 |
INTRODUCTION |
Lysyl oxidase catalyzes the oxidative deamination of
peptidyl-lysine in elastin precursors and lysine and hydroxylysine
residues in collagen to form peptidyl-
-aminoadipic-
-semialdehyde
and peptidyl-
-hydroxy-
-aminoadipic-
-semialdehyde
residues, respectively. These aldehydes then undergo
nonenzymatic reactions resulting in the cross-linkages known to be
critical in the formation of mature functional insoluble elastin and
collagens (1, 2). Lysyl oxidase is a copper-dependent
enzyme and is specifically inhibited by
-aminopropionitrile.
Connective tissue abnormalities known as osteolathyrism result from
in vivo inhibition of lysyl oxidase activity by
-aminopropionitrile feeding (3). The abnormalities include malformed
and weak bones, as well as increased development of hernias (4). Lysyl
oxidase is synthesized as a 50-kDa glycoprotein and is processed
extracellularly to produce the ~30-kDa molecular form known to be
active (5). The sequence of the proteolytic processing site in
pro-lysyl oxidase resembles that of the fibrillar procollagen
C-terminal pro-peptide processing sites cleaved by procollagen
C-proteinase (PCP)1 (6).
Moreover, preparations highly enriched in PCP activity have been shown
to process pro-lysyl oxidase at the correct physiological site (7).
In mammals, PCP activity is provided by products of the Bmp1
gene (8-10), which encodes alternatively spliced mRNAs for the proteins bone morphogenetic protein 1 (BMP-1) and mammalian Tolloid (mTLD) (11). The mTLD protein product contains a longer C terminus than
BMP-1, resulting in a domain structure identical to that of the
Drosophila protein Tolloid. The domains in BMP-1, mTLD, and
Drosophila Tolloid include the astacin-like proteinase
domain and epidermal growth factor-like domains and CUB domains that may mediate binding to other extracellular proteins (12, 13). Drosophila Tolloid plays an important role in pattern
formation during embryogenesis by cleaving the secreted protein SOG
(short gastrulation), which
forms latent complexes with the transforming growth factor
-related
protein DPP (decapentaplegic) (14). BMP-1 may play a similar role in mammalian embryogenetic patterning, because it cleaves the SOG vertebrate homologue Chordin, which binds
and inactivates the DPP vertebrate homologues BMP-2/BMP-4 (10, 15).
Although mammalian BMP-1 and mTLD both are PCPs, only BMP-1
hydrolyzes Chordin, indicating that Bmp1 gene products have
different substrate specificities and some different biological functions (10).
The mammalian Bmp1 gene is a member of a multigene family,
and two genetically distinct mammalian Tolloid-related proteinases, mammalian Tolloid-like 1 and 2 (mTLL-1 and mTLL-2), have recently been
described (10). Interestingly, despite highly similar sequences and
domain structures, the substrate specificities of BMP-1, mTLD, mTLL-1,
and mTLL-2 differ. BMP-1, mTLD, and mTLL-1 all have readily detectable
PCP activity. By contrast, mTLL-2 does not appear to process fibrillar
procollagens. BMP-1 and mTLL-1 readily cleave Chordin, whereas mTLD and
mTLL-2 do not cleave Chordin (10). BMP-1 and mTLL-1 process
probiglycan, whereas mTLL-2 does not (16). It has yet to be determined
whether differences in specificity occur with other substrates, such as
pro-lysyl oxidase. Because lysyl oxidase is critical for production of
mature and functional extracellular matrices, it is important to
understand whether a single member of the BMP-1-related proteinases
predominates in processing pro-lysyl oxidase in connective tissues and
cells or whether functional redundancy for this activity exists among BMP-1-related proteins.
Mice homozygous null for the Bmp1 gene have impaired
ossification of calvaria and herniation of the gut and do not survive beyond birth but have grossly normal axial and appendicular skeletons (17). In vitro, fibroblasts from Bmp1-null
embryos accumulate less insoluble collagen, and collagen fibrils are
not normal in appearance. Procollagen processing is diminished,
resulting in accumulation of intermediates that retain the C-propeptide
and in low amounts of fully processed native collagen monomers (17). These results indicate that in Bmp1-null embryos and
fibroblasts, related proteinases are able to partially, but not fully,
compensate for the loss of the PCP activity normally provided by BMP-1
and mTLD. Interestingly, the Bmp1-null phenotype that
includes gut herniation and abnormalities in calvaria maturation
appears similar to lathyrism (4). As noted above, lathyrism is caused
by a deficiency in lysyl oxidase enzyme activity (1, 3). These observations raise the possibility that lysyl oxidase enzyme activity may be abnormally low in the absence of a functional Bmp1
gene. Tll1-null animals have been recently created, and the
homozygous null genotype is embryonic lethal (18). In contrast to
Bmp1-null embryos, the Tll1-null embryonic
phenotype has abnormalities predominantly in vascular and cardiac
tissues. These differences in phenotypes, along with differences in the
tissue-specific expression patterns of the different
Bmp1-related genes, suggest differences in functions (18),
although the absence of more widespread defects in Bmp1-null and Tll1-null embryos suggests some functional overlap
between the cognate products of the two genes.
