Characterization of the oligosaccharide structures on recombinant human prorenin expressed in Chinese hamster ovary cells.
Prorenin was isolated by immunoprecipitation from the culture medium of Chinese hamster ovary cells transfected with a human prorenin cDNA. The N-linked oligosaccharide structures on the in vivo [3H]mannose-labeled, purified protein were characterized using a combination of serial lectin affinity chromatography, high-pressure liquid chromatography, ion-exchange chromatography, and size-exclusion chromatography and treatment with specific glycosidases and methylation analysis. Approximately 61% of the oligosaccharides on the molecule are complex type, in the form of tetraantennary (2%), 2,6-branched triantennary (13%), 2,4-branched triantennary (3%), and biantennary (43%) structures. The majority of all complex type structures are core-fucosylated. Sialic acids are linked at the C-3 position of terminal galactose, and the degree of sialylation of the bi- and triantennary structures varies between nonsialylated and fully sialylated; no tetraatennary structure contains more than three sialic acid residues. Recombinant prorenin contains 4% hybrid-type structures, all of which carry a terminal sialic acid residue. The remaining 35% of the structures on the molecule are high mannose type, composed of 5, 6, or 7 mannose residues. Approximately 6% of the high mannose type structures and 10% of the hybrid structures are phosphorylated, as judged by their susceptibility to treatment with alkaline phosphatase. Compositional analysis of an unlabeled preparation of the protein suggested the presence of approximately 1.4 oligosaccharide units per molecule.
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Upjohn Laboratories, Upjohn Company, Kalamazoo, Michigan 49001.
Guido
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Mathews
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Elhammer
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D000818:Animals; D016466:CHO Cells; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002845:Chromatography; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D006224:Cricetinae; D004247:DNA / Q000235:genetics; D004792:Enzyme Precursors / Q000737:chemistry* / Q000302:isolation & purification; D006026:Glycoside Hydrolases / Q000378:metabolism; D006801:Humans; D008358:Mannose / Q000032:analysis / Q000737:chemistry; D008745:Methylation; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000032:analysis / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012083:Renin / Q000737:chemistry* / Q000302:isolation & purification; D012794:Sialic Acids / Q000032:analysis / Q000737:chemistry; D014162:Transfection
D004792:Enzyme Precursors; D009844:Oligosaccharides; D011994:Recombinant Proteins; D012794:Sialic Acids; D004247:DNA; D006026:Glycoside Hydrolases; D012083:Renin; D008358:Mannose