is not necessary for proper translocation or function of the EGF receptor, the addition of N-linked oligosaccharides is required.     

Characterization of lipoprotein lipase activity, secretion, and degradation at different sites of post-translational processing in primary cultures of rat adipocytes.

The regulation of adipose tissue lipoprotein lipase (LPL) by feeding and fasting occurs through post-translational changes in the LPL protein. In addition, LPL activity and secretion are decreased when N-linked glycosylation is inhibited. To better understand the role of oligosaccharide processing in the development of LPL activity and in LPL secretion, primary cultures of rat adipocytes were treated with inhibitors of oligosaccharide processing. LPL catalytic activity from the heparin-releasable fraction of adipocytes was inhibited by more than 70%, with similar decreases in LPL mass, when cells were cultured for 24 h in the presence of either tunicamycin or castanospermine. On the other hand, deoxymannojirimycin (DMJ) and swainsonine had no effect on LPL activity. LPL secretion was examined after pulse-labeling cells with [35S]methionine. The appearance of 35S-labeled LPL in the medium was blocked by treatment of cells with tunicamycin and castanospermine, whereas secretion was not affected by DMJ or swainsonine. To examine the effect of oligosaccharide processing on LPL intracellular degradation, adipocytes were treated with tunicamycin, castanospermine, and DMJ and then pulse-labeled with [35S]methionine, followed by a chase with unlabeled methionine for 120 min. The unglycosylated [35S]LPL that was synthesized in the presence of tunicamycin demonstrated essentially no intracellular degradation. In the presence of castanospermine and DMJ, the half-life of newly synthesized LPL was increased to 81 and 113 min, as compared to 65 min in control cells. Thus, castanospermine-treated adipocytes demonstrated a decrease in LPL activity and secretion, suggesting that the glucosidase-mediated cleavage of terminal glucose residues from oligosaccharides is a critical step in LPL maturation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biosynthetic precursors and in vitro translation products of the glucose transporter of human hepatocarcinoma cells, human fibroblasts, and murine preadipocytes

Antisera to the human erythrocyte Glc transporter immunoblotted a polypeptide of Mr 55,000 in membranes from human hepatocarcinoma cells, Hep G2, human fibroblasts, W138, and murine preadipocytes, 3T3-L1. This antisera immunoprecipitated the erythrocyte protein which had been photoaffinity labeled with [3H]cytochalasin B, immunoblotted its tryptic fragment of Mr 19,000, and immunoblotted the deglycosylated protein as a doublet of Mr 46,000 and 38,000. This doublet reduced to a single polypeptide of Mr 38,000 after boiling. When Hep G2, W138, and 3T3-L1 cells were metabolically labeled with L-[35S]methionine for 6 h, a broad band of Mr 55,000 was immunoprecipitated from membrane extracts. In pulse-chase experiments, two bands of Mr 49,000 and 42,000 were identified as putative precursors of the mature transporter. The t1/2 for mature Glc transporter was 90 min for Hep G2 cells that had been starved for methionine (2 h) and pulsed for 15 min with L-[35S]methionine. Polypeptides of Mr 46,000 and 38,000 were immunoprecipitated from Hep G2 cells that had been metabolically labeled with L-[35S]methionine in the presence of tunicamycin. This doublet reduced to the single polypeptide of Mr 38,000 after boiling. In the absence of tunicamycin, but not in its presence, mature polypeptide of Mr 55,000 was immunoprecipitated from Hep G2 cells metabolically labeled with D-[3H]GlcN. A polypeptide of Mr 38,000 was observed in boiled immune complexes from the in vitro translation products of Hep G2, W138, and 3T3-L1 cell RNA. Dog pancreatic microsomes cotranslationally, but not posttranslationally, converted this to a polypeptide of Mr 35,000. A model for Glc transporter biogenesis is proposed in which the primary translation product of Mr 38,000 is converted by glycosylations to a polypeptide of Mr 42,000. The latter is then processed via heterogeneous complex N-linked glycosylations to form the mature Glc transporter, Mr 55,000.     

