CD44 substituted with heparan sulfate and endo-beta-galactosidase-sensitive oligosaccharides: a major proteoglycan in adult human epidermis

Abstract

CD44 is a group of cell surface glycoproteins that is generated from a single gene by mRNA splice variation. Its functions in matrix adhesion and tumor invasion are strongly influenced by glycosylation. We studied the glycosylated tissue forms of CD44 from extracts of normal adult human epidermis by using western blotting and immunoprecipitation from short-term skin organ cultures. An antibody for CD44 (Hermes 3) precipitated 7-17% of all 35SO4-labeled proteoglycans (PGs) synthesized in epidermis. Immunoprecipitates digested with heparitinase lost 40-68% of incorporated 35SO4 and 24-40% of [3H]glucosamine, indicating that heparan sulfate was the predominant glycosaminoglycan in epidermal CD44. Chondroitinase ABC released 10-25% and 6-12% of 35SO4 and [3H]glucosamine, respectively. Less than 5% of both isotopes were susceptible to keratanase. Five to 33% of 35SO4 and 26-37% of [3H]glucosamine, however, was released by endo-beta-galactosidase, implying marked substitution by oligosaccharides with N-acetyllactosamine repeats. Heparitinase pretreatment retarded, whereas endo-beta-galactosidase enhanced the mobility of the > or = 180-kDa polydisperse CD44 on agarose gel electrophoresis. On SDS-polyacrylamide gel electrophoresis, however, western blotting and fluorographs of 35SO4-labeled immunoprecipitates showed the main CD44 isoform at > or = 250 kDa and a shift to 180-200 kDa after heparitinase treatment. Keratanase, keratanase II, and chondroitinase ABC had minor effects. A less abundant form of CD44, with a core of 100 kDa, partly substituted with chondroitinase ABC- and endo-beta-galactosidase-sensitive chains, was also present. Therefore, the large heparan sulfate-substituted CD44 forms a significant part of all proteoglycans in normal human epidermis. Both the large and the 100-kDa variant of epidermal CD44 contain endo-beta-galactosidase-sensitive oligosaccharides not previously noted in other cells or tissues.