We report cDNA cloning and primary structure of a new metalloproteinase inhibitor (ChIMP-3) produced by chicken embryo fibroblasts. ChIMP-3, formerly.
Vol. 267, No . 24, Issue of’ August 25, pp. 17321-17326,1992 Printed in U.S.A.
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.
A New Inhibitor of Metalloproteinases from Chicken: ChIMP-3 A THIRD MEMBER OF THE TIMP FAMILY* (Received for publication, March 18, 1992)
Nadine Pavloff, Paul W. Staskus*, Narendra S. Kishnani, and Susan P. HawkesQ From the Departmentof Pharmacy, Schoolof Pharmacy, Universityof California, San Francisco, California 94143-0446
We report cDNA cloning and primary structure of a disease, and tumor metastasis, are proposed to involve alternew metalloproteinase inhibitor (ChIMP-3) produced ations in the balance between active enzymes and theirinhibby chicken embryo fibroblasts. ChIMP-3, formerly itors (Emonard and Grimaud, 1990;Lennarz and Strittmatter, called the 21-kDa protein, is one of five ChIMPs 1991; Murphy et al., 1991a; Page, 1991). Current information (Chicken Inhibitor of Metallogroteinases). In this pa- suggests that thereis a family of metalloproteinase inhibitors per, wereport that of the three most abundant ChIMPs, containing at least two members, TIMP-1 (tissue inhibitorof ChIMP-3 and ChIMP-a are extracellular matrix com- metalloproteinases-1) and TIMP-2. ThecDNAs of these proponents, whereas ChIMP-2 is found in the media con- teins have been isolated from a number of species and seditioned by the cells. Treatment of ChIMP-3 and quenced. They include TIMP-1 from human (Docherty et al., ChIMP-a with N-glycosidase-Findicates that ChIMP- 1985; Gasson et al., 1985; Carmichael et al., 1986), bovine a is N-glycosylated whereas ChIMP-3 is not. The deduced aminoacid sequence of ChIMP-3 predicts a pro- (Freudenstein et al., 1990), rabbit (Horowitz et al., 1989), and tein whose properties are consistent with experimental murine (Edwards et al., 1986; Gewert et al., 1987; Johnson et measurements. Analysis of sequence alignments with al., 1987) andTIMP-2 from human (Boone et al., 1990; the two previously described members of the TIMP Stetler-Stevenson et al., 1990) and bovine (Boone et al., 1990) (tissue inhibitor of metalloproteinases) family, TIMP- sources. Previously, we reporteda 21-kDa ECM protein from 1 and TIMP-2, from various species indicates that ChIMP-3 is a related but distinct protein. This conclu- chicken embryo fibroblasts whose synthesis was stimulated sion is supported by lack of significant binding with during the early stages of transformation initiated by Rous anti-TIMP-1 and anti-TIMP-2 antibodies. Based on sarcoma virus (Blenis and Hawkes, 1983). Synthesis of this these data, its unusual solubility properties, and its protein was also stimulated by treatment of normal, uninexclusive location in the matrix, we propose that fected cells with the tumor promoter phorbol myristate aceChIMP-3 is a new member of this family of metallo- tate (Blenis and Hawkes, 1984). These observations impliproteinase inhibitors, a TIMP-3. cated the protein in the development of transformation. Recently,electrophoretic purification andpartial sequence analysis strongly suggested that the21-kDa protein is a member of the family of metalloproteinase inhibitors which inThe extracellular matrix (ECM)’ is a complex structure cludes TIMP. Furthermore, the purified 21-kDa protein disthat contains collagens, proteoglycans, glycosaminoglycans, played inhibitoractivitycharacteristic of these molecules glycoproteins (fibronectin,chondronectin,laminin) and, in (Staskus et al., 1991). Based on anumber of criteria, including some tissues, elastin (Hay, 1981). Matrix metalloproteinases NH2-terminal sequence, statistical analysis of amino acid (MMPs) constitute the major group of enzymes that degrade composition, size, andapparent lack of glycosylation, we extracellular proteins during remodeling of connective tissue proposed that the 21-kDa protein was either a variant of accompanying normal biological processes. The MMPs are TIMP-1 or a third member of the TIMP family (Staskus et secreted from cells as inactive zymogens and their activity in al., 1991). the extracellular environment is regulated by