Glucosidase II, a protein of the endoplasmic reticulum with high mannose oligosaccharide chains and a rapid turnover

Glucosidase II is regarded as a resident protein of the endoplasmatic reticulum. The enzyme removes alpha-1-3-linked glucose from high mannose oligosaccharides N-linked to asparagine residues of glycoproteins. Monospecific antibodies raised against the pig kidney enzyme are used to study the metabolism of the enzyme in a rat hepatoma cell line. These antiglucosidase II antibodies specifically immune precipitate glucosidase II as a 100,000-Da species from [35S]methionine-labeled cells. In addition, protein blotting and immune staining of cell extracts from both rat liver and human and rat hepatoma cell lines show identity in apparent Mr (100,000). Glucosidase II synthesized in the presence of tunicamycin is approximately 94,000 Da, indicating the presence of one or more N-linked oligosaccharide chains. Cell-free protein synthesis of rat hepatoma total RNA demonstrates that glucosidase II is synthesized as a slightly higher molecular weight species as compared to the polypeptide synthesized in whole cells in the presence of tunicamycin, indicating that the enzyme has a cleavable signal sequence. Using a pulse-chase protocol, the apparent molecular weight does not change upon longer chase periods. In addition, the 100,000-Da protein remains sensitive to endo-beta-N-acetylglucosaminidase H regardless of prolonged chase periods. The cells incorporate [3H]mannose into the enzyme; after release with endo-beta-N-acetylglucosaminidase H, most of the radioactivity comigrates with Glc1-Man9-GlcNAc on a gel filtration column. Phase separation in Triton X-114 shows a partition between the aqueous and the Triton phase, the major portion being separated in the aqueous phase. In rat hepatoma cells glucosidase II has a half-life of 50 min. This value is not altered if the cells are grown in the presence of monensin nor of methyl-deoxynoijirimycin. However, tunicamycin and low concentrations or primaquine (raising the pH of acidic compartments) causes a 100% increase in half-life of glucosidase II. We conclude that glucosidase II is a hydrophilic, probably not a transmembrane membrane, protein with a short half-life. It is the first example of an oligosaccharide-processing enzyme not being an integral membrane protein.     

Secretion of rat hepatic lipase is blocked by inhibition of oligosaccharide processing at the stage of glucosidase I

Rat hepatic lipase is a glycoprotein bearing two N-linked oligosaccharide chains. The importance of glycosylation in the secretion of hepatic lipase was studied using freshly isolated rat hepatocytes. Various inhibitors of oligosaccharide synthesis and processing were used at concentrations that selectively interfere with protein glycosylation. Secretion of hepatic lipase activity was abolished by tunicamycin, castanospermine, and N-methyldeoxynojirimycin. No evidence was found by ELISA or Western blotting for secretion of inactive protein. Inhibition of secretion became apparent after a 30-min lag, corresponding to the time of intracellular transport of pre-existing protein. Simultaneously, intracellular hepatic lipase activity ws depleted. Secretion of hepatic lipase protein and activity was not affected by deoxymannojirimycin and swainsonine. Upon SDS-polyacrylamide gel electrophoresis, hepatic lipase secretion by deoxymannojirimycin- or swainsonine-treated cells showed an apparent Mr of 53 kDa and 55 kDa, respectively, which was distinct from hepatic lipase secreted by untreated cells (Mr = 58 kDa). We conclude that glycosylation and subsequent oligosaccharide processing play a permissive role in the secretion of hepatic lipase. As secretion is prevented by the glucosidase inhibitors castanospermine and N-methyldeoxynojirimycin, but not by inhibitors of subsequent oligosaccharide trimming, the removal of glucose residues from the high-mannose oligosaccharide intermediate in the rough endoplasmic reticulum appears the determining step.     

