(Received for publication, August 1, 1994; and in revised form, October 31, 1994)
From the
Type XIV collagen is a newly described member of the
fibril-associated collagens with interrupted triple helices (FACITs).
Expression of this collagen has been localized to various embryonic
tissues, suggesting that it has a functional role in development. All
FACITs thus far described (types IX, XII, XIV, and XVI) contain a
highly homologous carboxyl-terminal triple helical domain designated
COL1. We have studied the capacity of various matrix metalloproteinases
(interstitial collagenase, stromelysin, matrilysin, and 92-kDa
gelatinase) to degrade the COL1 domain of collagen XIV. We found that
only 92-kDa gelatinase cleaves COL1. Furthermore, digestion of whole
native collagen XIV by the 92-kDa gelatinase indicates that this enzyme
specifically attacks the carboxyl-terminal triple helix-containing
region of the molecule. COL1 is cleaved by 92-kDa gelatinase at 30
°C, a full 5-6 °C below the melting temperature (T) of this domain; native collagen XIV
is also degraded at 30 °C. In comparison to interstitial
collagenase degradation of its physiologic native type I collagen
substrate, the 92-kDa enzyme cleaved COL1 (XIV) with comparable
catalytic efficacy. Interestingly, following thermal denaturation of
the COL1 fragment, its susceptibility to 92-kDa gelatinase increases,
but only to a degree that leaves it several orders of magnitude less
sensitive to degradation than denatured collagens I and III. These data
indicate that native COL1 and collagen XIV are readily and specifically
cleaved by 92-kDa gelatinase. They also suggest a role for 92-kDa
gelatinase activity in the structural tissue remodeling of the
developing embryo.
The collagen family of extracellular matrix proteins can be
divided into several groups, each with distinct structural and
functional characteristics(1) . The historically oldest group,
the fibrillar collagens (types I, II, III, V, and XI), contains members
that form banded collagen fibrils. Type XIV collagen (2, 3) belongs to the more recently recognized and
consequently less understood group of fibril-associated collagens with
interrupted triple helices (FACITs). ()FACITs share regions
of high sequence homology (3, 4, 5) and
distinct structural characteristics. By definition, molecules in this
class contain more than one triple helical domain separated by
nontriple helical segments(6, 7) . FACITs do not
require proteolytic processing from a procollagen form as the fibrillar
collagens do. Furthermore, the FACITs do not form homopolymers but
rather are associated with collagen fibrils. In fact, their multidomain
structure and tissue distribution suggest that FACITs constitute
molecular bridges linking together the fibrils of a tissue (8, 9) . Four FACITs are known at present, types IX,
XII, XIV, and XVI, the best characterized being type IX, associated
with type II collagen fibrils(10, 11) , and a major
constituent of hyaline cartilage. Type XII collagen is found in dense
connective tissues such as tendons, ligaments, and
perichondrium(12, 13, 14) . The homology
between types IX and XII collagens is highest in the short
carboxyl-terminal triple helical domain designated COL1(15) .
This triple helical domain has been used as a ``signature''
to define FACIT molecules(16) . The structure of COL1 is unique
to FACITs and is characterized by the presence of a precisely conserved
pair of cysteine residues and by imperfections in the triple
helix(3) . While cysteine residues and helix imperfections have
been found in other collagen types (e.g. type IV
collagen(17) ; type VI collagen(18) , and type XIII
collagen(19) ), their locations are completely different from
those observed in FACITs.
Recently described FACITs with COL1-like domains include types XIV and XVI collagens(3, 20, 21, 22) . Type XIV collagen was isolated from tissues containing predominantly type I collagen, such as skin and tendon(3) . The biologic function of type XIV collagen is not yet clearly understood. However, aside from a role in linking together fibrils of a tissue, collagen XIV has been shown to interact with heparin sulfate proteoglycan, collagen VI and the dermatan sulfate side chain of decorin and may be important in cell-matrix interactions(23, 24) . As determined by cDNA analysis, collagen XIV has a number of sequences in its nonhelical domains that are homologous to other structural proteins and has two RGD potential cell recognition sequences in its COL2 domain (7, 25) . Collagen XIV has also been localized around growing hair follicles, which suggests a role for this molecule in the formation of skin appendages(26) . Interactions between epidermis and dermis are vital for hair follicle initiation and development(27) . The prominent location of collagen XIV in developing hair follicle mesenchyme may be important for such epidermal-mesenchymal interactions(26) .