various activaIn this paper, we report that the 21-kDa protein is one of tors andinhibitors (Matrisian, 1990; Murphy et al., 1991a). A five ChIMPs (Chicken inhibitor of MetalloProteinases) pronumber of human diseases, including arthritis, periodontal duced by chickenembryo fibroblasts. Of thethree most abundant andwell characterized inhibitors, ChIMP-3 (the21-kDa * This work was supported in partby National Institutes of Health protein) and ChIMP-a are ECM components and ChIMP-2 Grant CA 39919 and the Elsa U. Pardee Foundation. The costs of publication of this article were defrayed in part by the payment of is found in the media conditioned by cells. ChIMP-b and page charges. This article must therefore be hereby marked “adver- ChIMP-c are minor inhibitors of the ECM and media, retisement’’ in accordance with 18 U.S.C. Section 1734 solely to indicate spectively. Here we describe cDNA clones which include the this fact. entire coding region of ChIMP-3 (212 amino acids). A total The nucleotide sequence(s) reported in this paperhas been submitted of 886 nucleotides have been determined including 5’ and 3‘ to theGenBankTM/EMBL Data Bankwith accession number(s) noncoding sequences. Biochemical data substantiate thelack M94531. $ Present address: Biosciences Division of General Atomics, P.O. of N-glycosylation of ChIMP-3. From the comparison of this sequence with the sequences of TIMP-1 and TIMP-2, and Box 85608, San Diego, CA 92186. § T o whom correspondenceshould be addressed. Tel.: 415-476- other supporting data, we propose that ChIMP-3 is a third 2318; Fax: 415-476-2744. member of this family, a TIMP-3.
’ The abbreviations used are: ECM, extracellular matrix; ChIMP, chicken inhibitor of metalloproteinases; EGTA, [ethylenehis(oxyethylenenitrilo)]tetraaceticacid; MMP, matrix metalloproteinase; PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate; TIMP, tissue inhibitor of metalloproteinases.
MATERIALSANDMETHODS
Cell Culture and Preparation of ECM and Conditioned MediaChicken embryo fibroblasts were prepared and cultured as described
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(Blenis and Hawkes, 1983), with the exceptions of storage in liquid nitrogen at theprimary stage and seeding of tertiary cells at 2 X lo6/ 100-mm culture dish (Falcon Labware). Cells were infected as secondary cultures with the temperature-sensitive mutant of Rous sarcoma virus, LA24, clone G2. For preparation of ECM, cells were maintained at 41 "C for 12-15 h before transfer to the permissive temperature for transformation (35 "C). Ten hours after temperature shift the ECM washarvested, basically as described previously (Blenis and Hawkes, 1983). The transforming cells were removed from the culture dishes following a 15-min incubation in Ca2'-, M e - f r e e phosphate-buffered saline, containing 5 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, and 1 mg/ml t-amino-n-caproic acid (pH 7.4). After several rinses in phosphate-buffered saline and water, the ECM wassolubilized in a small volume of electrophoresis sample buffer without reducing agent (Laemmli, 1970). Conditioned media samples were prepared from LA24-infected cultures which had been maintained at the permissive temperature from the time of seeding. After 38 h the medium was removed, the cell monolayer was rinsed twice with phosphate-buffered saline and the cells cultured for an additional 22 h in serum-free medium. This conditioned medium was removed from the cells, centrifuged at 10,000 x g for 20 min and analyzed by protease/substrate gel electrophoresis. Protease/Substrate Gel Electrophoresis-Samples of ECM and conditioned media were assayed for metalloproteinase inhibitor activity by electrophoresis on protease/substrate gels as described earlier (Staskus etal., 1991).Basically, the substrate gel technique (Heussen and Dowdle,1980; Herron et al., 1986) was modified to include a source of metalloproteinase in addition to gelatin, during polymerization of the gel. Phosphorylase b ( M , = 97,400), bovine serum albumin ( M , = 66,200), ovalbumin (M,= 45,000), and carbonic anhydrase (Mr= 31,000) from Bio-Rad were usedas molecular weight standards. Treatment of Proteins with N-Glycosidase-F-The ECM harvested from 