IMMUNOLOCALIZATION OF THE CLEAVAGE OF THE AGGRECAN CORE PROTEIN AT THE ASN(341)-PHE(342) BOND, AS AN INDICATOR OF THE LOCATION OF THE METALLOPROTEINASES ACTIVE IN THE LYSIS OF THE RAT GROWTH-PLATE

In view of the extensive lysis of hyaline cartilage known to take plac e during endochondral bone formation, the current study was designed t o test the hypothesis that metalloproteinases are the agents that medi ate this lysis. Since these enzymes have been shown in vitro to cleave the core protein of the major proteoglycan of cartilage, aggrecan, at the Asn(341)-Phe(342) bond, an immunohistochemical method has been de veloped to find out whether or not there are sites in the growth plate of the rat tibia where cleavage of this bond takes place. The cleavag e of aggrecan by metalloproteinases is followed by the retention of th e fragment known as G1, for it includes the G1 domain. Since the G1 fr agment terminates in the amino acid residues ...FVDIPEN, we prepared a n antiserum against FVDIPEN, confirmed its specificity, then applied i t to the growth plate of 21-day-old rat tibia in the hope of localizin g the GI fragments. The antiserum specificity was shown by its recogni tion of the ...FVDIPEN sequence at the C-terminus of peptides and of G 1 fragments produced by aggrecan cleavage. When the antiserum was appl ied to Western blots of guanidinium chloride extracts prepared from ep iphyseal growth plate, it recognized two species (56 and 52 kDa), whic h differed only in the degree of glycosylation. These fragments were c omparable in size to the G1 fragments generated by the action of recom binant metalloproteinase in vitro, thus confirming antiserum specifici ty for these fragments. Applying the antiserum to cryosections of 21-d ay-old rat tibiae revealed immunostaining at two intensities within th e growth plate matrix: a strong staining was observed in a 1-5 mu m-wi de layer designated ''peripheral'' matrix, which borders the epiphysea l and metaphyseal marrow spaces as well as the perichondrium, while a weak staining was found in the rest of the plate, designated ''central '' matrix. The abundance of G1 fragments terminating in ...FVDIPEN in the peripheral matrix indicates that this is where the growth plate is lysed to achieve longitudinal and latitudinal bone growth. The site w here metalloproteinases exert their main lytic activity is a thin laye r of matrix separating central from peripheral matrix. (C) 1998 Wiley- Liss, Inc.