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Structure-Function Relationship of Human Neutrophil Collagenase

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Structure-Function Relationship of Human Neutrophil Collagenase Proc. Natl. Acad. Sci. USA Vol. 90, pp. 2569-2573, April 1993 Biochemistry Structure-function relationship of human neutrophil collagenase: Identification of regions responsible for substrate specificity and general proteinase activity (matrix metalloproteinase/stromelysin/extracellular macromolecule) TOMOHIKO HIROSE*, CHRISTY PATTERSON*, TAYEBEH POURMOTABBEDt, CARLO L. MAINARDIt, AND KAREN A. HASTY*t Departments of *Anatomy and Neurobiology, and of tMedicine, University of Tennessee, Memphis, TN 38163 Communicated by Jerome Gross, December 16, 1992 (received for review September 10, 1992) ABSTRACT The family of matrix metalloproteinases is a identity and 70% chemical similarity with human neutrophil family ofclosely related enzymes that play an inportant role in collagenase (MMP-8; NC). Preliminary data from other lab- physiological and pathological processes of matrix degrada- oratories have pointed toward the COOH-terminal domain as tion. The most distinctive characteristic of interstitial collage- a major determinant in substrate specificity. Windsor et al. nases (fibroblast and neutrophil collagenases) is their ability to (16) have demonstrated that the cysteine residue immediately cleave interstitial coilagens at a single peptide bond; however, adjacent to the COOH terminus was essential for colla- the precise region of the enzyme responsible for this substrate genolytic activity of fibroblast collagenase (FC). Recently, specificity remains to be defined. To address this question, we Murphy et al. (17) have suggested that both COOH- and generated truncated mutants of neutrophil collagenase with NH2-terminal domains ofthe active enzyme contribute to the various deletions in the COOH-terminal domain and chimeric substrate specificity of collagenase and proposed a possible molecules between neutrophil collagenase and stromelysin and role of the COOH-terminal domain in substrate binding. Our assayed the expressed enzymes against type I collagen and the data presented herein support the concept that the COOH- general substrate, casein. Our data suggest that substrate terminal domain of NC plays a major role in substrate specificity for interstitial collagen is determined by a 16-aa specificity. In addition, we have identified two loci in the sequence in the COOH-terminal domain of neutrophil colla- COOH-terminal domain, one governing collagenolytic activ- genase and is influenced by the integrity of a disulfide-defined ity and the other essential for general proteinase activity. loop at the COOH terminus for maximal activity. It was found that a relatively large region of 62-aa residues influenced the relative efficiency of collagenolytic activity. In addition to the MATERIALS AND METHODS region that conferred this specificity, a site at the COOH side Plasmid Construction ofTruncated Mutants ofNC (TrNCs). ofthe presumptive zinc-binding locus was found to be necessary The NC 7.2 cDNA containing a full-length coding region for for general catalytic activity. Mutation of a critical aspartic NC was used to create TrNCs with various deletions in the residue at position 253 within this area resulted in complete loss COOH-terminal sequence (6). The size of the TrNC is of proteolytic activity, suggesting that Asp-253 might function identified by amino acid residue numbers starting from the as one ofthe ligands for divalent cations, which are essential for initiating Met (Fig. 1). A premature stop codon was intro- enzymatic activity. duced by PCR. The primer (5'-GCTCGAATTCGGGC- TCGCCAGGGAAGGGCCCTACCC-3') complementary to The family of matrix metalloproteinases (MMPs) is a family the 5' end of NC 7.2 incorporated a unique EcoRI restriction of closely related enzymes that play an important role in a site and was used for construction of all the mutants. Primers variety ofphysiological and pathological processes, including at the 3' end contained sequences for a stop codon at various embryonic development (1), tumor invasion (2), and arthritis intervals and a unique Not I restriction site. The isolated (3, 4). The human MMP gene family contains at least two fragments were digested with EcoRI and Not I and then distinct interstitial collagenases (5, 6), three types of strome- ligated into these sites in the expression vector pcDNA I lysins (7-9), putative metalloproteinase 1 (10), and two ge- (Invitrogen, San Diego). The plasmid DNA was purified from latinases, 72-kDa type IV collagenase (11, 12) and 92-kDa transformed MC1061.P3 by CsCl gradient ultracentrifuga- type V collagenase (13, 14). When the primary structures of tion, and each PCR fragment was sequenced by the dideox- MMPs are compared, it is apparent