The lysyl oxidase pro-enzyme processing activities of the different
BMP-1-related proteinases have not previously been compared, nor has
the major enzyme responsible for processing of pro-lysyl oxidase
in vivo been identified. The present study compares
pro-lysyl oxidase processing in vitro by all four known
mammalian BMP-1 proteinase family members (BMP-1, mTLD, mTLL-1, and
mTLL-2). In addition, lysyl oxidase enzyme activity and pro-enzyme
biosynthetic processing are determined in mouse embryo fibroblasts
containing null alleles for Bmp1, for the mTLL-1 gene
Tll1, or for both genes. Results from in vitro
studies show that BMP-1, mTLD, mTLL-1, and mTLL-2 all process pro-lysyl
oxidase at the correct physiological site but that the BMP-1 and mTLL-1
enzymes are the most efficient. Moreover, data from in vitro
assays and from cell culture experiments support the important
conclusion that the Bmp1 and Tll1 genes together
provide the majority of activity for the biosynthetic processing of
pro-lysyl oxidase and the generation of lysyl oxidase enzyme activity.
The studies also identify pro-lysyl oxidase as the first known
substrate for mTLL-2.
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EXPERIMENTAL PROCEDURES |
Recombinant Enzymes--
Recombinant human BMP-1, mTLD, mTLL-1,
and mTLL-2 were produced as C-terminal FLAG-tagged proteins in
transfected 293-EBNA cells and purified by affinity chromatography
using the same methodology as in Ref. 10. Each enzyme exhibited a
single major band on SDS-PAGE gels stained with Coomassie Blue, and
purity of each enzyme preparation was estimated to be 90% or greater.
The concentration of enzyme solutions was determined by subjecting
serial dilutions of enzymes and known amounts of standard proteins to
SDS-PAGE and Coomassie Blue staining (10). The relative specific
activities of enzymes against known substrates type I procollagen and
Chordin were verified prior to initiating experiments with pro-lysyl
oxidase (10).
Substrate and Antibodies--
The maltose-binding protein/lysyl
oxidase fusion protein was produced and purified as described
previously (19). Rabbit anti-bovine lysyl oxidase antibodies were
raised against purified 30-kDa mature bovine aorta lysyl oxidase as
described (20) and were provided by Dr. Herbert M. Kagan (Boston
University School of Medicine). The antibodies were affinity purified
utilizing immobilized lysyl oxidase fusion protein (19). Affinity
purification of anti-lysyl oxidase was accomplished utilizing
recombinant rat lysyl oxidase fusion protein covalently attached to
Sulfolink (Pierce), and the specific antibodies were purified according to the manufacturer's protocol. The titer of the purified lysyl oxidase antibody was determined by Western blotting against the lysyl
oxidase fusion protein. Alkaline phosphatase-coupled goat anti-rabbit
antibodies and Western Blue detection reagents were purchased from
Promega. Although there is no obvious sequence similarity between lysyl
oxidase and the maltose-binding protein, the affinity purified
anti-lysyl oxidase antibody recognizes both mature 30-kDa lysyl oxidase
itself and the maltose-binding protein (New England BioLabs, Beverly,
MA) on Western blots. Thus, by chance, shared epitopes between lysyl
oxidase and the maltose-binding protein appear to exist. This provides
for recognition of both the lysyl oxidase portion and the
maltose-binding protein portions, respectively, of the recombinant
fusion protein substrate by this antibody preparation.
