J Biol Chem, Vol. 275, Issue 12, 8749-8759, March 24, 2000
The Pro-
3(V) Collagen Chain
COMPLETE PRIMARY STRUCTURE, EXPRESSION DOMAINS IN ADULT AND
DEVELOPING TISSUES, AND COMPARISON TO THE STRUCTURES AND EXPRESSION
DOMAINS OF THE OTHER TYPES V AND XI PROCOLLAGEN CHAINS*
Yasutada
Imamura
,
Ian C.
Scott, and
Daniel S.
Greenspan§
From the Department of Pathology and Laboratory
Medicine, University of Wisconsin, Madison, Wisconsin 53706
 |
ABSTRACT |
The low abundance fibrillar collagen type V is
widely distributed in tissues as an 
1(V)22(V)
heterotrimer that helps regulate the diameters of fibrils of the
abundant collagen type I. Mutations in the 1(V) and 2(V) chain
genes have been identified in some cases of classical Ehlers-Danlos
syndrome (EDS), in which aberrant collagen fibrils are associated with
connective tissue fragility, particularly in skin and joints. Type V
collagen also exists as an 1(V)2(V)3(V) heterotrimer that has
remained poorly characterized chiefly due to inability to obtain the
complete primary structure or nucleic acid probes for the 3(V) chain
or its biosynthetic precursor, pro-3(V). Here we provide human and
mouse full-length pro-3(V) sequences. Pro-3(V) is shown to be
closely related to the 1(V) precursor, pro-1(V), but with marked
differences in N-propeptide sequences, and collagenous domain features
that provide insights into the low melting temperature of
1(V)2(V)3(V) heterotrimers, lack of heparin binding by 3(V)
chains and the possibility that 1(V)2(V)3(V) heterotrimers are
incorporated into heterotypic fibrils. In situ
hybridization of mouse embryos detects 3(V) expression primarily in
the epimysial sheaths of developing muscles and within nascent
ligaments adjacent to forming bones and in joints. This distribution,
and the association of 1(V), 2(V), and 3(V) chains in
heterotrimers, suggests the human 3(V) gene COL5A3 as a
candidate locus for at least some cases of classical EDS in which the
1(V) and 2(V) genes have been excluded, and for at least some
cases of the hypermobility type of EDS, a condition marked by gross
joint laxity and chronic musculoskeletal pain. COL5A3 is
mapped to 19p13.2 near a polymorphic marker that should be useful in
analyzing linkage with EDS and other disease phenotypes.
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INTRODUCTION |
Monomers of the low abundance fibrillar collagen types V and XI
are incorporated into fibrils of the abundant collagen types I and II,
respectively (1, 2). In vitro fibrillogenesis experiments
(3, 4) and analysis of a type V mutation in transgenic mice (5) have
indicated that type V collagen helps regulate the size and shape of
type I/V heterotypic fibrils. Further evidence that type V collagen
plays a role in regulating type I collagen fibrillogenesis
in vivo comes from the heritable connective tissue disorder
classical Ehlers-Danlos syndrome
(EDS),1 in which type I
collagen fibrils of abnormal shape and diameter have been shown to
result from mutations in type V collagen genes (6-10). Similar
evidence for an in vivo role for type XI collagen in
regulating type II collagen fibrillogenesis comes from a study of
chondrodysplasia, in which abnormal type II collagen fibrils were shown
to result from defects in a type XI collagen gene (11).
Type V collagen is widely distributed in vertebrate tissues as an
1(V)22(V) heterotrimer (12, 13). However, other forms of type V collagen include an 1(V)3 homotrimer that is
secreted by a line of Chinese hamster cells (14) and which may also
exist in normal tissues (15, 16), and a poorly characterized
1(V)2(V)3(V) heterotrimer, isolated primarily from placenta
(17, 18), but also reported in uterus, skin, and synovial membranes
(12, 19-21). Type XI collagen, in the form of an
1(XI)2(XI)3(XI) heterotrimer (22), was first characterized
as a minor collagen of cartilage. However, findings of type XI
chains in noncartilaginous tissues (23), of type V chains in
cartilage (24), and of cross-type heterotrimers composed of
2(V) and 1(XI) chains (25, 26) now suggest that type V and type
XI chains constitute a single collagen type in which different
combinations of chains associate in a tissue-specific manner.
Fibrillar collagens are synthesized as procollagen precursors with N-
and C-propeptides that are proteolytically processed to yield mature
monomers. Complete primary structures of the type V/XI procollagen
chains pro-1(V), pro-1(XI), pro-2(XI), and pro-2(V) are now
known (27-35). In addition, the primary structure of the pro-3(XI)
chain is known, in that it is thought to be an alternatively spliced
product of the gene that encodes the pro-1 chain of type II collagen
(13, 24). Full-length cDNA sequences have provided not only the
inferred primary structure of each chain, but have also provided probes
that have allowed fine mapping of the expression domains of cognate
mRNAs (27, 36-41). Such studies are important, as the low levels
of collagen type V/XI chains have limited biochemical and histochemical
analyses of expression in developing and adult tissues. Nucleic acid
probes have also enabled those studies which established the causal
links between defects in type V/XI chains and genetic diseases (6-11). The only known type V/XI procollagen chain, or fibrillar procollagen chain, for which neither complete primary structure nor nucleic acid
probes have been available is pro-3(V).
Thus, although NH2-terminal sequencing of proteolytic
fragments of 3(V) chains has yielded a third of the amino acid
sequence of the major collagenous domain (42), the nature of this chain has remained relatively obscure. Nevertheless, a true understanding of
the nature of collagen type V/XI and its roles in development, physiology, and disease requires characterization of the very low
abundance and hitherto elusive pro-3(V) chain, the limited distribution of which may reflect a more specialized role than those of
the other type V/XI chains.
Here we report full-length pro-3(V) cDNA sequences for human and
mouse, use nucleic acid probes to analyze pro-3(V) expression in
developing and adult tissues, and map the chromosomal locations of the
cognate mouse Col5a3 and human COL5A3 genes.
Implications of pro-3(V) sequences and expression domains are
discussed in the context of type V/XI biology and the possible
involvement of pro-3(V) defects in human disease.
