A Study of the Collagen-binding Domain of a 116-kDaClostridium histolyticum Collagenase

Abstract

The Clostridium histolyticum 116-kDa collagenase consists of four segments, S1, S2a, S2b, and S3. A 98-kDa gelatinase, which can degrade denatured but not native collagen, lacks the C-terminal fragment containing a part of S2b and S3. In this paper we have investigated the function of the C-terminal segments using recombinant proteins. Full-length collagenase degraded both native type I collagen and a synthetic substrate, Pzpeptide, while an 88-kDa protein containing only S1 and S2a (S1S2a) degraded only Pz-peptide. Unlike the fulllength enzyme, S1S2a did not bind to insoluble type I collagen. To determine the molecular determinant of collagen binding activity, various C-terminal regions were fused to the C terminus of glutathione S-transferase. S3 as well as S2bS3 conferred collagen binding. However, a glutathione S-transferase fusion protein with a region shorter than S3 exhibited reduced collagen binding activity. S3 liberated from the fusion protein also showed collagen binding activity, but not S2aS2b or S2b. S1 had 100% of the Pz-peptidase activity but only 5% of the collagenolytic activity of the fulllength collagenase. These results indicate that S1 and S3 are the catalytic and binding domains, respectively, and that S2a and S2b form an interdomain structure.Collagens are the major protein constituents of the extracellular matrix and the most abundant proteins in all higher organisms (1). The tightly coiled triple helical collagen molecule assembles into water-insoluble fibers or sheets which are cleaved only by collagenases, and are resistant to other proteinases. Various types of collagenases, which differ in substrate specificity and molecular structure, have been identified and characterized. Bacterial collagenases differ from vertebrate collagenases in that they exhibit broader substrate specificity (2, 3). Clostridium histolyticum collagenase is the best studied bacterial collagenase (4) and is widely used as a tissuedispersing enzyme (5, 6). This enzyme is unique in that it can degrade both water-insoluble native collagens and water-soluble denatured ones, can attack almost all collagen types, and can make multiple cleavages within triple helical regions (4). Kinetic studies of collagenases have provided insight into the high-ordered structure of collagens (7,8). However, the structure-function relationship of this unique enzyme is not known.

Substrate Recognition by the Collagen-binding Domain ofClostridium histolyticum Class I Collagenase

“…However, an isolated C-terminal 1 The abbreviations used are: ColG, C. histolyticum type I collagenase; ColH, C. histolyticum type II collagenase; CBD, collagen-binding domain; MALDI-TOF MS, matrix-assisted laser desorption time of flight mass spectrometry; CD, circular dichroism; HPLC, high performance liquid chromatography; BSA, bovine serum albumin; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; aa, amino acid(s); GST, glutathione S-transferase; PCR, polymerase chain reaction; BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis; CB buffer, collagen binding buffer; Hyp and O, three-letter code and one-letter code of 4-hydroxy-L-proline residue. fragment, S2bϩS3 from ColH, bound to lyophilized insoluble collagen to the same extent as full-length ColH (6). This observation lead us to conclude that this C-terminal fragment contains the collagen-binding domain (CBD).…”

“…1A (see below). A truncated ColH consisting of only S1 showed full hydrolytic activity on a peptide substrate (6). When a consensus motif for zinc proteases, HEXXH, present in S1 was altered, catalytic activity was drastically reduced (7).…”

“…The water phase was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). When necessary, the salt concentration in CB buffer was modified ( Semi-quantitative measurements of binding were carried out as described previously (6), where the amount of the unbound fusion protein was quantitated by GST assay using 1-chloro-2,4-dinitrobenzene as the substrate.…”

The FAXWXXT motif in the carboxyl terminus of Vibrio mimicus metalloprotease is involved in binding to collagen

“…To date, the collagen-binding regions of bacterial collagenases have been well characterized in the ColG and ColH collagenases from Clostridium histolyticum [22]. According to Matsushita et al [28], clostridial collagenases contained four segments in their molecules and were classified into class I and class II by homology comparison of each segment.…”

“…The collagen-binding assay was performed following the methods of Matsushita et al [22]. For each replicate sample, approximately 10 mg of insoluble type I collagen from bovine achilles tendon (Sigma Chemical Co., St. Louis, MO) were pre-swelled in 50 mM Tris-HCl, pH 8.0, containing 5 mM CaCl 2 and then centrifuged at 10 000 · g for 10 min.…”