Pro-lysyl Oxidase Processing Assays--
Maltose-binding
protein/lysyl oxidase fusion protein (7 µg) was incubated with 30 ng
of recombinant FLAG-tagged BMP-1, mTLD, mTLL-1, or mTLL-2 for either 45 min or 4 h at 37 °C in a final volume of 200 µl of 50 mM Tris, 150 mM NaCl, 5 mM
CaCl2, pH 7.5. The reactions all contained 40 µg/ml
soybean trypsin inhibitor, 10 µg/ml leupeptin, and 0.4 mM
phenylmethylsulfonyl fluoride to inhibit nonspecific proteinases. The
reactions were stopped by adding an equal volume of SDS-PAGE sample
buffer and boiling for 3 min. Fifty-µl aliquots were subjected to
10% PAGE and Western blotting (21) to polyvinylidene difluoride
membranes (PerkinElmer Life Sciences), and the blots were blocked with
1% bovine serum albumin in 20 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween 20. The blots were incubated with
affinity purified anti-lysyl oxidase antibody at a dilution of 1:10,000
at room temperature overnight. The blots were washed and then incubated
with alkaline phosphatase goat anti-rabbit IgG according to the
manufacturer's instructions. The bands were detected using stabilized
nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate Western Blue
reagent (Promega). The relative amount of 86-kDa fusion protein
substrate and 30-kDa lysyl oxidase product in each lane was determined
by densitometry measurements performed in triplicate utilizing a
Bio-Rad Fluor-S MultiImager equipped with Quantityone software (version
4.01), and the percentages of the conversions were determined. The
semi-quantitative pro-lysyl oxidase conversion activity/pmol proteinase
was then calculated from reactions in which conversion of 86-kDa fusion
protein to 30-kDa product was 50% or less. Previous studies have shown
that pro-lysyl oxidase processing activity assays analyzed as described
above are linear as a function of time of incubation in reactions in
which up to 50% conversion of 86-kDa fusion protein substrate to
30-kDa product is observed (19).
N-terminal Amino Acid Sequence Analyses--
For N-terminal
sequence analyses of 30-kDa products, reactions containing 7 µg of
fusion protein, as described above, were incubated at 37 °C for
12 h. The reactions did not contain soybean trypsin inhibitor but
did contain all other proteinase inhibitors. The reactions were
concentrated by lyophilization, subjected to SDS-PAGE, transferred to
polyvinylidene difluoride membranes (Bio-Rad Trans-Blot), and stained
with Coomassie Blue. Visualized 30-kDa protein bands were excised and
subjected to N-terminal analyses by Edman degradation by Bill Lane and
colleagues at the Harvard Microchemistry Facility (Cambridge, MA).
Cell Culture--
Fibroblasts from Bmp1 +/+,
Bmp1 +/
, Bmp1
/
, Tll1 +/+,
Tll1 +/
, Tll1
/
, and
Bmp1/Tll1 homozygous double null (Bmp1
/
and Tll1
/
) embryos were isolated as described
(17, 18). The cells were passaged no more than seven times and were
grown in 100-mm tissue culture plates in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and antibiotics.
Lysyl Oxidase Enzyme Activity--
Lysyl oxidase enzyme activity
was measured in conditioned media using a recombinant tritiated
tropoelastin substrate (22). Media samples (0.5 ml) were assayed in
quadruplicate in a final volume of 1 ml, containing 0.1 M
borate, 0.15 M NaCl, pH 8.0, and 320,000 dpm of tritiated
tropoelastin in the presence and absence of 5 × 10
4
M
-aminopropionitrile. The reactions were incubated for
90 min at 37 °C, followed by distillation under vacuum. The
level of radioactivity in tritiated water in 0.5-ml aliquots of
distillate was determined by scintillation spectrometry. Units of
enzyme activity are defined as dpm released above
-aminopropionitrile controls. Determinations of DNA to normalize
enzyme activity measurements were performed from each cell layer
(23).
Pulse Labeling/Immunoprecipitation Studies--
Three 100-mm
tissue culture plates each of wild type, Bmp1
/
,
Tll1
/
, and double null (Bmp1
/
,
Tll1
/
) fibroblasts were grown to near confluence. The
cells were then placed in serum-free and methionine/cysteine-free
Dulbecco's modified Eagle's medium for 20 min. The cultures were then
refed with this same medium containing 50 µCi/ml methionine/cysteine
(PerkinElmer catalogue number NEG-072) and cultured for 18 h.
