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1.
The production of chicken myelomonocytic growth factor (cMGF) can be rapidly induced by bacterial lipopolysaccharide from the macrophage cell line HD11. Immunoprecipitation analysis of lipopolysaccharide-induced HD11 cells labeled with various radioactive precursors showed the secretion of a variety of cMGF forms. The precursor-product relationships of the different cMGF forms were studied by pulse-chase experiments, by long-term metabolic labeling in the presence or absence of glycosylation- and oligosaccharide-processing inhibitors, as well as by glycosidase treatment of immunoprecipitates. Our results show that the half-time for intracellular processing/secretion is less than 10 min, making cMGF one of the most rapidly processed proteins. The different forms of the factor are generated from a 24-kDa polypeptide precursor by co- and post-translational acquisition of one or two N-linked oligosaccharides and by O-linked glycosylation. In addition, a fraction of cMGF is modified by long chain, chondroitinase-sensitive, sulfated glycans. This modification is tunicamycin-sensitive, suggesting that the sulfated glycans are attached to N-linked rather than to O-linked oligosaccharides.  相似文献   

2.
In human fibroblasts, the receptor for low density lipoprotein (LDL) is synthesized as a precursor of apparent Mr = 120,000 which is converted to a mature form of apparent Mr = 160,000, as determined by migration in sodium dodecyl sulfate (SDS)-polyacrylamide gels (Tolleshaug, H., Goldstein, J. L., Schneider, W. J., and Brown, M. S. (1982) Cell 30, 715-724). The current paper describes the relationship of N- and O-glycosylation to this post-translational modification. Oligosaccharides were analyzed from precursor and mature forms of LDL receptors that had been immunoprecipitated from cells grown in media containing radioactive sugars. In human epidermoid carcinoma A-431 cells, the receptor precursor appears to contain one N-linked high mannose oligosaccharide and approximately 6-9 N-acetylgalactosamine residues linked O-glycosidically to Ser/Thr residues. In the mature receptor, the O-linked oligosaccharides are mono- and disialylated species having the core structure of galactose leads to N-acetylgalactosamine leads to Ser/Thr. The single N-linked oligosaccharide of the mature receptor can either be a tri- or tetraantennary complex-type species. Similar results were obtained with normal human fibroblast receptor except that the O-linked oligosaccharides on the precursor are neutral disaccharides, of which one component is GalNAc and the N-linked complex type unit on the mature receptor is less branched. Since the addition of GalNAc residues to Ser/Thr residues precedes the conversion of N-linked high mannose-type oligosaccharides to complex-type structures, the transfer of N-acetylgalactosamine must occur prior to the entry of glycoproteins into the region of the Golgi containing the processing enzyme alpha-mannosidase I. We also studied the receptor from tunicamycin-treated cells and after treatment with neuraminidase. In addition, we analyzed the receptor synthesized by a lectin-resistant clone of Chinese hamster ovary cells that is deficient in adding galactose residues to both N- and O-linked oligosaccharides. These studies suggest that the apparent differences in molecular weight between the precursor and mature forms of the LDL receptor are largely, if not entirely, due to the addition of sialic acid and galactose residues to the O-linked GalNAc residues.(ABSTRACT TRUNCATED AT 400 WORDS)  相似文献   

3.
Characterization of the human melanoma nerve growth factor receptor   总被引:13,自引:0,他引:13  
Monoclonal antibodies to the human nerve growth factor receptor have been used to biochemically characterize the receptor in the human melanoma cell line A875. Labeling of A875 cell proteins by culture with [35S]cysteine or labeling of cell surface proteins with 125I followed by immunoprecipitation with anti-nerve growth factor receptor antibody reveals a receptor protein with an apparent Mr of 70,000-75,000 and an isoelectric point of 4.9-5.2. Incorporation of [3H]glucosamine into this species indicates it is a glycoprotein. The receptor becomes phosphorylated on serine residues in intact cells and in isolated membranes incubated with [gamma-32P]ATP. The receptor appears to exist, at least partially, in the form of a disulfide-linked oligomer (probably a dimer) of Mr = 75,000 subunits. Kinetic [35S]cysteine labeling studies reveal an Mr = 59,000 core protein which is glycosylated via N-linked and probably also O-linked sugar moieties to produce the mature (Mr = 70,000-75,000) receptor.  相似文献   