that they are structurally ynucleotide chain termination method (Sequenase kit; homologous molecules consisting of defined functional do- United States Biochemical) to confirm that there was no error mains (13, 15). All of these enzymes contain an essential introduced by PCR. catalytic zinc-binding domain and an NH2-terminal domain, Plasmid Construction of Chimeric Enzymes. Junctional which functions to preserve the latent state of the enzyme. A sites between collagenase and Strom in the chimeras are variable COOH-terminal domain is present in all but putative identified by numbers referring to the last amino acid residue metalloproteinase 1. of NH2-terminal collagenase. A plasmid containing an insert Despite the high degree ofsimilarity among these enzymes, of cDNA for Strom (the kind gift of Constance E. Brincker- the MMPs studied to date are distinct with respect to their hoff, Dartmouth Medical School, Hanover, NH) was used to substrate specificity. Interstitial collagenases (MMP-1 and construct chimeric mutants between NC and Strom. A re- MMP-8) cleave native interstitial collagens. By comparison, striction enzyme site shared by the two enzymes at the same human stromelysin 1 (MMP-3; abbreviated as Strom) does locus was used for splicing their cDNA fragments. For not degrade interstitial collagens although it shares 52% Abbreviations: MMP, matrix metalloproteinase; NC, human neu- The publication costs of this article were defrayed in part by page charge trophil collagenase; Strom, human stromelysin 1; TrNC, truncated payment. This article must therefore be hereby marked "advertisement" mutant of NC; FC, fibroblast collagenase. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 2569 Downloaded by guest on September 26, 2021 2570 Biochemistry: Hirose et al. Proc. Natl. Acad. Sci. USA 90 (1993) Cys 91 Cys 279 Cys 464 demonstrated by casein zymograms as described, with the NC i (467 aa) -.-m-M c exception that casein (2 mg/ml) was incorporated into the polyacrylamide gel (4). TrNC 464 i., RESULTS I Substrate Specificity of TrNCs. In our initial experiments, TrNC 463 111101111 we constructed TrNCs with various deletions in the COOH terminus and transfected these mutants into COS-7 cells. The molecular mass of wild-type NC and the mutants TrNC 464 TrNC 262 _ and TrNC 463, lacking three and four terminal amino acid residues, respectively, was 66 kDa under reducing condi- FIG. 1. Schematic representation of the truncated mutants of tions. In contrast, TrNC 262, which lacks the COOH- NC. The size ofthe TrNC is identified by amino acid residue numbers starting from the initiating Met. The black boxes represent the terminal half of the molecule, migrated at 51 kDa. All the presumptive zinc-binding region, and the Cys residues (Cys-91, mutants showed catalytic activity against the general sub- Cys-279, and Cys-464) are indicated by vertical bars. strate casein; however, significant reduction in collagenolytic activity was seen with progressive deletion of the COOH- chimera NC 273-Strom, a 3' Aha II restriction site in NC was terminal sequence. TrNC 262 lost all collagenase activity generated by PCR. although it retained proteinase activity against casein (data Mutant NC-Strom-NC, in which the amino acid sequence not shown). TrNC 464 and TrNC 463 preserved collagenase of NC at positions 259-274 was replaced with the corre- activity as well as caseinase activity. Control cells trans- sponding polypeptide of Strom, was generated as follows. fected with the pcDNA I vector alone did not show any The primer 5'-ATGCCTGACGCCCAAACCCTGTGAC- collagenase or caseinase activities and were negative by CCCAGTTTGACA-3' was designed to create an Aha II site, Western blot analysis. To compare the specific activity, and the reverse primer was complementary to the 3'-flanking mutants and wild-type collagenases were quantitated by region of NC 7.2 cDNA. The amplified cDNA fragments image analysis ofWestern blots (Fig. 2). The activity ofTrNC coding for the COOH-terminal collagenase were digested 464 was essentially identical to wild-type NC; in contrast, with Aha II and Sph I and ligated with the 83-bp Bsm I-Aha removal of the COOH-terminal cysteine (TrNC 463) resulted II Strom fragment and with the 838-bp EcoRI-Bsm I se- in a loss of nearly 62% of collagenase activity. quence in pUC19 coding for the NH2-terminal half of NC. Substrate Specificity of the Recombinant Chimeric En- Plasmid Construction for Site-Directed Mutants of NC. zymes. The diminished collagenolytic activity of TrNC 262 Site-directed mutagenesis of Asp-252 and Asp-253 to Gly or and TrNC 463 confirmed the importance of the COOH- Glu was performed using PCR with mutated oligonucleotide terminal region. To preserve the tertiary configuration of the primers and cut with EcoRI and Bsm I to give an 838-bp COOH terminus, we constructed
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