20 culture dishes was used to isolate ChIMP-3 and ChIMP-a. Unreduced material was first isolated from preparative polyacrylamide gels by cutting bands from the gel lanes after staining with cetyltrimethylammonium bromide to visualize ChIMP-3 (Staskus et al.,1991). As ChIMP-a could not be clearly located using this technique, several narrow bands were cut behind ChIMP-3 in the gel lanes, one of which contained ChIMP-a. After electroelution of samples (Staskus et al., 1991),without reducing agent, small aliquots were electrophoresed a second time on protease/substrate gels to identify those band eluates which contained separatedChIMP-3 or ChIMP-a activities. These two samples were dialyzed extensively against 20 mM sodium phosphate, pH 6.8, containing 0.01% (w/v) SDS at 4 "C using Spectropor tubing ( M , 3500 cut-off, Spectrum Diagnostics). When necessary, sample volumes werereduced by coating the dialysis tubing with carboxymethylcellulose(Aquacide,Calbiochem)to absorb water. The samples were then dialyzed for 2 days against 0.2 M sodium phosphate, pH 8.0, containing 10 mM EDTA, 0.05% (w/v) SDS. To promote thorough exchange of SDS, some samples of electroeluted ChIMP-3and ChIMP-a were also dialyzed against 6 M urea containing 0.1% (w/v) Brij 35, before dialysis against the phosphate buffers, as described above. To 38 p1 of each sample was added 2 pl of 10% (w/v) octyl glucoside in the phosphate buffer, for a final glucoside concentration of 0.5% (w/v). Each sample was split into20p1 aliquots. One aliquot was incubated with 0.5 units of recombinant N-glycosidase-F (N-glycanase, Genzyme Corp.) added in a volume of 2 pl. The samples were incubated overnight at 35 "C and then electrophoresed on protease/substrate gels. Oligonucleotide Synthesis-Oligonucleotide primers for PCR were synthesized on a Milligen Biosearch automated DNA synthesizer (model 8600; Biosearch) and purified by 7 M urea, polyacrylamide gel electrophoresis. The oligonucleotide bands were visualized byUV shadow casting, excised and electroeluted by use of an Elutrap apparatus (Schleicher & Schuell), and desalted on Sep-Pak (Millipore Corp.) using standard protocols. Primer I was designed to bind to the noncoding strand of ChIMP-3 cDNA corresponding to the NH2terminal amino acids 6-11 of the mature protein (IHPQDA, Staskus et al., 1991) (nucleotides 200-216, underlined in Fig. 3). This sequence was chosen for the relatively small codon degeneracy (96-fold) and for the least possible sequence similarity to other TIMPs. The sequence of the 24-base primer consists of 17 bases specific for ChIMP3, a 6-base Sal1 restriction site, and an extra base a t the 5' end (underlined), as follows: 5'-GGTCGACATA(or C or T)CAC(or T)CCA(or Cor G or T)CAA(or G)GAC(or T)GC-3'. Primer 11, which incorporates an XbaI restriction site and an extrabase at the 5' end (underlined), was designed to bind to polyadenylated sequences.
Primer I1 has the following sequence: 3'-(T),,AGATCTC-5'. These primers contained restriction sites for use as an alternative to blunt end ligation for subsequent cloning. Two additional oligonucleotides (22-mers) were designed to screen the Xgtll library. These primers flank the Xgtll EcoRI cloning site. Primer 111, the 5' Xgtll oligonucleotide, is upstream of the cloning site: 5"GGTGGCGACGACTCCTGGAGCC-3' and primer IV, the 3' X g t l l oligonucleotide, is on the complementary strand downstream of the cloning site: 3'GTAATGGTCAACCAGACCACAG-5'. Primer V: 5"TGCTCTCCAACTTCGGCCACT-3' was designed on the basis of partial sequence information on ChIMP-3 and corresponds to nucleotides 618-638of ChIMP-3 cDNA. It was used to clone the 3' end of ChIMP-3. Primer VI was designed to anneal to the coding sequence of ChIMP-3 (nucleotides 640-663) in order to characterize the 5' end of ChIMP-3 cDNA. Its sequence: 3"TCCTGTGGTTCGCTTCGTGATACGGACGTCAC-5', includes 24 nucleotides specific for ChIMP-3, one PstI restriction site, and two extra bases at the5' end (underlined). PCR (Polymerase Chain Reaction) and Cloning-A Xgtll cDNA library derived from 10-day old chicken embryos was purchased from Clontech. The library was screened using the PCR as described by Friedman et al. (1990). Initially, primer I, coding for