Matrix metalloproteinase 2: involvement in keratoconus

PURPOSE. The activation of matrix metalloproteinase-2 (MMP-2) is postulated to be a crucial pathogenic factor behind progressive and chronic diseases in which basement membranes are disrupted. An ocular example is keratoconus . The purpose of the present enquiry was therefore to investigate and compa re the activities of the MMP-2 secreted by keratocytes of normal and kerato conic corneas. METHODS. The spectrum of MMP-2 activities secreted by cultures of keratocyt es derived from normal and keratoconic corneas was analysed by zymography. Subsequently, selected preparations were assayed for peptidase activity usi ng Type I, Type III, Type IV and Type V collagen as substrate, under native conditions and after treatment with a variety of putative activating reage nts. RESULTS. Although MMP-2 of M-r 65,000 on SDS gelatin polyacrylamide gels is the major protease secreted by keratocytes of normal corneas, the keratocy tes of early-phase keratoconic corneas secrete an additional zymographic ac tivity of M-r 61,000. From their N-terminal amino acid sequences, both thes e proteins were shown to be conformers of proMMP2. Treatment with SDS follo wed by protein fractionation was required to achieve in vitro activation of the MMP-2 secreted by normal corneal keratocytes. Treatment with SDS alone partially activated the enzyme produced by early-phase keratoconic corneal keratocytes. This procedure and autocatalysis, yielded an enzyme of M-r 43 ,000 that selectively hydrolysed Type IV and denatured Type 1 collagen. CONCLUSIONS. The zymographic gelatinase activities of apparent Mr 65,000 an d 61,000 are conformers of corneal proMMP-2. Activated enzyme, of M-r 43,00 0, is more readily generated from protein preparations of the culture media of early phase keratoconic corneal keratocytes than from protein preparati ons of the culture media of normal corneal keratocytes.