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EXPERIMENTAL PROCEDURES |
Determination of Full-length Human and Mouse Pro-3(V) cDNA
Sequences--
A BLAST search of the dbEST data base of expressed
sequences tags, using query sequence
LGPPGEDGAXGSVGPTGLPGDLGPPGDPGVSGIDG from a human 3(V)
peptide TSK5/K1 (42), located 459-bp of 3(V) triple helix-encoding
sequences from a mouse mammary gland EST (GenBank accession number
AI021711). Primers 5'-GGTCCCACAGGACTCCCTGGAGATCT-3' (forward, nt
3853-3878 of the full-length mouse pro-3(V) cDNA sequences
reported in the present study, AF176645) and
5'-TAGCCCAGGAGGTCCCAGGAGACCTG-3' (reverse, nt 4209-4184),
corresponding to EST sequences, amplified a 357-bp PCR product, using a
mouse 17-day postcoitus (dpc) embryo cDNA 5' stretch 
gt10
library (CLONTECH) as template. This product was
used to screen the same gt10 library, yielding one positive clone
(ME7) with a 1742-bp insert. The EST clone, IMAGE clone 1366609, was
obtained from the IMAGE consortium, sequenced in its entirety, and
found to contain an insert of 2259-bp corresponding to roughly the
3'-most third of the final full-length mouse pro-3(V) cDNA
sequence (nt 3850-6108). Sequences of clone ME7 overlapped those of
the EST clone and contained an additional 422-bp at the 5'-end. A
304-bp EcoRI fragment from the 5'-portion of the clone ME7
insert was used as a probe for further screening of the 17 dpc embryo
library, yielding two additional clones, ME8-11 (1059-bp insert) and
ME3-5 (876-bp insert), with 606 and 423 bp of additional 5' sequences,
respectively. Next, 5' RACE was performed with two nested
pro-3(V)-specific reverse primers,
5'-CCTTCAAACCAATGGGTCCTGGGTCT-3' (nt 3061-3036) and
5'-CAATGCCACCAGAGGGGCCTACAGGA-3' (nt 3142-3117), corresponding to
sequences near the 5'-end of clone ME8-11, using the Marathon cDNA
Amplification Kit and mouse brain Marathon-Ready cDNA template,
according to the manufacturer's protocol
(CLONTECH). This nested 5'-RACE produced a 613-bp
product. To obtain further mouse sequences, two pro-3(V)-specific
reverse primers corresponding to sequences near the 5'-end of the
613-bp 5'-RACE product, 5'-CTTTCTCCCCCAGTGGTCCCAAGGGT-3' (primer MSP3,
nt 2530-2505) and 5'-CCGGTGTGCCGCGTTCTCCTTCCTCT-3' (primer
MSP4, nt 2584-2559), were used both for a further nested 5'-RACE, performed as above, but in addition using Advantage-GC cDNA Polymerase Mix (CLONTECH); and for nested
PCR using 17 dpc embryo gt10 library cDNA as template and a
gt10 vector-specific primer, 5'-TCCCCACCTTTTGAGCAAGTTCAGCCT-3'.
Nested PCR with the gt10 primer and library yielded a product with
898 bp of pro-3(V) sequences. The 5'-RACE products were subcloned
into the pGEM-T vector (Promega). A forward PCR primer,
5'-GTGACAGGGAGTGATGGCGCACCA-3' (nt 1930-1953), corresponding to
sequences within the 898-bp PCR product, and reverse primer MSP3 (see
above) were used as a primer set for PCR screening of the 5'-RACE
product, pGEM-T clones. One clone, which contained a 2530-bp PCR
insert, was found to contain the remainder of mouse pro-3(V) coding
sequences plus 81-bp of the 5'-untranslated region (UTR).
To obtain human pro-3(V) sequences, a human placenta cDNA
gt11 library (CLONTECH) was screened with a
562-bp EcoRI cleavage fragment of the mouse IMAGE clone,
roughly corresponding to the complete pro-3(V) C-propeptide coding
sequences. One positive clone (HP3--2) was obtained, and this had a
3382-bp insert that corresponded to the 3'-half of human pro-3(V)
coding sequences plus 820-bp of 3'-UTR. A BLAST search of the dbEST
data base, using mouse pro-3(V) C-propeptide sequences as the query
sequence, located human retina EST pro-3(V) sequences (GenBank
accession number AA317772). The EST clone (EST19755, clone HARAL32) was obtained from the American Type Culture Collection (ATCC number 118234), sequenced in its entirety, and found to have an insert of
1316-bp that overlapped the 3'-end of clone HP3-2 and included an
additional 34-bp of 3'-UTR extending to a poly(A) tail.
Pro-3(V)-specific reverse primers 5'-TCACCTAGAGGTCCCACTTCTCCTGTCT-3'
(nt 2884-2857 of the full-length human pro-3(V) cDNA sequences
reported in the present study, AF177941) and
5'-AGTTCTCCTCTCTGTCCAGGGTGCCCT-3' (nt 2797-2771), corresponding to
sequences near the 5'-end of gt11 clone HP3-2, were used for nested
5'-RACE with Marathon-Ready human fetal brain cDNA as template,
resulting in a product containing 366-bp of pro-3(V) sequences. A
subsequent nested PCR with pro-3(V)-specific reverse primers
5'-GCTGCCCTGTCTTTCCCGACTTCCCT-3' (nt 2562-2537) and
5'-ACCGGGAAATCCAATAGATCCCTTAGGT-3' (nt 2513-2486), corresponding to sequences near the 5'-end of the 366-bp RACE product, and using a
gt10 vector-specific primer 5'-AGATTGGGGGTAAATAACAGAGGTGGCT-3' and
gt10 human fetal heart cDNA library template, produced a product
containing 774-bp of pro-3(V) sequences. Next, nested 5'-RACE with
pro-3(V)-specific reverse primers
5'-ACCCTTCTCCCCAGGAGTGCCAATGAGT-3' (nt 2081-2054) and
5'-ACCCATGGTTTCCCTGCTGTCCCGGA-3' (nt 2028-2003), corresponding to
sequences near the 5'-end of the 774-bp product, and using
Marathon-Ready human heart cDNA template, yielded a 1532-bp
product. This was followed by another nested 5'-RACE with pro-3(V)-specific reverse primers 5'-TCACAAGCCTGGAAGGCGGCCTGAGGA-3' (nt 739-713) and 5'-GGGTCCCCAGCACAGTGAGTCCAGCTA-3' (nt 654-628), and
using Marathon-Ready human heart cDNA template, which yielded a
551-bp product. A final nested 5'-RACE with pro-3(V)-specific reverse primers 5'-AGTTCTCAGGAAAGTGGCCTTCTGGAA-3' (nt 354-328) and
5'-GCACACCCAGGGCCTTCAGGACATCCA-3' (nt 207-181),
corresponding to sequences near the 5'-end of the 551-bp product, and
using Marathon-Ready human placenta cDNA template and Advantage-GC
cDNA Polymerase Mix (CLONTECH), produced a
207-bp product that contained remaining pro-3(V) coding sequences
plus 86-bp of 5'-UTR.