The effect of inhibitors of glycoprotein synthesis and processing on the phagocytosis of rod outer segments by cultured retinal pigment epithelial cells

Retinal pigment epithelial cells selectively phagocytize rod outer segments by a process that may be mediated by specific cell surface receptors. Since many receptors are glycoproteins, we have studied the effect of tunicamycin, an inhibitor of N-linked oligosaccharide synthesis, and of castanospermine and swainsonine, which are inhibitors of oligosaccharide processing, on the ability of cultured retinal pigment epithelial cells to phagocytize rod outer segment. Tunicamycin inhibits the glycosylation of newly synthesized glycoproteins by 85-90%; concomitantly, the phagocytosis of rod outer segments is inhibited by 70-80%. The effect of tunicamycin is to initially reduce rod outer segments binding, and therefore the subsequent ingestion of rod outer segments. SDS-PAGE analysis and autoradiography of [35S]methionine labelled extracts of tunicamycin-treated cells, demonstrates the disappearance of a number of glycoprotein bands, and the appearance of a number of protein bands of lower Mr. Kinetic analysis of the disappearance and reappearance of specific glycoproteins suggests that the lower Mr bands are the non-glycosylated forms of the higher Mr bands. By contrast, castanospermine and swainsonine have no effect on the ability of retinal pigment epithelial cells to phagocytize rod outer segments, or on the SDS-PAGE pattern of treated cells, although they were shown to inhibit oligosaccharide processing as expected. These results support the hypothesis that rod outer segment phagocytosis by retinal pigment epithelial cells is mediated by specific glycoprotein receptors. N-Glycosylation of these receptors is required for their function, or for their insertion into the plasma membrane, whereas processing of the N-linked oligosaccharide chains of these receptors is not crucial for rod outer segment phagocytosis by retinal pigment epithelial cells.     

Isolation and amino terminal sequencing of a novel melanoma-associated antigen

The biochemical characteristics are described for a melanoma-associated glycoprotein antigen, whose expression depends on stage of tumor progression and melanocytic differentiation. This antigen, identified using a monoclonal antibody which specifically stains melanoma cells in immunoperoxidase assay of fixed tissue sections, is synthesized as a 30,000-Da precursor and then processed to a 30,000- to 60,000-Da sialylated glycoprotein. Two-dimensional gel electrophoresis of the antigen resolved more than 20 forms, heterogeneous in both charge and molecular weight. The kinetics of post-translational processing, the sensitivity of processing to tunicamycin, and the molecular weight of the oligosaccharide chains indicate that the oligosaccharides are N-linked. Amino acid sequencing of the antigen purified by immunoaffinity chromatography and by high-pressure liquid chromatography or polyacrylamide gel electrophoresis allowed the assignment of the first 20 acids.     

Effects of tunicamycin on the expression and function of formyl peptide chemotactic receptors of differentiated HL-60 cells

We previously showed that formyl peptide chemotactic receptors (FPCR) of human phagocytic cells contain at least two asparagine-linked oligosaccharide chains located at the distal end of the receptor. The requirement of these N-linked oligosaccharide chains for expression and function of FPCR was investigated in HL-60 cells induced to differentiate by N6,O2-dibutyryladenosine 3',5'-monophosphate (Bt2cAMP) in the presence or absence of 5 micrograms/ml tunicamycin. Tunicamycin did not prevent the changes in morphology associated with Bt2cAMP-induced differentiation of HL-60 cells. Autoradiographic analysis after SDS-PAGE of FPCR affinity labeled with N-formyl-Nle-Leu-Phe-Nle-[125I]iodo-Tyr-Lys (formyl 125I-hexapeptide) and ethylene glycol bis(succinimidyl succinate) demonstrated that greater than 95% of FPCR expressed by tunicamycin-treated cells completely lacked N-linked oligosaccharide (Mr 32,000), and no fully glycosylated FPCR (Mr 62,000 to 85,000) was detectable. Scatchard analysis of formyl 125I-hexapeptide binding indicated the presence of two classes of binding sites for both control and tunicamycin-treated cells (control cells, 82,000 +/- 32,000 sites/cell with Kd 10.0 +/- 4.3 nM and 520,000 +/- 40,000 sites/cell with Kd 250 +/- 80 nM; tunicamycin-treated cells, 11,000 +/- 5000 sites/cell with Kd 3.0 +/- 1.9 nM and 470,000 +/- 70,000 sites/cell with Kd of 500 +/- 140 nM). Both control and tunicamycin-treated cells augmented superoxide anion release, exhibited a migratory response, and showed a transient rise in intracellular free Ca2+ upon stimulation with N-formyl-Nle-Leu-Phe. However, the responses of the tunicamycin-treated cells were less than that of the control cells. The present studies demonstrate that N-glycosylation of FPCR is not essential for cell surface expression or for several FPCR-mediated cell responses.     

