MOLECULAR AND BIOCHEMICAL CHARACTERIZATIONS OF A MATRIX METALLOPROTEINASE GENE FROM Xenopus laevis

Abstract

Matrix metalloproteinases (MMPs) are extracellular enzymes that are capable of degrading various components of the extracellular matrix. They participate in extracellular matrix remodeling and degradation and have been implicated as important for amphibian metamorphosis. The process of tadpole-frog transition during frog development systematically transforms all tissues and organs of a tadpole to those of an adult frog and is regulated by the endogenous thyroid hormone (TH). A number of genes which are regulated by TH have been cloned from metamorphosing Xenopus laevis tadpoles. One of these genes encodes a putative MMP. Deduced amino acid sequence comparison with other known MMPs showed homologies with different MMPs, and a slightly higher level of homology with human collagenase-1. In order to determine its true identity, the region which encodes the proenzyme of this putative MMP was subcloned into an expression vector with an N-terminal polyhistidine tag. High levels of expression of the fusion protein were achieved in E. coli. The fusion protein was purified under denaturing conditions from a total cellular protein lysate and then renatured by dialysis in a series of renaturing buffers. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that the purified full-length protein was >85% pure with the expected size of 56 Kd. In vitro biochemical studies have demonstrated that the purified protein had identical enzymatic activity on type I collagensubstrates like human interstitial collagenase. Since this Xenopus collagenase only shares 54% identity with human collagenase-1, it is designated as a new member of the subclass of collagenases, i.e. Xenopus collagenase-4 (xCo14). The xCol4 is the first cloned amphibian MMP that has been biochemically demonstrated to be a collagenase.