The matrix
metalloproteinases ()are a gene family of enzymes that
contribute to the degradation and remodeling of connective
tissues(28) . These enzymes are important in normal physiologic
remodeling such as wound healing (29) and uterine
involution(30, 31) , but they have also been
implicated in destructive pathologic processes including rheumatoid
arthritis (32) and tumor invasion(33) . Interstitial
collagenase, the most thoroughly studied member of this gene family,
catalyzes the initial and rate-limiting step in the cleavage of native
types I, II, and III collagens(34, 35, 36) .
The 92-kDa and 72-kDa gelatinases are structurally related enzymes that
are produced by different cell types. In addition to denatured
collagens (gelatins) of all genetic types, both gelatinases degrade
native types IV and V collagens, fibronectin, and insoluble elastin (37, 38) . Stromelysins-1 and -2 are highly related
metalloproteinases with the capacity to attack a broad range of matrix
substrates including laminin, fibronectin, proteoglycans, and types IV
and IX collagens in their nonhelical
domains(28, 37, 39) . Matrilysin is the
smallest metalloproteinase, but it exhibits potent catalytic activity
against noncollagenous matrix components including proteoglycans,
fibronectin, laminin, entactin, and
elastin(37, 40, 41) .
The susceptibility of most FACITs to the matrix metalloproteinases has not been studied. Yet, these collagens must be degraded during the turnover of fibrillar collagens with which they are so closely associated. The purpose of this study was to examine the susceptibility of one such FACIT, collagen XIV, and in particular its COL1 domain, to cleavage by matrix metalloproteinases.
Figure 1: Trypsin susceptibility of the COL1 domain of type XIV collagen as a function of temperature. COL1 (2 µg) was incubated at the temperatures indicated at the top of the figure and treated with trypsin as described under ``Materials and Methods.'' The reaction mixtures were resolved by nonreducing SDS-polyacrylamide gel electrophoresis (15% gel), and protein bands were stained with Coomassie Blue. Electrophoretic mobility of CNBr fragments from type I collagen are indicated as standards on the left of the figure. Electrophoretic mobility of the COL1 domain is shown by the arrow on the right of the figure.
Figure 2:
Degradation of the COL1 domain of type XIV
collagen by matrix metalloproteinases. The COL1 domain of type XIV
collagen (2.8 µg) was incubated with the indicated
metalloproteinases at 30 °C for 18 h. The enzymes were all used at
a final concentration of 6.5 10
M.
Reactions were stopped by boiling in SDS sample buffer containing
dithiothreitol and then applied to a 15% polyacrylamide
gel.
Degradation of
the COL1 domain of type XIV collagen demonstrated significant
temperature dependence. As shown in Fig. 3, at 32 °C and pH
7.4, 92-kDa gelatinase (6.3 10
M)
degraded approximately 90% of the COL1 fragment. However, at 25 °C,
an identical amount of enzyme cleaved only a trivial proportion of the
substrate.
Figure 3:
Temperature dependence of the degradation
of the COL1 domain of type XIV collagen by 92-kDa gelatinase. COL1 (3.0
µg) was incubated with 92-kDa gelatinase (6.3
10
M) at the various temperatures indicated
for 24 h. The enzyme-free control was incubated at 25 °C. Reactions
were stopped by boiling in SDS sample buffer containing dithiothreitol
and then applied to a 4-20% gradient
gel.
Figure 4:
Comparison of 92-kDa gelatinase
degradation of COL1 with interstitial collagenase cleavage of native
type I collagen. Incubation of equimolar concentrations of COL1 (XIV)
and type I collagen (4 10
M) were
performed with the target enzymes at 30 and 32 °C for 1 h, as
described under ``Materials and Methods.'' Molarities of
92-kDa gelatinase used to degrade COL1 at 30 °C were 6.6
10
M, 3.3
10
M, and 1.6
10
M.
Molarities at 32 °C were 5.3
10
M, 1.3
10
M, and 4.4
10
M. Molarities of collagenase
used to degrade type I collagen at 30 °C were 3
10
M, 1.5
10
M, and 7.5
10
M.
Molarities at 32 °C were 1.0
10
M, 3.3
10
M, and 1.1
10
M. Reaction mixtures using COL1
were separated on a 23% polyacrylamide gel. Type I collagen products
were separated on an 8% polyacrylamide gel. Degradation was determined
by densitometric scanning of substrate
disappearance.