Media proteins were recovered by precipitation with trichloroacetic
acid and dissolved in 2% SDS. Duplicate samples containing equivalent
levels of radiolabeled media proteins from each cell culture were
immunoprecipitated with anti-bovine lysyl oxidase and with preimmune
serum, respectively (5, 19), and then subjected to SDS-PAGE and autoradiography.
 |
RESULTS |
In Vitro Pro-lysyl Oxidase Processing Assays--
Recombinant
maltose-binding protein/lysyl oxidase fusion proteins were created and
used as substrate in assays to assess pro-lysyl oxidase proteolytic
processing activity (7, 19). As shown in Fig.
1, the N-terminal end of the intact
fusion protein consists of the maltose-binding protein, and the
C-terminal end is the rat lysyl oxidase proenzyme. Following
proteolytic processing by PCP, two products are generated: 30-kDa
mature lysyl oxidase and a 56-kDa maltose-binding protein/lysyl oxidase
propeptide product. Immunoblotting of products utilizing anti-30-kDa
lysyl oxidase antibodies allows qualitative and quantitative assessment of lysyl oxidase processing activity (19).

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Fig. 1.
Scheme showing the structure of the
maltose-binding protein/lysyl oxidase fusion protein and products
following processing by enzymes with PCP activity.
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Fusion protein processing by purified recombinant BMP-1, mTLD, mTLL-1,
and mTLL-2 was assayed. Thirty ng of each purified enzyme was incubated
with 7 µg of fusion protein for 4 h at 37 °C, and products
were subjected to SDS-PAGE and Western blotting with lysyl oxidase
polyclonal antibodies as described under "Experimental Procedures."
Incubations containing fusion protein and no added proteinase exhibited
a single major 86-kDa band and sometimes an additional minor 84-kDa
band when probed with the lysyl oxidase antibody (Fig.
2A, lane 1) (19).
No 30-kDa product was observed in the absence of added enzymes. By
contrast each reaction containing a recombinant enzyme resulted in
production of a 30-kDa product recognized by the lysyl oxidase
antibody. Under these conditions, BMP-1 and mTLL-1 caused near complete
to complete loss of 86-kDa fusion protein and production of large
amounts of 30-kDa product, whereas mTLD and mTLL-2 resulted in slight
loss of 86-kDa fusion protein and production of small amounts of 30-kDa
product. Semi-quantitative densitometric analyses of the relative
intensity of 86- and 30-kDa bands in each lane indicate 93-100%
conversion by BMP-1 and mTLL-1 and 10-15% conversion by mTLD and
mTLL-2, respectively. Similar results were obtained in a total of four
experiments performed with two different preparations of enzymes.

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Fig. 2.
Western blots of incubations of
maltose-binding protein/lysyl oxidase fusion protein alone or with
BMP-1, mTLD, mTLL-1, or mTLL-2. For both blots fusion protein was
incubated with no enzyme (lanes 1), 30 ng of mTLD
(lanes 2), 30 ng of BMP-1 (lanes 3), 30 ng of
mTLL-2 (lanes 4), 30 ng of mTLL-1 (lanes 5), or
sample buffer alone (lane 6). A, 4-h incubations
probed with anti-lysyl oxidase. B, 45-min incubations probed
with anti-lysyl oxidase. In A, 56-kDa maltose-binding
protein/lysyl oxidase propeptide was detected with anti-lysyl oxidase
because of cross-reactivity of anti-lysyl oxidase with the
maltose-binding protein. The asterisk marks the position of
an artifact band detected with the lysyl oxidase antibody also found in
sample buffer alone (lane 6, A) as noted
previously (37).
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Semi-quantitative assessment of lysyl oxidase fusion protein conversion
is linear with respect to both the time of incubation and the amount of
activity if the percentage of conversion is 50% or less under the
experimental conditions utilized in the present study and as previously
reported (19). Thus, incubations were carried out for shorter times
with the goal of obtaining valid relative semi-quantitative activity
estimates for all four enzymes. Incubations carried out for 45 min
resulted in lower percentage conversions for all enzymes, as expected
(Fig. 2B). BMP-1 converted 50% of the maltose-binding
protein/lysyl oxidase fusion protein, and mTLL-1 converted 15%.
Conversion activity per hour was then calculated for each enzyme, and
the results were expressed as micrograms of fusion protein converted
per hour per picomole of enzyme (Table
I). The values in Table I are derived
from blots shown in Fig. 2A (mTLD and mTLL-2) and Fig. 2B (BMP-1 and mTLL-1). The results clearly show that BMP-1
processes the lysyl oxidase fusion protein with the highest efficiency
compared with the other enzymes. As noted above, blots from four
independent experiments in which incubations were performed for 2 or
4 h all exhibited higher conversion of 86-kDa fusion protein to
30-kDa lysyl oxidase by BMP-1 compared with the other enzymes, with
results similar to the blot shown in Fig. 2A. The data in
Table I are derived from assays performed in the linear responsive
range of the assay, and relationships identified by these
semi-quantitative assay results demonstrate that BMP-1 is the most
efficient pro-lysyl oxidase processing enzyme. These results are
representative of all of the experiments performed.
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Table I
Pro-lysyl oxidase processing activity by recombinant BMP-1, mTLD,
mTLL-1, and mTLL-2 enzymes
Seven µg of maltose binding protein/lysyl oxidase fusion protein was
incubated with 30 ng of recombinant enzyme for either 45 min or 4 h at 37 °C, and aliquots were subjected to Western blotting and
probed with anti-bovine lysyl oxidase (Fig. 2). The percentage of
conversion of 86-kDa fusion protein to 30-kDa lysyl oxidase was
determined by densitometry of both bands in each lane. Pro-lysyl
oxidase processing activity/pmol enzyme was then calculated from
reactions in which conversion was 50% or less (19). Errors are ± S.E. from triplicate densitometric analyses of blots shown in Fig. 2.
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N-terminal sequence analyses were performed on the 30-kDa products
obtained from lysyl oxidase fusion protein and BMP-1, mTLD, mTLL-1, and
mTLL-2, respectively. The expected N terminus of DDPYN corresponding to
the known N terminus of the tissue form of mature active 30-kDa lysyl
oxidase was obtained with the highest level of confidence from all four
reactions. No 30-kDa product was obtained from reactions incubated
without added proteinase. Thus, although the rate of processing of the
lysyl oxidase fusion protein by the four enzymes differed, the 30-kDa
product generated in all cases corresponds to the physiologic cleavage
product of pro-lysyl oxidase.
Cell Culture Studies with Bmp1- and Tll1-null Fibroblastic
Cells--
The data presented above indicate that the two products of
the Bmp1 gene, BMP and mTLD, and products from the
genetically distinct Tll1 and Tll2 genes encoding
mTLL-1 and mTLL-2, respectively, all process pro-lysyl oxidase in
vitro. Nevertheless, similarities of the phenotype of
Bmp1-null animals to lathyrism suggest that the phenotype
might be largely due to diminished lysyl oxidase activity, at least in
certain tissues such as calvaria and ventral body wall. The latter
consideration, plus the relatively robust pro-lysyl oxidase processing
activity of BMP-1, suggest that lysyl oxidase processing and activity
in vivo may depend to a large extent on products of the
Bmp1 gene and to BMP-1 in particular. To explore this
question, levels of lysyl oxidase enzyme activity were first determined
from cultures of fibroblasts derived from wild type, Bmp1
+/
, Bmp1
/
, Tll1 +/
, Tll1
/
, and homozygous double null (Bmp1
/
,
Tll1
/
) embryos. The results indicate that lysyl oxidase
enzyme activity secreted by the double null embryonic fibroblasts is
about 30% of that found in cells from wild type, Bmp1
/
, Bmp +/
, Tll1
/
, and Tll
+/
cells, respectively (Table II).
Thus, only cells homozygous null for both the Bmp1 and
Tll1 genes produced a readily detectable reduction in levels of lysyl oxidase enzyme activity. Assessment of accumulating molecular forms of lysyl oxidase was then performed by pulse
labeling/immunoprecipitation analyses in wild type, Bmp1
/
, Tll1
/
, and double null cells (Bmp1
/
, Tll1
/
) to determine whether 50-kDa pro-lysyl
oxidase processing was diminished only in the double null cells. The
cells were cultured to near confluence and then cultured in serum-free medium containing [35S]methionine for 18 h. Media
proteins were then immunoprecipitated with anti-lysyl oxidase antibody
or preimmune serum and subjected to SDS-PAGE and autoradiography. As
shown in Fig. 3, unprocessed 50-kDa lysyl
oxidase proenzyme accumulated at higher levels in media from double
null cells compared with the other cultures. Densitometric analyses of
the 50-kDa lysyl oxidase pro-enzyme band indicate that the double null
cells accumulated 3.9-, 3-, and 4-fold more 50-kDa lysyl oxidase
pro-enzyme than wild type, Bmp1
/
, and
Tll1
/
cells, respectively. The pro-lysyl oxidase protein immunoprecipitated from cell cultures made from all genotypes is predominantly the 50-kDa N-glycosylated form of this
protein but also includes the 45-kDa nonglycosylated proenzyme (Fig.