IHPQDA, was used along with primer 11, modified oligo(dT) sequence, on 1pl of the library (1-9 X lo9 phages/ml). The PCR was run in a DNA thermal cycler (Perkin-Elmer Cetus) for 30 cycles. Each cycle consisted of heating at 98 "C for 1s, annealing at 50 "C for 15 s, and polymerization at 60 "C for 4 min. This reaction consistently yielded a single 483base pair product (P483) detected on a 1%agarose gel representing a partial ChIMP-3 cDNA. After treatment with the Klenow enzyme, P483 was cloned into the HincII site of pUC19 (GIBCO/BRL) as a blunt-end fragment. Six independent subclones were selected. After sequence analysis of P483, a specific primer (V) was designed to determine the 3'-end sequence of the cDNA. This primer along with primer I11 or IV (the 5' or 3' Xgtll oligonucleotides described above) were used to amplify cDNA from the Xgtll library. The PCR was run for 30cycles. Each cycle consisted of heating at 94 "C for 30 s, annealing at 61"C for 2 min, and polymerization at 72 "C for 5 min. The resulting single 269-base pair fragment (P269) was cloned as described before in pUC19. Two independent subclones were analyzed. A 32P-labeledprobe, P483, was generated by PCR and end labeling for subsequent screening of the Xgtll library. The PCR was run using primer I only (see "Results") for 30 cycles with each cycle consisting of heating at 94 "C for 30 s, annealing at 61 "C for 2 min, and polymerization a t 72 "C for 5 min. The resulting product was gelpurified and labeled with [ ( u - ~ * P ] ~ Cusing T P the multiprimer labeling system from Amersham Corp. Incorporated nucleotides were separated from unincorporated nucleotides on a Sephadex (2-50 column (Boehringer Mannheim). Screening the cDNA Library-Approximately lo6 phages were grown on six 150-mm plates, lifted in duplicate onto supported nitrocellulose transfer membrane (BAS-NC from Schleicher & Schuell), and hybridized to thepartial cDNA probe, 32P-labeledP483, described above. Hybridizations were performed overnight at 42 "C in 5 X Denhardt's solution (Denhardt, 1966),5 X SSC (SSC is 15 mM sodium citrate, 150 mM NaCl), 50 mM sodium phosphate (pH 6.5), 0.1% SDS (w/v), 250 pg/ml fish sperm DNA, 50% deionized formamide, 1%dextran sulfate (w/v). The filters were washed in 0.1 X SSC containing 0.1% SDS (w/v) at 60 "C. Five positively hybridizing plaques were purified. Two independent clones, C and D, were chosen. After extraction and purification, the DNA was analyzed by PCR using both 5' and 3' Xgtll oligonucleotides (primers I11 and IV). The phage DNA from clone C was amplified by PCR after the first round of purification using primer VI and either primer I11 or IV in order to characterize the 5' end of ChIMP-3 cDNA. The amplified DNA from clones C and D was subsequently cloned into pUC19, as described before, and sequenced. Standard protocols for cDNA library screening, X phage purification, agarose gel electrophoresis, and plasmid cloning were employed (Maniatis et al., 1982). DNA Sequencing-Double-stranded cDNA cloned into pUC19 was sequenced by the dideoxy terminator method (Sanger et al., 1977) using sequencing kits purchased from Pharmacia LKB Biotechnology Inc. or U. S. Biochemical Corp. (Sequenase version 2.0). Each cDNA subclone was sequenced using an M13 universal primer, a reverse sequencing primer (Pharmacia LKB), or internal primers. The sequencing strategy used is presented in Fig. 4. In all cases, both strands were analyzed in order to confirm the sequence results. For this purpose, in some cases, smaller fragments were subcloned into pUC19
Metalloproteinase Chicken Inhibitor: using the restriction sitesindicated in Fig. 4. At least two independent subclones were sequenced to identify possible errors caused by PCR. Sequence Analysis-DNA and deduced amino acid sequence analyses were performed using the EuGene Sequence Analysis Package from the MBIR Molecular Biology Information Resource, Department of Cell Biology,Baylor College of Medicine. Comparison of the deduced amino acid sequence of ChIMP-3 with other sequences in the data bank was performed using thePattern-Induced Multisequence Alignment (PIMA) algorithm of Smith and Smith (1990, 1992) which employs secondary structure-dependent gap penalties for comparative protein modeling. RESULTS
