(Received for publication, August 17, 1995; and in revised form, January 9, 1996)
From the
Lysyl oxidase is secreted from fibrogenic cells as a 50-kDa
proenzyme that is proteolytically processed to the mature enzyme in the
extracellular space. To characterize the secreted proteinase activity,
a truncated, recombinant form of lysyl oxidase was prepared as a
proteinase substrate containing the sequence of the propeptide cleavage
region. The processing proteinase activity secreted by cultured
fibrogenic cells resists inhibitors of serine or aspartyl proteinases
as well as tissue inhibitor of matrix metalloproteinases-2 (MMP-2) but
is completely inhibited by metal ion chelators. Known
metalloproteinases were tested for their activity toward this
substrate. Carboxyl-terminal procollagen proteinase (C-proteinase),
MMP-2, and conditioned fibrogenic cell culture medium cleave the lysyl
oxidase substrate to the size of the mature enzyme. The
NH-terminal sequence generated by arterial smooth muscle
conditioned medium and the C-proteinase but not by MMP-2, i.e. Asp-Asp-Pro-Tyr, was identical to that previously identified in
mature lysyl oxidase isolated from connective tissue. The C-proteinase
activity against the model substrate was inhibited by a synthetic
oligopeptide mimic of the cleavage sequence
(Ac-Met-Val-Gly-Asp-Asp-Pro-Tyr-Asn-amide), whereas this peptide also
inhibited the generation of lysyl oxidase activity in the medium of
fetal rat lung fibroblasts in culture. In toto, these results
identify a secreted metalloproteinase activity participating in the
activation of prolysyl oxidase, identify inhibitors of the processing
activity, and implicate procollagen C-proteinase in this role.
Lysyl oxidase (protein-lysine 6-oxidase, EC 1.4.3.13) is a
copper-dependent amine oxidase that oxidatively deaminates the
-amino group of specific peptidyl lysine and hydroxylysine
residues of collagen and of lysine in elastin. The resultant peptidyl
aldehydes can spontaneously condense with other vicinal peptidyl
aldehydes or with unreacted
-amino groups to form inter- and
intramolecular cross-linkages stabilizing the fibrous forms of these
connective tissue structural proteins(1) . Evidence has been
presented that the inhibition of lysyl oxidase action toward collagen
molecules results in the accumulation and ultimate proteolytic
degradation of soluble collagen monomers, thus preventing the formation
of insoluble collagen fibers(2) . The participation of this
enzyme is critical, therefore, to the development and repair of
structurally sound connective tissues as in the respiratory,
cardiovascular, and skeletal systems of the body.
Recent studies of the pathway by which lysyl oxidase is produced in arterial smooth muscle cells revealed that the protein is translated as a 46-kDa preproenzyme. Following signal peptide cleavage, the proenzyme undergoes N-glycosylation apparently within the propeptide region, and the resulting 50-kDa proenzyme is then secreted into the extracellular space. The secreted proenzyme is then proteolytically converted to the functional catalyst derived from the COOH-terminal sequence of the proprotein(3) . The molecular mass of the mature, functional catalyst, as isolated from various connective tissues, ranges from 28 to 32 kDa(1) . The involvement of a proenzyme processing protease in the biosynthetic pathway of lysyl oxidase is of particular interest, because this may represent a previously unexpected extracellular mechanism for the regulation of the production of this cross-linking enzyme. In turn, such a protease could profoundly influence the rate and/or extent of connective tissue fiber formation. The nature of this proteolytic activity is explored in the present study.
The precursor of lysyl oxidase was biosynthetically
produced by RS485 cells, as described previously(3) . Briefly,
these cells were derived from NIH 3T3 fibroblasts by stable
transfection with the Ha-ras oncogene(6) . The
resulting RS485 cell line was then stably cotransfected with the lysyl
oxidase expression vector pSV40poly(A)COD and pSV2neo to yield the S4
cell line that secretes the 50-kDa lysyl oxidase precursor but does not
process it to the mature enzyme(3) . These cells were
maintained in DMEM containing 4 g/liter glucose, 10% fetal bovine
serum, 50 units/ml penicillin, 50 µg/ml streptomycin, 1 mM sodium pyruvate, and 0.1 mM nonessential amino acids. The
S4 cells were pulse-labeled for 3 h with 50 µCi/ml
[S]methionine in the same medium lacking
[
S]methionine and fetal bovine serum. The
conditioned medium containing the 50-kDa glycosylated prolysyl oxidase
was then used as a substrate for assay of prolysyl oxidase processing
protease activity of conditioned smooth muscle cell medium. Following
incubation of the S4 medium containing the
S-labeled
substrate with the conditioned medium as the source of proteinase
activity, the reaction mixture was immunoprecipitated with rabbit
anti-lysyl oxidase, and the immunoprecipitates were analyzed by
SDS-PAGE (7) and autoradiography, as described(3) .
