Monoclonal Antibodies with Defined Recognition Sequences in the Stem Region of CD44: Detection of Differential Glycosylation of CD44 between Tumor and Stromal Cells in Tissue

CD44 is an enigmatic cell adhesion molecule acting as a major receptor for hyaluronan and playing roles in many biological and pathological processes such as lymphocyte homing, T-cell activation, wound healing, angiogenesis, and metastatic spread of tumor cells. However, the complexity of the molecule, with its alternatively spliced variants, extensive glycosylation, and processing by different proteases, has hampered detailed analysis. In this study, we prepared four monoclonal antibodies (285-2F12, 284-43F1, 268-1F5, and 294-6F2) and one polyclonal antibody (C6) that recognize defined sequences in the stem region of CD44H. Interestingly, two of the monoclonal antibodies, 268-1F5 and 294-6F2, failed to recognize the CD44 expressed in five of the seven human tumor cell lines examined by Western blotting. Treatment of the samples with a combination of neuraminidase and O-glycosidase as well as the expression of mutants with site-directed mutations at possible modification sites rendered the CD44 reactive to the antibodies. Thus, the reactivity of the antibodies is sensitive to O-glycosylation presumably near the recognition site. Glycosylation of CD44 that affects reactivity to the antibodies was found to be regulated differentially between tumor and stromal cells in two breast and three oral carcinoma tissues. Antibody 268-1F5 reacted to the tumor cells, but not to the cells in the surrounding stroma. On the other hand, the reactivity of 294-6F2 to the cells was opposite between the two tumor types. Thus, these sets of antibodies are useful to detect and analyze the as-yet-unknown roles of site-specific glycosylation of CD44, particularly in tumors.