Biosynthesis of human sialophorins and analysis of the polypeptide core.
Biosynthesis was examined of sialophorin (formerly called gpL115) which is altered in the inherited immunodeficiency Wiskott-Aldrich syndrome. Sialophorin is greater than 50% carbohydrate, primarily O-linked units of sialic acid, galactose, and galactosamine. Pulse-labeling with [35S]methionine and chase incubation established that sialophorin is synthesized in CEM lymphoblastoid cells as an Mr 62,000 precursor which is converted within 45 min to mature glycosylated sialophorin, a long-lived molecule. Experiments with tunicamycin and endoglycosidase H demonstrated that sialophorin contains N-linked carbohydrate (approximately two units per molecule) and is therefore an N,O-glycoprotein. Pulse-labeling of tunicamycin-treated CEM cells together with immunoprecipitation provided the means to isolate the [35S]-methionine-labeled polypeptide core of sialophorin and determine its molecular weight (58,000). This datum allowed us to express the previously established composition on a "per molecule" basis and determine that sialophorin molecules contain approximately 520 amino acid residues and greater than or equal to 100 O-linked carbohydrate units. A recent study showed that various blood cells express sialophorin and that there are two molecular forms: lymphocyte/monocyte sialophorin and platelet/neutrophil sialophorin. Biosynthesis of the two forms was compared by using sialophorin of CEM cells and sialophorin of MOLT-4 cells (another lymphoblastoid line) as models for lymphocyte/monocyte sialophorin and platelet/neutrophil sialophorin, respectively. The time course of biosynthesis and the content of N units were found to be identical for the two sialophorin species. [35S]Methionine-labeled polypeptide cores of CEM sialophorin and MOLT sialophorin were isolated and compared by electrophoresis, isoelectrofocusing, and a newly developed peptide mapping technique.(ABSTRACT TRUNCATED AT 250 WORDS)
D000118:Acetylglucosaminidase / Q000494:pharmacology; D015703:Antigens, CD; D002460:Cell Line; D004586:Electrophoresis; D006801:Humans; D007525:Isoelectric Focusing; D051917:Leukosialin; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008715:Methionine / Q000378:metabolism; D008985:Monensin / Q000494:pharmacology; D010449:Peptide Mapping; D010455:Peptides / Q000032:analysis*; D012795:Sialoglycoproteins / Q000032:analysis / Q000096:biosynthesis*; D013462:Sulfur Radioisotopes / Q000378:metabolism; D013997:Time Factors; D014415:Tunicamycin / Q000494:pharmacology
D015703:Antigens, CD; D051917:Leukosialin; D010455:Peptides; C465014:SPN protein, human; D012795:Sialoglycoproteins; D013462:Sulfur Radioisotopes; D014415:Tunicamycin; D008985:Monensin; D008715:Methionine; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase