Identification of the 183RWTNNFREY191Region as a Critical Segment of Matrix Metalloproteinase 1 for the Expression of Collagenolytic Activity

Hideaki Nagase,Linda Chung,Ken-ichi Shimokawa,Deendayal Dinakarpandian,Gregg B. Fields,Frank Grams

doi:10.1074/jbc.m004039200

Abstract

Matrix metalloproteinase 1 (MMP-1) cleaves types I, II, and III collagen triple helices into (3/4) and (1/4) fragments. To understand the structural elements responsible for this activity, various lengths of MMP-1 segments have been introduced into MMP-3 (stromelysin 1) starting from the C-terminal end. MMP-3/MMP-1 chimeras and variants were overexpressed in Escherichia coli, folded from inclusion bodies, and isolated as zymogens. After activation, recombinant chimeras were tested for their ability to digest triple helical type I collagen at 25 degrees C. The results indicate that the nine residues (183)RWTNNFREY(191) located between the fifth beta-strand and the second alpha-helix in the catalytic domain of MMP-1 are critical for the expression of collagenolytic activity. Mutation of Tyr(191) of MMP-1 to Thr, the corresponding residue in MMP-3, reduced collagenolytic activity about 5-fold. Replacement of the nine residues with those of the MMP-3 sequence further decreased the activity 2-fold. Those variants exhibited significant changes in substrate specificity and activity against gelatin and synthetic substrates, further supporting the notion that this region plays a critical role in the expression of collagenolytic activity. However, introduction of this sequence into MMP-3 or a chimera consisting of the catalytic domain of MMP-3 with the hinge region and the C-terminal hemopexin domain of MMP-1 did not express any collagenolytic activity. It is therefore concluded that RWTNNFREY, together with the C-terminal hemopexin domain, is essential for collagenolytic activity but that additional structural elements in the catalytic domain are also required. These elements probably act in a concerted manner to cleave the collagen triple helix.

Highlights

Matrix metalloproteinase 1 (MMP-1) cleaves types I, II, and III collagen triple helices into 3⁄4 and 1⁄4 fragments
Similar attempts were made by Hirose et al [22] using matrix metalloproteinase (MMP)-8 as a model collagenase. Those studies showed the importance of the hemopexin domain in expressing collagenolytic activity in both MMP-1 and MMP-8
Studies by Hirose et al [22] indicated that the correct proline-rich linker is another important region for collagenolysis, because collagenolytic activity was not detected when the linker region of MMP-8 was replaced with that of MMP-3

Summary

EXPERIMENTAL PROCEDURES

Materials—4-Aminophenylmercuric acetate (APMA) was purchased from ICN Biochemicals. Chymotrypsin and phenylmethylsulfonyl fluoride were from Sigma. Using the sense T7 promoter primer and an antisense chimeric junction primer containing the MMP-3 sequence at the 5Ј-end and the MMP-1 sequence at the 3Ј-end (see Table I), the N-terminal part of the chimera containing primarily the MMP-3 sequence was made. Using an antisense primer annealing to the vector (pET3a primer) and a sense chimeric junction primer containing the MMP-1 sequence at the 5Ј-end and the MMP-3 sequence at the 3Ј-end (overlapping with the previous primer), the C-terminal part of the chimera containing primarily the MMP-1 sequence was made. A final polymerase chain reaction was performed using these two first round products as the template and the two flanking primers (T7 promoter and pET3a primers). Collagenase Assays—All chimeras were tested for their ability to digest pepsin-treated type I collagen at 25 °C, and products were analyzed on SDS-PAGE stained with Coomassie Brilliant Blue R250. Where [S0] and [E0] are the initial concentrations of substrate and enzyme, respectively

RESULTS

DISCUSSION

TABLE II Substrate specificities of MMP chimeras and mutants