4.
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.  相似文献   

5.
Antibodies to phosphotyrosine were used in immunoprecipitation experiments to determine if post-translational modification of the platelet-derived growth factor (PDGF) receptor was required for the acquisition of ligand-induced tyrosine kinase activity. In intact Balb/c 3T3 fibroblasts, only the fully processed 180-kDa receptor was activated (tyrosine-phosphorylated) by PDGF. In a cell-free assay, however, the tyrosine-phosphorylated forms of the 160- and 145-kDa PDGF receptor precursors were also detected. These activated precursors were immunoprecipitated after brief (5-15 min) metabolic labeling periods. Thus the receptor could bind PDGF and induce tyrosine kinase activity shortly after translation. Unlike the mature form of the receptor, the 160-kDa receptor precursor was resistant to digestion with endo-alpha-N-acetylgalactosaminidase and thus did not contain O-linked oligosaccharides. Since this receptor precursor was activated by PDGF in the cell-free assay, the addition of O-linked sugars must not be necessary for ligand binding activity. Incubation of cells with tunicamycin completely inhibited N-linked glycosylation of the PDGF receptor. Nevertheless, PDGF still induced tyrosine phosphorylation of the 130-kDa aglycoreceptor in lysates of tunicamycin-treated cells. Thus, the addition of N-linked oligosaccharides was also not required for receptor activation. These findings show that the PDGF receptor acquired the ability to be activated by ligand cotranslationally or immediately after translation and that the addition of N- or O-linked oligosaccharides was not required for ligand binding and tyrosine kinase activities.  相似文献   

6.
The biosynthesis and post-translational processing of the insulin-like growth factor II (IGF-II) receptor has been studied in H-35 hepatoma cells using a specific polyclonal anti-receptor immunoglobulin preparation. Cells were pulse-labeled with [35S]methionine followed by incubation with excess unlabeled methionine (chase). Gel electrophoresis of the immunoadsorbed receptors shows that the receptor is first synthesized as a 245-kDa precursor which is transformed to the mature 250-kDa form with a half-time of about 2 h. The 245-kDa precursor could also be labeled biosynthetically with [3H]mannose, only one-half of which was ultimately found associated with the 250-kDa product. Neuraminidase converts the 250-kDa receptor species to a 245-kDa form. Whereas the 250-kDa receptor is insensitive to detectable cleavage by endoglycosidase H, digestion of the 245-kDa species with this enzyme produces a 232-kDa form. A similar 232-kDa receptor species accumulates in H-35 cells incubated with tunicamycin (2 micrograms/ml). This tunicamycin-induced aglyco-receptor is not further processed to the 250-kDa form. Monensin (50 nM) blocks receptor processing at the 245-kDa stage. Endoglycosidase H treatment of the monensin-induced 245-kDa species indicates that this is a mixture of partially processed precursors having equivalent Mr. No evidence was obtained for the presence of O-linked oligosaccharides on the IGF-II receptor. The IGF-II binding activity of the three different biosynthetic forms of the receptor was assessed by affinity cross-linking of 125I-IGF-II to the receptors using disuccinimidyl suberate. Both the mature 250-kDa receptor and the neuraminidase-digested 245-kDa form specifically bound 125-I-IGF-II. However, the 232-kDa aglyco-receptor had no detectable IGF-II binding activity using this method. In summary, these studies show: 1) that the H-35 cell IGF-II receptor is synthesized first as a 245-kDa precursor having 4-6 high-mannose oligosaccharide side chains, 2) processing of the receptor oligosaccharides by mannose removal and terminal sialylation converts the 245-kDa precursor to the 250-kDa mature product which has been previously identified as the functional receptor in the plasma membrane, 3) the apparent molecular mass of the receptor in the absence of N-glycosylation is 232-kDa, and 4) glycosylation of the IGF-II receptor is required for the acquisition of IGF-II binding activity.  相似文献   