First rounds of nested RACE PCRs were performed in 50-µl reactions
with 20 pmol of each primer, 5 µl of Marathon cDNA, and 1 µl of
Advantage cDNA Polymerase Mix (CLONTECH) at
95 °C/3 min followed by 40 cycles of 95 °C/20 s, 68 °C/30 s,
72 °C/2-4 min and final extension at 72 °C/7 min. When
Advantage-GC cDNA Polymerase Mix was used, GC-Melt was added to a
final concentration of 1 M per reaction. First rounds of
nested PCRs using gt10 primers were performed the same way as first
round RACE PCRs, except that the annealing temperature was 70 °C,
and template was 5 µl of a gt10 library that had been
diluted 12-fold with water and heat-denatured by boiling for 10 min.
The second nested rounds of RACE PCRs and second nested rounds of PCRs
using gt10 primers were performed the same way as first rounds,
except that 25, rather than 40, cycles were used and template was 5 µl of first round PCR products diluted 50-fold with water.
Generation of Probes for RNA Blots and in Situ
Hybridization--
The 1.6-kb probe for human 3(V), corresponding
to 3'-UTR and C-propeptide sequences, was generated by restricting
clone HP3-2 (see above) with EcoRI and FspI. The
human 1(V) probe was the 1815-bp EcoRI insert of cDNA
clone CW32 (27) and contains main triple helical and C-propeptide
sequences. The human 2(V) probe was a 564-bp
EcoRI-HindIII fragment, corresponding to
C-propeptide sequences, derived from cDNA clone pBSL18 (43).
Template for PCR amplification of human 1(XI) and 2(XI) probes
was human heart Marathon cDNA. Amplification of the 1,004-bp
1(XI) probe, corresponding to C-propeptide and 3'-UTR sequences, was
with primers 5'-TGATCCTAACCAAGGTTGCTCAGG-3' (forward) and
5'-GAGTCAGCGGAATTCAGGGACACG-3' (reverse) using Advantage cDNA
polymerase mixture and conditions of 95 °C/3 min followed by 35 cycles of 95 °C/20 s, 58 °C/30 s, 72 °C/3 min, and final
extension at 72 °C/7 min. Amplification of the 890-bp 2(XI)
probe, corresponding to C-propeptide and 3'-UTR sequences, was by
nested PCR. The first round was with primers
5'-AGGCGAGGTGATCCAGCCACTGC-3' (forward) and
5'-GCTCTCTAACGGGTAACAGGCTCC-3' (reverse) using the same conditions used
for PCR amplification of the 1(XI), except that annealing was at
55 °C. The second, nested round was with primers
5'-ATGCAGGAAGATGAGGCCATACC-3' (forward) and
5'-GCTCTCTAACGGGTAACAGGCTCC-3' (reverse), using 5 µl of
a 1/50 dilution of the first round PCR product as template, and conditions of 95 °C/3 min followed by 25 cycles of 95 °C/20 s, 58 °C/30 s, 72 °C/3 min, and final extension at 72 °C/7 min.
PCR generated probes were cloned into pGEM-T, sequenced to confirm identity, and excised by restriction with SpeI and
ApaI.
Template for PCR amplification of mouse probes was 17-dpc mouse embryo
Marathon cDNA, except for the 3(V) Northern blot probe for which
template was EST IMAGE clone 1366609 (see above). General PCR
conditions for amplifying mouse probes employed Advantage cDNA
polymerase mixture at 94 °C/3-5 min, followed by 30-35 cycles of
94 °C/30 s, 55-70 °C/30 s, 72 °C/2 min, with final extension at 72 °C/10 min. All PCR products were cloned into pGEM-T. Primers for the 784-bp 3(V) Northern blot probe, which corresponded to 3'-UTR sequences, were 5'-TGAAGTTGTGAGGTGGGAAGGAAGCT-3' (forward) and
5'-GAGCACAGTTCCTTGGTTTATTCT-3' (reverse), with the probe excised from
pGEM-T with SacII and SpeI. Primers for the
1,480-bp 3(V) in situ hybridization probe (nt 34-1513),
which corresponded to N-propeptide/telopeptide sequences, were
5'-AGACCAGTCCACATCCCCCTTGGCCT-3' (forward) and
5'-CTTTCATGGACAGCTGAGCCTGTTGCA-3' (reverse). Riboprobes were generated
from this template by linearizing with ApaLI and transcribing with polymerase SP6 (antisense) or by linearizing with
NotI and transcribing with polymerase T7 (sense). Primers for the 1,206-bp 1(V) Northern blot probe, which corresponded to
C-propeptide and 3'-UTR sequences, were 5'-GGAGAGCTACGTGGATTATGC-3' (forward) and 5'-CCATCGGAAAGGCACGTGTGG-3' (reverse), with the probe excised from pGEM-T with SpeI and ApaI.
Primers for the 475-bp 1(V) in situ hybridization probe,
which corresponded to 3'-UTR sequences, were
5'-TGAGCCCACCGGTCTCCAGAGC-3' (forward) and 5'-CCATCGGAAAGGCACGTGTGG-3'
(reverse). Antisense and sense riboprobes were generated by
linearizing with NotI and transcribing with T7 two different
subclones in which the insert was in opposite orientations. Primers for
the 524-bp 2(V) Northern blot probe, which corresponded to 3'-UTR
sequences, were 5'-CTTCAAGACACCTGCTCTAAGCG-3' (forward) and
5'-ACATACCCCATCATGTAAGCTACC-3' (reverse), with the probe gel-purified,
direct-sequenced to check identity, and random-primed for blotting.
Primers for generating the 948-bp 1(XI) Northern blot and in
situ hybridization probes, corresponding to C-propeptide and
3'-UTR sequences, were 5'-GTTTGGATTTGAAGTCGGTCCAGC-3' (forward) and 5'-TGGCATTACTGAAGCACGCTGAGG-3' (reverse), with the probe for Northern blots excised from pGEM-T with SpeI and
ApaI and antisense and sense riboprobes generated by
linearizing with NotI and transcribing with T7 two different
subclones in which the insert was in opposite orientations. Primers for
generating the 611-bp 2(XI) Northern blot and in situ
hybridization probes, corresponding to N-propeptide/telopeptide sequences, were 5'-ATGTGGCTTACCGTGTGGCACG-3' (forward) and
5'-GCTCTGTGGCTTATGAAGTCTTGC-3' (reverse), with the probe for
Northern blots excised from pGEM-T with SpeI and
ApaI and riboprobes generated by linearizing the template
with NotI and transcribing with T7 (antisense) or by linearizing with NcoI and transcribing with SP6 (sense).
