The oligosaccharide component of alpha 1-adrenergic receptors from BC3H1 and DDT1 muscle cells. Studies with glycosidases and photoaffinity labelling of intact cells.

In this study, we clarify the structural aspects of the oligosaccharides associated with the alpha 1-adrenergic receptor in two muscle cell lines. Photoaffinity labelling of intact BC3H1 or DDT1 muscle cells with 2-[4-(4-azido-3-[125I]iodobenzoyl)piperazin-1-yl]-4-amino-6, 7-dimethoxyquinazoline ([125I]azidoprazosin) followed by SDS/polyacrylamide-gel electrophoresis (PAGE) and autoradiography revealed specifically labelled proteins of molecular mass = 87,000 and 81,000, respectively. Treatment of photoaffinity-labelled receptors in DDT1 cells with 33 u. of endoglycosidase F/ml for 24 h resulted in the loss of the 81 kDa receptor and the appearance of a 52.5 kDa protein. When lower concentrations of glycosidase or shorter incubation times were used, the 81 kDa receptor was converted to a 66 kDa protein. Treatment of the photoaffinity-labelled BC3H1 receptor with endoglycosidase F resulted in the appearance of a 50.5 kDa protein. Neither alpha-mannosidase nor endoglycosidase H had an effect on the photoaffinity labelling patterns of the receptor from the two cell types. alpha 1-Adrenergic receptors, solubilized from membranes prepared from BC3H1 and DDT1 cells, bound to wheat germ agglutinin-Sepharose and were displaced by N-acetylglucosamine. Taken together, these results indicate that alpha 1-adrenergic receptors in BC3H1 and DDT1 cells contain complex, but not high, mannose oligosaccharide chains; differences in the composition or number of chains partially accounts for the different molecular mass of the receptor in the two cell lines. The results further indicate that the oligosaccharide chains contribute substantially to the apparent molecular mass of alpha 1-adrenergic receptors, as detected by SDS/PAGE, and that the protein backbone of these receptors is likely to be approximately 50 kDa.     

The cell surface receptor for immunoglobulin E. Effect of tunicamycin on molecular properties of receptor from rat basophilic leukemia cells

Cell surface receptors for immunoglobulin E were isolated by repetitive affinity chromatography from rat basophilic leukemia cells biosynthetically labeled with L-[35S]methionine and D-[3H]mannose. Native immunoglobulin E receptor appeared as a very broad band in the 45,000 to 62,000 Mr region in sodium dodecyl sulfate polyacrylamide gels. However, from cells cultured in the presence of tunicamycin, a relatively narrow band with an apparent Mr of 38,000 was isolated. The 38,000 Mr band rebound to immunoglobulin E-Sepharose, was immunoprecipitated with antibodies to immunoglobulin E receptor, shared tryptic peptides with native receptor, and was labeled with L-[35S]methionine but not D-[3H]mannose, and thus appears to be immunoglobulin E receptor lacking N-linked oligosaccharides. It is demonstrated that N-linked oligosaccharides account for much of the apparent heterogeneity of native receptor in sodium dodecyl sulfate polyacrylamide gels and in two-dimensional gel electrophoresis. A receptor-associated protein with apparent Mr = 30,000, prominently labeled with L-[35S]methionine but not with D-[3H]mannose, did not have altered molecular properties when isolated from tunicamycin-cultured cells, and did not share tryptic peptides with receptor.     

Biosynthesis and glycosylation of the carcinoembryonic antigen.

Human gastric adenocarcinoma MKN-45 cells were found to synthesize actively carcinoembryonic antigen (CEA). The biosynthesis and carbohydrate processing of CEA were studied in these cells by means of metabolic labelling followed by immunoadsorption with a specific polyclonal-antibody preparation and gel electrophoresis. Pulse-chase studies with [14C]leucine and [3H]mannose (shortest pulse 3 min) showed that N-linked oligosaccharide side chains are added to the protein co-translationally, producing a high-mannose immature CEA; the average molecular mass of this form is 145 kDa. The protein is later translocated to the Golgi apparatus and here undergoes additional processing; these modifications are visible in our system as a broadening of the CEA band and require about 4 h. The upper limit of mature CEA band reaches 200 kDa, but radioactivity is maximally incorporated at 168 kDa. The extent of co-translational glycosylation was measured by treating the cells with tunicamycin; in the presence of this inhibitor, a 74 kDa aglyco-CEA was produced and was still recognized by the antibody. Monensin, an ionophore which interferes with glycoprotein maturation and terminal sugar addition, blocked broadening of the CEA band, producing a sharp 141 kDa peak. In conclusion, CEA appears to be synthesized as a 145 kDa high-mannose immature form, the protein core accounting for about half of its molecular mass. Full maturation results in a broad band at 168 kDa.     

