Structural requirements for collagenolytic activity of matrix metalloproteinase 1 (MMP-1)

Abstract

Interstitial collagen types I, II and III are the major structural proteins in connective tissues such as skin, bone, cartilage, tendon and blood vessels. They consists of three chains with repeating Gly-X-Y triplets that adapts a left-handed poly-Pro II-like helical conformation, and each chain winds around each other in a gentle right-handed twist to form a triple helical structure. This triple helical conformation, about 3000 A long and 15 A in diameter, makes interstitial collagens resistant to most proteolytic enzymes in vertebrates, but it is readily cleaved by collagenases [1]. Collagenases are synthesized by many cell types such as fibroblasts, chondrocytes, keratinocytes, osteoblasts, endothelial cells, and macrophages and act on collagen at a neutral pH. These collagenolytic enzymes consist of collagenases (MMP-1, MMP-8, MMP-13, and MMP-18) [1], gelatinase A (MMP-2) [2], and MT1MMP (MMP-14) [3], all of which are members of the matrix metalloproteinase (MMP) family. They cleave interstitial collagens at a single site approximately 3/4 away from the N-terminus of the collagen molecule. Once collagen is cleaved, the resulting triple helical fragments denature at body temperature and are degraded by non-specific proteinases. Thus, the initial cleavage of triple-helical collagen is critical for the initiation of collagenolysis. A typical collagenase consists of an N-terminal propeptide of about 80 amino acids, a catalytic domain of about 170 amino acids, and a C-terminal hemopexin-like domain (CTD) of about 190 amino acids. The catalytic domain of MMP-1 lacking the CTD [MMPhas proteolytic activity but it cannot cleave triple-helical collagens [4]. This suggests that the CTD is essential for the expression of collagenolytic activity of MMP-1. On the other hand, MMP-3 (stromelysin 1) which shares 54% identity in amino acid sequence with MMP-1 does not cleave interstitial collagens even in its full-length form. Furthermore, when the catalytic domain of MMP-3 was linked with the CTD of MMP-1, it failed to express collagenolytic activity [5]. Since the catalytic domain of MMP-3 can cleave synthetic substrates harboring the collagenase cleavage sites of types I collagen at the same site as MMP-1, it is not clear why such a chimera fails to cleave collagen. Furthermore, the three-dimensional structure of collagenases [6] indicated that the substrate binding site of the enzyme is too narrow to accommodate the scissile bond of the triple helical collagen in the active site unless the triple helical collagen is locally unwound. To investigate regions or domains that participate in collagenolytic activity of collagenases we have attempted to transform non-collagenolytic MMP-3 to a collagenolytic enzyme by introducing various segments of MMP-1 sequence from the Cterminus and examined a functional gain in MMP-3. The ability of MMP-1 to unwind triple helical type I collagen was investigated by the generation of typical 3⁄4 and 1⁄4 fragments of the collagen by or in the presence of inactive MMP-1 whose Glu200 is mutated to Ala [MMP-1(E200A)].