Prolysyl oxidase was also biosynthetically labeled and prepared from
RFL6 cells by these pulse labeling and immunoprecipitation procedures
described for the S4 cells. Unlike the S4 cells, which secrete but do
not process prolysyl oxidase, the RFL6 cells both secrete and
proteolytically process the 50-kDa precursor to the 30-kDa mature
enzyme. These cells accumulate readily assayed levels of lysyl oxidase
activity in the medium to a greater extent than the smooth muscle cell
cultures, thus facilitating the assessment of proenzyme activation. The
RFL6 cells were used to assess the effect of a proteinase inhibitor on
proenzyme processing during enzyme biosynthesis.
The recombinant COOH-terminally truncated form of human stromelysin (MMP-3) was expressed in Escherichia coli expression system. PGEMEX-1 vector (Promega) containing the truncated version of the protein-coding region of MMP-3 (obtained from Dr. Henning Birkedal-Hansen at the University of Alabama, Birmingham) was transformed into E. coli strain BL21 and recombinant protein purified according to Refs. 12, 14, and 16.
MMP-9 (92-kDa gelatinase) was expressed in mammalian p98 cells according to (15) . The stable cell line expressing proMMP-9 was obtained from Dr. G. Goldberg (Washington University School of Medicine, St. Louis, MO). The expression system and purification of the recombinant protein has been described(15) .
The P5128 stable cell line expressing proMMP-1 (interstitial collagenase) (15) was obtained from Dr. G. Goldberg (Washington University School of Medicine, St. Louis, MO). The expression system and purification of the recombinant enzyme is as described(13, 15) .
All matrix metalloproteinases
were expressed and purified as proenzymes. ProMMPs were activated by
incubation with 1 mM 4-aminophenylmercuric acetate following
which excess 4-aminophenylmercuric acetate was removed by
gel-filtration. Activities of MMP-3, MMP-9, and MMP-1 were assayed
according to Knight et al.(17) with a
coumarin-labeled synthetic peptide substrate:
(7-methoxycoumarin-4-yl)acetyl-Pro-Leu-Gly-Leu-(3-[2,4-dinitrophenyl]-L-2,3-diaminopropionyl)-AlaArg-NH.
The specific activities were: 172, 2560, and 1000 pmol of substrate
hydrolyzed µg
min
for MMP-3,
MMP-9, and MMP-1, respectively.
The expected 76-kDa fusion protein (based on the molecular masses of the 43-kDa maltose-binding protein, 0.7 kDa derived from the intervening residues encoded by the cDNA sequence linking the two proteins, and the truncated 32-kDa recombinant lysyl oxidase protein) was identified (band FP, Fig. 1). Close inspection of this band indicated that it represented two closely spaced bands at 76 ± 1 kDa, possibly reflecting the tendency of products expressed in this E. coli system to undergo some degree of proteolysis (New England BioLabs manual). A second band appearing at 65 kDa appeared to be a nonspecific, cross-reacting protein because preincubation of the polyclonal antibody with pure bovine aorta lysyl oxidase did not reduce its appearance in Western blots. In contrast to the fusion protein at 76 ± 1 kDa, this cross-reacting band was not affected by any of the protease treatments used in this study, and its presence did not influence the conclusions of this study. Moreover, attempts at purifying the antibody fraction from the polyclonal rabbit serum did not eliminate the reactivity with this band. The 76-kDa fusion protein preparation was cleaved by factor Xa to produce a 43- and a 33-kDa protein, indicating that the factor Xa Arg-Ile cleavage site within the intervening sequence of the fusion protein is accessible to this protease (not shown).
