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

Abstract

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 post-translational modifications of the CD44 standard receptor could play a pivotal role in regulating CD44-mediated functions in melanoma. Using metabolic inhibitors of N- and 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.

Sialylated Core 1 O-Glycans Influence the Sorting of Pmel17/gp100 and Determine Its Capacity to Form Fibrils

“…We hypothesize that sialic acid, when added to N-glycans on Pmel17, makes it hydrophilic and leads to the exposure of hydrophobic protein domains, as occurs after the cleavage of Pmel17; this would then favor the formation of polymers through hydrophobic interactions. In contrast, sialic acid added to O-glycans may have a dual role as follows: 1) carrying plasma membrane sorting signals, and 2) protecting Pmel17 against early cleavage and degradation, a function that has been reported previously in other cell lines (41,42). Several studies support these roles as follows.…”

“…Several studies support these roles as follows. The extent and type of O-glycans modified with sialic acid in the RPT domain of Pmel17 play key roles to determine the proper glycoform of Pmel17 destined to form fibrils (19) or to be targeted to the plasma membrane (41,42). Interestingly, the cNTF fragment reconstituted from E. coli has been shown to be amyloidogenic (58).…”

“…Because there is evidence that sialylated O-glycans can carry plasma membrane apical sorting information (41,42), we analyzed the intracellular distribution of Pmel17 in melanoma cells treated with DANA using immunofluorescence (Fig. 5C).…”

Noninvasive In Vivo Imaging and Biologic Characterization of Thyroid Tumors by ImmunoPET Targeting of Galectin-3

“…Cells were cultured in complete RPMI1640 culture medium in standard condition (29) and tested for integrity, Gal-3 expression, and tumorigenicity before each experimental procedure as reported elsewhere. All in vitro experiments using the aforementioned cell lines were repeated at least four times.…”

“…FRO82-1, WRO82-1, and BCPAP cell lysates were prepared using RIPA buffer (NaCl 150 mmol/L, Tri-HCl 50 mmol/L, Triton-100 2%, SDS 0.1%, EDTA 1 mmol/L) supplemented with 1% protease inhibitors (Sigma) as reported (29). Protein concentration was determined (BCA assay) and aliquots of cell lysate (38 mg) were loaded and resolved on 10% SDS-Precast gel (Bio-Rad Laboratories) under nonreducing conditions and then blotted onto polyvinylidene difluoride membrane (PVDF; Millipore).…”