Glycosylation is not needed for the intracellular transport of phytohemagglutinin in developing Phaseolus vulgaris cotyledons and for the maintenance of its biological activities

Approximately 10% of the total protein contained in Phaseolus vulgaris L. cv. Greensleeves seeds is composed of the glycoprotein lectin, phytohemagglutinin. We have investigated whether the presence of N-linked oligosaccharide side chains is a prerequisite for the correct intracellular transport of this protein and whether unglycosylated phytohemagglutinin maintains its biological activities. Excised developing cotyledons were incubated in the presence of tunicamycin to prevent glycosylation "in vivo", and the fate of the unglycosylated protein synthesized in such cotyledons determined. It was found that unglycosylated phytohemagglutinin reaches its normal site of accumulation, the protein bodies, and maintains erythro-agglutinating and mitogenic activities.     

Tunicamycin inhibits proteoglycan synthesis in rat ovarian granulosa cells in culture

The effects of tunicamycin, an inhibitor of N-linked oligosaccharide biosynthesis, on the synthesis and turnover of proteoglycans were investigated in rat ovarian granulosa cell cultures. The synthesis of proteoglycans was inhibited (40% of the control at 1.6 micrograms/ml tunicamycin) disproportionately to that of general protein synthesis measured by [3H]serine incorporation (80% of control). Proteoglycans synthesized in the presence of tunicamycin lacked N-linked oligosaccharides but contained apparently normal O-linked oligosaccharides. The dermatan sulfate and heparan sulfate chains of the proteoglycans had the same hydrodynamic size as control when analyzed by Sepharose 6B chromatography. However, the disulfated disaccharide content of the dermatan sulfate chains was reduced by tunicamycin in a dose-dependent manner, implying that the N-linked oligosaccharides may be involved in the function of a sulfotransferase which is responsible for sulfation of the iduronic acid residues. When [35S]sulfate and [3H]glucosamine were used as labeling precursors, the ratio of 35S/3H in chondroitin 4-sulfate was reduced to approximately 50% of the control by tunicamycin, indicating that the drug reduced the supply of endogenous sugar to the UDP-N-acetylhexosamine pool. Neither transport of proteoglycans from Golgi to the cell surface nor their turnover from the cell surface (release into the medium, or internalization and subsequent intracellular degradation) was affected by the drug. Addition of mannose 6-phosphate to the culture medium did not alter the proteoglycan turnover. When granulosa cells were treated with cycloheximide, completion of proteoglycan diminished with a t1/2 of approximately 12 min, indicating the time required for depleting the core protein precursor pool. The glycosaminoglycan synthesizing capacity measured by the addition of p-nitrophenyl-beta-xyloside, however, lasted longer (t1/2 of approximately 40 min). Tunicamycin decreased the core protein precursor pool size in parallel to decreased proteoglycan synthesis, both of which were significantly greater than the inhibition of general protein synthesis. This suggests two possibilities: tunicamycin specifically inhibited the synthesis of proteoglycan core protein, or more likely a proportion of the synthesized core protein precursor (approximately 50%) did not become accessible for post-translational modifications, and was possibly routed for premature degradation.     

