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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 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.
Insights
Matrix metalloproteinase-2 (MMP-2) activation is key in diseases like keratoconus. Keratoconus corneal cells more readily activate MMP-2, suggesting its role in corneal basement membrane disruption.
Area of Science:
- Ophthalmology
- Biochemistry
- Cell Biology
Background:
- Matrix metalloproteinase-2 (MMP-2) activation is implicated in progressive diseases involving basement membrane disruption.
- Keratoconus, an ocular disease, involves corneal basement membrane abnormalities.
Purpose of the Study:
- To investigate and compare the activities of MMP-2 secreted by keratocytes from normal and keratoconic corneas.
- To understand the pathogenic role of MMP-2 in keratoconus.
Main Methods:
- Zymography was used to analyze MMP-2 activity in keratocyte cultures from normal and keratoconic corneas.
- Peptidase activity assays were performed on collagen substrates (Types I, III, IV, V) under various activation conditions.
Main Results:
- Keratocytes from early-phase keratoconic corneas secreted an additional proMMP-2 conformer (Mr 61,000) compared to normal corneal keratocytes (Mr 65,000).
- Activated MMP-2 (Mr 43,000) was more readily generated from keratoconic corneal keratocyte cultures than from normal ones.
- The activated enzyme selectively hydrolyzed Type IV and denatured Type I collagen.
Conclusions:
- The observed gelatinase activities represent conformers of corneal proMMP-2.
- Easier activation of MMP-2 from keratoconic corneal keratocytes suggests its significant role in keratoconus pathogenesis.
- Findings highlight MMP-2's potential as a therapeutic target in keratoconus.