title: 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.
abstract: OBJECTIVE
  We have observed that Western blot analysis with an anti-G1 antibody detects G1-NITEGE product in a disintegrin and metalloprotease with thrombospondin motifs-4 (ADAMTS4)-digested fetal and mature human and bovine aggrecan, but the neoepitope-specific anti-NITEGE antibody only detects this product in digests of mature aggrecan. Our objective was to determine whether enzymatic removal of O- and/or N-linked oligosaccharides from the fetal products would enable detection of the NITEGE neoepitope with anti-NITEGE antibody.
  
  
  METHODS
  Aggrecan was purified from fetal and mature human and bovine cartilage and digested with: (1) ADAMTS4, (2) ADAMTS4, sialidase II, and N-glycanase, (3) ADAMTS4, sialidase II, and O-glycanase, or (4) ADAMTS4, sialidase II, and both N- and O-glycanases. Western blot analysis was performed using anti-G1 and anti-NITEGE antibodies.
  
  
  RESULTS
  When fetal G1-NITEGE products were treated with a combination of ADAMTS4, sialidase II, O-glycanase and N-glycanase, the resultant products migrated faster on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the NITEGE neoepitope was rendered detectable.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610-0136, USA.
authors.forename: J E
authors.initials: JE
authors.lastname: Frank
authors.forename: V P
authors.initials: VP
authors.lastname: Thompson
authors.forename: M P
authors.initials: MP
authors.lastname: Brown
authors.forename: J D
authors.initials: JD
authors.lastname: Sandy
mesh_terms: D051722:ADAM Proteins / Q000378:metabolism*; D000071121:ADAMTS4 Protein; D000368:Aged; D053674:Aggrecans / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002358:Cartilage, Articular / Q000187:drug effects / Q000378:metabolism; D002417:Cattle; D010450:Endopeptidases / Q000378:metabolism*; D016326:Extracellular Matrix Proteins; D005333:Fetus; D006801:Humans; D009844:Oligosaccharides / Q000378:metabolism*; D011348:Procollagen N-Endopeptidase / Q000378:metabolism*; D011509:Proteoglycans; D000099249:Proteoglycan Link Protein
chemical_list: D051722:ADAM Proteins; D000071121:ADAMTS4 Protein; D053674:Aggrecans; D010450:Endopeptidases; D016326:Extracellular Matrix Proteins; D009844:Oligosaccharides; D011348:Procollagen N-Endopeptidase; D011509:Proteoglycans; D000099249:Proteoglycan Link Protein; C000603918:ADAMTS4 protein, human; C083641:aggrecanase