Removal of O-linked and N-linked oligosaccharides is required for optimum detection of NITEGE neoepitope on ADAMTS4-digested fetal aggrecans: implications for specific N-linked glycan-dependent aggrecanolysis at Glu373-Ala374

It appears that the NITEGE neoepitope is blocked on Western blots by oligosaccharide structures present on Asn368 and Thr370 of fetal human and bovine aggrecans. Such masking structures do not appear to be present on mature aggrecans from these species. We suggest that when anti-NITEGE antibody is used in Western analysis, enzyme-linked immunosorbent assay (ELISA), fluorescence-activated cell sorting (FACS), and immunohistochemistry (IHC), removal of oligosaccharides with appropriate glycosidases may unmask reactivity that would otherwise go undetected. The implications of these findings for the much-studied effect of Asn368-linked keratan sulfate (KS)-based structures on ADAMTS4 and ADAMTS5 activity are discussed.