Matrix metalloproteinase 2: involvement in keratoconus.
OBJECTIVE
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 compare the activities of the MMP-2 secreted by keratocytes of normal and keratoconic corneas.


METHODS
The spectrum of MMP-2 activities secreted by cultures of keratocytes derived from normal and keratoconic corneas was analysed by zymography. Subsequently, selected preparations were assayed for peptidase activity, using Type I, Type III, Type IV and Type V collagen as substrate, under native conditions and after treatment with a variety of putative activating reagents.


RESULTS
Although MMP-2 of Mr 65,000 on SDS gelatin polyacrylamide gels is the major protease secreted by keratocytes of normal corneas, the keratocytes of early-phase keratoconic corneas secrete an additional zymographic activity of Mr 61,000. From their N-terminal amino acid sequences, both these proteins were shown to be conformers of proMMP-2. Treatment with SDS followed 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 Mr 43,000 that selectively hydrolysed Type IV and denatured Type 1 collagen.


CONCLUSIONS
The zymographic gelatinase activities of apparent Mr 65,000 and 61,000 are conformers of corneal proMMP-2. Activated enzyme, of Mr 43,000, is more readily generated from protein preparations of the culture media of early phase keratoconic corneal keratocytes than from protein preparations of the culture media of normal corneal keratocytes.
Smith
V A
VA
Department of Ophthalmology, Bristol Eye Hospital, University of Bristol, UK.
Easty
D L
DL
D002478:Cells, Cultured; D002850:Chromatography, Gel; D003315:Cornea / Q000201:enzymology*; D046508:Culture Techniques / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D007640:Keratoconus / Q000201:enzymology*; D020778:Matrix Metalloproteinase 2 / Q000737:chemistry / Q000378:metabolism*; D008970:Molecular Weight; D020539:Sequence Analysis, Protein
D020778:Matrix Metalloproteinase 2