Blots were hybridized to random primed probes in ExpressHyb
(CLONTECH) at 65 °C. Northern blots were washed
in 2× SSC, 0.1% SDS at 65 °C, followed by 0.1× SSC, 0.1% SDS at
55 °C; and dot blots were washed following the manufacturer's
instructions (CLONTECH). For in situ
hybridization, uniform labeling of riboprobes with [35S]UTP, tissue preparation, and hybridization were
performed as described (44), except that sections were 5 µm thick and
mounted two to six/slide. For histological analysis, sections were
prepared and stained with hematoxylin, eosin, and Alcian blue as
described previously (45). Slides were analyzed using light- and
dark-field optics of a Zeiss Axiophot 2 microscope.
Chromosomal Mapping of the Human COL5A3 and Mouse Col5a3
Genes--
Mapping of the human COL5A3 gene was by
radiation hybrid mapping (46), using PCR analysis of the Genebridge 4 radiation hybrid panel (Research Genetics). Primers (50 pmol each) were 5'-CTGCTTCAGCAGCTGAGAGTGTCC-3' (forward, nt 5309-5332) and
5'-ACCACCTGGCATGGCAAGGTGAGC-3' (reverse, nt 5946-5923), in 50-µl
reactions with 100 ng of template DNA and 2.5 units of Taq
polymerase (Sigma) at 95 °C/5 min followed by 30 cycles of
94 °C/30 s, 60 °C/45 s, 72 °C/2 min, and final extension at
72 °C/10 min. These conditions amplified a 615-bp product from human
genomic DNA template, corresponding to 3'-UTR sequences. Scoring was
submitted to the WICGR Mapping Service at the Whitehead Institute/MIT
Center for Genome Research. The murine Col5a3 gene was
mapped by PCR analysis of 94 progeny of the C57BL/6J X Mus
spretus (BSS) backcross from the Jackson Laboratory (47). Primers
(20 pmol each) were 5'-CCTGGCAAGAGGGTGAGTGGTCTTCCA-3' (forward) and
5'-GCATCCAGGTTTATGTCAAGAGTGGGCT-3' (reverse), in 20-µl
reactions with 25 ng of template DNA and 0.4 µl of Advantage cDNA
polymerase mixture (CLONTECH) at 95 °C/3 min
followed by 30 cycles of 94 °C/30 s, 65 °C/45 s, 72 °C/30 s
and final extension at 72 °C/5 min. These conditions amplified 315- (C57BL/6J) and 285-bp (M. spretus) products, corresponding
to Col5a3 intronic sequences with differences in length
mostly due to different alleles of a CA polymorphic repeat (25 and 9 CA
repeats, respectively). Segregation of these products in the 94 BSS
backcross progeny showed linkage of Col5a3 to proximal
chromosome 9.
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RESULTS AND DISCUSSION |
The Primary Structure of the Pro-3(V) Collagen Chain--
The
full-length mouse and human prepro-3(V) collagen chain sequences,
inferred from cDNA clones and PCR products described under
"Experimental Procedures," are presented in Fig.
1. The human and mouse prepro-3(V)
chains comprise 1745 and 1739 amino acid residues, respectively. This
includes, for each, a 1011-amino acid major collagenous domain (COL1),
which is shorter than the COL1 domains of the other known vertebrate
fibrillar collagen chains. The latter COL1 domains range in length from
1014 amino acids, for the 1(I), 2(I), 1(II), 1(XI),
2(XI), and 1(V) chains; to 1017 amino acids, for the 2(V)
chain; to 1029 amino acids, for the 1(III) chain. As predicted,
based on partial amino acid sequences obtained from proteolytic
fragments of the human 3(V) COL1 region (42), the pro-3(V) chain
is most similar among fibrillar procollagens to the pro-1(V),
pro-1(XI), and pro-2(XI) chains. The latter three chains form a
subgroup among fibrillar procollagen chains on the basis of sequence
similarities, structures of cognate genes, and size and configuration
of N-propeptides (27, 28, 30, 33, 34, 42, 48, 49). As in the
pro-1(V), pro-1(XI), and pro-2(XI) chains, the pro-3(V)
NH2-terminal region that lies between the signal peptide
and COL1 domain is relatively large (comprising 412 amino acid residues
in both mouse and human) and can be divided into four subdomains (Figs.
1 and 2A). Immediately
upstream of the COL1 domain is a short non-collagenous linker region,
and immediately NH2-terminal of this is a short collagenous
domain. In the pro-1(V), pro-1(XI), and pro-2(XI) chains these
regions have been referred to as the NC2 (noncollagenous 2) and COL2
domains, respectively (33). Although COL2 has been described as being
divided into three small triple helical motifs by two short
noncollagenous interruptions, in the pro-1(V), pro-1(XI), and
pro-2(XI) chains (13), it has also been argued (34) that the short
length of the most COOH-terminal 3 Gly-X-Y triplets and low
imino acid content makes it unlikely that this last set of repeats
participates in triple helix formation in the pro-1(XI) COL2 domain.
In the case of the human pro-3(V) chain, this COOH-terminal set of
repeats is reduced to a single Gly-X-Y triplet devoid of imino acids, and thus quite unlikely to participate in triple helix
formation. In the mouse pro-3(V) chain, even this final single
Gly-X-Y triplet is missing. The most
NH2-terminal set of COL2 repeats, comprising 4 Gly-X-Y repeats in pro-1(XI) and pro-2(XI) and 5 Gly-X-Y repeats in pro-1(V), is reduced to 3 Gly-X-Y triplets in both human and mouse pro-3(V) (Figs.
1 and 2A). Thus, the triple helix formed by the pro-3(V)
COL2 domain is likely to be shorter than those formed by the COL2
domains of the other procollagen chains of this subfamily.
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Fig. 1.
Complete amino acid sequences of human and
mouse prepro-3(V) collagen.
First and second rows show murine (accession
number AF176645) and human (accession number AF177941) prepro-3(V)
amino acid residues, respectively, predicted by corresponding cDNA
sequences. Murine sequences are shown only where they differ from the
human. Vertical arrows mark signal peptide cleavage sites
predicted by the method of Nielsen et al. (82).
SP denotes the signal peptide. Collagenous domains
COL1 and COL2 are shaded. A
noncollagenous interruption in COL2 is double
underlined. The PARP and variable
(VAR) subdomains of noncollagenous domain 3 (NC3)
are labeled, as are noncollagenous domain 2 (NC2) and the
C-propeptide or noncollagenous domain 1 (NC1).
Cysteines are circled. Human sequences which align with
previously reported human 3(V) peptide fragments (42) are
underlined. Residues which differ from those reported for
the peptide fragments or which could not be resolved by amino acid
sequencing of peptide fragments by Mann (42), are boxed.
Murine and human sequences were aligned with the GAP program from
Genetics Computer Group (83). Dots represent gaps introduced
by the program for optimal alignment of sequences.
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Fig. 2.