46-kDa mannose 6-phosphate-specific receptor: biosynthesis, processing, subcellular location and topology

Synthesis of the cation-dependent mannose 6-phosphate-specific receptor was followed in cells of human (fibroblasts, Hep G2 cells, U937 monocytes, blood-derived macrophages) or rat (Morris hepatoma 7777 cells) origin. The mature form of the receptor has an apparent molecular size of 46 kDa except in fibroblasts, where the apparent molecular size was 43 kDa. The receptor contains 7-8 N-linked oligosaccharide chains, about 5 of which are converted into endo H-resistant forms within 2 h of synthesis. A small fraction of the receptor (about 3% of total in U937 monocytes) is located at the cell surface while the bulk of the receptor resides in internal membranes. Part of the internal receptors (20% in fibroblasts) resides in membranes of the endocytic pathway. The receptor was not detectable in dense lysosomes. The receptor is a hydrophobic transmembrane protein partitioning with Triton X-114. The cytosolic portion of the receptor comprises a molecular size of about 5 kDa and contains the C-terminus. The luminal (or external) portion of the receptor comprises a molecular size of greater than or equal to 37.5 kDa, of which more than half is represented by carbohydrate. Cross-linking experiments suggest that the mature receptor exists in membranes as a dimer.     

The oligosaccharide moieties of the epidermal growth factor receptor in A-431 cells. Presence of complex-type N-linked chains that contain terminal N-acetylgalactosamine residues

The receptor for epidermal growth factor (EGF) in the human epidermoid carcinoma cell line A-431 is a glycoprotein of apparent molecular weight = 170,000. During biosynthesis, the receptor is first detected as a precursor of apparent Mr = 160,000. In this report we describe our studies on the structures of the oligosaccharide moieties of the mature receptor and its precursor. A-431 cells were grown in medium containing radioactive sugars and the radiolabeled receptors were purified by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radiolabeled glycopeptides were prepared from the purified receptor by proteolysis, and their structures were examined by a variety of techniques. The mature EGF receptor contains both complex-type and high mannose-type Asn-linked oligosaccharides in the approximate ratio of 2 to 1, while the precursor contains only high mannose-type chains. A number of experimental results demonstrate that the mature receptor does not contain oligosaccharides in O-linkage through N-acetylgalactosamine to either serine or threonine. The high mannose-type oligosaccharides in both precursor and mature receptor can be cleaved by endo-beta-N-acetylglucosaminidase H and occur in the mature receptor as Man9GlcNAc2 (6%), Man8GlcNAc2 (49%), Man7GlcNAc2 (25%), and Man6GlcNAc2 (20%), whereas, in the receptor precursor the high mannose chains occur primarily as Man8GlcNAc2 (70%). The complex-type oligosaccharides in the mature receptor are predominantly tri- or tetraantennary species and are unusual in several respects. (i) Many of the chains do not contain sialic acid, while the remaining chains contain 1-2 sialic acid residues. (ii) Half of the [3H] mannose-derived radioactivity was recovered as [3H] fucose and the remaining half as [3H] mannose, indicating that there may be an average of 3 fucose residues/chain. (iii) About one-third of the [3H] glucosamine-derived radioactivity in these glycopeptides was recovered as N-acetylgalactosamine and these residues are all alpha-linked and occur at the nonreducing termini. These data demonstrate that the complex-type Asn-linked oligosaccharides in the EGF receptor from A-431 cells contain sugar residues related to human blood type A. In light of other recent studies, these results suggest that in A-431 cells blood group determinants in surface glycoproteins are contained in Asn-linked but not O-linked oligosaccharides.     

