©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Gene Structure of Tissue Inhibitor of Metalloproteinases (TIMP)-3 and Its Inhibitory Activities Define the Distinct TIMP Gene Family (*)

Suneel S. Apte (1)(§), Bjorn R. Olsen (1), Gillian Murphy (2)

From the (1)Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115 and the (2)Cell and Molecular Biology Department, Strangeways Research Laboratory, Worts Causeway, Cambridge CB1 4RN, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) play a critical role in extracellular matrix homeostasis. We have previously cloned human and mouse TIMP-3 cDNAs and mapped their chromosomal loci (Apte, S. S., Mattei, M-G., and Olsen, B. R.(1994) Genomics 19, 86-90; Apte, S. S., Hayashi, K., Seldin, M. F., Mattei, M-G., Hayashi, M., and Olsen, B. R.(1994) Dev. Dynam. 200, 177-197); the identification of TIMP3 mutations in Sorsby's fundus dystrophy has underscored the functional importance of TIMP-3. We now report that TIMP-3 is encoded by five exons spanning over 30 kilobase pairs of mouse genomic DNA. In the attribution of protein domains to specific exons, as well as exon structures, the Timp-3 and Timp-1 genes are similar, confirming the common evolutionary origin of the TIMPs and defining a distinct gene family. We have expressed human and mouse TIMP-3 in mouse NSO myeloma cells. In each case, an N-glycosylated 27-kDa protein was generated, that, like TIMP-1 and TIMP-2, inhibited collagenase-1, stromelysin-1, and gelatinases A and B. TIMP-3 and TIMP-1 inhibition were quantitatively similar, implying that all TIMPs are equally efficient in MMP inhibition. Instead, differential regulation of the TIMP genes or divergent C-terminal protein sequences may underlie distinct biological functions for each TIMP.


INTRODUCTION

Remodeling of the extracellular matrix (ECM)()is an important process during normal development, and deregulated remodeling may have a role in the etiology of diseases such as cancer and arthritis (Pelletier et al., 1990; Alexander and Werb, 1991; Liotta and Stetler-Stevenson, 1990). The matrix metalloproteinases (MMPs), a family of secreted, zinc-containing neutral proteases (including collagenases, stromelysins, gelatinases, matrilysin, a metalloelastase, and membrane-type MMP), are believed to play a significant role in this process (Matrisian, 1992). The complex regulation of these enzymes occurs at three levels, transcriptional regulation of the genes, activation of secreted proenzymes, and via specific inhibitors, the tissue inhibitors of metalloproteinases (TIMPs),()which bind to activated MMPs with 1:1 molar stoichiometry. Two TIMPs, TIMP-1 and TIMP-2, are relatively well characterized. The most recently isolated member of this family, TIMP-3, is unique in having an affinity for the ECM (Blenis and Hawkes, 1983; Leco et al., 1994). cDNA cloning of TIMP-3 from chicken (Pavloff et al., 1992), humans (Apte et al., 1994a; Wick et al., 1994; Silbiger et al., 1994) and mouse (Leco et al., 1994; Apte et al., 1994b; Sun et al., 1994) has been reported.

The TIMPs show an overall peptide sequence identity of only about 25%. However, 12 cysteine residues are highly conserved and are believed to form six intra-chain disulfide bonds (Williamson et al., 1990). These, other conserved residues (especially a highly conserved N-terminal domain) and the general inhibition by TIMP-1 and TIMP-2 of all members of the MMP family establish the TIMPs as a distinct family of proteins. It is possible that the TIMP genes (like the MMP and collagen gene families) have been generated by duplication and diversification of a primitive MMP inhibitor gene.

We have previously cloned human and mouse TIMP-3 cDNA and assigned the chromosomal locus for the TIMP-3 genes in both species (Apte et al., 1994a, 1994b). Our in situ hybridization studies of TIMP-3 mRNA expression during mouse development and Northern analysis of human and mouse tissues have shown that expression of the Timp-3 gene is distinct from that of the Timp-1 gene (Apte et al., 1994a, 1994b; Reponen et al., 1995; Flenniken and Williams, 1990; Nomura et al., 1992; Leco et al., 1994). While Timp-3 is strongly expressed in the uterine deciduum in the peri-implantation period (Reponen et al., 1995), it is primarily expressed in cartilage, placental trophoblast, various epithelia, and muscle in the latter half of gestation (Apte et al., 1994b). That TIMP-3 has a critical function in the eye has been demonstrated by the presence of TIMP-3 mutations in Sorsby's fundus dystrophy (Weber et al., 1994) and by elevated retinal expression of TIMP-3 mRNA in simplex retinitis pigmentosa (Jones et al., 1994). The structure of the TIMP 3 gene, mechanisms of its regulation, the inhibition of specific MMPs by TIMP-3, and the molecular basis of ECM binding by TIMP-3 are presently unknown.