7.
Using human-specific antibody reagents, we have examined the biosynthesis of the epidermal growth factor receptor in human epidermoid carcinoma-derived A431 cells. Four Mr species (Mr = 70,000, 95,000, 135,000, and 145,000) are detected when cells are subjected to a brief pulse of L-[35S]methionine; an Mr = 165,000 species is detected after 45-60 min of exposure of cells to radiolabel. In pulse-chase experiments, the four lower Mr species appear to bear a precursor relation to the Mr = 165,000 protein. The molecule acquires N-linked oligosaccharide cotranslationally, and two of the species (Mr = 95,000 and 145,000) are susceptible to digestion with endo-beta-N-acetylglucosaminidase H. The Mr = 145,000 and Mr = 165,000 proteins, which become labeled with 125I-epidermal growth factor after treatment of intact cells with a bifunctional cross-linking reagent, are phosphorylated at serine and threonine on identical tryptic peptides.  相似文献   

8.
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.  相似文献   

9.
These studies were initiated to determine whether the soluble, truncated form of the nerve growth factor (NGF) receptor arises from post-translational processing of the intact, membrane-bound receptor or from an alternatively spliced mRNA. Pulse-chase analysis of cultured primary rat Schwann cells coupled with immunoprecipitations using antibodies to the intracellular and extracellular domains of the receptor were used to monitor receptor production. Three forms of the NGF receptor (80, 83, and 85 kDa) displaying a precursor product relationship were detected over the 2-h chase period; only the 85-kDa species was detected on the cell surface. Truncated receptors (50 and 52 kDa) were detected in conditioned media 5 h after cell labeling but were never observed intracellularly. Polymerase chain reaction and RNase protection analyses of NGF receptor mRNA targeted toward the coding region for the transmembrane domain detected no splice variants that could generate truncated receptor, and media conditioned by fibroblasts transfected with rat receptor cDNA, in which splicing cannot occur, nonetheless contained the truncated receptor protein. Taken together, these results suggest that the truncated NGF receptor does not arise as a distinct translation product but rather from a post-translational modification of the intact, surface-bound form of the protein.  相似文献   

10.
We previously showed that the epidermal growth factor (EGF) receptor in human A431 epidermoid carcinoma cells undergoes a slow post-translational modification whereby it acquires (t1/2 = 30-40 min) EGF binding capacity (Slieker, L.J., et. al. (1986) J. Biol. Chem., 261, 15233-15241). This activation occurs in the endoplasmic reticulum and requires core N-linked glycosylation. By employing both anti-EGF receptor and anti-phosphotyrosine antibodies to immunoprecipitate receptor pulse-labeled with [35S]methionine, we demonstrate here that the EGF receptor also acquires tyrosine kinase autophosphorylation activity post-translationally (t1/2 = 10-15 min). The acquisition of tyrosine kinase activity is independent of the acquisition of EGF binding capacity, since it precedes the latter process and does not require N-linked glycosylation.  相似文献   

11.
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.  相似文献   

12.
We have developed a monoclonal antibody, designated PR7212 (IgG1), which specifically recognizes the platelet-derived growth factor receptor (PDGFR) of primate cells. The antibody recognizes an extracellular epitope of the receptor, demonstrated by its ability to bind to intact cells. Using this antibody, we have detected three forms of PDGFR of approximately 180, 164, and 130 kDa. All three of the forms were detected by Western blot analysis of human dermal fibroblasts. Immunoprecipitates of 32P-labeled membrane extracts of human dermal fibroblasts demonstrate that phosphorylation of all three forms of the receptor is stimulated by PDGF. In addition, several smaller molecules were detected, ranging in size from 113 to 49 kDa, which are also phosphorylated in response to PDGF addition. These smaller molecules may be either PDGFR kinase substrates or partially degraded PDGFR. Only the 180- and the 164-kDa forms of the receptor are detectable from immunoprecipitates of soluble extracts of 35S-metabolically labeled cells. Pulse-chase experiments demonstrate that the 164-kDa form is a precursor of the 180-kDa molecule. After PDGF binding at 37 degrees C, the 180-kDa form disappears from the cell surface in parallel with a decrease in 125I-PDGF binding, providing evidence that occupation results in internalization of PDGFR rather than inactivation.  相似文献   