Figure 5:
Degradation of native and denatured COL1
and of native and denatured types I and III collagens by 92-kDa
gelatinase. A, native type I collagen (3.0 µg) was
incubated at 30 °C for 18 h with either no(-) or 2.6
10
M 92-kDa gelatinase. Denatured type I
collagen was incubated with either no(-) or 10-fold dilutions of
92-kDa gelatinase starting in lane1 with 2.6
10
M enzyme to 2.6
10
M enzyme in lane5. Products were
separated on a 10% polyacrylamide gel. B, native type III
collagen (3.0 µg) was incubated at 30 °C for 18 h with either
no (-) or 2.6
10
M 92-kDa
gelatinase. Denatured type III collagen was incubated with either
no(-) or 10-fold dilutions of 92-kDa gelatinase starting in lane1 with 2.6
10
M enzyme to 2.6
10
M enzyme in lane5. Products were separated on a 10%
polyacrylamide gel. C, native COL1 fragment of type XIV
collagen (3.0 µg) was incubated at 30 °C for 18 h with either
no(-) or 2.6
10
M 92-kDa
gelatinase. Denatured COL1 (XIV) was incubated with either no(-)
or 10-fold dilutions 92-kDa gelatinase starting in lane1 with 2.6
10
M enzyme to 2.6
10
M enyzme in lane5. Products were separated on a 20% polyacrylamide
gel.
Figure 6:
TIMP
inhibits degradation of the COL1 domain of type XIV collagen by 92-kDa
gelatinase. COL1 (3.0 µg) was incubated with 92-kDa gelatinase (2.6
10
M) at 30 °C for 24 h. In the
reaction mixtures indicated, TIMP (1.6 or 8.1 µg) was added to the
gelatinase as described under ``Materials and Methods.''
Products were resolved on a 4-20% polyacrylamide gradient
gel.
Figure 7: Cleavage of whole native type XIV collagen by 92-kDa gelatinase. Two preparations of type XIV collagen (4.0 µg/lane) were used in this experiment. Lanes1-3 contain a mixture of type I and type XIV collagens from the carboxymethyl-cellulose pool obtained as described under ``Materials and Methods.'' Lanes4-7 contain pure bovine tendon type XIV collagen, which was obtained following concanavalin A-Sepharose chromatography as described under ``Materials and Methods.'' Lanes1 and 4 represent the respective collagen preparations that were not incubated. Lanes2 and 5 were incubated with buffer alone at 30 °C for 18 h. Lanes3 and 6 were incubated with 92-kDa gelatinase (0.5 µg) at 30 °C for 18 h. Lane7 contains the pure NC3 domain of the type XIV collagen, obtained as described (4) . The arrowheads mark partially digested forms of type XIV collagen, which must retain the disulfide bridge in the NC2 domain to exhibit this pattern of electrophoretic migration. After incubation, the samples were acetone-precipitated before being analyzed by 5% SDS-polyacrylamide gel electrophoresis.
In this study, we have examined the capacity of several human matrix metalloproteinases to degrade the COL1 fragment of type XIV collagen. Our data indicate that this important helical component of FACITs is susceptible only to 92- and 72-kDa gelatinases. The structure of COL1 is unique to FACITs and is characterized by the presence of a pair of cysteines, one at the carboxyl-terminal end of the triple helix and one 5 residues inside the amino-terminal NC1 domain. The COL1 domain of all FACITs has two imperfections of triple helical structure located at similar positions relative to the cysteines, one Gly-X-Gly-X-Y imperfection and one Gly-X-Y-X-Y imperfection. Disulfide bridges and helix imperfections have been found in other collagen types such as type IV collagen(17) , type VI collagen(18) , and type XIII collagen(19) ; of these collagens, only type IV is susceptible to 92-kDa gelatinase. In addition to type IV collagen, 92-kDa gelatinase is known to degrade a number of other matrix proteins including native collagen types V, VII, and X, as well as denatured collagens of all genetic types, fibronectin, and insoluble elastin(38, 46, 54) .