Analysis of cells, ECM, and conditioned media from cultures of normal and transforming chicken embryo fibroblasts by protease/substrate gel electrophoresis indicates five distinct IMP activities which range in size from M, 20,000 to M, 28,000.’ Until we have firmly established the relationship of these proteins to one another and to thetwo existing members of the TIMPfamily we propose to call them ChIMP2 and -3 and ChIMP-a through -c. In this paper we report that the three most abundant are ChIMP-2, ChIMP-3, and ChIMP-a. ChIMP-2 is a major inhibitor found in the conditioned media and ChIMP-3 and ChIMP-a are found in the ECM (Fig. 1).ChIMP-b and ChIMP-c are minor inhibitors detected in the ECM and media, respectively.’ ChIMP-3 was formerly called the 21-kDa protein and was studied because its synthesis is stimulated during oncogenic transformation. Early characterization of this protein indicated that it was probably not N-glycosylated (Blenis and Hawkes, 1983). To examine this question further, isolated ChIMP-3 was incubated with N-glycosidase-F and then analyzed by protease/substrate gel electrophoresis. As shown in Fig. 2 (left panel), the electrophoretic mobility of ChIMP-3 was not altered by treatment with N-glycosidase-F. Under identical conditions, the apparent relative mass of ChIMP-a was decreased by approximately 5 kDa after enzyme treatment. These data support our earlier proposal that ChIMP-3 is not N-glycosylated whereas ChIMP-a isan N-glycoprotein and, like TIMP-1 (Stricklin, 1986), does not require carbohydrate for its activity. ChIMP-3 and deglycosylated ChIMPa have slightly different relative mobilities in SDS-polyacrylamide gels, with ChIMP-3 migrating slower than ChIMP-a (Fig. 2, right panel). Thus ChIMP-3 appears to differ from ChIMP-a by more than just the absence of N-linked carbohydrate, suggesting that ChIMP-3 is a distinct protein. We have previously determined the primary structure of the NH2 terminus of ChIMP-3 by direct amino acid sequencing(Staskus et al., 1991). This information was used to prepare a mixture of synthetic oligonucleotides (primer I) coding for amino acids 6-11, IHPQDA (numbering of residues from the NH, terminus of the mature protein, Staskus et al. (1991)). This sequence was chosen for the relatively low degeneracy of its codons and because it exhibits the most differences with published sequences of TIMP-1 and TIMP2. Primer I and anoligo(dT) primer (11)were used in a PCR on the Xgtll chicken cDNA library. A single 483-base pair product (P483) resulted. This was cloned into pUC19 and sequenced. Sequence analysis of P483 revealed that only primer I was used in the amplification of partial ChIMP-3 cDNA (nucleotides 200-682, Fig. 3). This primer, designed to anneal to the noncoding strand of the sequence (nucleotides 200-216, Fig. 3), was also able to anneal to thecoding strand (nucleotides 666-689, Fig. 3) with a total of six mismatches, of which two werein the sequence specific for ChIMP-3 (Fig.
-
-
* P. W. Staskus, F. R. Masiarz, N. S. Kishnani, and S. P. Hawkes, submitted for publication.
ChIMP-3
17323 1 2 3
p-
I
97.4 66.2 45.0
- 31.0
ChlMP-a + ChIMP-3 a ChIMP-2 -+
,
:.r-+:z, 9.0.3 Thus, on the basis of NH2-terminal amino acid sequence, size, potential disulfide bonding, and PI, we conclude that the nucleotide sequence presented in Fig. 3 encodes ChIMP-3. TIMP-1 is a glycoprotein (Mr 28,000) with two proposed oligosaccharide chains (Murphy and Werb, 1985; Carmichael et al., 1986; Stricklin, 1986) whereas proteins in the TIMP-2 group aresmaller with M, 18,000-23,000 under nonreducing conditions (Murrayet al., 1986; DeClerck et al., 1989; StetlerStevenson et al., 1989) and based on their inability to bind concanavalin A are proposed to be unglycosylated (Murray et al.,1986; De Clerck et al., 1989). ChIMP-3 exhibits metalloproteinase inhibitoractivity (Staskus et al., 1991) and in terms of molecular mass and apparentlack of glycosylation it resembles TIMP-2. Polyclonal antibodies to either human TIMP1 or human TIMP-2 do not demonstrate significant binding toChIMP-3and antibody toChIMP-3 does notbind to human TIMP-1or TIMP-2 (Staskus et al., 1991). It is clear that ChIMP-3is related to but distinctfrom the groups of TIMP-1 and TIMP-2 proteins whose deduced amino acid sequences are compared in Fig. 5. In common with other TIMPs, the ChIMP-3 sequence predicts a hydrophobic leader sequence consistent with it being a secreted protein and 12 cysteine residues which are conserved among all members of this family. The mature ChIMP-3 (188 amino acids) is intermediate in size between TIMP-1 and TIMP-2(181-184 and 196 amino acids, respectively). As discussed under "Results," the region of greatest similarity for all TIMPs and ChIMP-3