In this paper, we have addressed the question of the structure of the protein encoding part of the Timp-3 gene and its relation to the Timp-1 gene. The Timp-1 gene has been described previously (Gewert et al., 1987; Coulombe et al., 1988) while the structures of the TIMP-2 (apart from the structure of the TIMP2 promoter and exon 1 (DeClerck et al., 1994)) and TIMP-3 genes are presently unknown.

To examine the activities of TIMP-3 against individual MMPs, and to find out whether there are any functional differences compared to the activities of TIMP-1, we have expressed mouse and human TIMP-3 cDNAs in a myeloma cell line. rTIMP-3 is expressed as a glycosylated 24-29-kDa protein with inhibitory activities against interstitial collagenase, stromelysin-1, and gelatinase A and B. Both the spectrum of inhibition and the specific activity are comparable to those of TIMP-1.

Based on these genetic and biochemical data, we have identified characteristics shared by TIMP-1, TIMP-2, and TIMP-3 and proposed that these be considered as the hallmarks of the TIMP family.


EXPERIMENTAL PROCEDURES

Isolation and Characterization of the Timp-3 Gene

The Timp-3 cDNA clone pSAmT39 (whose 1.5 kbp insert encodes the complete TIMP-3 protein as well as 41 bp of the 5`-UT region and 810 bp of the 3`-UT region (Apte et al., 1994b)) was labeled with [P]dCTP by the random-primed method and used as a probe to screen a mouse genomic library from the 129SV strain (Stratagene, La Jolla, CA). 3 10 plaques were screened. A number of positive plaques were purified, and phage DNA was isolated for further analysis from six of these. Restriction mapping and Southern analysis with specific probes was used to identify putative exons. Appropriate fragments were subcloned into pBluescript SK+ (Stratagene) for further characterization, including partial sequence analysis. Sequencing of both strands in selected regions was carried out using the chain termination method with internal or flanking primers, and the sequence data was analyzed using the Lasergene software package (DNASTAR, Madison, WI). Exon-intron boundaries were assigned based on a comparison of the sequences of the Timp-3 cDNA and genomic sequences.

Expression of Mouse and Human TIMP-3

The Timp-3 cDNA pSAmT39 (Apte et al., 1994b) was subcloned into the expression vector pEE12 (Murphy et al., 1991) as an EcoRI fragment. The TIMP3 cDNA pSAhT3 (Apte et al., 1994a) had an incomplete 5` end which was extended by ligation of the 5` end of pSAmT39, including the 5`-untranslated region and the coding sequence, up to a common AccI site (coding for Tyr of the mature protein). Sequencing of this chimeric cDNA (pSAmhT3) confirmed that it would generate an amino acid sequence identical to that of mature human TIMP-3. pSAmhT3 was subcloned as an EcoRI-BstXI fragment into pEE12. Both plasmids were transfected into NSO mouse myeloma cells, and TIMP-3 producing clones were selected and expanded as described previously (O'Shea et al., 1992).

Purification of Mouse and Human TIMP-3

TIMP-3-producing clones were grown in serum-free defined medium, and the inhibitors were purified from 500-1000-ml batches. Conditioned medium was concentrated approximately 5-fold using an Amicon hollow fiber concentrator (10,000 molecular weight cut off) and adjusted to 25 mM MES, pH 6.0, and 0.025% brij 35 with 0.02% sodium azide. The medium was applied to a column of S-Sepharose (Pharmacia LKB Biotechnology, fast flow; 4.0 5 cm) at 200 mlh and the column subsequently washed with 25 mM MES, pH 6.0, 0.025% brij, 100 mM NaCl, 0.02% sodium azide. TIMP-3 activity was then eluted using the same buffer containing 200 mM NaCl. Mouse and human TIMP-3 were further purified after concentration (Amicon YM10 concentrator) by chromatography on a Sephacryl S200 (Pharmacia) column (1.5 100 cm) equilibrated with 25 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.025% brij 35, and 0.02% sodium azide. Alternatively, human TIMP-3 was chromatographed on zinc-iminodiacetic acid Sepharose (0.6 6 cm) equilibrated with 25 mM MES, pH 6.5, 500 mM NaCl, and 0.02% sodium azide. The column was washed with the same buffer and TIMP-3 activity eluted with 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM imidazole, 0.02% sodium azide.

