Matrix metalloproteinases are active following guanidine hydrochloride extraction of cartilage: generation of DIPEN neoepitope during dialysis.
We have recently observed marked increases in MMP-derived aggrecan fragments in extracts of cartilage stimulated with IL-1. The fragments were detected with an anti-DIPEN neoepitope antibody that is specific for fragments generated by MMP cleavage at the DIPEN(341) F(342)FGVG site. Because our results contrasted with another study, we systematically compared our methods with other published methods. We now report that DIPEN(341) neoepitope can be generated post-culture, by dialysing GuHCl(1)-denatured samples against unbuffered, deionized water at 4 degrees C. We show that EDTA must be included in the GuHCl extractant, as well as the dialysis buffer, in order to block post-culture processing of aggrecan by MMPs.
Stanton
Heather
H
University of Melbourne, Department of Paediatrics, Cell & Matrix Biology Research Unit and Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, 3052, Australia.
Fosang
Amanda J
AJ
D053674:Aggrecans; D000818:Animals; D002356:Cartilage / Q000737:chemistry / Q000201:enzymology*; D046508:Culture Techniques; D003956:Dialysis; D000939:Epitopes / Q000096:biosynthesis / Q000276:immunology; D016326:Extracellular Matrix Proteins; D019791:Guanidine; D037181:Lectins, C-Type; D020782:Matrix Metalloproteinases / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis / Q000096:biosynthesis; D011509:Proteoglycans / Q000032:analysis* / Q000096:biosynthesis; D013552:Swine; D014020:Tissue Extracts / Q000737:chemistry*
D053674:Aggrecans; D000939:Epitopes; D016326:Extracellular Matrix Proteins; D037181:Lectins, C-Type; D010446:Peptide Fragments; D011509:Proteoglycans; D014020:Tissue Extracts; D020782:Matrix Metalloproteinases; D019791:Guanidine