Biosynthesis pathway of gp90MEL-14, the mouse lymph node-specific homing receptor.
The mouse lymph node specific homing receptor gp90MEL-14 is a 95-kDa molecular mass ubiquitinated cell surface molecule involved in the binding of lymphocytes to high endothelial venules in peripheral lymph nodes. The molecule is thought to consist of a core protein to which ubiquitin side chains are covalently bound. Recently we cloned the cDNA encoding the core protein; this cDNA clone encodes for a polypeptide with an estimated molecular mass of 37 kDa. We have studied the biosynthesis of gp90MEL-14 in an effort to explain the difference in molecular mass between the core protein and the 95-kDa mature molecule. Pulse labeling experiments show a rapid synthesis of a 70-kDa precursor form that contains high-mannose N-linked oligosaccharides. On processing of the high-mannose oligosaccharides into complex N-linked oligosaccharides, the precursor matures in a single step into the 95-kDa form. Experiments using deglycosylating enzymes and inhibitors of N-linked glycosylation demonstrate that the molecular mass of deglycosylated gp90MEL-14 is 45 kDa; extensive N-linked glycosylation is responsible for the difference in molecular mass with the mature 95-kDa form. The core protein molecular weight of in vitro transcribed and translated gp90MEL-14 cDNA is consistent with the estimated molecular mass of 37 kDa, calculated from the cDNA sequence of the core protein, and 8 to 10 kDa less than the protein molecular mass of gp90MEL-14 translated in vivo in the presence of tunicamycin (45 kDa). Inasmuch as we have ruled out glycosylation as accounting for this discrepancy, this is consistent with the addition of one ubiquitin moiety to the core protein during biosynthesis. Limited proteolysis confirms the similarity between in vitro transcribed gp90MEL-14 cDNA and the tunicamycin form of gp90MEL-14.
van de Rijn
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M
Department of Pathology, Stanford University School of Medicine, CA 94305.
Weissman
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IL
Siegelman
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D000818:Animals; D000954:Antigens, Surface / Q000096:biosynthesis* / Q000235:genetics; D006026:Glycoside Hydrolases / Q000494:pharmacology; D006031:Glycosylation; D006596:Hexosaminidases / Q000494:pharmacology; D066298:In Vitro Techniques; D008198:Lymph Nodes / Q000378:metabolism*; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins / Q000096:biosynthesis*; D008698:Mesylates / Q000494:pharmacology; D051379:Mice; D008970:Molecular Weight; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011956:Receptors, Cell Surface / Q000096:biosynthesis*; D016201:Receptors, Lymphocyte Homing; D014158:Transcription, Genetic; D014415:Tunicamycin / Q000494:pharmacology; D014452:Ubiquitins / Q000378:metabolism
D000954:Antigens, Surface; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D008698:Mesylates; D011498:Protein Precursors; D011956:Receptors, Cell Surface; D016201:Receptors, Lymphocyte Homing; D014452:Ubiquitins; D014415:Tunicamycin; D006026:Glycoside Hydrolases; D006596:Hexosaminidases; C020668:glycanase; C012077:trifluoromethanesulfonic acid