From the
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.
Remodeling of the extracellular matrix (ECM)
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.
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.
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
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.
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.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
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.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)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.
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
ml
h
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.
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, ).
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.
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.
Table: The
exon-intron boundaries of the Timp-3 gene
Table: Specific activities of human and mouse TIMP-3
and human TIMP-1 against the matrix metalloproteinases
/EMBL Data Bank with accession number(s) L19622, U26433,
U26434, U26435, U26436, U26437.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.