Figure 1: Western blotting assay of proteolysis of recombinant maltose binding-lysyl oxidase FP by SMC medium. Lanes 1 and 2, probed with anti-lysyl oxidase; lane 3, probed with anti-maltose-binding protein. FP (0.5 µg) was incubated at 37 °C for 2 h with 15 µl of nonconditioned medium (lane 1) or with 15 µl of conditioned SMC medium (lane 2). Lane 3 shows the same sample as in lane 2 reprobed with anti-maltose-binding protein. LO, lysyl oxidase.
Incubation of the affinity purified fusion protein (Fig. 1, lane 1) with conditioned smooth muscle cell medium resulted in the proteolytic conversion of the 76-kDa fusion protein into 30- and 46-kDa fragments (Fig. 1, lanes 2 and 3, respectively). This reaction can be assayed with this substrate simply by Coomassie Blue staining of protein bands resolved by SDS-PAGE without the need for isotopic labeling of the precursor, because of the relative purity and greater quantities of the protease substrate (not shown). The 46-kDa fragment reacted with a polyclonal antibody specific for the maltose-binding protein (MBP, lane 3) (New England Biolabs) but did not react with polyclonal anti-lysyl oxidase as observed by Western blotting (lane 2). The 30-kDa band reacted with anti-lysyl oxidase but not with the anti-maltose-binding serum (LO, lane 2).
Figure 2:
Effect of smooth muscle cell protease
activity against prolysyl oxidase substrate of S4 cells. Conditioned
medium (3 ml) of S-4 cells labeled in culture with
[S]methionine was incubated for 3 h at 37 °C
with 3 ml of nonlabeled medium conditioned by SMC. The reaction
mixtures were immunoprecipitated by anti-lysyl oxidase and resolved by
SDS-PAGE (see ``Materials and Methods''). S4 substrate
incubated with: control (nonconditioned SMC medium, lane 1);
conditioned SMC medium (lane 2); heat-denatured (100 °C, 5
min) conditioned SMC medium (lane 3); conditioned SMC medium
in the presence of 0.2 mM PMSF (lane 4), 2 µg/ml
of aprotinin (lane 6), or 5 mM EDTA (lane
6). LO, lysyl oxidase.
In the course of these studies, it became apparent that the availability of larger quantities of a more conveniently prepared substrate for the assay of the SMC protease was desirable. For that purpose, a recombinant 76-kDa protein was expressed in E. coli consisting of maltose-binding protein fused to that portion of the lysyl oxidase proenzyme suspected to contain the proprotein proteolytic processing sites contiguous with the remaining COOH-terminal sequence representing the mature portion of lysyl oxidase (Fig. 1; also see Fig. 8and ``Discussion''). The presence of the maltose-binding domain within this fusion protein permitted the facile purification of this product from the bacterial lysate by affinity chromatography (see ``Materials and Methods''). As shown in Fig. 3, the cleavage of the fusion protein by SMC medium was not prevented by inhibitors of serine (PMSF and aprotinin), thiol (leupeptin), or acid proteases (pepstatin) but was inhibited by EDTA, EGTA, and prior heat inactivation of the SMC medium, consistent with the results presented in Fig. 2. It is important to note that Western blot analysis of the conditioned smooth muscle cell medium alone did not reveal the presence of lysyl oxidase protein bands, consistent with our repeated observations that lysyl oxidase produced by these cells in culture associates predominantly with the insoluble cell layer/matrix fraction. The results presented at Fig. 2and Fig. 3indicate that full-length or truncated recombinant precursor forms of lysyl oxidase prepared in mammalian or bacterial cells, respectively, can be cleaved to the same apparent size by the SMC-conditioned medium and that the conversion of the proprotein to the mature enzyme species is catalyzed by a heat-labile secreted metalloproteinase.
Figure 8:
Predicted amino acid sequences of
preprolysyl oxidase of different species. Sequences of lysyl oxidase of
the rat(34, 35) ; human (Hum; (36) and (37) ); mouse (Mse; Refs. 6, 38, and
39); and chick (Chk; (40) ) are shown. Left-facing
arrows, Maltose-binding protein is fused to serine 133 and
replaces lysyl oxidase residues 1-132 in the truncated
recombinant fusion protein. , residue(s) corresponding to other
lysyl oxidase species shown are absent. The GDD site is in bold and doubly underlined.
