Human Neutrophil Collagenase A DISTINCT GENE PRODUCT WITH HOMOLOGY TO OTHER MATRIX METALLOPROTEINASES*

We have identified and sequenced a cDNA encoding human neutrophil collagenase from a Agtll cDNA library constructed from mRNA extracted from the peripheral leukocytes of a patient with chronic gran- ulocytic leukemia. The library was screened with an oligonucleotide probe constructed from the putative zinc-binding region of fibroblast collagenase.

between residues 771 and 772 into TC* and TCB degradation products that further denature at 37 "C and become susceptible to degradation by a variety of other proteases (1). Skin fibroblast and synovial cell interstitial collagenases are well characterized enzymes that are identical (2, 3) while neutrophil collagenase has been reported to differ in substrate specificity (4) and immunological cross-reactivity (5). We have investigated the substrate specificity of neutrophil collagenase for the interstitial collagens and have shown the preference of the neutrophil enzyme for type I collagen in contrast with the greater susceptibility of type III collagen to digestion by fibroblast collagenase (6). Our immunological data suggest that while there are epitopes present in neutrophil collagenase which are distinct to this proteinase, other epitopes are shared with fibroblast collagenases (7). Although we have shown that polyclonal antibodies prepared to neutrophil collagenase cross-react significantly with fibroblast collagenase, we have identified a specific inhibitory monoclonal antibody to neutrophil collagenase which did not recognize synovial cell or fibroblast collagenase (8).
In contrast to human skin or synovial cells which constitutively synthesize and release collagenase, in the neutrophil the enzyme is synthesized and stored intracellularly in specific granules during the maturation of the neutrophil in the bone marrow (9). We have purified the secreted neutrophil collagenase and have shown the enzyme to have a molecular mass of 75 kDa (10). In addition, a smaller 57-kDa form of the enzyme, presumably the result of proteolytic processing, is often present.
In this communication, we present definitive evidence that neutrophil collagenase is a unique member of the family of matrix metalloproteinases. Analysis of the cDNA sequence and the derived amino acid sequence confirm the previous reports from ours and other laboratories that neutrophil collagenase is a distinct enzyme from fibroblast collagenase. Red column was eluted with a linear gradient 50 mM to 2 M NaCl, 50 mM Tris-HCl, pH 7.5. The fractions were dialyzed against 50 mM Tris-HCl, pH 7.2, buffer and assayed for activity using the collagen fibril assay described previously (15) and Western blotting with antibodies to neutrophil collagenase (10). Active fractions were pooled and applied onto a CM-52 cellulose column (1 X 5 cm). The void through this column was activated with 1 mM p-chloromercuribenzoate and applied onto a Pro-Leu-Gly-NHOH peptide affinity column. Briefly, the column was equilibrated with 50 mM Tris-HCl, pH 7.2, at 4 "C, 10 mM CaC&, 150 mM NaCl. The activated enzyme was then eluted with 100 mM Tris-HCl, pH 9, buffer containing 100 mM CaClz and 500 mM NaCl.

AND DISCUSSION
From the nucleotide sequence analysis of the 2.2-kb cDNA NC 7.2 clone we have deduced a 467-amino acid sequence (Fig. 1). This amino acid sequence contains the presumptive zinc-binding sequence VAAHEFGH which has 88% identity to a similar region in the metalloproteinases, fibroblast collagenase and stromelysin (16). Similar to other proteins, a signal peptide of 18 amino acids is present at the amino terminus. In addition to the coding sequences, the clone contains 70 nucleotides at the 5'-end of the cDNA and 751 nucleotides at the 3'-end which are untranslated. The identity of the clone as neutrophil collagenase was confirmed by comparison with the NHp-terminal sequences of isolated tryptic peptides of the purified enzyme.
In order to identify the species of mRNA complementary to this cDNA, we utilized mRNA extracted from human bone marrow cells, since granular constituents are synthesized during the maturation of the neutrophil. As a comparison we Human Neutrophil Collagenase performed parallel analyses using mRNA extracted from differentiated U937 cells which have been shown to express fibroblast collagenase in culture (13). Northern blot analysis of RNA extracted from human bone marrow cells and U937 cells showed that the NC 7.2 clone hybridized to a 3.3-kb mRNA from bone marrow but did not bind to mRNA isolated from U937 cells (Fig. 2). By contrast, a 1.7-kb cDNA probe specific for human synovial collagenase did not hybridize to bone marrow mRNA but clearly recognized a 2.2-kb mRNA in U937 mRNA in agreement with previously published data.
To confirm that the cDNA isolated was indeed collagenase, the cDNA was transfected into COS-7 cells using the NC 7.2 cDNA subcloned into a pCDNA1 vector. Medium from the cells transfected with the pCDNA1 vector bearing the insert degraded the collagen substrate into the characteristic al*, ~u2*, al", and (~2~ degradation products typical of interstitial collagenase activity (Fig. 3 Whole-cell RNA was harvested from human bone marrow cells and U937 cells which had been previously incubated at 37 "C for 24 h with phorbol 12-myristate 13-acetate. 15 rg of total RNA was electrophoresed in a 1% agarose-formaldehyde gel and transferred to nitrocellulose. The RNA on the blot was hybridized with either (A) insert cDNA from the NC7.2 clone or (B) a 1.7-kb insert cDNA for human synovial collagenase which was radiolabeled by random primer extension and [3LP]dCTP. pCDNA1 vector alone did not show significant collagenase activity. Interestingly, the majority of the activity was present in the medium and not in cell lysates (data not shown), suggesting that these cells constitutively secrete the collagenase and do not store it intracellularly.
Although neutrophil collagenase is similar to fibroblast collagenase in initiating degradation of native interstitial collagen molecules at a single site in the triple helix, there is only 57% identity and 72% chemical similarity (Fig. 4) (2). By comparison, neutrophil collagenase has 52% identity to human stromelysin, a metalloproteinase with a broader range of substrates. Many of the residues identical with those of both collagenase and stromelysin are conserved in other members of the family of known metalloproteinases. This conservation may relate to properties shared by all metalloproteinases such as zinc and calcium binding or may govern the conversion of latent enzyme to active enzyme. The sequence PRCGVPD located between residues 89 and 95 in neutrophil collagenase is highly conserved among the characterized matrix metalloproteinases and may play a critical role in activation of these enzymes. Mutations of this sequence in transin, the rat homolog to stromelysin, enhanced spontaneous activation of the enzyme (17). Regions of conserved residues for neutrophil and fibroblast collagenases would explain the significant immunological cross-reactivity between these two enzymes that we have observed with antibody prepared to purified neutrophil collagenase. The presence of unique epitopes for neutrophil collagenase as demonstrated by a discriminating monoclonal antibody may be due to additional epitopes resulting from a more extensive degree of glycosylation. Neutrophil collagenase was found to contain six potential sites for N-linked glycosylation (Fig. 1); only one of these sites is present in fibroblast collagenase. Increased amounts of glycosylation would also account for the molecular weight of 75,000 that we have observed for the purified neutrophil enzyme. The functional significance of increased glycosylation is unknown. Conceivably, extra glycosylation may serve a protective role in the extracellular milieu of inflammation in which the neutrophil functions. Alternatively, glycosylation may aid in