Post-translational acquisition of insulin binding activity by the insulin proreceptor. Correlation to recognition by autoimmune antibody

The post-translational acquisition of ligand binding activity by the insulin receptor was examined in 3T3-L1 adipocytes. In pulse-chase experiments with [35S] methionine, labeled receptor species were separated into "active" and "inactive" forms by affinity chromatography on insulin-agarose and then were characterized and quantitated. It was found that the newly translated high molecular weight proreceptor lacks the capacity to bind insulin. The acquisition of binding activity is relatively slow (t1/2 = 45 min) and occurs prior to conversion of the proreceptor to the mature alpha- and beta-subunits by proteolytic cleavage and maturation of its N-linked oligosaccharide chains (t1/2 = 3 h). Glycosylation appears to be required for this activation since the aglycoproreceptor, synthesized in the presence of tunicamycin, does not acquire insulin binding activity. However, once the proreceptor has acquired ligand binding activity, removal of its N-linked oligosaccharide chains with endoglycosidase H has no effect on the ability of the proreceptor to bind insulin. The modification of the proreceptor to bind insulin. The modification of the proreceptor that gives rise to insulin binding activity most likely involves a conformational change in the binding domain. A human autoimmune antibody that recognizes only the active insulin binding site does not interact with the inactive proreceptor, whereas a rabbit polyclonal antireceptor antibody recognizes all forms. Thus, the autoimmune antibody must recognize a new epitope created during conversion of the inactive proreceptor to the active form.     

Biosynthesis and glycosylation of the epidermal growth factor receptor in human tumor-derived cell lines A431 and Hep 3B.

Biosynthesis of the receptor for epidermal growth factor was investigated in two human tumor-derived cell lines, Hep 3B and A431. When grown in the presence of tunicamycin, both cells expressed a receptor-related species p135, the presumptive aglycosylated form of the biosynthetic precursor, gp145, of the mature form of the receptor, gp165, expressed at the cell surface. Two additional receptor-related species, p115 and p70, were detected when A431, but not Hep 3B, cells were treated with tunicamycin. Furthermore, digestion of the A431 receptor-related proteins with endoglycosidase F resulted in the detection of these three aglycosylated species. P70 appears to be the aglycosylated form of gp95, the presumptive intracellular precursor of the receptor-related species gp120 that is secreted by A431 but not Hep 3B cells; gp120 has a complex pattern of N-linked glycosylation, with consequent molecular weight and charge heterogeneity. P115 may be the aglycosylated form of a third biosynthetic intermediate, possibly a gp135 species detected in the early time points of pulse-chase labeling. Alternatively, p115 and gp135 may be derived co- or post-translationally by Ca2+-mediated proteolysis from p135 and gp145, respectively. The implications of the complexity of the biosynthesis of this molecule with regard to the multiple opportunities it affords the cell to modulate cell proliferation are discussed.     

The effects of processing inhibitors of N-linked oligosaccharides on the intracellular migration of glycoprotein E2 of mouse hepatitis virus and the maturation of coronavirus particles

We have studied the effects of tunicamycin and inhibitors of the processing of N-linked glycans including N-methyl-1-deoxynojirimycin, castanospermine, mannodeoxynojirimycin, and swainsonine on the transport of glycoprotein E2 and the intracellular maturation of the coronavirus mouse hepatitis virus A59. Indirect immunofluorescence staining with monoclonal antibodies revealed that glycoprotein E2 exhibits different antigenic properties depending on the presence and on the structure of the N-linked oligosaccharides and that efficient transport of glycoprotein E2 to the plasma membrane requires the removal of glucose residues. In the presence of tunicamycin in the nonglycosylated E2 apoprotein was synthesized in normal amounts and readily acylated throughout the infectious cycle. This E2-species could not be detected on the surface of mouse hepatitis virus A59-infected cells with indirect immunofluorescence staining or lactoperoxidase labeling. N-Methyl-1-deoxynojirimycin and castanospermine, both of which selectively inhibited the processing glucosidases, caused a drop in virion formation by two log steps and a drastic delay in the surface expression of glycoprotein E2. The E2 species synthesized under such conditions was acylated but accumulated intracellularly in a compartment distinct from the Golgi. Concomitantly, synthesis of the matrix glycoprotein E1 of mouse hepatitis virus A59 was drastically impaired. Mannodeoxynojirimycin and swainsonine, which block later stages of the processing pathway, had less or no effect on the transport of glycoprotein E2 and the formation of virus particles.     