Inhibitor Assays

The inhibitory activity of TIMP-3 preparations was routinely assayed using rabbit interstitial collagenase in a C-labeled collagen diffuse fibril assay, as described previously (Murphy et al., 1981). The ability of TIMP-3 to inhibit individual human MMPs including gelatinase B, gelatinase A, collagenase-1, and stromelysin-1 was assessed using purified recombinant enzymes and C-labeled gelatin, collagen, or -casein assays (Murphy et al., 1991). One unit of inhibitor activity is defined as the amount required to give 50% inhibition of 2 units of enzyme (1 unit of enzyme is 1 µg of substrate cleavage/minute).

N-terminal Amino Acid Sequencing

Samples of recombinant human and mouse TIMP-3 were sequenced directly using a 470A protein sequencer (Applied Biosystems).

Gel Electrophoresis

Samples of inhibitor were analyzed by SDS-polyacrylamide gel electrophoresis using 12% gels (Laemmli and Favre, 1973). Gels were run under reducing conditions and silver stained (Merril et al., 1981); TIMP-3 activity was also detected by gelatin reverse zymography (Ward et al., 1991).

Deglycosylation

Mouse or human TIMP-3 (0.5 µg) were denatured in the presence of 0.5% SDS and 1% 2-mercaptoethanol at 25 °C for 60 min and then treated with N-glycosidase F (Boehringer Mannheim, 1.6 units) using the manufacturer's suggested protocol for 15 h at 37 °C. The deglycosylated inhibitors were analyzed by polyacrylamide gel electrophoresis.


RESULTS AND DISCUSSION

Characterization of the Timp-3 Gene

Six genomic clones were isolated that spanned a distance of approximately 50 kbp (Fig. 1). The sequence of the Timp-3 cDNA pSAmT39 was represented in five exons, presently numbered 1-5 from the 5` to 3` end of the gene. Although we have not defined the 5` limit of the Timp-3 mRNA and the transcription start site, this numbering is based on an analysis of the TIMP3 gene which indicates that the first protein coding exon in the human gene also contains the transcription start site and is exon 1 of the gene.()Exon 1 of the Timp-3 gene contains the 5`-UT sequence (that of pSAmT39 as well as additional published 5`-UT sequences; Leco et al., 1994) and the translation initiation codon. It encodes the signal peptide and the N-terminal region of the mature protein. The 5` limit of this exon and the location of the Timp-3 promoter are presently unknown. The translation termination codon and the 3`-UT region of the cDNA pSAmT39 are located in exon 5. The 3`-EcoRI terminus in pSAmT39 coincides with an EcoRI site in the genomic clone T3g5, suggesting that the actual 3` end of the mRNA extends beyond this EcoRI site. We have used polymerase chain reaction to confirm that exon 5 contains the complete 3`-UT sequence isolated by Sun et al. (1994, GenBank accession no. Z30970), up to the polyadenylation signal (data not shown). This exon is thus approximately a maximum of 3 kbp in size, although the exact size varies depending on polyadenylation signal usage. Exon-intron boundaries have consensus splice site sequences ().


Figure 1: A, structure of the Timp-3 gene. The exons are indicated by the solid boxes with the respective numbers above them; introns are indicated by the thin line. Since the size of exon 5 varies depending on polyadenylation signal usage, this is shown by the hatched box beyond the translation stop codon. A break in the overlap of genomic clones and restriction maps is indicated by the double slashes. The translation start codon (ATG) and translation stop codon (TGA) are shown. Restriction sites for selected restriction endonucleases within the genomic clones are shown at the top of the figure. Solid lines above the gene structure indicate the relative positions and sizes of the genomic clones from which the gene structure was deduced. The scale marker indicates 4 kbp. B, structure of the mouse TIMP-3 protein. The signal peptide is represented by the solid box, and the mature protein is shown by the open box. The projection of protein domains onto their respective exons is indicated above the protein. The conserved cysteine residues are shown by solid circles. The scale bar indicates 20 amino acids.