------~ ----
163
SWyrgWappDKTllN ~~
is at the NH2 terminus of the mature protein.Indeed, Murphy et al. (1991b) have shown that theactivity of TIMP-1 resides in the NH2-terminal half of the protein. Some features of the ChIMP-3 sequence resemble the TIMP-1 sequences, others resemble the TIMP-2sequences, and -44% of the amino acid residues of ChIMP-3 areunique. The mechanism of inhibition of MMPs by their inhibitors has not yet been elucidated. However, Woessner (1991) has noted two fairly conserved regions of TIMP-1 and TIMP-2 where a negatively charged residue precedes several hydrophobic residues in the primary structure. He has speculated that binding of the hydrophobic amino acids to the Sl'-S2'S3' region of MMPs would place the negatively charged residue in a position to interact with the zinc and render the enzymes inactive. The first sequence, beginning at Asp-16 of mature TIMP-1 and TIMP-2,is also conserved in ChIMP-3; however, the second, beginning at Glu-82, is not conserved in the chicken protein. The Glu is replaced by Gln in ChIMP-3. Thus, if such a mechanism is operative in the inhibition of MMPs by TIMPs, our data support the potential role of Asp16 rather than Glu-82 in thisprocess. The two sites of potential N-linked glycosylation in TIMP1(Docherty et al., 1985; Carmichael et al., 1986) are not found in the ChIMP-3sequence. The lack of incorporation of D-[23H]mannose into ChIMP-3 (Blenis and Hawkes, 1983) and the absence of any change in the electrophoretic mobility of ChIMP-3treated with N-glycosidase-F or synthesized by tunicamycin-treated cells: indicate that ChIMP-3is probably not N-glycosylated. Although one potential site of N-linked glycosylation is apparent in the deduced amino acid sequence at the carboxyl terminus of ChIMP-3, not every sequence of this type is glycosylated in secreted proteins (Beeley, 1985). The carbohydrate cannot contribute significantly to the molecular mass of the protein because estimates of the molecular weight of ChIMP-3 determined by electrophoretic analysis and calculation from the deduced amino acid sequence show a close correspondence, as indicated under "Results." Previously, we proposed, on the basis of amino acid composition data,thatChIMP-3 was more closely related to members of the TIMP-1group of proteins than the TIMP-2 group (Staskus et al., 1991). This analysis, and additional C. J. Henrich and S. P. Hawkes, unpublished data.
Metalloproteinase Chicken Inhibitor:
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data, led us to speculate that ChIMP-3 was either a variant of TIMP-1 or a third, new member of the TIMP family. However, the deduced amino acid sequence, which is presumably more accurate than chemical measurements of amino acid composition, indicates that ChIMP-3is more like TIMP2 than TIMP-1, but clearly a distinct protein. ChIMP-3 has 42% identity with a consensus sequence of TIMP-2 and 28% identity with a consensus of TIMP-1. The only TIMP-2 sequences reported so far (human and bovine) are highly conserved (94% identity). One could reasonably argue that TIMP-2 from mammalian species would not be expected to share the same degree of identity with an avianprotein. However, we have recently purified another protein, ChIMP2, which is undoubtedly the chicken equivalent of TIMP-2. This conclusion is based upon a number of criteria including its co-purification in a complex with pro-MMP-2, NHp-terminal amino acid sequence, amino acid composition and binding with anti-TIMP-2 antibody.2 Thus we propose thatChIMP-3 is neither TIMP-1or TIMP-2 but a new member of the TIMP family, TIMP-3. Although in some respects ChIMP-3 is similar to