Alignments of NH2-terminal
sequences, heparin-binding domains and C-propeptides of
pro-3(V), pro-1(XI),
pro-1(V), and
pro-2(XI) chains.
NH2-terminal (A) and C-propeptide (C)
sequences and sequences corresponding to the 1(V) heparin-binding
site (B) were aligned for murine (Mur3(V)) and human
(Hu3(V)) pro-3(V) chains, and human pro-1(V) (Hu1(V)) (27,
28), pro-1(XI) (Hu1(XI)) (29, 34), pro-2(XI) (Hu2(XI)) (30,
33), and pro-2(V) (Hu2(V)) (32) chains using the Pileup program
from Genetics Computer Group (83). Dots represent gaps
introduced by the program for optimal alignment of sequences. A
vertical arrow marks the murine pro-3(V) signal peptide
cleavage site predicted by the method of Nielsen et al.
(82). SP, PARP, NC3, VAR, COL1, NC2, and COL1 are as
described in the legend to Fig. 1. The extent of the COL1
domain and possible extents of COL2 domains are marked by
brackets. Noncollagenous interruptions in COL2
domains are underlined (A) as are potential
cleavage sites for furin-like proprotein convertases (C).
Cysteines are circled. Tyrosines which lie between the PARP
and COL2 domains, many of which have been shown to be sulfated in the
pro-1(V) chain (12), are boxed, as are potential
Asn-linked glycosylation sites (A) and basic residues in the
region corresponding to the 1(V) heparin-binding domain
(B). Residues found at the pro-1(V) BMP-1-cleavage site
and conserved in the other chains of the alignment are in bold
face type (A) as are acidic residues in the region
corresponding to the 1(V) heparin-binding domain (B). An
asterisk marks a conserved lysine 24 resides
NH2-terminal of COL1 (A). Residues conserved at
the same position in all chains in an alignment are
shaded.
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Between the pro-3(V) COL2 domain and signal peptide is a large
globular region. Similar globular regions in the pro-1(V), pro-1(XI), and pro-2(XI) chains have been referred to as NC3 domains (33) and, as in these other procollagen chains, the pro-3(V)
NC3 domain can be roughly divided into two subdomains (Figs. 1 and
2A). The amino-terminal portion of NC3, which extends from
the signal peptide to the vicinity of two clustered cysteines in all 4 chains, was first designated the PARP (proline/arginine-rich protein)
domain for the pro-2(XI) chain (33, 50). PARP domains show some
conservation of sequences between pro-3(V), pro-1(V), pro-1(XI), and pro-2(XI) chains (Fig. 2A) and also
have homologies to similar modules found in FACIT collagens, such as
types IX and XII, and to the NH2-terminal heparin-binding
domain of thrombospondin (51). Four cysteines, which have been shown to
form two intramolecular disulfide bonds in the pro-2(XI) PARP domain
(50), are perfectly conserved in the PARP domains of pro-1(V),
pro-1(XI), and pro-3(V) (Figs. 1 and 2A) suggesting
similar conformations for this module in the different chains. However,
the highly basic pI predicted by the sequence of the pro-2(XI) PARP
domain (10.4) is replaced by somewhat acidic pI values predicted by the
sequences of the PARP domains of pro-1(XI) and pro-1(V) (6.0 and
5.9, respectively) and by a markedly acidic pI of 4.4 predicted by the
sequence of the PARP domain of pro-3(V). Thus, despite similarities,
the PARP domains of the various type V/XI procollagen chains are
predicted to differ in physical properties which may reflect functional differences. Subsequent to cleavage from the rest of the pro-2(XI) chain, the pro-2(XI) PARP domain persists intact at relatively high
concentrations in some cartilage (50), suggestive of a physiological
role for this isolated module. The NC3 domains of pro-1(XI) (52) and
pro-1(V) (12, 53, 54) also appear to be processed at a site just
downstream of the PARP domain and, thus, the pro-1(XI) and
pro-1(V) PARP regions may also be released as intact modules that
serve functional roles in the extracellular compartment. In
vitro assays have suggested that the PARP domain may be cleaved
from the pro-1(V) chain by the astacin-like protease bone
morphogenetic protein-1 (BMP-1), and various residues found at the
BMP-1 cleavage site of the human pro-1(V) chain are conserved at the
same positions in the pro-1(V), pro-1(XI), and pro-2(XI) chains of various species (53). However, most of these residues, with
the exception of a proline residue at what corresponds to the P3'
position of the pro-1(V) BMP-1 cleavage site, are not conserved at
similar positions in either the mouse or human pro-3(V) chain (Fig.
2A). Astacin-like proteases are generally not highly specific for residues immediately flanking cleavage sites (55) and,
thus, it is possible that the pro-3(V) PARP domain is cleaved by
BMP-1. Alternatively, the unique string of basic residues immediately COOH-terminal to the pro-3(V) PARP region (Figs. 1 and
2A), suggests the possibility that cleavage may occur via a
furin-like proprotein convertase (56, 57). Further insights into the
nature of processing of the pro-3(V) N-propeptide region may be
obtained from in vitro assays similar to those used to study
NH2-terminal processing of the pro-1(V) chain (53).
Between PARP and COL2 is a region of the NC3 domain that has been
designated the variable region (33), since little to no homology exists
in this region between pro-1(V), pro-1(XI), and pro-2(XI)
chains (27, 28, 33, 34) (Fig. 2A). Unlike the procollagen
chains of collagen types I-III, type V/XI procollagen chains retain
some NH2-terminal globular sequences (12, 15, 52, 54,
58-61), and a number of studies suggest that retained NH2-terminal sequences include the variable regions of the
pro-1(V), pro-1(XI), and pro-2(XI) chains (12, 33, 50,
52-54). These retained sequences appear to be of functional importance
since, as shown for type V collagen, they protrude beyond the surface of heterotypic fibrils and may directly control fibrillogenesis by
sterically hindering the further addition of collagen monomers to the
fibril surface (54). These protruding sequences may also help modulate
interactions between heterotypic collagen fibrils and other components
of the extracellular matrix. Thus, the divergence of sequences in
variable regions may reflect differences in the biological activities
of the pro-1(V), pro-1(XI), and pro-2(XI) chains. The
diversity of variable region sequences is further increased by a
complex and tissue-specific pattern of alternative splicing of
sequences in the variable domains of the pro-1(XI) (62, 63) and
pro-2(XI) (31, 62) chains, predicted to produce pro-1(XI) and
pro-2(XI) chains with variable regions in which highly acidic
stretches of amino acid residues are either present or absent or, in
the case of pro-1(XI), are replaced by stretches of basic residues.