Regulation of low density lipoprotein receptor synthesis in cultured luteinized human granulosa cells by human chorionic gonadotropin and 8-bromo-cyclic AMP

We investigated the regulation of synthesis of low density lipoprotein (LDL) receptor in cultured luteinized human granulosa cells using a monoclonal antibody recognizing the human LDL receptor (IgG-C7). Cells cultured under serum-free conditions were treated with human chorionic gonadotropin (hCG) or 8-bromo-cAMP alone or in combination with aminoglutethimide (to block conversion of cholesterol to steroid hormones) and 5-cholesten-3 beta, 25-diol (25-hydroxycholesterol, a potent suppressor of LDL receptor expression in human fibroblasts) and pulse-labeled with [35S]methionine. A labeled protein immunoisolated with IgG-C7 was identified as the mature LDL receptor in 7.5% sodium dodecyl sulfate-polyacrylamide gels on the basis of an apparent molecular mass of 160 kDa, absence of the protein from immunoisolates prepared with a monoclonal antibody against an irrelevant antigen, and an apparent decrease in molecular weight of the mature receptor upon treatment with neuraminidase or electrophoresis under nonreducing conditions. hCG and 8-bromo-cAMP consistently increased the incorporation of radioactivity into the mature LDL receptor by 2-6-fold. The effect of hCG on LDL receptor synthesis was observed with as little as 10 mIU of hCG/ml and was apparent within 2 h of addition of the hormone. A combination of 25-hydroxycholesterol and aminoglutethimide resulted in a 60% suppression of label incorporation into mature LDL receptor compared to untreated cells. This would suggest some regulation of LDL receptor synthesis by negative feedback of sterol. However, both hCG and 8-bromo-cAMP increased label incorporation into the LDL receptor in the face of these agents. We conclude that in human granulosa cells, hCG, through the intermediacy of cAMP, rapidly increases LDL receptor synthesis by a mechanism which is, at least in part, independent of alterations in cellular cholesterol balance.     

The bovine mannose 6-phosphate/insulin-like growth factor II receptor. Localization of mannose 6-phosphate binding sites to domains 1-3 and 7-11 of the extracytoplasmic region.

The extracytoplasmic region of the 270-kDa mannose 6-phosphate/IGF-II receptor is composed of 15 repeating domains and is capable of binding 2 mol of mannose 6-phosphate (Man-6-P). To localize the Man-6-P binding domains, bovine receptor was subjected to partial proteolysis with subtilisin followed by affinity chromatography on pentamannosyl phosphate-agarose. Eleven proteolytic fragments ranging in apparent molecular mass from 53 to 206 kDa were isolated. Sequence analysis of six of the fragments localized their amino termini to either the beginning of domain 1 at the amino terminus of the molecule or the beginning of domain 7, according to the alignment of Lobel et al. (Lobel, P., Dahms, N. M., and Kornfeld, S. (1988) J. Biol. Chem. 263, 2563-2570). The smallest fragment, with an apparent molecular mass of 53 kDa, is predicted to encompass domains 1-3. Another fragment, with an apparent molecular mass of 82 kDa, is predicted to encompass domains 7-10 or 7-11. The Man-6-P binding site contained within domains 1-3 was further defined by expressing truncated forms of the receptor in Xenopus laevis oocytes and assaying their ability to bind phosphomannosyl residues. A soluble polypeptide containing domains 1-3 exhibited binding activity, whereas a polypeptide containing domains 1 and 2 did not. This indicates that domain 3 is a necessary component of one of the Man-6-P binding sites of the receptor.     

Prostaglandin E1 receptor from mouse mastocytoma P-815 cells couples to 60 kDa GTP-binding protein.

The stable [3H]prostaglandin E1 (PGE1)-bound receptor, which couples to 60 kDa GTP-binding protein, from membranes of mouse mastocytoma P-815 cells has been purified and characterized. When the membranes were preincubated with [3H]PGE1 for 60 min at 37 degrees C, the dissociation of the ligand from the receptor was remarkably decreased, even in the presence of GTP gamma S. The stable [3H]PGE1-bound receptor complex was solubilized with 6% digitonin. The solubilized [3H]PGE1 receptor was eluted with [35S]GTP gamma S bindings activity from an Ultrogel AcA44 column. The fractions containing activities of both [3H]PGE1 and [35S]GTP gamma S bindings were further purified by column chromatographies on wheat germ agglutinin (WGA)-agarose and phenyl-Sepharose CL-4B. The partially purified [3H]PGE1-bound receptor was affinity-labeled with [14C]5'-p-fluorosulfonylbenzoylguanosine and a protein with a molecular mass of 60 kDa was detected. These results suggest that the ligand-bound PGE1 receptor of P-815 cells associates with a novel GTP-binding protein with a molecular mass of 60 kDa.     