TIMP-1 and TIMP-2, in others it is unique. In particular, ChIMP-3 is relatively insoluble (Blenis and Hawkes, 1984) and found exclusively in the ECM (Staskus et al., 1991), unlike most members of the TIMP-1 and TIMP-2 groups of proteins that are isolated from tissue fluids or cell culture media. In contrast to TIMP-2 (and ChIMP-2) ChIMP-3 is not isolated in a complex with aparticularpro-MMP. Since most of the chicken MMPs have not yet been purified, the inhibitory specificity of ChIMP-3 remains to be determined. We have not yet characterized a TIMP-1 from the avian system. One possible candidate for this assignment is ChIMP-a, which is clearly an N-glycosylated metalloproteinase inhibitor. Relative to ChIMP-3, ChIMP-a is a minor activity in the ECM, and its possible localization in conditioned media has not been rigorously examined. Evidence for the existence of a family of TIMP-like proteins has been accumulating for a number of years. Only in two species (human andbovine) have both TIMP-1 and TIMP-2 been completely sequenced. Three metalloproteinase inhibitors have been reported in rabbit brain capillary endothelial cells: TIMP-1 (Mr= 30,000), IMP-1 (Mr= 22,000), and IMP2 (Mr= 19,000) (Herron et al., 1986). The same threeinhibitorsandan additional IMP-3 ( M , = 16, 500) have been detected in human glioma cell lines (Apodaca et al., 1990). We originally reported four (Staskus and Hawkes, 1989) and nowfive inhibitor activities in chicken embryo fibroblasts. Recently, Chen et al. (1991) reported the purification of a chicken 70-kDa gelatinase associated with a 22-kDa protein. Based on this association the protein was assumed to be TIMP-2. Craig et al. (1991) have reported the detection of three discrete metalloproteinase inhibitors in the culture media conditioned by 11-day chick tibiae. Since two of these activities were not produced by skin fibroblasts or cultures of calvariae, the relationship of these proteins to the five ChIMPS discussed above remains to be clarified. The third inhibitor ( M , 23,000), which was not characterized further, was assumed to be chicken TIMP-2. As discussed earlier we propose to call the chicken inhibitors ChIMPs (Chicken inhibitor of MetalloEroteinases). A letter designation (ChIMP-a, -b, and -c) will beused for those whose relationship to other ChIMPs and TIMPsremains to be clarified. The letter will be converted to a number desig-
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ChIMP-3 nation (as for ChIMP-2 and ChIMP-3) when the identity of the inhibitor is established. In summary, based on its primary structure andbiochemical properties, we propose that ChIMP-3is a new, matrix-specific TIMP-3. It is of particular interest because its synthesis is stimulatedduring oncogenic transformation and purified ChIMP-3 hasbeen shown to promote some of the phenotypic properties of transformed cells (Yang and Hawkes, 1989, 1992). Acknowledgments-We thank Dr. Wolfgang Sadee for useof equipment, Leslie Taylor for help with the computer analysis, and Dr. Thomas Meehan, Alan Wolfe, and Joelle Thomas for critical review of the manuscript. REFERENCES Apodaca, G., Rutka, J. T., Bouhana, K., Berens, M. E., Giblin, J. R., Rosenblum, M. L., McKerrow, J. H., and Banda, M. J. (1990) Cancer Res. 50,2322-2329 Beeley, J. G. (1985) in Laboratory Techniques in Biochemistry and Molecular Biology; Glycoprotein and Proteoglycan Techniques (Burdon, R. H., and van Knippenberg, P. H., eds) Vol. 16, p. 15, Elsevier Science Publishers B.V., Amsterdam Blenis, J., and Hawkes, S. P. (1983) Pmc. Natl. Acad.Sci. U. S. A. 80,770-774 Blenis, J., and Hawkes, S. P. (1984) J. Biol. Chem. 259,11563-11570 Boone, T. C., Johnson, M. J., De Clerck, Y. A,, and Langley, K. E. (1990) Proc. Natl. A d . Sei. U. S. A. 87, 2800-2804 Brawerman, G. (1981) Crit. Reu. Biochem. 10, 1-38 Carmichael, D. F., Sommer, A. Thompson, R. C., Anderson, D. C., Smith, C. G., Welgus, H. G., and Stricilin, G. P. (1986) Proc. Natl. Acad. Sci. U. S. A. 83,2407-2411 Chen, J.-M., Aimes, R. T., Ward, G. R., Youngleib, G. L., and Quigley, J. P. (1991) J. Biol. Chem. 266,5113-5121 Craig, F. M., Archer, C.W., and Murphy, G. (1991) Biochim. Biophys. Acta
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