Comparison of the Timp-3 and Timp-1 Genes

Both in terms of the number of protein coding exons, and the allocation of protein domains to exons, the Timp-3 gene is remarkably similar to the Timp-1 gene (Gewert et al., 1987; Coulombe et al., 1988). Thus a comparison of the protein domains encoded by corresponding exons shows considerable homology using the conserved cysteine residues as reference points (Fig. 2). Although, (apart from exon 2 of the Timp-3 gene) the exons are not of identical size (), the splice junctions between exons are at the same relative locations with reference to the amino acid encoded at the splice junctions, so that splice junctions in the mRNA affect codons in an identical manner in both the Timp-1 and Timp-3 genes (Fig. 2). The different sizes of the protein coding exons reflect the differences in the primary structure of the TIMP polypeptides, outside of the region of high N-terminal homology.


Figure 2: Comparison of the structures of the Timp-3 (top row) and Timp-1 genes (bottom row). All features of the Timp-3 gene are indicated in bold type, whereas those of the Timp-1 gene are italicized. The exons are represented by the amino acid sequences they encode and are enclosed in boxes. Conserved cysteine residues are in bold type and indicated by solid circles. Arrowheads indicate that the splice junction splits the codon and that the split is after the first nucleotide in this codon. The asterisk indicates the only published splice site for the TIMP2 gene (DeClerck et al., 1994); this splits the relevant codon of TIMP-2 in an identical fashion. Other splice junctions are between codons. Potential N-linked glycosylation sites in TIMP-1 are underlined, while a single site in TIMP-3 is overlined. The sequences of murine TIMP-3 and TIMP-1 (Gewert et al., 1987) were aligned using the DNASTAR software with gaps introduced for optimal alignment.



In contrast to the small size of the Timp-1 gene (the entire Timp-1 gene is contained within a 4.3-kbp genomic fragment) (Gewert et al., 1987), the Timp-3 gene is quite large (at least 30 kbp in size) (Fig. 1, ). Comparison of corresponding introns shows that they are widely divergent in size () and the size of the Timp-3 gene can primarily be attributed to its large introns. In particular, intron 1, which has not been completely defined, appears to be at least 15 kbp in size. Characterization of the TIMP3 gene shows that it is approximately 30 kbp in size. It is possible that the large introns may contain regulatory information, although this needs to be ascertained by experimental studies. One contrast between the Timp-3 and Timp-1 genes is in the size of their untranslated regions. Those of Timp-1 are relatively small (Gewert et al.; 1987; Coulombe et al., 1988), but the Timp-3 gene has a very long 3`-UT region (Apte et al., 1994b; Leco et al., 1994; Sun et al., 1994). In the 5` region of the Timp-1 gene, exon 1 encodes only 5`-UT sequence (Coulombe et al., 1988), while exon 2 is the first protein coding exon and corresponds to exon 1 of the Timp-3 gene. The TIMP2 gene is homologous to the Timp-3 and TIMP3 genes in the limited structural data available; exon 1 of the TIMP2 gene is also the first protein coding exon, and the location of its 3` splice site (DeClerck et al., 1994) and the manner in which it splits the relevant codon are identical to the Timp-1 and Timp-3 genes (Fig. 2, ).

Our data suggest that the Timp-1 and Timp-3 genes are members of a distinct gene family and predict that other members of the family such as TIMP-2 will have a similar gene structure. Definition of the structure of this gene family is useful for understanding the relationship that related genes in other species may have to these genes, for the prediction and facilitated characterization of gene structure of new TIMPs, and for cloning of new members of this family from genomic DNA using polymerase chain reaction. We hypothesize that the TIMP genes arose from a common precursor gene but have diverged to cope with the increasing complexity of the ECM and the increasingly complex mechanisms for its degradation.