Synthesis and processing of arylsulfatase A in human skin fibroblasts

Biosynthesis of arylsulfatase A in normal and mutant human fibroblasts was studied by growing cells in the presence of L-[4,5-3H] leucine or [2-3H] mannose, isolation of labelled arylsulfatase A by immune precipitation and visualization of electrophoretically separated polypeptide by fluorography. Arylsulfatase A was synthesized as a precursor with a mean apparent molecular mass of 62 kDa. Intracellularly the precursor was converted into a 60.5 kDa polypeptide within a chase period of 1 to 7 days. The 60.5 kDa product in polyacrylamide corresponded to one of two polypeptides present in arylsulfatase A isolated from human placenta. In fibroblasts from a patient with metachromatic leukodystrophy no immune precipitable polypeptides of arylsulfatase A were detected. In normal fibroblasts less than 10% of the precursor of arylsulfatase A was secreted into the medium, whereas in mucolipidosis II fibroblasts and in control fibroblasts grown in the presence of NH4Cl up to 90% of the precursor of arylsulfatase A, appeared in the medium and remained there without change in the apparent molecular mass for at least 7 days. Arylsulfatase A polypeptides appear to contain two carbohydrate side chains. In about 90% of the polypeptides both side chains are cleaved by endo-beta-N-acetylglucosaminidase H, whereas in the remaining chains one of the two oligosaccharides is not cleaved.     

Direct evidence for intra- and intermolecular disulfide bond formation in the human glucocorticoid receptor. Inhibition of DNA binding and identification of a new receptor-associated protein

We have investigated the potential for the steroid affinity-labeled human glucocorticoid receptor to form both intramolecular and intermolecular disulfide bonds. Glucocorticoid receptors labeled in intact HeLa S3 cells with the covalent affinity label [3H]dexamethasone mesylate ([3H]DM) were analyzed on denaturing 5-12% polyacrylamide gels under both nonreducing and reducing conditions. Under nonreducing conditions the affinity-labeled receptor migrated as a heterogeneous species having an average molecular mass of approximately 96 kDa whereas, under reducing conditions, the receptor migrated as a more discrete form. These data suggest that a reducing environment can influence the structure of the glucocorticoid receptor monomer and further imply that sulfhydryl groups within the affinity-labeled receptor are available for modification. To pursue this observation in greater detail, we tested the effect of oxidizing conditions on the structure of the glucocorticoid receptor. The presence of low concentrations (0.125-0.5 mM) of three oxidizing reagents (sodium tetrathionate, disulfiram, and iodosobenzoate) altered the migration of the affinity-labeled receptor resulting in forms of apparent lower molecular mass (as low as 78 kDa). This altered migration, not seen with most other cytosolic proteins, is consistent with the formation of intramolecular disulfide bonds within the receptor which presumably cause it to assume a folded conformation and migrate faster through the gel. At higher concentrations of these reagents (up to 5.0 mM), we also detect a saturably labeled [3H]DM band which has a higher molecular mass (approximately 140 kDa), indicating the formation of intermolecular disulfide bonds between the [3H]DM-labeled receptor and another closely associated protein(s) having a molecular mass of approximately 40 kDa. The effects which these oxidizing reagents have on glucocorticoid receptor structure are completely reversed upon the addition of dithiothreitol, indicating that the observed changes in migration do not reflect receptor proteolysis but rather a folding and unfolding within the receptor monomeric protein. We have also analyzed the effect of this oxidation/reduction on the function of the glucocorticoid receptor. Oxidation of the [3H]DM-labeled receptor complex with 0.5 mM sodium tetrathionate inhibited activation of receptor to a form capable of binding to DNA-cellulose. This inhibition can be reversed with dithiothreitol at 25 degrees C but not at 0 degrees C, suggesting that these oxidizing reagents are inhibitory at the transformation and/or activation steps.(ABSTRACT TRUNCATED AT 400 WORDS)     

Stimulation of the biosynthesis of lactosamine repeats in glycoproteins in differentiating U937 cells and its suppression in the presence of NH4Cl.

