Structural variability of CD44v molecules and reliability of immunodetection of CD44 isoforms using mAbs specific for CD44 variant exon products

CD44 can be considered structurally and functionally one of the most variab le surface molecules. Alternative splicing of variant exons as well as post translational modifications of the molecule (differences in glycosylation) generate a rich repertoire of CD44 isoforms (CD44v), some of which seem to play a key role in tumor growth and progression. Immunodetection of CD44 is oforms in vivo, using mAbs specific for CD44 variant exon products, is larg ely used to identify those CD44 molecules involved in tumor growth and prog ression and to interfere with CD44-mediated processes, In the present work we demonstrate that the immunoreactivity of some mAbs directed to CD44 exon -specific epitopes can be impaired by the structural variability of the mol ecule. Our findings demonstrate that (1) specific exon assortment and/or po sttranslational modifications of CD44v molecules can mask CD44 exon-specifi c epitopes; (2) glycosaminoglycan side chains, carried by some CD44v isofor ms of high molecular weight, may play a critical role in determining the ex act conformation of the molecule, which is necessary for the detection of C D44 variant epitopes by specific mAbs; and (3) in a panel of stable transfe ctants expressing CD44 N-glycosylation site-specific mutants, generated in the constant region of CD44 extracellular domain, asparagine-isoleucine sub stitution is sufficient per se to impair the immunoreactivity of several mA bs to pan-CD44. Thus, conformational changes due to the alternative splicin g of CD44 variant exons and/or posttranslational modifications of the molec ule (different degree of glycosylation), which are cell type-specific, are Likely to generate CD44 variants that elude immunodetection. These findings strongly suggest that immunohistochemical analysis of CD-44 expression ill vitro and in vivo, using mAbs specific for CD44 variant exon epitopes, can potentially be impaired by a large number of false negative results.