Biochemical characterization of Matrix Metalloproteinase-9 Binding interactions with two small non-peptide inhibitors and the proteoglycan serglycin & Processing of serglycin

Abstract

The present work has a focus on biochemical characterization of the human matrix metalloproteinase-9. First part of the investigation focuses on the enzymes ability to form a complex with the small proteoglycan serglycin. Here the focus was on which amino acids and regions in both proMMP-9 and serglycin are important for the complex formation. For that, recombinant full-length proMMP-9 and various deletion variants of proMMP-9 were produced and purified as was intact serglycin from a cancer cell line. Methods used involved in vitro reconstitution of the complex, peptide arrays, docking and molecular dynamic studies. The second part has a focus on the catalytic site in MMP-9 and to which extent activation with various activators that generates not only different N-terminal ends of the activated enzyme, but also different C-terminal ends affect the enzymes binding of small non-peptide inhibitors. It was shown that the differences in the N- and C-terminal residues of the activated enzyme did not affect the binding of two small active site inhibitors. Methods used were inhibition kinetics and molecular modelling. The third study has investigated if the proteoglycan serglycin is a substrate for MMP-9. This was tested on a commercial his-tagged serglycin (Ht-SG) produced in E-Coli and hence lacked CS-chains and on intact and cABC treated serglycin from THP-1 cells. cABC degrades CS-chains and leaves a short stub on the core protein. It was shown that active MMP-9 cleaved the core protein of intact serglycin with and without CS-chains, although the cleavage pattern was different. Cleavage of Ht-SG with different deletion variants of MMP-9 showed that one variant produced a different cleavage pattern. This suggested that there is an exosite for serglycin in MMP-9, which presents the substrate to the catalytic site of the enzyme.