We prepared a mouse monoclonal antibody, 2D5, which recognized a highly glycosylated human lysosomal membrane antigen. The apparent molecular mass of this antigen was cell type dependent and ranged between 100 kDa and 130 kDa. The difference was due to a variation in the carbohydrate moiety, since upon removal of the N-linked oligosaccharides the size of the glycoprotein was reduced to approximately 50 kDa in all cases. The high carbohydrate contents, subcellular localization and N-terminal sequence indicated a high similarity or identity of this antigen with the lamp-2 protein. In U937 cells several agents known to elicit differentiation induced synthesis of a larger form of the lamp antigen. Thus, treatment of cells with calcitriol resulted in a shift in its average molecular mass from 115 kDa to 130 kDa. The difference was due to an increase in the contents of lactosamine repeats. In subcellular membranes from calcitriol-treated cells the specific activity of the UDP-N-acetylglucosamine: N-acetyllactosamine N-acetylglucosaminyltransferase was enhanced 3-fold. The enhancement was accompanied with an elongation of lactosamine repeats in N-linked oligosaccharides in the 46 kDa mannose 6-phosphate receptor and the homing receptor, the leucocyte antigen CD44. In contrast, the apparent size of the leucocyte antigen CD43 which bears numerous O-linked oligosaccharides was not changed indicating a selectivity in the modulation of the formation of lactosamine repeats in N- and O-linked carbohydrates. It is shown further that the synthesis of lactosamine repeats in U937 cells is impeded in the presence of NH4Cl.     

Characterization of the intracellular processing and secretion of hepatic lipase in FU5AH rat hepatoma cells

The processing and secretion of newly synthesized hepatic lipase was characterized in FU5AH rat hepatoma cells. Pulse-chase experiments revealed two immunoreactive species with apparent molecular weights of 55,400 and 57,600. The 55.4 kDa species was detectable only in cell extracts, whereas the 57.6 kDa species was present in both cell extracts and media. Following a 5 min pulse with L-[35S]methionine and a 10 min chase, these two species represented only 0.003% of the total labelled protein. Quantitation of the 55.4 kDa and 57.6 kDa species in a chase time course taken together with their respective sensitivity and resistance to digestion with endo-beta-N-acetylglucosaminidase H indicates that the 55.4 kDa species is a high mannose precursor to the mature 57.6 kDa enzyme which contains only complex N-linked oligosaccharides. From a time course of endo-beta-N-acetylglucosaminidase H digestion, it was determined that hepatic lipase contains a minimum of two N-linked oligosaccharides. Treatment of the 55.4 kDa species with endo-beta-N-acetylglucosaminidase H yields a protein with a kDa value similar to that observed after treatment of the mature secreted enzyme with endo-beta-N-acetylglucosaminidase F or trifluoromethanesulfonic acid. Therefore, processing of N-linked oligosaccharides is probably the only post-translational modification responsible for the observed change in the apparent molecular weight of hepatic lipase. The half-residence times of hepatic lipase in the endoplasmic reticulum-cis Golgi region and in the cell were estimated at 34 min and 57 min, respectively. Newly synthesized hepatic lipase in Fu5AH cells is secreted constitutively and is not stored in an intracellular pool. Finally, little of the newly synthesized enzyme is degraded during the course of a 1 h chase.     

Effect of swainsonine on the processing of the asparagine-linked carbohydrate chains of alpha 1-antitrypsin in rat hepatocytes. Evidence for the formation of hybrid oligosaccharides

The biosynthesis of the proteinase inhibitor alpha 1-antitrypsin has been studied in rat hepatocyte primary cultures. Newly synthesized alpha 1-antitrypsin was found in hepatocytes as a glycoprotein of an apparent molecular weight of 49,000 carrying oligosaccharide side chains of the high mannose type. In the hepatocyte medium a secreted alpha 1-antitrypsin of an apparent molecular weight of 54,000 could be identified as a glycoprotein with carbohydrate chains of the complex type. Pulse-chase experiments revealed a precursor-product relationship for the two forms of alpha 1-antitrypsin. When the hepatocytes were treated with swainsonine, an intracellular form of alpha 1-antitrypsin with an apparent molecular weight of 49,000 indistinguishable from that of control cells was found. However, the alpha 1-antitrypsin secreted from swainsonine-treated hepatocytes was different from that present in control media. It was characterized by a lower apparent molecular weight (51,000), a higher amount of [3H]mannose incorporation, half as much incorporation of [3H]galactose, and the same amount of [3H]fucose incorporation compared to alpha 1-antitrypsin of control media. In contrast to the 54,000 complex type alpha 1-antitrypsin from control media the 51,000 alpha 1-antitrypsin from the medium of swainsonine-treated cells was found to be susceptible to the action of endoglucosaminidase H, even when fucose was attached to the proximal GlcNAc residue. alpha 1-Antitrypsin secreted from swainsonine-treated cells combines features usually associated with either high mannose or complex type oligosaccharides and therefore represents a hybrid structure. In spite of its effect on the carbohydrate part of alpha 1-antitrypsin swainsonine did not impair the secretion of the incompletely processed glycoprotein.     