Such alternative splicing does not appear to occur within the
pro-1(V) variable region (27, 62), which has a highly acidic
predicted pI of 3.4, and which is rich in tyrosines that are sulfated
(12), further acidifying this domain. Variants of pro-1(XI) and
pro-2(XI) chains with variable regions that retain stretches of
acidic amino acids are also rich in tyrosine residues (Fig.
2A). In contrast to these other chains, the pro-3(V)
variable domain has a highly basic predicted pI (e.g. 10.3 for the human sequence) and a total absence of tyrosines (Figs. 1 and
2A). Moreover, PCR with a number of different primer sets in
this region of mouse and human pro-3(V), using various templates
from different tissues and developmental stages, gave no evidence for
alternative splicing (data not shown). The basic and acidic predicted
pI values of the pro-3(V) and pro-1(V) variable regions,
respectively, indicate that the retained NH2-terminal
sequences of 1(V)2(V)3(V) heterotrimers will have far
different charge properties than those of 1(V)22(V)
heterotrimers, providing heterotypic fibrils which incorporate the
different molecules with far different surface characteristics.
It has previously been shown that homotypic covalent cross-links
between type V/XI collagen chains involves lysines that are 24 residues
NH2-terminal of COL1, within the NC2 domain, and at residue
924 of the major collagenous domain (COL1), in the pro-1(V), pro-1(XI), and pro-2(XI) chains (64, 65). Heterotypic
cross-linking of type V/XI chains to type I or type II collagen chains
involves lysines at residue 84 of the pro-1(V) and pro-1(XI) COL1
domains (64, 65). Lysyl residues are conserved at the same three
positions within the pro-3(V) chain (Figs. 1 and 2A),
suggesting that the pro-3(V) chain may participate in the same types
of homo- and heterotypic cross-linking already characterized for the
other members of this subfamily of procollagen chains. Interestingly, the indication that 3(V) chains may be involved in heterotypic cross-links, further suggests that 1(V)2(V)3(V)
heterotrimers, like 1(V)22(V) heterotrimers, may be
incorporated into heterotypic fibrils. An RGD sequence juxtaposed to
the lysine at COL1 position 84 in the pro-1(V), and pro-2(XI)
chains, is conserved at the same position in pro-3(V) (Fig. 1). Such
RGD sequences are conceivably involved in interactions with cell
surfaces, as it has been reported that cells may adhere to type V
collagen via RGD-integrin interactions (66). A second RGD is found in
the mouse pro-3(V) sequence at COL1 position 360, but is not
conserved in the human pro-3(V) sequence, or in any of the other chains.
The pro-3(V) COL1 domain, which is 1011 amino acids in length,
is shorter by one Gly-X-Y triplet repeat than the 1014-amino acid pro-1(V), pro-1(XI), and pro-2(XI) COL1 domains.
Alignment of the four chains shows this to be due to substitution of an Ala for a Gly in what is the most COOH-terminal Gly-X-Y
triplet in the pro-1(V), pro-1(XI) and pro-2(XI) COL1 domains,
but which becomes part of the pro-3(V) C-telopeptide (Fig.
2C). The pro-3(V) COL1 domain is also shorter than that
of the pro-2(V) chain (1017 residues). This shortening of the
pro-3(V) COL1 domain, together with a lower number of imino acid
residues (215 codons for Pro) compared with the COL1 domains of the
pro-1(V) and pro-2(V) chains (249 and 223 codons for Pro,
respectively), helps explain the lower melting temperature of
pepsinized 1(V)2(V)3(V) heterotrimers compared with that of
pepsinized 1(V)22(V) heterotrimers (18, 67). A
comparison of the COL1 amino acid sequences of the various fibrillar
procollagen chains confirms that the pro-3(V) COL1 domain is most
similar to that of pro-1(V) (76% similarity, 71% identity),
but only slightly less similar to that of pro-1(XI) (74%
similarity, 70% identity) and only somewhat less similar to that of
pro-2(XI) (72% similarity, 67% identity) (comparison was via the
Genetics Computer Group GAP program, Ref. 83).
The COL1 domain of type V collagen has been shown to possess a
site which binds heparin/heparan sulfate under physiological conditions
(68, 69). This site has been located within the NH2-terminal half of the 1(V) COL1 domain and contains a
cluster of basic residues which appear to be necessary for
heparin/heparan sulfate binding (69, 70). Unlike isolated 1(V)
chains, 2(V) and 3(V) chains do not bind heparin under
physiological or denaturing conditions (69-71). Similarly, triple
helical type V collagen trimers bind to heparin with decreasing
affinity in the order 1(V)3 > 1(V)22(V) > 1(V)2(V)3(V), indicating
that 1(V) chains mediate heparin binding, while 2(V) and 3(V)
chains do not (70, 71). It has been suggested that 2(V) chains do
not bind heparin because the region which corresponds to the
1(V)-binding site is less basic (69, 70). It has similarly been
suggested that type XI collagen binds heparin due to high basicity in
the corresponding region in type XI chains (69). However, the
corresponding 3(V) sequence has not previously been available for
comparison. In Fig. 2B, alignment of the cluster of basic
amino acids in the heparin-binding domain of 1(V) with the
corresponding regions of the 3(V), 1(XI), 2(XI), and 2(V)
chains shows that 3(V), like 2(V), has less basic residues in
this region than do 1(V), 1(XI), or 2(XI). Moreover, 3(V),
like 2(V), has more acidic residues in this region than do the other
chains (Fig. 3B), further reducing localized basicity. Thus, the 3(V) sequence is consistent with predictions (69-71) that basicity in the region depicted in Fig.
2B is a determinant of heparin/heparan sulfate binding in type V/XI collagen chains.
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Fig. 3.
Dot-blot comparison of the expression
patterns of pro-3(V),
pro-1(V), pro-2(V),
pro-1(XI), and
pro-2(XI) RNA in adult and fetal human
tissues. Probes for pro-3(V), pro-1(V), pro-2(V),
pro-1(XI), pro-2(XI), and a ubiquitin control probe were
hybridized to a human RNA multiple tissue expression array
(CLONTECH) of dot-blotted poly(A)+ from
whole brain (1A), cerebral cortex (1B), frontal
lobe (1C), parietal lobe (1D), occipital lobe
(1E), temporal lobe (1F), paracentral gyrus of
cerebral cortex (1G), pons (1H), cerebellum left
(2A), cerebellum, right (2B), corpus callosum
(2C), amygdala (2D), caudate nucleus
(2E), hippocampus (2F), medulla oblongata
(2G), putamen (2H), substantia nigra
(3A), accumbens nucleus (3B), thalamus
(3C), pituitary gland (3D), spinal cord
(3E), heart (4A), aorta (4B), atrium,
left (4C), atrium, right (4D), ventricle, left
(4E), ventricle, right (4F), interventricular
septum (4G), apex of heart (4H), esophagus
(5A), stomach (5B), duodenum (5C),
jejunum (5D), ileum (5E), ileocecum
(5F), appendix (5G), colon, ascending
(5H), colon, transverse (6A), colon, descending
(6B), rectum (6C), kidney (7A),
skeletal muscle (7B), spleen (7C), thymus
(7D), peripheral blood leukocyte (7E), lymph node
(7F), bone marrow (7G), trachea (7H),
lung (8A), placenta (8B), bladder
(8C), uterus (8D), prostate (8E),
testis (8F), ovary (8G), liver (9A),
pancreas (9B), adrenal gland (9C), thyroid gland
(9D), salivary gland (9E), mammary gland
(9F), leukemia HL-60 (10A), HeLa S3
(10B), leukemia K-562 (10C), leukemia MOLT-4
(10D), Burkitt's lymphoma, Raji (10E),
Burkitt's lymphoma, Daudi (10F), colorectal adenocarcinoma,
SW480 (10G), lung carcinoma, A549 (10H), fetal
brain (11A), fetal heart (11B), fetal kidney
(11C), fetal liver (11D), fetal spleen
(11E), fetal thymus (11F), and fetal lung
(11G).