13.
Human T-cell growth factor (TCGF) has been isolated from conditioned media of the Jurkat T-leukemia cell line. Using a high-efficiency isolation procedure involving hollow fiber concentration, gel filtration and 3 steps of reverse-phase HPLC we obtained 100 to 600 pmol TCGF per liter of conditioned medium. Jurkat cell-derived TCGF (jTCGF) has a molecular weight of 15,750. The amino acid composition of jTCGF agrees well with that derived from the cDNA sequence coding for this protein (Taniguchi et al, Nature 302, 305, 1983). jTCGF is highly active in vitro in stimulating the proliferation of T-cells as measured by 3H-thymidine incorporation into DNA (half-maximal stimulation with 3 fmol/100 microliters well).  相似文献   

14.
NIH-3T3 cells expressing the human epidermal growth factor (EGF) receptor were used in experiments to determine the fate of the EGF receptor in cells continuously exposed to EGF. EGF receptor was immunoprecipitated from cells labeled for 12 h with [35S] methionine in the absence or presence of 10 nM EGF. As expected, a single Mr = 170,000 polypeptide representing the mature EGF receptor was immune-precipitated from control cells. Surprisingly, immune precipitates from EGF-treated cells contained a prominent Mr = 125,000 receptor species, in addition to the Mr = 170,000 mature receptor. The Mr = 125,000 species was shown to be derived from the Mr = 170,000 form by pulse-chase experiments, in which the Mr = 170,000 receptor chased into the Mr = 125,000 form when EGF was included during the chase and by partial proteolysis. Both proteins became extensively phosphorylated on tyrosine residues in immune precipitate kinase assays. Treatment of immune precipitates with endoglycosidase F changed the apparent molecular weight of the Mr = 170,000 receptor to Mr = 130,000 and of the Mr = 125,000 form to Mr = 105,000, indicating that the appearance of the Mr = 125,000 protein was probably due to proteolysis. Antibody against the carboxyl terminus of the mature EGF receptor recognized the Mr = 125,000 protein, whereas antibody against the amino terminus did not. Incubation of cells with leupeptin prior to and during EGF addition inhibited processing to the Mr = 125,000 species. Methylamine and low temperature also inhibited the EGF-induced processing to the Mr = 125,000 form. These data suggest a possible role for proteolysis of the EGF receptor in receptor function.  相似文献   

15.
The post-translational processing and maturation of the receptor for IgE (Fc epsilon R) on murine hybridoma B cells were studied to determine the carbohydrate content and the importance of processing events in cell surface expression and ligand (IgE) binding ability. Endo and exoglycosidase treatment demonstrated that the mature receptor is composed of two to three complex-type N-linked oligosaccharides and contains sialic acid. Pulse-chase experiments indicated that the receptor is synthesized as a 44,000 dalton precursor that begins to be processed by 1 hr to the mature 49,000 dalton form, and the latter is expressed at the cell surface by 2 hr. It was determined that the processing included the conversion of N-linked oligosaccharides to the complex type as well as an additional processing event, because in the presence of tunicamycin, the receptor is synthesized as a 36,000 dalton precursor that is processed to a 38,000 dalton species. Analysis of the effects of tunicamycin treatment and endo F digestion on soluble Fc epsilon R isolated from cell supernatants demonstrated the existence of several m.w. species of Fc epsilon R fragments, and indicated that only the higher m.w. fragments were N-glycosylated. The use of several inhibitors of the N-linked carbohydrate processing pathway demonstrated that the addition of core N-linked side-chains, but not their processing to the complex type, is required for cell surface expression of Fc epsilon R. Also, processing of N-linked carbohydrate is not required for ligand binding activity. Finally, IgE affinity chromatography indicated that the 49,000 and 38,000 dalton (tunicamycin) Fc epsilon R bind IgE more effectively than their precursor forms, 44,000 and 36,000 daltons, respectively, indicating that a processing event independent of N-linked glycosylation is necessary for optimal ligand binding activity.  相似文献   