Pharmacological and Biochemical Properties of the γ-Aminobutyric Acid–Benzodiazepine Receptor Protein from Codfish Brain

The gamma-aminobutyric acidA (GABAA) receptor of codfish brain has been purified to homogeneity and contains a single polypeptide band of 56 kDa molecular mass. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE) of codfish GABA receptor photoaffinity-labeled by both [3H]flunitrazepam ([3H]Flu) and [3H]muscimol showed a single radioactive peak with molecular mass of 56 kDa, in contrast to the multiple subunits found in other vertebrate species. The codfish receptor, purified using benzodiazepine (BZ, Ro 7-1986/1) affinity chromatography, contains an apparent single band both by isoelectric focussing and on a silver-stained SDS gel. The receptor density and affinity constants for [3H]muscimol and [3H]Flu binding are comparable to those in mammalian brain, and the specific activity (greater than 1,000 pmol/mg of protein) is comparable to that of preparations purified from those sources. The pharmacological specificity of the codfish GABA-BZ receptor is generally similar to that of mammalian brain, including GABA-BZ coupling. The BZ binding exhibits homogeneous kinetic properties resembling those of the mammalian BZ2 receptor type, and shows strong GABA enhancement of [3H]Flu binding and weaker pentobarbital potentiation. This is consistent with other observations of an earlier phylogenetic, as well as ontogenetic, emergence in mammals of the BZ2 receptor subtype than the BZ1. Codfish GABA receptor is postulated to be a homo-oligomer in which the conformation of GABA and BZ recognition sites is very similar to that in the mammalian hetero-oligomeric GABAA receptor. The codfish receptor appears to be encoded by an ancestral gene and indicates an early development of BZ-GABA coupling.     

Processing of the platelet-derived growth factor receptor. Biosynthetic and degradation studies using anti-receptor antibodies

Studies of platelet-derived growth factor (PDGF) receptor biosynthesis and degradation have been limited by the lack of anti-receptor antibodies. In this study, peptides based on the cDNA-predicted amino acid sequence of the PDGF receptor were used to produce antisera that specifically immunoprecipitated the receptor. PDGF receptor biosynthesis was examined by pulse-chase labeling of cultured fibroblasts with [35S]methionine followed by immunoprecipitation. In BALB/c 3T3 fibroblasts the receptor was synthesized as a 160-kDa precursor that was converted to a mature 180-kDa form within 30-45 min. Removal of high mannose oligosaccharides by endo-beta-N-acetylglucosaminidase H treatment reduced the apparent molecular weight of the 160-kDa precursor but did not affect the migration of the 180-kDa mature receptor. When mannosidase II was inhibited by swainsonine, the 160-kDa precursor failed to mature; instead a 168-kDa form of the receptor was observed. Nevertheless, swainsonine-treated cells responded mitogenically to PDGF. The mature 180-kDa form of the receptor had a half-life of approximately 3 h in the absence of ligand. Addition of PDGF reduced the receptor half-life to 45 min. These studies define and characterize a PDGF receptor precursor, show that receptor degradation is enhanced by PDGF, and demonstrate the functional integrity of incompletely processed PDGF receptors.     

Alterations in lipid-linked oligosaccharide metabolism in human melanoma cells concomitant with induction of stress proteins

Challenge of human A375 melanoma cells with sodium arsenite induced the synthesis of stress proteins and stimulated [3H]mannose incorporation into a novel component migrating on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular mass of 14 kDa (designated M14). Enhanced M14 expression was elicited by heavy metals (zinc, copper, cadmium, and nickel), thiol-reactive agents (iodoacetamide and auranofin), and hyperthermia. The kinetics of M14 induction and recovery from stress were similar to those of the stress proteins, but M14 half-life was only 15 min. Incorporation of [3H]mannose into M14 was inhibited by tunicamycin but not by cycloheximide or actinomycin D. M14 was metabolically labeled with [32P]orthophosphate but not by [35S] methionine or [3H]asparagine. Further studies revealed that M14 was selectively soluble in chloroform/methanol/water (10:10:3) and sensitive to both endo-beta-N-acetylglucosaminidase H digestion and mild acid hydrolysis. The latter released a water-soluble mannose-labeled moiety which eluted from Bio-Gel P-6 in a manner similar to Glc3Man9GlcNAc2. Together, these data suggest that M14 is a lipid-oligosaccharide intermediate of N-linked protein glycosylation and that enhanced expression of this class of molecule in response to chemical insults and hyperthermia is a newly described cellular reaction to stress.     

