Biosynthesis of human sialophorins and analysis of the polypeptide core

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Abstract
Biosynthesis was examined of sialophorin (formerly called gpL115) which is altered in the inherited immunodeficiency Wiskott-Aldrich syndrome. Sialophorin is > 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 to express the previously established composition on a "per molecule" basis and determine that sialophorin molecules contain .apprx. 520 amino acid residues and .gtoreq. 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 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. The polypeptides were indistinguishable, strongly indicating that the two sialophorin species contain identical polypeptide cores and suggesting that their differences arise through the action of Golgi region enzymes.