Identification of Structural Elements Important for Matrix Metalloproteinase Type V Collagenolytic Activity as Revealed by Chimeric Enzymes

Abstract

Digestion of type V collagen by the gelatinases is an important step in tumor cell metastasis because this collagen maintains the integrity of the extracellular matrix that must be breached during this pathological process. However, the structural elements that provide the gelatinases with this unique proteolytic activity among matrix metalloproteinases had not been thoroughly defined. To identify these elements, we examined the substrate specificity of chimeric enzymes containing domains of gelatinase B and fibroblast collagenase. We have found that the addition of the fibronectin-like domain of gelatinase B to fibroblast collagenase is sufficient to endow the enzyme with the ability to cleave type V collagen. In addition, the substitution of the catalytic zinc-binding active site region of fibroblast collagenase with that of gelatinase B increased the catalytic efficiency of the enzyme 3- to 4-fold. This observation led to the identification of amino acid residues, Leu(397), Ala(406), Asp(410), and Pro(415), in this region of gelatinase B that are important for its efficient catalysis as determined by substituting these amino acids with the corresponding residues from fibroblast collagenase. Leu(397) and Ala(406) are important for the general proteolytic activity of the enzyme, whereas Asp(410) and Pro(415) specifically enhance its ability to cleave type V collagen and gelatin, respectively. These data provide fundamental information about the structural elements that distinguish the gelatinases from other matrix metalloproteinases in terms of substrate specificity and catalytic efficiency.

Milk-borne lactocrine-acting factors affect gene expression patterns in the developing neonatal porcine uterus

“…Gelatinases MMP2 and MMP9 digest type IV and V collagens in basement membranes to facilitate tissue growth and remodeling (Zeng et al 1999, O'Farrell & Pourmotabbed 2000. Both MMP2 and MMP9 were detected in the murine uterus (Hu et al 2004) and human endometrial cells (Martelli et al 1993, Iwahashi et al 1996.…”

Matrix metalloproteinase 9 modulates collagen matrices and wound repair

“…SHG imaging reveals Mmp9-dependent collagen remodeling in the hai1 morphants It is known that Mmp9 alters the ECM through the cleavage of matrix components, including collagen (Collier et al, 1988;O'Farrell and Pourmotabbed, 2000;Van den Steen et al, 2002). To determine whether Mmp9 modulates the collagen matrix in hai1 morphants we performed SHG imaging (Campagnola et al, 2002;Mohler et al, 2003).…”

The Treatment of Breast Cancer Tumor Growth and Metastasis With an Anti-MMP9 DNAzyme

“…The Kcat/Km values for the fluorogenic peptide substrate were then obtained by assaying the enzymes at 23ºC using 1.5 M peptide substrate as previously described [99]. Assays were conducted under the condition where K m >> [S], and K cat /K m values were calculated from the following equation:…”

“…MMP-9 has an additional domain known as the gelatin binding domain composed of 3 homology fibronectin-type II modules of 58 amino acids inserted into the catalytic domain ( Figure 5-1b) [98]. We have shown that this domain is responsible for the type V collagen activity of the enzyme [40,99]. The carboxyl-terminal domain of MMP-9 is involved in anchoring the enzyme to CD44 [100][101][102] and binds to heparin [103].…”

“…The 5' primer used in all of the above PCR reactions was 5'-AAG GAG ATA TAC ATA TGC GGG CGA AAC TCC TAG GAA TAG-3'. All the PCR products were sequenced by Dideoxy method [99] and were found to be free of any secondary mutations. The resulting PCR products were digested with XbaI and SalI and ligated to NheI /SalI fragment of pET-12C expression vector.…”