, sites of cleavage by
MMP-2 ( . . . N-LR . . . ) and procollagen C-proteinase ( . . . G-DD .
. . ) are indicated. The cysteine 21-alanine 22 bond is a
putative signal peptide cleavage site (34) .
Figure 3: Western blotting assay of effect of proteinase inhibitors on SMC proteolytic activity. FP (0.5 µg) was incubated for 2 h at 37 °C with nonconditioned medium (lane 1) or with conditioned SMC medium in the presence of 2 µg/ml of aprotinin (lane 2), 0.2 mM PMSF (lane 3), 5 µg/ml leupeptin (lane 4), 0.7 µg/ml pepstatin (lane 5), 5 mM EGTA (lane 6), 5 mM EDTA (lane 7), or 0.2 mM dithiothreitol (lane 8). Heat-denatured SMC medium incubated with FP in the absence of added proteinase inhibitors (lane 9). LO, lysyl oxidase.
The profile of inhibitor
sensitivity and the cleavage site specificity of the SMC medium
proteolytic activity pointed toward candidate extracellular
metalloproteinases that may participate in the extracellular maturation
of prolysyl oxidase. Among possible candidates, procollagen
C-proteinase has been purified from chick calvaria and conditioned
medium of cultures of mouse fibroblasts and of organ cultures of chick
embryo tendons and sterna(18, 22) . This
metalloproteinase normally processes procollagen, cleaving the
pro-1(I) and pro-
2(I) chains at an Ala-Asp bond and
pro-
1(III) at a Gly-Asp bond to release the corresponding
COOH-terminal extension peptides(18, 22) . This enzyme
operates at nearly neutral pH, is Ca
-dependent, and
may require a second metal in addition to Ca
. It is
inhibited by metal chelators but not by inhibitors of serine or
cysteine proteinases. Additional consideration was given to 72-kDa
gelatinase (type IV collagenase; matrix metalloproteinase-2), which is
secreted by mammalian cells of various tissue origins (23, 24) and has been reported to be the major
secreted metalloproteinase constitutively expressed by human vascular
smooth muscle cells(25, 26) . In this regard, analyses
in the present study of the conditioned medium of neonatal rat aorta
SMC medium by gelatin zymography revealed a single gelatinolytic
activity that appeared to be 72-kDa MMP-2 both by molecular mass and by
its sensitivity to antibody specific for MMP-2 (data not shown). Other
matrix metalloproteinases, including MMP-9 (92-kDa gelatinase) and
stromelysin are synthesized and secreted upon stimulation of human
vascular SMC by specific cytokines(26) .
Thus, the following enzymes were screened for their potential to cleave the fusion protein substrate: procollagen C-proteinase, purified from the medium of organ cultures of chick embryo tendons; MMP-2, partially purified from neonatal rat SMC conditioned medium; and purified, recombinant human MMP-2; purified recombinant 92-kDa gelatinase, stromelysin, and MMP-1 (interstitial matrix metalloprotease), each in their catalytically active forms. As shown (Fig. 4), among these, only MMP-2 and procollagen C-proteinase cleaved the fusion protein substrate to yield a 30-kDa product the apparent size of which was indistinguishable from that of the product resulting from cleavage by conditioned SMC medium. As noted in the legend of Fig. 4, approximately equal amounts of activity units of each of the various metalloproteinases were individually incubated with the fusion protein, with the exception that a lesser quantity of the C-proteinase was employed. The results indicate that the C-proteinase is the most effective of the proteinases employed in this assay. When incubated with 10-fold larger quantities of each of these metalloproteinases, the fusion protein substrate was again selectively cleaved to yield proportionately larger quantities of the 30-kDa product by the C-proteinase and by MMP-2, whereas the 92-kDa gelatinase and interstitial collagenase nonspecifically degraded fusion protein to a various products of different sizes (not shown).
Figure 4: Processing of the fusion protein substrate by various metalloproteinases. Western blotting assay. FP (0.5 µg) was incubated for 1 h at 37 °C with nonconditioned medium (lane 1), with 15 µl of conditioned SMC medium (lane 2), with 0.01 nM C-proteinase (lane 3), 5 nM MMP-2 partially purified from SMC medium (lane 4), or with activated recombinant (each of the following) 0.8 nM MMP-2 (lane 5), 0.6 nM 92 kDa gelatinase (lane 6), 2 nM interstitial collagenase (lane 7), or 28 nM stromelysin (lane 8). The partially purified preparation of MMP-2 produces a slightly visible band at 30,000 Da, more readily observed in the original autoradiogram.