Effect of inhibiting N-glycosylation on the stability and binding activity of the low density lipoprotein receptor

Tunicamycin, a specific inhibitor of N-glycosylation, was used to study the function of asparagine-linked oligosaccharides of the low density lipoprotein (LDL) receptor in cultured human skin fibroblasts. When cells were preincubated in the presence of 0.5 micrograms/ml of the drug the incorporation of [3H]mannose into the receptor was completely prevented and that of [3H]glucosamine was reduced to approximately 41% of the control value. The [35S]methionine radioactivity detected in receptor core protein of tunicamycin-treated cells was about 52% of that measured in the receptor of control cells. The decrease in the radioactivity was similar in both the mature receptor as well as in its precursor form, and it was significantly greater than that found in total protein. The rates of receptor degradation in control- and tunicamycin-treated cells were comparable. Neither cell surface appearance of the newly synthesized LDL receptor nor its recycling were affected by tunicamycin. However, the LDL receptor produced in tunicamycin-treated cells was smaller in molecular size, and it exhibited an about 50% lower binding capacity when compared with its counterpart synthesized in control cells. This indicates that there is a relationship between N-glycosylation and the ligand binding activity of the LDL receptor. The possible role of asparagine-linked oligosaccharides in optimizing the biological activity of the LDL receptor is discussed.     

Synthesis, intracellular processing and secretion of thrombospondin in human endothelial cells

The biosynthesis of thrombospondin, a glycoprotein first described in platelets, has been studied in human endothelial cells. This glycoprotein has a molecular mass of 450 kDa. It is secreted and incorporated into the extracellular matrix of several cell types in culture. Pulse-chase experiments with [3H]leucine were performed and the synthesis and secretion of the glycoprotein was studied by immunoprecipitation and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The results of these experiments show that the three subunits of thrombospondin are identical in molecular mass. During synthesis there is a small but significant increase in molecular mass within 20 min after pulse labeling. The early form of thrombospondin is sensitive to endoglucosaminidase H treatment, indicating that a transformation of the oligosaccharide structures from 'high-mannose' to 'complex' structures takes place. Within 60 min after synthesis only the mature form of the glycoprotein is secreted into the medium. In the presence of tunicamycin, an inhibitor of N-glycosylation, there is a reduction in molecular mass of the subunit from 165 kDa to 155 kDa. Pulse-chase experiments in the presence of tunicamycin supported the conclusion that the carbohydrate part is processed during biosynthesis. Inhibition of glycosylation had a pronounced effect on the secretion of thrombospondin. The decreased occurrence of thrombospondin in the culture medium seemed to be due to a high intracellular degradation rate of unglycosylated thrombospondin. Characterization of the glycopeptide structures of thrombospondin metabolically labeled with [3H]mannose by Bio-Gel P-4 and concanavalin-A-Sepharose column chromatography revealed that the oligosaccharide structures of the cellular and secreted forms of thrombospondin differ in their composition.     

Effect of glycosylation inhibitors on the structure and function of the murine transferrin receptor