Human and Mouse TIMP-3 Are Expressed as Glycosylated Proteins in the NSO Myeloma Cell Line

Human and mouse TIMP-3 were expressed by cloned NSO myeloma cells to a level of up to 20 µg/ml. Since these cells do not produce an extracellular matrix, both inhibitors were largely free in the culture medium, allowing efficient purification of milligram amounts of material. Purification of TIMP-3 from both species was effected using a combination of S-Sepharose and Sephacryl S200 gel filtration chromatography. The inhibitors were considerably retarded by the Sephacryl even in the presence of high salt and detergent and eluted well after the total volume of the column. By SDS-polyacrylamide gel electrophoresis analysis a major protein of 27 kDa was obtained in each case (Fig. 3A). N-terminal sequencing of purified mouse TIMP-3 gave the sequence XTXSPSHPQDA, and of human TIMP-3, gave the sequence XTXSPSHPQDAFXN (where X is an unidentified residue, presumably cysteine, which was not identifiable in the sequencing protocol used), confirming the identity of these proteins. Human TIMP-3 was also purified in a detergent-free form using S-Sepharose and zinc iminodiacetic acid Sepharose and yielded a major band of 27 kDa as well as two further bands of 29 kDa (minor) and 24 kDa. Reverse zymographic analysis of the inhibitor preparations in gelatin-polyacrylamide gels showed that all three bands had metalloproteinase inhibitor activity, corresponding to bands of activity in the original conditioned culture medium (Fig. 3B).


Figure 3: Analysis of recombinant human and mouse TIMP-3 by polyacrylamide gel electrophoresis. A, purified human recombinant TIMP-3 (lane 1) and mouse TIMP-3 (lane 2) were electrophoresed in an 11% polyacrylamide gel under reducing conditions before staining with Coomassie Brilliant Blue. B, crude and purified samples of recombinant TIMP-3 were electrophoresed in a 12% polyacrylamide gel containing gelatin under non-reducing conditions before development of inhibitory activity as described under ``Experimental Procedures.'' Purified human TIMP-3 (lanes 1 and 2) and mouse TIMP-3 (lanes 3 and 4) and partially purified (S-Sepharose) human TIMP-3 (lane 7) are shown and compared with rTIMP-1 (lane 5) and rTIMP-2 (lane 6). Lanes 8 and 9 show culture medium from NSO myeloma cells expressing mouse and human TIMP-3, respectively. The mobilities of standard molecular mass markers (kDa) are indicated to the right of each gel. The arrow indicates nonspecific inhibition by bovine serum albumin in the culture medium.



Treatment of human and mouse TIMP-3 with N-glycosidase F reduced the 27 kDa form to 24 kDa, corresponding to the lower molecular mass inhibitor (Fig. 4), indicating that recombinant TIMP-3 is largely N-glycosylated. 29-kDa TIMP-3 occurred at very low levels but was reduced to a 24 kDa form by N-glycosidase F treatment (data not shown). To assess the nature of the carbohydrate chains, we examined the binding of both human and mouse TIMP-3 to lectin-coupled Sepharose columns. Both TIMP-3s were only lightly retarded by passage through concanavalin A-Sepharose and did not bind to Helix pomatia lectin-Sepharose. However, they both bound largely (90%) to wheat germ agglutinin-Sepharose and could be eluted by 25 mg/ml N-acetyl glucosamine. It was concluded that the TIMP-3 preparations were probably heterogeneously substituted with variable length carbohydrate chains containing N-acetylglucosamine and were not the complex glycoprotein, poly-mannose variety like TIMP-1 (Murphy and Werb, 1985).


Figure 4: Deglycosylation of human and mouse TIMP-3. N-Linked sugar chains were removed from denatured samples of recombinant purified mouse and human TIMP-3 and recombinant TIMP-1 and analyzed by polyacrylamide gel electrophoresis on 12% gels under reducing conditions followed by silver staining. A preparation of purified human TIMP-3 containing a significant amount of the 24 kDa form (lane 1) is compared with a predominantly 27 kDa preparation (lane 2), with the same preparation after incubation for 15 h at 37 °C alone (lane 3) or with N-glycosidase F (lane 4). Complete conversion of the 27-kDa TIMP-3 to 24 kDa occurred. Similarly, unincubated purified mouse TIMP-3 (lane 5) is compared with the same preparation after incubation for 15 h at 37 °C alone (lane 6) or in the presence of N-glycosidase F (lane 7). Human TIMP-1 (lane 8), incubated at 37 °C for 15 h alone (lane 9) and with N-glycosidase F (lane 10) were run on the same gel to demonstrate the similar molecular masses of deglycosylated TIMP-3 and TIMP-1. Lanes 4, 7, and 10 contain an extra band at about 32 kDa which is the N-glycosidase F. The mobilities of standard molecular mass markers (kDa) are indicated to the right of the gel.