The effect of swainsonine and castanospermine on the sulfation of the oligosaccharide chains of N-linked glycoproteins

MDCK (Madin-Darby canine kidney) cells infected with the NWS strain of influenza virus incorporate 35SO4 into complex types of oligosaccharides of the N-linked glycoproteins. On the other hand, when these virus-infected MDCK cells are incubated in the presence of swainsonine, an inhibitor of the processing mannosidase II, approximately 40-80% of the total [35S]glycopeptides were of the hybrid types of structures. Thus, these sulfated, hybrid types of glycopeptides were completely susceptible to digestion by endoglucosaminidase H, whereas the sulfated glycopeptides from infected cells incubated without swainsonine were completely resistant to endo-beta-N-acetylglucosaminidase H. When virus-infected MDCK cells were incubated in the presence of castanospermine, an inhibitor of the processing glucosidase I, the N-linked glycopeptides contained mostly oligosaccharide chains of the Glc3Man7-9GlcNAc2 types of structures, and these oligosaccharides were devoid of sulfate. Structural analysis of these abnormally processed oligosaccharides produced in the presence of swainsonine or castanospermine indicated that they differed principally in the processing of one oligosaccharide branch as indicated by the structures shown below. They also differed in that only the swainsonine-induced structures were sulfated. These data indicate that removal of glucose units and perhaps other processing steps are necessary before sulfate residues can be added. (Formula: see text).     

Nonionic detergents increase the stoichiometry of ligand binding to the rat hepatic galactosyl receptor

When digitonin is used to expose intracellular galactosyl (Gal) receptors in isolated rat hepatocytes, only about half of the binding activity for 125I-asialoorosomucoid (ASOR) is found as compared to cells solubilized with Triton X-100. The increased ligand binding in the presence of detergent is not due to a decrease in Kd but could be due either to an increase in the number of ASORs bound per receptor or to exposure of additional receptors. Several experiments support the former explanation. No additional activity is exposed even when 80% of the total cell protein is solubilized with 0.4% digitonin. It is, therefore, unlikely that receptors are in intracellular compartments not permeabilized by digitonin and inaccessible to 125I-ASOR. Digitonin-treated cells are not solubilized by Triton X-100 if they are first treated with glutaraldehyde under conditions that retain specific binding activity. 125I-ASOR binding to these permeabilized/fixed cells increases about 2-fold in the presence of Triton X-100 and a variety of other detergents (e.g., Triton X-114, Nonidet P-40, Brij-58, and octyl glucoside) but not with the Tween series, saponin, or other detergents. When these fixed cells are washed to remove detergent, 125I-ASOR binding decreases almost to the initial level. Affinity-purified Gal receptor linked to Sepharose 4B binds approximately twice as much 125I-ASOR in the presence of Triton X-100 as in its absence. The results suggest that the increase in Gal receptor activity in the presence of nonionic detergents is due to an increase in the valency of the receptor rather than to exposure of additional receptors.     

Antibody-induced receptor loss. Different fates for asialoglycoproteins and the asialoglycoprotein receptor in HepG2 cells

The human asialoglycoprotein receptor (ASGP-R) is a membrane glycoprotein which participates in receptor-mediated endocytosis and delivery of its ligands to lysosomes for degradation. In order to examine the pathways and mechanisms responsible for the turnover and degradation of the ASGP-R we have followed the fate of the ASGP-R in HepG2 cells during exposure to anti-receptor antibody as well as inhibitors of lysosomal processing and receptor recycling. Incubation of cells at 37 degrees C with anti-ASGP-R antibody results in the rapid (t 1/2 30 min) loss of mature 46,000-Da ASGP-R (control, t 1/2 20 h). This process requires whole IgG, since Fab fragments do not induce loss of receptor. Furthermore, this antibody-induced loss is specific, since incubation with antibody to the transferrin receptor does not alter cellular ASGP-R content. Of note, weak bases (e.g. primaquine) abrogate this antibody-induced loss of ASGP-R. Inhibitors of lysosomal proteases (EC64 and leupeptin) do not alter this antibody-mediated loss. Furthermore, this effect occurs at 18 degrees C, a temperature at which delivery of ligand to the lysosome is blocked. Thus, the present observations suggest a unique pathway for antibody-induced ASGP-R loss which is distinct from the pathway of lysosomal delivery of ligand.     