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In contrast to type I-III procollagen chains, in which C-propeptides
are cleaved by BMP-1 (72), the pro-1(V) C-propeptide is not cleaved
by BMP-1, but instead appears to be cleaved by a furin-like proprotein
convertase (53). This cleavage occurs just COOH-terminal of the COL1
domain, immediately downstream of the sequence RTRR (53), a canonical
RX(K/R)R furin cleavage site (56, 57). As can be seen (Fig.
2C), both human and mouse pro-3(V) chains have canonical
RX(K/R)R sites that align with that of the pro-1(V)
chain, and with the sequence KKTRR in pro-1(XI) and pro-2(XI)
chains, which is also suitable for cleavage by furin-like proprotein
convertases (56). Thus, the C-propeptides of the
1/3(V)/1/2(XI) subfamily of procollagen chains may all be
cleaved by the same, or by similar furin-like proprotein convertases.
Within the pro-3(V) C-propeptide, or NC1 domain, are 7 cysteine
residues conserved at similar positions in the C-propeptides of all
previously characterized fibrillar procollagen chains (Fig.
2C). It has been predicted (29) that fibrillar procollagen
chains capable of forming homotrimers will have 8, rather than 7 C-propeptide cysteines, as is the case for the pro-1(V) chain. If
so, the presence of 7 C-propeptide cysteines would be consistent with
reports that 3(V) chains are found in tissues only in the context of
1(V)2(V)3(V) heterotrimers (17, 18, 71), but inconsistent with
reports of 3(V)3 homotrimers (73). Alignment of
sequences shows that the pro-3(V) C-telopeptide is shortened
compared with those of the pro-1(V), pro-1(XI), and pro-2(XI)
chains, as is the portion of the pro-3(V) C-propeptides immediately
adjacent to the C-telopeptide (Fig. 2C). Both of these regions have previously been noted as areas of relative sequence variability between procollagen chains (74). A potential site for
Asn-linked glycosylation that precedes cysteine 6 of the C-propeptide is highly conserved between members of the
pro-1(I)/pro-2(I)/pro-1(II)/pro-1(II)/pro-2(V) subfamily
of procollagen chains, in which it is thought to be of some functional
significance (74), and is also conserved in the pro-1(V) and
pro-1(XI) chains (27-29). However, it is absent from the pro-2(XI)
chain (30) and, although it is found in the human pro-3(V) sequence,
it is absent in mouse. Thus, this site would not seem to be of great
functional significance for either pro-2(XI) or pro-3(V) chains.
In contrast, a potential glycosylation site (NQT) that is conserved in
mouse and human pro-3(V) sequences, between C-propeptide cysteines 6 and 7, is not found in any other fibrillar procollagen C-propeptide
and, thus may be of specific importance to the structure/function of pro-3(V) chains. Availability of the pro-3(V) sequence also demonstrates that the potential glycosylation site NFT, which occurs immediately downstream of C-propeptide cysteine 4, is conserved in all members of the pro-1(V)/pro-1(XI)/pro-2(XI)/pro-3(V) subfamily of procollagen chains. This site is not conserved in any
member of the
pro-1(I)/pro-2(I)/pro-1(II)/pro-1(II)/pro-2(V) subfamily
of chains, and thus may represent some fundamental
structural/functional difference between the C-propeptides of the two
subclasses of fibrillar procollagen chains.
Distributions of Expression of Pro-3(V) RNA in Adult and
Developing Tissues--
To begin characterizing distributions of
pro-3(V) expression in adult and developing tissues, patterns of
pro-3(V) mRNA expression were first examined in a dot-blot array
of poly(A)+ RNA from a variety of adult and fetal human
tissues and compared with the distributions of mRNAs for the
pro-1(V), pro-2(V), pro-1(XI), and pro-2(XI) chains in the
same array (Fig. 3). Particularly high pro-3(V) expression was
detected in mammary gland, correlating with the initial isolation of
pro-3(V) sequences as a mouse mammary gland EST (see "Experimental
Procedures") and suggesting a role for pro-3(V) chains in this
tissue in humans and mice. Relatively high pro-3(V) mRNA levels
were also seen in placenta and uterus, consistent with the
results of previous protein studies (12, 17-19). In addition, high
expression of pro-3(V) mRNA was found in fetal heart and lung,
and moderately high levels were detected in certain structures of adult
human heart (Fig. 3). Relatively high levels of pro-1(V) and
pro-2(V) RNA were found in most of the same tissues just noted for
pro-3(V) expression, suggesting the presence of
1(V)2(V)3(V) heterotrimers in these tissues. An exception was
adult brain, in which relatively high levels of pro-3(V) mRNA
expression were not matched by high levels of either pro-1(V) or
pro-2(V) mRNA. The significance of this finding is unknown,
although these data are consistent with the possibility that
pro-3(V) chains may combine with other procollagen chains or form
homotrimers in these regions of adult human brain. In the same
dot-blot array, highest levels of pro-1(XI) and pro-2(XI)
mRNA were seen in trachea, probably reflecting the hyaline
cartilage content of this structure. Surprisingly high levels of
pro-1(XI) and especially high levels of pro-2(XI) mRNA were
also found in structures of adult human brain. However, although this
may suggest the possibility of heterotrimer formation between
pro-3(V) and one or both type XI procollagen chains in brain, it
must be noted that distributions of both type XI procollagen mRNAs
in the different brain structures are quite different from that of
pro-3(V) mRNA.
