Glycoproteins in culture medium: a comparison from cystic fibrosis and control skin fibroblasts

Scanlin, T.F.; Voynow, J.A.; Thomas, E.J.; Glick, M.C.

Biochemistry 21(3): 491-497


ISSN/ISBN: 0006-2960
PMID: 7066301
DOI: 10.1021/bi00532a012
Accession: 005530420

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High MW glycoproteins were partially purified by 4 methods from the medium after growth of human skin fibroblasts. One of these methods, precipitation by heparin, yields one major glycoprotein, MW 220,000, when examined by polyacrylamide gel electrophoresis under denaturing conditions. The glycoprotein was labeled with L-[3H]fucose or D-[3H]glucosamine and yielded glycopeptides after digestion with Pronase. Using this method of precipitation, a comparison was made of matched cystic fibrosis (CF) and control human skin fibroblast media with and without 10% fetal calf serum, a nutritive requirement for growth. The profilies of the [3H]fucose-labeled material from media of both cell types were similar after polyacrylamide gel electrophoresis regardless of the procedure used to obtain them. In contrast, differences were seen in the composition of the material obtained by precipitation with heparin, which suggested differences in glycosylation by CF fibroblasts. In addition, differences were noted which were related to the presence or absence of fetal calf serum in the growth medium. The analysis showed that when the CF and control materials were compared, the heparin precipitate of the CF medium without fetal calf serum contained less protein and carbohydrate, 30-35% less radioactivity/ml of protein, a lowered fucose content, higher sialic acid content in confluency and lower turnover when expressed as cpm/nmol of fucose. When the medium from the CF fibroblasts contained fetal calf serum, the heparin-precipitated material had more radioactivity and a higher ratio of fucose to other monosaccharides than that from control cells. Thus, there was either a differential response of the CF and control fibroblasts grown in the presence of 10% fetal calf serum or a differential recruiment of other glycoproteins during the precipitation of the serum-containing medium. The results are discussed in relation to previous findings of the monosaccharide content of CF fibroblast membranes and CF secretions.