Biosynthesis and secretion of M- and Z-type alpha 1-proteinase inhibitor by human monocytes. Effect of inhibitors of glycosylation and of oligosaccharide processing on secretion and function

The biosynthesis and secretion of M-type and Z-type alpha 1-antitrypsin was studied in human monocytes. In monocytes of PiMM individuals alpha 1-antitrypsin represented 0.08% of the newly synthesized proteins and 0.44% of the secreted proteins. Two molecular forms of alpha 1-antitrypsin could be identified: a 51-kDa intracellular form, susceptible to endoglucosaminidase H, thus representing the high-mannose type precursor form and a 56-kDa form resistant to endoglucosaminidase H which was secreted into the medium. Inhibition of de novo glycosylation by tunicamycin impaired the secretion of M-type alpha 1-antitrypsin by about 75% whereas inhibition of oligosaccharide processing by the mannosidase II inhibitor swainsonine did not alter the secretion of M-type alpha 1-antitrypsin. alpha 1-Antitrypsin secreted by human monocytes was functionally active as measured by complex formation with porcine pancreatic elastase. Even unglycosylated alpha 1-antitrypsin secreted by human monocytes treated with tunicamycin formed a complex with elastase. In monocytes of PiZZ individuals the secretion of alpha 1-antitrypsin was decreased. 72% of newly synthesized M-type alpha 1-antitrypsin, but only 35% of newly synthesized Z-type alpha 1-antitrypsin were secreted during a labeling period of 3 h with [35S]methionine. The 51-kDa form of Z-type alpha 1-antitrypsin accumulated intracellularly, whereas the 56-kDa form was secreted. Inhibition of oligosaccharide processing by swainsonine did not alter the decreased secretion of Z-type alpha 1-antitrypsin, whereas inhibition of de novo glycosylation by tunicamycin blocked the secretion of Z-type alpha 1-antitrypsin completely.     

Biosynthesis of proteodermatan sulfate in cultured human fibroblasts

Biosynthesis and secretion of proteodermatan sulfate produced by cultured human skin fibroblasts were investigated employing immunological procedures. During an incubation period of 10 min in the presence of [3H]leucine, two core protein forms of Mr = 46,000 and 44,000, respectively, were synthesized. They were converted to mature proteodermatan sulfate with a half-time of approximately 12 min. Fifty per cent of total mature proteodermatan sulfate were found in the culture medium after a 35-min chase. Six to eight per cent remained associated with the cell layer after a chase of 6 h. In the presence of tunicamycin, fibroblasts synthesized a single core protein of Mr = 38,000 that was converted to mature proteodermatan sulfate and secreted with similar kinetics as the N-glycosylated species. Subtle differences in the molecular size of core proteins were noted when cell-associated and secreted proteodermatan sulfate were degraded with chondroitin ABC lyase, but core proteins free of N-linked oligosaccharides were identical. Labeling with [3H]mannose revealed that secreted proteodermatan sulfate contains two or three complex-type or two complex-type and one high-mannose-type N-linked oligosaccharide chains. The N-glycans are bound to a 21-kDa fragment of the core protein. After incubation in the presence of [3H]glucosamine, the [3H]galactosamine/[3H]glucosamine ratio was 3.76 and 3.30 for secreted and cell-associated proteodermatan sulfate, respectively. Evidence for the presence of O-linked oligosaccharides could not be obtained. Small amounts of core protein free of dermatan sulfate chains were secreted when the cultures were treated with p-nitrophenyl-beta-D-xyloside.