16.
Biosynthetic processing of the T-cell surface receptor for interleukin-2 was investigated in a cultured human T-cell line MT-1 by means of metabolic and cell surface radiolabeling followed by immunoprecipitation with a monoclonal anti-receptor antibody (anti-Tac) and analysis by one- and two-dimensional polyacrylamide gel electrophoresis. The nascent precursor of the receptor (Mr = about 40,000, pI = 6.2-6.5) underwent a post-translational modification giving rise to the mature receptor (IL-2R; Mr = 60,000-65,000, pI = 4.2-4.7) within 2-4 hr. The post-translational processing of IL-2R caused a 20,000-25,000 increase in apparent molecular weight and a 2.0-2.5 acidic shift in the isoelectric point. The increase in molecular weight was attributable mainly to addition of sugar residues including glucosamine and galactose, and the charge shift to the addition of sialic acids. A carboxylic ionophore monensin completely blocked the maturation of IL-2R at the mid-stage of the processing. Fatty acid attachment appeared to comprise one of the steps of the post-translational modification. Two-dimensional analyses of IL-2R biosynthesis enabled identification of the precursor of IL-2R and its intermediate forms, from which it was partially possible to estimate reactions involved in the maturation of the precursor molecule.  相似文献   

17.
Maltase-glucoamylase (MGA) was immunoprecipitated from detergent extracts of brush border membranes of the human small intestinal mucosa. Electrophoretic analysis of the precipitates under denaturing conditions revealed a single polypeptide of Mr = 335,000 in the presence or absence of reducing agents. Cross-linking of brush border membranes with the homobifunctional reagent dithiobis(succinimidylpropionate) did not result in considerable changes in the electrophoretic pattern of MGA. In contrast, aminopeptidase N, used in these studies as a control glycoprotein of the brush border membrane revealed dimeric structures of its single subunit in the presence of dithiobis(succinimidylpropionate). These data suggest that MGA is expressed in the human small intestinal brush border as a monomeric polypeptide. The biosynthesis of MGA was studied by pulse-labeling of human intestinal biopsy specimens or mucosal explants in organ culture. Continuous labeling with [35S]methionine for 30 min revealed a single polypeptide high mannose precursor of Mr = 285,000 (MGAh) which matures after 4 h of labeling to the Mr = 335,000 as judged by the susceptibility of these two forms to endo-beta-N-acetylglucosaminidase H. Owing to the absence of pancreatic secretions in the culture medium and the isolation of an identical species from nonlabeled mucosa, this result indicates that the Mr = 335,000 does not undergo an in situ extracellular cleavage by intraluminal proteases. Further, biosynthetically labeled, intracellularly cleaved polypeptides corresponding to the high mannose precursor or mature forms of MGA were not detected. The mature form of MGA (MGAm) bears in addition to N-linked glycans also O-glycosidically linked oligosaccharides. In fact, endo-beta-N-acetylglucosaminidase F/glycopeptidase F treatment of MGAm followed by chemical deglycosylation with trifluoromethanesulfonic acid revealed approximately 35,000 daltons of O-linked sugars. Furthermore, MGAm as well as its N-linked sugars-depleted form bound to Helix pomatia lectin which has specificity toward Gal-GalNAc structures. In addition, the data were suggestive of a post-translational O-glycosylation of the molecule since (i) the high mannose precursor of MGA did not bind to H. pomatia lectin and (ii) its endo-beta-N-acetylglucosaminidase H or endo-beta-N-acetylglucosaminidase F/glycopeptidase F form displayed an apparent molecular weight similar to that obtained upon endo-beta-N-acetylglucosaminidase F/glycopeptidase F/trifluoromethanesulfonic acid deglycosylation. Finally, pulse-chase experiments revealed a relatively slow rate of post-translational processing of MGA in comparison to aminopeptidase N.(ABSTRACT TRUNCATED AT 400 WORDS)  相似文献   