So far, most studies of naturally occurring TIMP-3 have been done in cultured chick fibroblasts (Blenis et al., 1983; Pavloff et al., 1992). Expression of rTIMP-3 into the medium of mouse myeloma cells contrasts with the observation that the natural form of TIMP-3 from chick fibroblasts in culture is essentially an extracellular matrix protein laid down by the cell in an insoluble, unglycosylated form (Blenis et al., 1983; Pavloff et al., 1992). Interestingly, expression of mouse TIMP-3 in COS-1 cells produced a non-glycosylated protein that was also bound to the ECM; furthermore, COS-1 cells produced an endogenous putative form of TIMP-3, also located in the ECM (Leco et al., 1994). The rTIMP-3 produced by the myeloma system was largely glycosylated, but in an apparently heterogenous fashion, increasing the expected molecular mass from 3-5 kDa (the 27 kDa and 29 kDa forms). Varying amounts of an unglycosylated form were also produced of comparable size (24 kDa) to the recombinant mouse TIMP-3 from COS-1 cells (Leco et al., 1994). All known TIMP-3 sequences show a conserved consensus sequence for N-linked oligosaccharide substitution (SI I/S NATDP; Apte et al., 1994b, Fig. 2), but this is the first instance that glycosylated forms of the inhibitor have been isolated. Glycosylation had the effect of modifying the predicted basic charge of the TIMP-3 protein (Pavloff et al., 1992; Apte et al., 1994; Leco et al., 1994). Whether glycosylated forms of naturally occurring TIMP-3 occur are presently unknown, but the conservation of the N-linked glycosylation site within the otherwise divergent C terminus suggests that this is a possibility. With other cDNAs, the expression system that we used here has consistently given glycosylation patterns comparable with those of the natural proteins.()

rTIMP-3 Has MMP Inhibitory Activity Comparable to TIMP-1

The specific activities of human and mouse TIMP-3 against human gelatinases A and B, collagenase-1, and stromelysin-1 were compared with those of human TIMP-1 using the appropriate macromolecular substrate assays and were found to be very similar (). However, neither species' TIMP-3 was recognized by antibodies to human TIMP-1 or TIMP-2 (data not shown). Since the MMP inhibition activity of TIMP-2 has been shown to be similar to that of TIMP-1, it follows that the specific activities of all the TIMPs are broadly similar. Further analysis of TIMP-3 specificity will require detailed kinetic studies. Preliminary data have shown that TIMP-3 binds to progelatinase A and is relatively fast-binding to active gelatinase A, comparable to TIMP-2 (Willenbrock et al., 1993).()Our data indicate that TIMP-3 is a fully functional MMP inhibitor, and its potential role in the regulation of these proteinases in vivo is equivalent to that of TIMP-1 and TIMP-2. Any difference in its mode of action may stem from its matrix location, tissue-specific expression, and differential regulation, as well as from its variant C terminus.

The TIMP Family of Genes and Proteins

Based on the present data and the existing knowledge of TIMP-1 and TIMP-2, the common characteristics of the TIMP family can be delineated.

1) Conserved gene structure. The structure of the protein coding exons is similar in the Timp-1 and Timp-3 genes and predicts the structure of the TIMP-2 exons. In this respect, the sizes of exons appear to be less conserved than the location of splice junctions (relative to codons) within the mRNA.

2) The proteins have inhibitory activities specifically against matrix metalloproteinases and are secreted extracellularly.

3) Twelve cysteine residues as well as their relative spacing are conserved in the three TIMPs. Although it is generally assumed that these form six intra-chain disulfide bonds, this has been categorically shown only for TIMP-1 (Williamson et al., 1990).

4) A highly conserved N-terminal domain. The N-terminal 126 amino acid residues of mature TIMP-1 (Murphy et al., 1991) and the N-terminal 127 residues of mature TIMP-2 (Willenbrock et al., 1993; DeClerck et al., 1993) have been shown to be adequate for the inhibition of MMPs, suggesting that this part of the proteins is functionally critical for inhibition of MMPs. This N-terminal region is the most conserved among the TIMPs, while the C terminus is more divergent (Pavloff et al., 1992; Apte et al., 1994b; Leco et al., 1994; Bodden et al., 1994).