To
further characterize the processing proteinase activity, the effects of
TIMP-2, a naturally occurring protein inhibitor of the MMP family (27) , and the effects of a thiol-based peptide,
HS-CH-CH(CH
-CH(CH
)
)-CO-Phe-Ala-NH
,
a potent inhibitor for the group of Zn
-dependent
matrix metalloproteinases(28) , were assessed. An S-acetylated derivative of the thiol peptide is inactive
toward these enzymes and serves as a control for the effect of the
noninhibitory components of the peptide. As shown (Fig. 5A), TIMP-2 inhibited processing of the fusion
protein by purified recombinant MMP-2 (lanes 1 and 2)
but failed to inhibit the cleavage by the conditioned SMC medium (lanes 3 and 4) or by C-proteinase (lanes 5 and 6). The thiol peptide, tested at 80 µM,
inhibited all three enzyme activities (lanes 8, 10,
and 12). The S-acetylated analog of this peptide was
not inhibitory (lanes 7, 9, and 11). To
assess the effect of the thiol peptide on the processing of prolysyl
oxidase during the biosynthesis of the enzyme by fibrogenic cells,
cultures of rat fetal lung fibroblasts were pulse-labeled with
[
S]methionine as described under
``Materials and Methods'' in the presence and the absence of
the thiol and acetylated thiol peptides. The prolysyl oxidase and lysyl
oxidase proteins appearing in the conditioned medium were
immunoprecipitated and resolved by SDS-PAGE, and the bands were
visualized by autoradiography. As shown (Fig. 5B), the
thiol but not the acetylated thiol peptide inhibited the conversion of
the 50-kDa prolysyl oxidase band to the 30-kDa enzyme in these RFL6
cell cultures. This result illustrates the potential for the controlled
prevention of lysyl oxidase processing by inhibitors of
metalloproteinase activity and supports the use of the fusion protein
substrate to assay prolysyl oxidase processing activity.
Figure 5:
Inhibition of processing activity by
TIMP-2 and HS peptide. A, Western blotting assay of inhibition
of FP cleavage. Lanes 1-6, effect of TIMP-2 on
proteolytic activity of 2.4 nM MMP-2 (lanes 1 and 2), 15 µl of SMC medium (lanes 3 and 4),
and 0.03 nM C-proteinase (lanes 5 and 6)
with FP as the substrate. FP was incubated in the absence (lanes
1, 3, and 5) or the presence (lanes 2, 4, and 6) of 300 nM TIMP-2. Lanes
7-12, effect of HS peptide on proteolytic activity of 2.4
nM MMP-2 (lanes 7 and 8), 15 µl of SMC
medium (lanes 9 and 10), and 0.01 nM C-proteinase (lanes 11 and 12) with FP as the
substrate. FP was incubated with 80 µMS-acetylated peptide (lanes 7, 9, and 11) or with 80 µM HS peptide (lanes 8, 10, and 12). B, inhibition of prolysyl
oxidase processing in cell culture by HS peptide. RFL6 cells were grown
to confluency in T-75 flasks and pulse-labeled with
[S]methionine for 2 h in the presence of 50
µMS-acetylated peptide (lane 1),
Me
SO carrier (lane 2), or 50 µM HS
peptide (lane 3). The conditioned medium of each incubation
was then immunoprecipitated with anti-lysyl oxidase as described. LO, lysyl oxidase.
The
NH-terminal amino acid sequences of the 30-kDa products
generated by MMP-2 and C-proteinase were determined to assess the site
of cleavage in the propeptide sequence within the fusion protein
substrate. As shown (Fig. 6), the NH
-terminal
sequence resulting from cleavage by MMP-2 indicates that this enzyme
hydrolyzes an Asn-Leu bond. Both conditioned SMC medium and the
purified C-proteinase yield the same Asp-Asp-NH
-terminal
sequence in the 30-kDa product, indicating that both are specific for
the same Gly-Asp bond in the propeptide region. The Asp-Asp sequence is
unique within the mature region of prolysyl oxidase and corresponds to
the NH
-terminal sequence recently identified in mature
lysyl oxidase purified from pig skin(29) .