3), consistent with studies performed in rat smooth muscle cells and murine MC3T3-E1 osteoblastic cells (5, 19). Surprisingly, similar
levels of ~30-kDa lysyl oxidase were found in all four cultures, but
in double null cells the mobility of this band is faster and more
diffuse than in the other three cultures. This suggests that the 30-kDa
band in the double null immunoprecipitates may contain abnormally
processed lysyl oxidase or a greater proportion of partially degraded
and inactive lysyl oxidase. This notion is consistent with lysyl
oxidase enzyme activity data that demonstrate diminished lysyl oxidase
activity in Bmp1/Tll1 double null cultures (Table II).
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Table II
Lysyl oxidase enzyme activity in cultures of wild type, Tll1 /+, and
Tll1 / , Bmp1 +/ , Bmp / , and double null (Bmp1 / , Tll1
/ ) murine embryonic fibroblasts
Lysyl oxidase enzyme activity was measured in 18-h media samples from
subconfluent cell cultures normalized to DNA measured from the same
culture plates. Numbers are the means ± S.E. of quadruplicate
determinations made from three independent cultures of each genotype.
Experiment 1 and experiment 2 utilized different preparations of
recombinant tropoelastin substrate. The data are representative of
experiments performed twice. For results of experiment 1, unpaired
t tests revealed no statistically significant differences in
lysyl oxidase activity between Tll1 / and wild type
cultures (p = 0.225), between Tll1 +/ and
wild type cultures (p = 0.414), or between Bmp1 +/
and wild type cultures. In experiment 2, double null (Bmp1
/ , Tll1 / ) lysyl oxidase enzyme activity was
significantly lower than wild type (p < 0.01), whereas
activity in Bmp1 / cultures was not significantly higher
than wild type (p = 0.04).
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Fig. 3.
Autoradiograms of 10% SDS-PAGE gel of
preimmune and lysyl oxidase immunoprecipitates from pulse-labeled
embryonic fibroblasts from mice with different genotypes:
Tll1 / (T); Bmp1
/ (B); Tll1 / ,
Bmp1 / (T/B); and wild type
(W). The markers are in vitro
transcription/translation of pro-lysyl oxidase in the presence
(+C) or the absence ( C) of canine pancreatic
microsomal membranes to produce a mixture of glycosylated 50-kDa and
nonglycosylated 45-kDa pro-lysyl oxidase (+C) and only
nonglycosylated 45-kDa pro-lysyl oxidase ( C),
respectively, to show the mobility of these proteins on SDS-PAGE (5).
Lane P shows the mobility of purified mature ~30-kDa
bovine aorta lysyl oxidase partially purified as described (38);
lane M shows the low molecular weight markers (Bio-Rad). The
~24-kDa protein that co-purifies with lysyl oxidase seen in
lane P has previously been identified as tyrosine-rich
acidic matrix protein (39, 40). For immunoprecipitation assays,
cells were cultured and pulse-labeled with
[35S]methionine, and media proteins were isolated and
immunoprecipitated with rabbit anti-bovine lysyl oxidase as described
under "Experimental Procedures." 0.6-2.45 × 106
cpm were utilized for immunoprecipitation experiments performed three
times with similar results.
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Taken together, the data support the conclusion that both
Bmp1 and Tll1 genes produce efficient pro-lysyl
oxidase processing enzymes, and that these enzymes contribute to
pro-lysyl oxidase processing and development of lysyl oxidase enzyme
activity in murine embryonic fibroblasts. These data support a
previously unrecognized significant role for mTLL-1 in pro-lysyl
oxidase processing and activation. In addition, results suggest that
mTLL-2 also contributes to pro-lysyl oxidase processing.
 |
DISCUSSION |
Two recent discoveries lead to the notion that proteinases with
PCP activity may be key in initiating and controlling a cascade of
extracellular transformations critical for extracellular matrix deposition. The two discoveries are (a) that enzymes with
PCP activity have multiple extracellular substrates and (b)
that the Bmp1 gene, which is related to genes that regulate
morphogenetic patterning in nonmammalian species, encodes BMP-1 and
mTLD, two products with PCP activity. In addition to the biosynthetic
processing of procollagen types I, II, and III, preparations highly
enriched in PCP activity proteolytically process pro-lysyl oxidase (7). Recently BMP-1 has been shown to inactivate Chordin to release growth
factors from latent complexes (10, 15) and may biosynthetically process
pro-
1(V) collagen (24), laminin-5 (25), and probiglycan (16). The
concept of a key proteinase activity that controls a series of
subsequent activities leading to a biological phenotype is well known
in catabolic cascades, in which extracellular matrix destruction is
initiated by collagenases (MMP-1, MMP-8, or MMP-13) or plasmin (26).