Helix imperfections
of the COL1 domain may represent the target of attack of 92-kDa
gelatinase. In this regard, we have previously shown that type X
collagen is susceptible at two loci to interstitial collagenase and
72/92-kDa gelatinases, both cleavage sites constituting the Gly-X bonds of Gly-X-Y-X-Y sequences. Since 92-kDa gelatinase is known to be an efficient
gelatinase, it was critical for us to determine the T of the COL1 fragment of type XIV collagen. Using trypsin
susceptibility as a probe, this T
was found to be
35-36 °C (Fig. 1). Therefore, we performed all
experiments at 30 °C, a full 5 °C less than the T
. Nevertheless, we found that 92-kDa cleavage of
COL1 at 30 °C (or even at 28 °C, data not shown) failed to
produce electrophoretically identifiable fragments (Fig. 2),
suggesting that the products of initial helical scission(s) rapidly
denature and are subsequently degraded to completion by the enzyme,
therefore making the determination of a turnover number unfeasible. To
place the rate of catalysis of the COL1 fragment by 92-kDa gelatinase
into biological perspective, we compared it to the rate of cleavage of
native type I collagen by interstitial collagenase. We found that at 30
°C, 92-kDa gelatinase degraded COL1 with approximately 50% the
efficacy, on a molar basis, of interstitial collagenase cleavage of
native type I collagen (Fig. 4). At 32 °C, 92-kDa gelatinase
degraded COL1 approximately 2-fold as rapidly as collagenase degrades
type I collagen. Therefore, 92-kDa gelatinase cleavage of the COL1
domain of type XIV collagen is approximately equivalent in rate to that
of the physiologically accepted degradation of type I collagen by
collagenase. It is important to note, however, that the specific
activity of stromelysin-activated 92-kDa gelatinase (used in Fig. 4) is severalfold greater than that of p-aminophenylmercuric acetate-activated 92-kDa gelatinase
(used in other figures of the manuscript). (
)Furthermore,
the degradation of COL1 shown in Fig. 2, Fig. 3, Fig. 5, and Fig. 6exceeded 75% and therefore greatly
underestimates initial velocity. In any event, the importance of 92-kDa
gelatinase cleavage of native COL1 is underscored by digestions of
whole native type XIV collagen (Fig. 7), which strongly suggest
that this helical domain represents a physiologic point of attack of
the enzyme upon this entire native collagen molecule.
Thermal denaturation of COL1 increased its sensitivity to degradation by 92-kDa gelatinase approximately 50-fold (Fig. 5), supporting the concept that the initial cleavage of this collagen at 30 °C indeed represents catalytic activity against a native helical substrate. However, cleavage of denatured COL1 by 92-kDa gelatinase was surprisingly poor as compared with this enzyme's degradation of denatured types I and III collagens (Fig. 5). The explanation for this interesting finding is not at all clear. While the presence of intramolecular disulfide bonds might be expected to preserve tertiary structure following denaturation or perhaps even enhance its reformation, type III collagen, as well as COL1, contains these stabilizing elements. Nevertheless, this resistance of denatured COL1 to proteolysis as compared with denatured types I and III collagens was maintained when trypsin was substituted for 92-kDa gelatinase (data not shown).
By performing our studies with recombinantly expressed 92-kDa gelatinase and thereby using enzyme without associated TIMP, we were able to avoid the problem of TIMP-inhibited enzyme, which necessarily accompanies the isolation of 92-kDa progelatinase from normal mammalian cell cultures(55, 56) . Since we have shown such enzyme to be almost 10-fold as active as 92-kDa gelatinase purified as a complex with TIMP (55) , this maneuver was critical to our studies. As expected, however, the addition of exogenous TIMP was able to largely ablate the capacity of 92-kDa gelatinase to degrade the COL1 fragment of type XIV collagen (Fig. 6).
Type XIV collagen mRNA has been found in many tissues including lung, heart, muscle, skin, stomach, tendon, sternum, and calvaria(57) . It has also been localized to various embryonic tissues, indicating a role in development. For example, using monoclonal antibodies, type XIV collagen has been found in the embryo in large bundles of collagen fibrils within tendons and, interestingly enough, also around growing hair follicles in basket-like structures(26) . The specific localization of type XIV collagen around hair follicles suggests a function for this protein during the formation of skin appendages. During the invagination of epithelial cells forming the hair follicle there is extensive association with underlying dermis, and this extracellular matrix is probably a site of protein turnover. In fact, TIMP gene expression has been shown in the sheath of follicles that form vibrissae(58) . Furthermore, epithelial cells from hair follicles have been reported to express 72-kDa gelatinase (59) as well as the 92-kDa gelatinase. It may therefore be speculated that these gelatinases play an important role in basement membrane remodeling in the embryo, especially during the morphogenesis of hair follicles. Finally, since FACITs link interstitial collagens, the involvement of more than one matrix metalloproteinase (e.g. interstitial collagenase and a gelatinase) may be required to effectively degrade collagen fibrils otherwise composed largely of types I, II, or III collagens.