Figure 6:
NH-terminal sequences of
30-kDa products cleaved from the FP substrate by different proteinase
sources. The arrows and underlining indicate the
location within the propeptide cleavage region of the NH
termini generated by proteolysis.
Figure 7:
Western blotting assay of C-proteinase
inhibition by Ac-Met-Val-Gly-Asp-Asp-Pro-Tyr-Asn-NH
peptide. FP (0.5 µg) was incubated with 0.01 nM of
C-proteinase for 1 h at 37 °C (lane 1) in presence of 0.1
mM (lane 2) or 1 mM (lane 3) of
Ac-Met-Val-Gly-Asp-Asp-Pro-Tyr-Asn-NH
peptide.
This peptide inhibitor, as well as the thiol and acetylated thiol peptide inhibitors, were then tested for their effects on the de novo generation of lysyl oxidase activity in cultures of RFL6 cells. RFL-6 fibroblasts were cultured to confluency, and the medium was changed to fresh DMEM/0.4% fetal bovine serum lacking or containing the peptide mimic of the cleavage site at 2.5 mM or the thiol or acetylated thiol peptides at 36 µM. Following 8 h of incubation, aliquots of the conditioned medium were assayed for lysyl oxidase activity. As shown in Table 1, the appearance of lysyl oxidase activity secreted by these fibroblasts is reduced by approximately two-thirds in the cultures incubated in the presence of the peptide containing the GDD cleavage sequence and in those containing the thiol peptide. The acetylated peptide control was not inhibitory. It is important to note that none of these synthetic peptides at the concentrations used in the cell culture experiments inhibited the endogenous lysyl oxidase activity of control RFL6 cell conditioned medium against the tropoelastin substrate (not shown). Consistent with the ability of the thiol peptide to inhibit the cleavage of the fusion protein in cell-free assays (Fig. 5A) and in cell culture (Fig. 5B), these data support the conclusion that the proenzyme is largely or completely catalytically inactive and that its proteolytic conversion to the 30-kDa species is necessary for the full unmasking of the catalytic activity of lysyl oxidase.
Previous studies have provided evidence that lysyl oxidase is
synthesized as a proenzyme that is secreted as a 50-kDa N-glycosylated protein. The secreted proenzyme is then
proteolytically processed in the extracellular space to a functional
30-kDa product. The present study indicates that the processing
protease is a metalloenzyme and, further, implicates procollagen
C-proteinase as a reasonable candidate to fulfill this role. This is
supported by the identity of the cleavage site specificity of
conditioned SMC medium and purified C-proteinase activities, both
yielding a product with an Asp-Asp NH-terminal sequence
deriving from a Gly-Asp-Asp sequence uniquely located in the region of
the expected proenzyme cleavage site of prolysyl oxidase. Consistent
with the specificity found here, an Asx residue has been identified at
the NH
terminus of each of the four ionic variants of
bovine aorta lysyl oxidase(31) , and, of further importance,
mature 30-kDa lysyl oxidase purified from pig skin contains the Asp-Asp
NH
-terminal sequence(29) .
The substrate
specificity of procollagen C-proteinase against the model lysyl oxidase
substrates found here is consistent with the known specificity of this
enzyme for type I procollagen in which this enzyme cleaves an Ala-Asp
bond in the pro-1(I) and pro-
2(II) chains and a Gly-Asp bond
is in the pro-
1(III) chain(22, 26) . Cronshaw et al.(29) have previously noted the similarity
between the known substrate specificity of procollagen C-proteinase and
the putative cleavage site predicted from their analysis of the
NH
-terminal of the isolated mature pig skin enzyme.
Additional although indirect support for the role of this proteinase in
prolysyl oxidase processing stems from: 1) the common susceptibility of
the rat fibroblast and procollagen C-proteinase processing activity to
inhibition by the synthetic peptide containing the GDD cleavage
sequence; 2) the common lack of susceptibility to TIMP-2 of the
processing activity of the SMC medium and procollagen C-proteinase; and
3) the selective cleavage by this proteinase of the prolysyl oxidase
model substrate discretely to the size of the mature enzyme.