Expression patterns of pro-3(V) mRNA in adult human tissues, and
comparison to the expression patterns of other type V/XI chains, were
further characterized by Northern analysis of poly(A)+ RNA
from a subset of the tissues analyzed by dot-blot array. As can be seen
(Fig. 4, A and B)
the distribution of pro-3(V) expression detected by the Northern
blots was generally consistent with that detected by the dot-blot
array, with particularly high levels of expression of a ~6.0-kb band
detected in heart, placenta, and uterus. Interestingly, pro-3(V)
mRNA in liver had a somewhat faster mobility (~5.5-kb) than that
detected in the other tissues just noted for pro-3(V) expression,
while the relatively high levels of pro-3(V) mRNA in brain were
found to be in the form of a considerably smaller ~4.2-kb band. The
reason for the smaller size of pro-3(V) transcripts in liver and
brain is, at present, unknown. In particular, the nature of the
~4.2-kb transcript in brain is rather mysterious, as the full-length
pro-3(V) coding sequence is 5235-bp, while PCR with a number of
different primer sets using mouse and human brain RNA as templates,
found no evidence for pro-3(V) N-propeptide alternative splicing
(data not shown). As in the dot-blot array, Northern blot analysis
found coexpression of pro-1(V), pro-2(V), and pro-3(V)
mRNAs in heart, placenta, and uterus, but low to undetectable
levels of both pro-1(V) and pro-2(V) mRNAs in brain, and
readily detectable levels of pro-1(XI) and pro-2(XI) mRNAs in
the latter tissue. Thus, the nature of pro-3(V) expression in brain
and the possible interaction of pro-3(V) chains with other type V/XI
procollagen chains in this tissue appears to be unique and will merit
further investigation. An interesting and, to our knowledge, novel
observation in both dot-blot array and Northern blot analysis was the
high expression of pro-1(XI) and pro-2(XI) mRNA in testis
(Figs. 3 and 4B), suggesting roles for these chains in that
tissue.
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Fig. 4.
Northern blot comparisons of the
spatiotemporal expression patterns of type V/XI procollagen
mRNAs. Probes for pro-3(V), pro-1(V), pro-2(V),
pro-1(XI), pro-2(XI), and a  -actin control probe were
sequentially hybridized to multiple tissue Northern (MTN) blots I
(A) and IV (B), or to a mouse embryo blot
(C) (CLONTECH) containing approximately
2 µg of poly(A)+ RNA per lane from various adult human
tissues (A and B), or from 7, 11, 15, and 17 dpc
mouse embryos (C).
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To begin characterizing the temporal expression pattern of pro-3(V)
during development, and to compare this pattern to the temporal
expression patterns of other type V/XI procollagen chains, pro-3(V)-, pro-1(V)-, pro-2(V)-, pro-1(XI)-, and
pro-2(XI)-specific probes were hybridized to a Northern blot
containing poly(A)+ RNA from 7, 11, 15, and 17 dpc mouse
embryos (Fig. 4C). The pro-3(V) probe hybridized to a
single ~6.3-kb band that was at readily detectable levels in the RNA
of 7 dpc mid-gastrulation mouse embryos. This pro-3(V) ~6.3-kb
mRNA disappears at 11 dpc (Fig. 4C) and was not visible
even upon prolonged exposure of the blot, nor was signal for
pro-3(V) RNA detectable at this stage by in situ hybridization of 11 dpc mouse embryos (not shown). Pro-3(V) mRNA reappears at 15 dpc and is further increased in abundance at 17 dpc,
during a period of post-organogenesis fetal growth and development. Strong expression of both pro-1(V) and pro-2(V) mRNAs
accompany that of pro-3(V) mRNA at 15 and 17 dpc. However,
although strong pro-2(V) mRNA expression is evident at 7 dpc,
expression of pro-1(V) is not readily detectable at this stage of
development (Fig. 4C), with low levels of pro-1(V)
mRNA just visible upon prolonged exposure of the blot (not shown).
Pro-1(XI) and pro-2(XI) mRNAs are also readily detectable at
15 and 17 dpc, but even prolonged exposure of the blot (not shown) did
not reveal detectable levels at 7 and 11 dpc. These results suggest a
role for type V, but not type XI collagen chains in mid-gastrulation
mouse embryos. The results are also consistent with the possibility
that pro-3(V) chains may exist either as homotrimers or in
heterotrimeric combination with pro-2(V) chains, in the absence of
pro-1(V) chains, at this time. However, the possibility that 3(V)
chains are found only in the context of 1(V)2(V)3(V)
heterotrimers at 7 dpc, despite wide differences in RNA levels for the
various chains, has certainly not been excluded.
To determine the distribution of expression of pro-3(V) during mouse
development, and to compare this to the expression domains of other
type V/XI procollagen chains, a series of in situ
hybridizations were performed on serial sagittal and parasagittal
sections of 13.5 and 15.5 dpc mouse embryos using antisense, and sense
control, riboprobes specific for pro-3(V), pro-1(V),
pro-1(XI), and pro-2(X) sequences. At 13.5 dpc pro-3(V) RNA
expression was barely detectable, although pro-1(V) RNA expression
was widely distributed throughout developing mesenchyme and intense
pro-1(XI) and pro-2(XI) signals were already visible in nascent
chondrified cartilaginous elements (data not shown). At 15.5 dpc,
however, pro-3(V) expression was readily discernible and the
pro-3(V) expression domain was seen to be a subset of that of
pro-1(V) (Figs. 5 and
6). Interestingly, although pro-1(V)
expression was widely distributed throughout developing connective
tissues, with especially high levels of expression seen in the
perichondrium associated with cartilaginous primordia of future bones,
expression of pro-3(V) was not detected in perichondrium or other
regions of bone primordia, but was instead most readily detectable in the superficial fascia and in the epimysia, or connective tissue sheaths, tracing the outlines of the developing muscles of the anterior
chest wall, the cutaneous panniculus carnosus muscle and the developing
musculature of the neck. In addition to its expression in epimysium,
pro-3(V) expression was also seen in the connective tissue sheath,
or epineureum, of some nerves (Fig. 6). Although pro-3(V) was not
expressed in perichondrium, high pro-3(V) expression was observed
closely apposed to the cartilage primordia of future bones in the soft
tissue associated with a number of joints, in what appeared to be
incipient ligamentous attachments (formation of ligaments and tendons
first begins in mouse development, as mesenchymal condensations at 14 dpc, Ref. 75). In Figs. 5 and 6, pro-3(V) expression in nascent
ligamentous attachments can be seen (i) between the cartilage primordia
of the basioccipital bone at the base of the skull and the first two
cervical vertebrae C1 (atlas) and C2 (axis), (ii) apposed to the
cartilage primordium of the exoccipital bone, and (iii) between the
cartilage primordia of the femoral head and acetabulum of the hip
joint. Pro-
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ABSTRACT
INTRODUCTION