The existence of anabolic or synthetic extracellular enzymatic cascades
now seems likely following characterization of developmentally
regulated enzymes such as those with PCP activity.
As with key catabolic proteinases such as the collagenases, a certain
degree of redundancy appears to exist for anabolic proteinases, where
key enzyme activities may be encoded by different genes in a
tissue-specific and developmentally regulated manner. The present study
investigates the ability of proteinases encoded by the Bmp1
gene itself and of two highly similar proteinases encoded by
Bmp1-related mammalian genes to process pro-lysyl oxidase in vitro. In addition, the effects of the absence of
proteinases encoded by the Bmp1 and Tll1 genes,
respectively, on lysyl oxidase activity and biosynthesis was studied in
fibroblasts cultured from wild type, Bmp1-null,
Tll1-null, and Bmp1/Tll1 double null mouse
embryos. This experimental approach, utilizing both in vitro assays with recombinant proteins and cultured cells derived from genetically altered animals, provides independent assays to assess the
roles of the different procollagen C-proteinases in lysyl oxidase
processing. In vitro data were obtained with human
recombinant enzymes, all of which were expressed in the same mammalian
expression system, all of which contained the same C-terminal epitope
tag, and all of which were purified by the same affinity chromatography methodology (10). Moreover, although the maltose-binding
protein/pro-lysyl oxidase fusion protein was made using a rat lysyl
oxidase cDNA (17), the proteolytic processing site MVG*DDPYN is
conserved between rat and human pro-lysyl oxidase (6). Thus, the use of
assays investigating relative efficiency of processing of the rat lysyl
oxidase substrate by human BMP-1-related proteinases, each of which was
prepared in the same manner, represents a valid experimental approach
to investigating pro-lysyl oxidase processing activity. The validity of
the experimental approach is further supported by the agreement between
the in vitro data and data obtained from studies of mouse
embryo fibroblasts, all of which support the conclusion that BMP-1 and
mTLL-1 together contribute the majority of processing leading to the
activation of lysyl oxidase.
The in vitro data indicate that BMP-1, mTLD, mTLL-1, and
mTLL-2 enzymes all process pro-lysyl oxidase at the physiological site
but differ in pro-lysyl oxidase processing efficiency. An important
finding is that BMP-1, which has the highest PCP activity, is also the
most efficient pro-lysyl oxidase processing enzyme. The high efficiency
of BMP-1 processing of both procollagens and pro-lysyl oxidase points
toward BMP-1 as a principal activity in the biosynthesis of functional
insoluble collagen. Previously reported expression patterns of BMP-1,
mTLD, mTLL-1, and mTLL-2 in developing hindlimbs indicate BMP-1 and
mTLD to be expressed at high levels throughout limb mesenchyme, whereas
mTLL-1 expression appeared more restricted to perichondrium or
periosteum, and mTLL-2 expression appeared principally in skeletal
muscle (10). The Bmp1 gene is also expressed at high levels
in developing dermis (27). Thus, patterns of expression of BMP-1 and
mTLD in highly collagenous tissues are also consistent with a principal
role in lysyl oxidase and mature collagen biosynthesis. The more
restricted expression patterns of mTLL-1 and mTLL-2 may indicate
tissue-specific roles for these products in the normal development of
collagenous extracellular matrices in vivo. It is
interesting, however, that mTLL-2 is able to process lysyl oxidase
slowly but does not appear to process procollagens or probiglycan at
all (16). Thus, of the procollagen C-proteinase substrates studied so
far, lysyl oxidase is unique in that it is processed by mTLL-2. We
speculate that because lysyl oxidase-dependent
cross-linking is critical for the biosynthesis of mature connective
tissues, evolution of greater redundancy providing for lysyl oxidase
activation may offer biological advantages.
As noted, two proteins are derived from the Bmp1 gene by
alternative splicing: BMP-1 and mTLD (28). It is of interest that mTLD
processes procollagens and pro-lysyl oxidase less efficiently than
BMP-1. It is also notable that mTLD does not cleave Chordin, whereas
BMP-1 efficiently cleaves Chordin (10). The mTLD protein is longer than
the BMP-1 protein and contains two additional CUB domains and an
additional epidermal growth factor domain. In addition to modulating
proteinase activity, these additional domains may provide binding sites
for other extracellular matrix components important for other functions
unique to mTLD (29). It is noteworthy that mTLD and BMP-1 proteins are
differentially expressed in human and mouse tissues (28).