The murine transferrin receptor is a disulphide-linked dimer with three N-glycosylation sites. We have investigated the structural and functional properties of the transferrin receptor from murine plasmacytoma cells (NS-1 cells) treated with the glycosylation inhibitor, tunicamycin and the glycosylation-processing inhibitors, swainsonine and castanospermine. 1. Tunicamycin (1 microgram/ml) inhibited mannose incorporation in NS-1 cells by greater than 90%, but also inhibited methionine incorporation by up to 50%. Both swainsonine (1 microgram/ml) and castanospermine (50 micrograms/ml) resulted in mannose incorporation greater than 100% of untreated cells and neither drug affected methionine incorporation. 2. Incubation of NS-1 cells with tunicamycin resulted in a shift in the apparent molecular mass of the transferrin receptor from 96 kDa and 94 kDa to approximately 82 kDa. 3. Peptide N-glycosidase F digestion of the receptor from untreated cells resulted in the fully deglycosylated 82 kDa component as well as an 87 kDa component which represents partially deglycosylated receptor resistant to peptide N-glycosidase F digestion. 4. The receptor from swainsonine-treated cells was equally sensitive to peptide N-glycosidase F and endo-beta-N-acetylglucosaminidase H (endo H; resulting in both 87-kDa and 82-kDa components), whereas the receptor from castanospermine-treated cells was only partially sensitive to endo H. 5. Analysis of mannose- and fucose-labelled cellular glycopeptides by concanavalin-A--Sepharose chromatography showed that swainsonine (1 microgram/ml) treatment resulted in approximately 90% inhibition of the synthesis of complex N-glycans and an accumulation of fucosylated hybrid structures. In contrast, castanospermine (100 micrograms/ml) treatment resulted in only partial inhibition (60%) of the synthesis of complex N-glycans. 6. Analysis of the receptor from tunicamycin, swainsonine and castanospermine treated cells under nonreducing conditions showed a single component corresponding to the dimer, indicating that dimerisation of newly synthesised murine receptor is independent of carbohydrate. 7. The non-glycosylated receptor from tunicamycin-treated cells appears to bind transferrin as demonstrated by interaction with transferrin-Sepharose. 8. Surface expression of the receptor was not significantly altered in the presence of either swainsonine or castanospermine as judged by flow cytometry.     

Preferential binding of insulin-like growth factor-II (IGF-II) to a putative alpha 2 beta 2 IGF-II receptor type in C2 myoblasts.

We have studied insulin-like-growth-factor (IGF) binding in two subclones of the C2 myogenic cell line. In the permissive parental subclone, myoblasts differentiate spontaneously into myotubes in medium supplemented with fetal calf serum. Unlike permissive myoblasts, inducible myoblasts require high concentrations of insulin (1.6 microM) or lower concentrations of IGF-I (25 nM) to differentiate, and expression of MyoD1 is not constitutive. IGF receptors were studied in microsomal membranes of proliferating and quiescent myoblasts and myotubes. IGF-II binding was also studied in inducible myoblasts transfected with the MyoD1 cDNA (clone EP5). Both inducible and permissive cells exhibited a single class of binding sites with similar affinity for IGF-I (Kd 0.8-1.2 nM). Affinity cross-linking of [125I]IGF-I to microsomal membranes, under reducing conditions, revealed a binding moiety with an apparent molecular mass of 130 kDa in permissive cells and 140 kDa in inducible cells, which corresponded to the alpha subunit of the IGF-I receptor. In permissive quiescent myoblasts, linear Scatchard plots suggested that [125I]IGF-II bound to a single class of binding sites (Kd 0.6 nM) compatible with binding to the IGF-II/M6P receptor. This was confirmed by affinity cross-linking experiments showing a labeled complex with an apparent molecular mass of 260 kDa and 220 kDa when studied under reducing and non-reducing conditions, respectively. In contrast, competitive inhibition of [125I]IGF-II binding to inducible quiescent myoblasts generated curvilinear Scatchard plots which could be resolved into two single classes of binding sites. One of them corresponded to the IGF-II/M6P receptor (Kd 0.2 nM) as evidenced by cross-linking experiments. The second was the binding site of highest affinity (Kd 0.04 nM) which was less inhibited by IGF-I than by IGF-II and was not inhibited by insulin. It migrated in SDS/PAGE at a position equivalent a molecular mass of 140 kDa, under reducing conditions, and at approximately 300 kDa, under non-reducing conditions. The labeling of this atypical binding moiety was not inhibited by anti(IGF-II/M6P-receptor) immunoglobulin. It was also observed in permissive and inducible myoblasts at proliferating stage. It was absent for permissive quiescent myoblasts and from permissive and inducible myotubes. Forced expression of MyoD1 in inducible cells (EP5 cells) dramatically reduced [125I]IGF-II binding to this atypical receptor. It emerges from these experiments that C2 cells express a putative alpha 2 beta 2 IGF-II receptor structurally related to the insulin/IGF-I receptor family. It is present in myoblasts but not in myotubes.(ABSTRACT TRUNCATED AT 400 WORDS)