In this region, the N-terminal most 22 amino acids of mature TIMP-3 share 72% identity with TIMP-1 and TIMP-2 and thus account for much of the 25% amino acid identity of the three TIMPs. The consensus sequence CXCXPXHPQXAFCNXDXVIRAK (single amino acid code; X = any amino acid) might be considered a diagnostic hallmark of the TIMPs' being present in all three proteins. Single residue mutations in this region (O'Shea et al., 1992) have significantly altered the properties of TIMP-1, and recent experiments have confirmed the importance of this sequence (Bodden et al., 1994). Other homologous regions between TIMP-1 and TIMP-2 noted by Bodden et al.(1994) are not present in TIMP-3. The tertiary structure of the N-terminal three loops of the TIMPs are expected to be similar, consisting of a five-stranded anti-parallel sheet that is rolled over on itself to form a closed -barrel (Williamson et al., 1994). This is thought to generate conserved surface regions of functional importance in MMP interaction.

We suggest that these characteristics of the TIMP family may be used as criteria for the inclusion in this family, of additional, novel proteins that might be identified in the future. Although additional members may differ in some respects from TIMP-1, TIMP-2, and TIMP-3, we expect that these would have a similar genomic organization, the conserved N-terminal domain, and show inhibition of at least one MMP. In view of the fact that the known TIMPs show broad spectrum inhibition of the MMPs, a conserved N-terminal region would likely result in a similar spectrum of inhibition by any novel TIMPs. These criteria permit the inclusion of proteins with more specific inhibitory activities and/or TIMPs with other dominant biological properties. Although MMP inhibition is the defining feature of the TIMPs, they have also been shown to have other properties, such as growth factor activity (Hayakawa et al., 1994). TIMP-3 has been implicated in transformation of chick embryo fibroblasts (Yang and Hawkes, 1994) and in progression of the cell cycle (Wick et al., 1994). It is possible that some of these activities may be independent of MMP inhibition. The determination of the Timp-3 gene structure and the production and isolation of rTIMP-3 facilitates further investigation of TIMP-3 function(s) at both the genetic and biochemical level.

  
Table: The exon-intron boundaries of the Timp-3 gene

Intron sequences are shown in lower case, exon sequences in upper case. Only the partial sequences of each exon and intron adjacent to the splice sites are shown. In the column for exon and intron sizes, the sizes of the corresponding exons and introns of the Timp-1 gene are shown in parentheses.


  
Table: Specific activities of human and mouse TIMP-3 and human TIMP-1 against the matrix metalloproteinases



FOOTNOTES

*
This work was supported by an Investigator Award from the Arthritis Foundation (to S. A.), National Institutes of Health Grants AR36819, AR36820, and HL33014 (to B. R. O.), and support from the Arthritis and Rheumatism Council, United Kingdom, and the Wellcome Trust (to G. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) L19622, U26433, U26434, U26435, U26436, U26437.

§
To whom correspondence should be addressed. Tel.: 617-432-2366; Fax: 617-432-0638; E mail: sapte@warren.med.harvard.edu.

The abbreviations used are: ECM, extracellular matrix; MMPs, matrix metalloproteinases; TIMPs, tissue inhibitors of metalloproteinases; kbp, kilobase pair(s); bp, base pair(s); UT, untranslated.

The following notations are used: TIMP-3, TIMP-2, and TIMP-1 designate the proteins, TIMP3 and TIMP2 are Human Genome Mapping Workshops approved symbols for the human TIMP-3 and TIMP-2 genes, respectively, and Timp-3 and Timp-1 are the approved symbols for the respective mouse genes. rTIMP-3 denotes recombinant TIMP-3.

M. Wick, R. Haronen, D. Murnberg, B. R. Olsen, M. Budarf, S. Apte, and R. Mueller, manuscript submitted for publication.

G. Murphy, unpublished data.

F. Willenbrock and G. Murphy, unpublished results.


ACKNOWLEDGEMENTS

We thank Drs. Andy Docherty and Mark Cockett for expert guidance in the expression studies, Mary Harrison for cell culture, and Dr. Bryan Smith for protein sequencing.


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