CD44s adhesive function spontaneous and PMA-inducible CD44 cleavage are regulated at post-translational level in cells of melanocytic lineage

Adhesion between the CD44s receptor and hyaluronic acid plays an important role in cell migration, tumour growth and progression. Although the alternative splicing of CD44 variant exons represents the principal regulatory mechanism of CD44-mediated functions, CD44v spliced variants are scantily expressed in melanoma cells. For this reason, we have investigated the possibility that posttranslational modifications of the CD44 standard receptor could play a pivotal role in regulating CD44-mediated functions in melanoma. Using metabolic inhibitors of Nand O-glycosylation, as well as melanoma transfectants expressing CD44s O-glycosylation site-specific mutants, we performed structural and functional analysis of N- and O-deglycosylated CD44s molecules expressed in melanoma cells. We discovered that complete N- and O-glycosylation is not required by CD44s to be correctly expressed on the melanoma cell surface. Indeed, variably glycosylated and functionally different CD44s molecules were constitutively expressed in primary and metastatic lesions. Furthermore, we observed that changes in N- and O-glycosylation of CD44s could modulate its cleavage. In fact, spontaneous CD44s shedding was dependent on the presence of partial or complete O-glycosylation of four serine-glycine motifs localized in the membrane-proximal CD44 ectodomain. Mutation of these serine residues, as well as an extensive metabolic O-deglycosylation, strongly impaired spontaneous CD44 shedding. Furthermore, an O-glycosylation-independent mechanism of CD44 cleavage has been identified. This alternative mechanism of receptor cleavage is phorbol 12-myristate-13-acetate (PMA) inducible, mediated by metalloproteinase and requires the presence of N-linked sugar residues. Our findings demonstrate that the post-translational modification of CD44s represents the principal regulatory mechanism of CD44s-mediated functions in melanoma.