Enzymatic analysis of matrix metalloproteinases using heterotrimeric and homotrimeric type I collagen as a substrate [abstract]

Abstract

The goals of this study are to determine if specific MMPs differentially degrade heterotrimeric and homotrimeric type I collagen, and the role of MMPs in the oim collagen glomerulopathy. Type I collagen is typically heterotrimeric, consisting of two proalpha1(I) chains and one proalpha2(I) chain. The oim model expresses homotrimeric type I collagen consisting of three alpha1 chains and is functionally null for the proalpha2(I) chain. This project is designed to examine the mechanisms involved in the abnormal deposition of type I collagen in the renal mesangium of oim mice. Production of type I collagen is regulated by synthetic mechanisms and matrix metalloproteinases (MMPs), which degrade type I collagen. The balance between synthesis and degradation of type I collagen is impaired and is hypothesized to be responsible for increased type I collagen deposition in the oim glomerulus. In this study, we compare heterotrimeric and homotrimeric collagen degradation by MMPs to determine whether specific MMPs differentially degrade heterotrimeric and homotrimeric type I collagen. MMPs found in the kidneys include MMP-1, 2, 3, 9, and 13. Zymography gels incorporating heterotrimeric collagen were produced to analyze enzymatic activity for specific MMPs. A large amount of collagen is required for use in zymography. We demonstrate greater collagen extraction is achieved from wildtype versus oim tails, and greater amounts are extracted from tails of younger animals, despite smaller amounts of tail tendon tissue. In addition, we evaluate the proteolytic activity of specific MMPs by in vitro analysis using homotrimeric and heterotrimeric type I collagen as substrates.