18.
Metabolic labeling of the murine T lymphoma cell line RDM-4 with [35S] sulfate results in intense incorporation into a cell-retained component of apparent Mr approximately 100,000. This macromolecule is identified as a glycoprotein by lectin chromatography. The sulfate is not incorporated as tyrosine sulfate. Release of the radiolabel by alkaline beta-elimination but not by endoglycosidase F is consistent with the sulfation of O- rather than N-linked oligosaccharides. The sulfated glycoprotein displays anomalous migration on SDS-PAGE in two respects: 1) the apparent Mr shifts from 115,000 to 87,000 on increasing the acrylamide concentration from 7 to 12%, and 2) on neuraminidase digestion migration is substantially reduced (apparent Mr 140,000). These properties indicate that the sulfated protein is both heavily glycosylated and extensively sialylated, and are characteristic of the lymphoid mucin, leukosialin (sialophorin, CD43). Specific labeling of the sialoglycoproteins of RDM-4 cells indicates that leukosialin, the most intensely labeled protein, comigrates with the sulfated protein on SDS-PAGE at varying acrylamide concentrations. Our data are therefore consistent with sulfation of at least some of the numerous O-linked oligosaccharides of this abundant glycoprotein in RDM-4 cells. No sulfation of CD43 in resting splenic T cells is observed.  相似文献   

19.
Biosynthesis of the epidermal growth factor receptor in A431 cells.   总被引:22,自引:6,他引:16       下载免费PDF全文
A monoclonal antibody R1 against the human epidermal growth factor receptor has been used to study biosynthesis in the carcinoma cell line A431. Two glycoproteins of apparent mol. wts. 95 000 and 160 000 were immunoprecipitated from cells labelled for short times with [35S]methionine or [3H]mannose. Pulse-chase studies show the 160 000 mol. wt. glycoprotein to be a precursor of the 175 000 mol. wt. receptor, but do not establish a precursor role for the 95 000 mol. wt. glycoprotein. Limited proteolysis, peptide mapping, endoglycosidase digestion and the use of monensin and tunicamycin show that the 95 000 mol. wt. glycoprotein is structurally related to the 160 000 mol. wt. glycoprotein and that both glycoproteins have approximately 22 000 - 28 000 mol. wt. of oligosaccharide side chains. Monensin blocks conversion of the 160 000 to the 175 000 mol. wt. mature receptor, a process which involves complexing several of its N-linked oligosaccharide chains. Pulse-chase studies showed that an immunoprecipitable polypeptide of 115 000 mol. wt., or 95 000 mol. wt., in the presence of monensin, was secreted into the medium at late chase times. The possible mechanisms for the origins of all the receptor-related polypeptides are discussed.  相似文献   

20.
Cellular membrane receptors for the immunostimulatory neuropeptide substance P have been previously identified on the cultured lymphoblast cell line, IM-9. The regulation of this receptor by ligand and the contribution to its molecular weight by N-linked sugars was studied by incubating IM-9 cells for 14 hr in the presence of [35S]met with or without substance P and tunicamycin, respectively. Cells were lysed and the receptor proteins were immunoprecipitated with an anti-receptor monoclonal antibody. SDS-PAGE analysis of untreated cellular lysates revealed specifically precipitated proteins of 38 kD and 33 kD, which were down-regulated by substance P. In tunicamycin-treated cells, whose substance P binding was not affected, the major immunoprecipitated protein had an apparent Mr of 29 kD. The time course of receptor processing was studied by pulse chase analysis. Three proteins of molecular weights 38 kD (mature receptor), 36 kD and 33 kD (receptor precursors) were identified for time periods of 30 min to 4 hr. The half life of the mature receptor and its precursors was approximately 1 hr and 0.5 hr, respectively. Results from the present studies suggest that the lymphocyte substance P receptor is translated as a precursor protein that is glycosylated.  相似文献   

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