Comparison of the primary structures predicted from the cDNA
sequences of human, rat, mouse, and chick prolysyl oxidase reveals that
the only Asp-Asp sequence occurs in the Gly-Asp-Asp sequence (residues
162-164 in the rat enzyme, Fig. 8) and that this sequence
is highly conserved in each species. The results uniquely obtained here
by analysis of the immediate product of proteolytic processing by
conditioned SMC medium and purified C-proteinase together with the
NH-terminal analysis of the mature enzyme species strongly
support the conclusion of the present study that the Gly-Asp bond is
the substrate site of a processing protease. The calculated molecular
mass of the COOH-terminal product of cleavage of prolysyl oxidase at
this site is 29,990 Da, in excellent agreement with the value
determined by SDS-PAGE of 30,000 Da.
The possibility that other
sites may also be cleaved in prolysyl oxidase catalyst certainly must
be considered. Notably, there are two polybasic sites in the rat
proenzyme, i.e. Arg-Arg-Arg at residues 62-64 and
Arg-Arg at residues 134-135. Such polybasic sequences are known
to be the sites of proteolytic processing of a variety of other
proproteins by enzymes such as furin(32) . Thus far, however,
evidence that cleavage may occur at these sites has not been seen. It
is of interest that minor immunoreactive bands are seen in Fig. 1, Fig. 3, and Fig. 5B at a
molecular mass of approximately 32,000 daltons. Efforts at determining
the NH terminus of this minor band have yet to be
successful. This may reflect intermediates or secondary products
generated during the proteolytic processing of prolysyl oxidase that do
not accumulate significantly. As noted, the 72-kDa MMP-2 is also able
to cleave the substrate to apparently the same size as obtained by
cleavage with the SMC medium and C-proteinase. However,
NH
-terminal sequencing showed that the cleavage site in
this case is at an Asn-Leu bond located 12 amino acids upstream from
Gly-Asp site specific for SMC medium and C-proteinase. Moreover, the
lack of inhibition of the SMC and procollagen C-proteinase activities
by TIMP-2, coupled with the inhibition of MMP-2 by TIMP-2, argues
against a role for MMP-2 the activation process.
Certainly, given
the anabolic role of procollagen C-proteinase and the catabolic role of
MMP-2 in collagen homeostasis, the C-proteinase would appear to be the
more reasonable of the two candidates for the processing of prolysyl
oxidase in view of the critical participation of lysyl oxidase in the
accumulation of insoluble collagen fibers. It is of further interest in
this regard that cleavage of the propeptides from procollagen by the
NH-terminal and C-proteinases is essential for the
subsequent, spontaneous assembly of the processed collagen molecule
into quarter-staggered, native fibrils. Moreover, as seen with type I
collagen, collagen molecules must be assembled into native fibrillar
aggregates to become an effective substrate for lysyl
oxidase(33) . Recent studies suggest that fibril formation of
collagen may be necessary for lysine oxidation by permitting the
intermolecular neutralization of an unfavorable anionic charge near the
potentially susceptible, N-telopeptide lysine by a cationic
site within the triple helical sequence of a neighboring, D-staggered molecule. Thus, procollagen C-proteinase may play
a doubly essential role in the cross-linking process by: 1) converting
procollagen to the collagen molecule that can then assemble into the
quaternary fibrillar structure essential to collagen lysine oxidation
and 2) by converting the latent prolysyl oxidase to the fully
functional catalyst capable of oxidizing lysine in the newly
preassembled native fibrils, thus, in essence, generating the active
forms of the collagen substrate and of the lysyl oxidase catalyst. More
definitive assessment of this intriguing possibility awaits the
development and/or discovery of specific chemical or macromolecular
inhibitors of the activity or expression of procollagen C-proteinase.
In view of the markedly different sequences surrounding peptidyl lysine in collagen and elastin substrates(41) , the possibility that different forms of lysyl oxidase may exist with specificity for one or another of these substrates has long been considered. However, there is no evidence presently available that this is the case. Indeed, purified lysyl oxidase readily oxidizes purified preparations of both elastin and collagen substrates (1) as well as elastin-like (42) and collagen-like synthetic polypeptides(41) . Thus, although procollagen C-proteinase plays an essential role in the post-translational processing of procollagen, the present study raises the possibility that this proteinase may be involved in the processing of an enzyme participating in the post-translational modification of both elastin and collagen.