Sialoglycoproteins of human mammary cells: partial characterization of sialoglycopeptides
Chandrasekaran, E.V.; Davidson, E.A.
Biochemistry 18(25): 5615-5620
ISSN/ISBN: 0006-2960 PMID: 518857 DOI: 10.1021/bi00592a015
Sialoglycopeptides were isolated and fractionated from cultured human mammary cell lines (1 control line, HBL-100 and 2 cancer lines, MDA-MB-231 and MCF-7) into 3 groups: neutral, less anionic and more anionic. Cells were cultured with [3H]glucosamine, [3H]glucosamine and [14C]galactose, or [14C]glucosamine and [3H]galactose, following which spent-medium and cell-associated components were analyzed. Exhaustive Pronase digestion and removal of glycosaminoglycans with cetylpyridinium chloride were followed by diethylaminoethylcellulose and wheat germ agglutinin (WGA)-Sepharose chromatography. The cancer lines produced 3-5 times greater amounts of the more anionic glycopeptides compared to the control line. Within this fraction, WGA-bound material was predominant in the cancer lines. Alkaline borohydride treatment of the WGA-binding fraction from both cancer lines gave rise to a major elimination product not observed with comparable material from HBL-100 cells. Analysis of the asialosaccharide chain gave the structure Gal .**GRAPHIC**. GlcNAc .**GRAPHIC**. Gal .fwdarw. GalNAcOH. The structural requirements for the binding of the glycopeptides to WGA were analyzed. The WGA-binding capacity is dependent on molecular size and sialic acid content. In addition, the saccharide moiety of the WGA-binding glycopeptides is purely O-glycosidically linked, whereas that from the WGA-nonbinding material is mainly N-glycosidic. The WGA-binding glycopeptides contain clustered oligosaccharide chains having the sequence NeuNAc .**GRAPHIC**. Gal .**GRAPHIC**. GlcNAc .**GRAPHIC**. Gal .fwdarw. GalNAc .fwdarw. Ser(Thr).