The in vitro finding that the BMP-1 proteinase is a highly
efficient pro-lysyl oxidase processing enzyme led to the notion that
BMP-1 might be the principal lysyl oxidase processing activity in both
cultured fibrogenic cells and in vivo. The connective tissue
defects in Bmp1
/
animals, including gut herniation and delayed development of skull bones, resemble lathyrism and, thus, may
be caused in part by below normal lysyl oxidase activity, in at least
some tissues in these animals (17). The results from our studies in
cultured cells were therefore surprising in that cells derived from
Bmp1-null embryos processed lysyl oxidase normally and
produced similar lysyl oxidase enzyme activity compared with wild type
cells. As noted, lysyl oxidase enzyme activity was not diminished, and
nearly wild type levels of 50-kDa pro-lysyl oxidase and of ~30-kDa
mature lysyl oxidase accumulated in Bmp1
/
cells. These
results indicate that other enzymes such as mTLL-1 and/or mTLL-2 might
almost fully compensate for the lack of BMP-1 and/or mTLD in these
fibroblast cultures, given that mTLL-1 and mTLL-2 mRNAs are
expressed in mouse embryo fibroblasts (16). Our data showing that
Bmp1/Tll1 double null fibroblastic cells produce levels of
lysyl oxidase activity diminished by 70% and highly elevated levels of
the 50-kDa lysyl oxidase pro-enzyme precursor support the notion that
both BMP-1 and mTLL-1 enzymes are together important in processing
pro-lysyl oxidase and that mTLL-1 significantly contributes to
pro-lysyl oxidase processing in the absence of BMP-1. Future studies
closely examining the expression and role of mTLL-2 in processing lysyl
oxidase will be important in determining whether mTLL-2 or a novel
lysyl oxidase processing enzyme may account for the residual lysyl
oxidase pro-enzyme processing and 30% lysyl oxidase enzyme activity
observed in the Bmp1/Tll1 double null murine
embryonic fibroblasts. This information combined with a greater
understanding of tissue- and cell-specific expression of different
Bmp1-related genes will enhance the understanding of the
roles of these different gene products in different tissues.
An additional contributing factor to the observed lysyl oxidase
activity in Bmp1/Tll1 double null cells may be that lysyl oxidase enzyme activity could be produced by at least one other gene.
In support of this possibility a recent study has shown that some
murine osteosarcoma cell clones that produce little or no
immunoreactive lysyl oxidase protein do produce lysyl oxidase activity
(30). Moreover, lysyl oxidase-like genes have been identified and
mapped to human chromosomes 15, 8, and 2, respectively (31-35),
whereas the lysyl oxidase gene itself maps to human chromosome 5 (36).
The cDNA sequences of all lysyl oxidase relatives predict proenzymes that require proteolytic processing. However, only the lysyl
oxidase sequence itself contains a G*DD sequence similar to the A*DD,
A*DQ, and G*DE sequences present in human types I, II, and III
procollagen cleavage sites, respectively (6). These observations raise
the possibility that lysyl oxidase activity derived from one or more
lysyl oxidase-like genes could develop independently of BMP-1-related
enzymes and could partially account for the residual lysyl oxidase
enzyme activity observed in cells cultured from Bmp1/Tll1
/
animals. It should be noted, however, that the BMP-1/mTLL-1
susceptible sequences in Chordin, A*DG, and S*DR differ from the
generally more highly anionic procollagen and pro-lysyl oxidase
sequences (10). The BMP-1-susceptible site in pro-
1(V) collagen also
has unique features and does not contain an Asp in the P' position
(24). Thus, the new pro-lysyl oxidase-like precursors may also require
processing by BMP-1 and/or BMP-1-related proteinases at a novel site.
Studies investigating the biosynthesis and structure/activity
relationships of lysyl oxidase and the lysyl oxidase-like proteins are
necessary to evaluate relatedness of functional roles. Such studies
could potentially involve identification of new pro-lysyl oxidase-like
processing enzymes distinct from the BMP-1 family. In addition, a
greater understanding of the substrate specificity of BMP-1 itself and of related enzymes will help to further clarify the biological roles of
this family of proteinases.