From the
Type XI collagen is an integral, although minor component of
cartilage collagen fibrils. We have established that alternative exon
usage is a mechanism for increasing structural diversity within the
amino-terminal non-triple helical domain of the pro-
The fibrillar collagens, types I, II, III, V, and XI, combine to
form the heterotypic interstitial collagen fibrils common to most
tissues
(1, 2) . These collagens are related by sequence
homology and genomic organization; the defining features of this family
of collagens include a major uninterrupted triple-helical domain,
flanked by propeptide domains
(1, 3, 4, 5) . In cartilage, the bulk of
the fibril is composed of type II collagen while type XI contributes a
relatively small amount to the mass of the fibril
(6) . In a
similar way, non-cartilaginous fibrils are mixtures of types I and V or
I, III and V, with type I the major component
(1) .
The
overall sequence and structural similarity among these collagen types
can mask specific structural differences which can vary from the subtle
to the dramatic. The carboxyl propeptides of collagens are thought to
guide the molecular associations leading to formation of a specific
subset of possible trimeric molecules from the mixture of fibrillar
collagen chains synthesized by the cell; this specificity is likely to
be imparted by structural variations among these homologous domains
(7) . The type XI heterotrimer is a particularly complex example
of this chain selectivity. It is composed of three distinct gene
products (
The greatest diversity lies
in the amino propeptide where dramatic variations in sequence as well
as the site and rate of proteolytic processing are observed. The amino
propeptides of all of the fibrillar collagen chains contain a minor
triple helix separated from the major triple helix by the amino
telopeptide. The region of diversity is found between the minor helix
and the signal peptide
(4, 5) . In the case of
One function of the amino propeptides of
fibrillar collagens appears to be an involvement in the process of
fibril formation
(27) . In vitro, varying the content
of pN-collagen
The pro-
Screening of the rat chondrosarcoma cDNA library led to the
identification of several overlapping clones encoding the
amino-terminal domain of pro-
The
identification of the exons and the differences observed within the rat
cDNA sequences and between rat and human forms of
Alternative exon splicing is now observed to be a relatively
common feature of the biosynthesis of extracellular matrix
macromolecules including fibronectin, elastin, aggrecan, and some of
the collagens (reviewed by Boyd et al. (43) ) and more
recently in fibrillin
(44) , fibulin 1
(45) , and fibulin
2
(46) , for example. It is clear from such studies that
alternative exon splicing can have important functional consequences.
Alternative splicing within the exon encoding the V region of
fibronectin regulates several properties of the protein
(43) .
The portion of this exon retained in the fibronectin dimer influences
fibronectin secretion from the cell, cell type-specific adhesion, and
incorporation of fibronectin into fibrin clots. Within the collagen
gene family, alternative splicing can involve triple-helical domains,
such as in type XIII, but more generally involves nontriple-helical
domains which has been shown for type VI
(43) and which has
been proposed for types XII and XIV
(25, 47) . Recently
it was shown that among the fibrillar collagens, the
Data presented in this report
demonstrate that the rat
Embryonic
noncartilaginous tissues have been shown to express mRNA transcripts of
the
Within cartilage, all six isoforms of the
amino-terminal domain are represented. Relative abundance cannot be
determined accurately by PCR. However, end labeling and analysis on
denaturing alkaline gels represents each isoform on a molar rather than
mass basis so that the observation of large differences in signal may
be relevant. Significant among the isoforms are the basic version, V1b
+ C2, and Vo. It is not known if a single chondrocyte expressing
The
51-amino acid peptide encoded by the V1b exon is a unique sequence
based on comparison to the sequence data base. The sequence is unusual
in that a majority of the basic residues (mostly lysines) are found in
clusters of three, four, or five residues. Such clusters might serve as
sites for proteolysis and generate a more readily processed form of the
In addition to the
alternative splicing reported here for the
Fetal and growth plate
cartilage collagen fibrils are relatively small in diameter, less than
25 nm. As a minor but integral component of these fibrils
(2, 52) , type XI collagen has been proposed as an
element involved in the regulation of fibril diameter. Evidence for
this function includes: very slow proteolytic processing within the
pro-
Based on
structural data, the proteolytic processing site(s) likely resides
between the PARP-like domain and the minor helix
(23, 24, 27) . This is precisely the location of
the variable region, portions of which could be retained after
processing. The PARP-like domains and the variable regions could
mediate interactions directly or indirectly between the proteoglycan
network and the collagen fibrils. The alternatively spliced exons, V1b,
V1a, and V2 could influence fibril formation and interactions by
modulating processing of the PARP-like domain or, independent of
processing, could, in themselves, provide sites for interaction with
the cartilage matrix.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank/EMBL Data Bank with accession number(s) U20116-U20121.
We thank Silvija Coulter, Babette Romancier, and
Xiaocun Chen for expert technical assistance. We also acknowledge Jay
Gambee and Jeff Bondar of the analytical core facility, Shriners
Hospital, Portland, OR, for the provision of automated sequencing and
oligonucleotides and Rich Watson for assistance with computer programs
and computer graphics.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
1(XI) collagen
gene. cDNA clones spanning the amino-terminal domain were selected from
a rat chondrosarcoma library, and were shown to contain two major
sequence differences from the previously reported human sequence. The
first difference was the replacement of sequence encoding an acidic
domain of 39 amino acids in length by a sequence encoding a 51-amino
acid basic domain with a predicted pI of 11.9. The second difference
was the absence of a sequence that would translate into a highly acidic
85amino acid sequence downstream from the first variation. These two
changes, expressed together, result in the replacement of most of the
acidic domain with one that is smaller and basic. These two sequence
differences serve to identify subdomains of a variable region,
designated V1 and V2, respectively. V1a is defined as the acidic
39-amino acid sequence element and V1b is defined as the 51-amino acid
basic sequence. Analysis of genomic DNA revealed that both V1a and V1b
are encoded by separate adjacent exons in the rat genome and V2 is also
encoded in a single exon downstream. Analysis of mRNA from
cartilage-derived sources revealed a complex pattern of
1(XI)
transcript expression due to differential exon usage. In non-cartilage
sources, the pattern is less complex; the most prevalent form is the
one containing the two acidic sequences, V1a and V2.
1,
2, and
3), where
3(XI) is the same
gene product as the
1(II) chain of type II collagen homotrimers
(8, 9, 10, 11) . In addition, type V/XI
hybrid molecules, with different combinations of the component chains,
have been inferred or directly demonstrated in bone
(12) ,
mature articular cartilage
(6) , vitreous
(13) , and in
the molecule formed by the rhabdomyosarcoma cell line A204
(14) . Structural differences have also been observed in the
triple-helical domain. Despite a primary structure which does not
significantly differ in the overall distribution of key stabilizing
amino acids such as proline and hydroxyproline, only collagens V and XI
show a complex melting behavior with several stable unfolding
intermediates
(15, 16) .
1(I),
1(III), and
2(V), a cystine-rich globular domain
comprises most this region (<100 amino acids) and this sequence is
lacking in the gene for
2(I). This domain is also found in the
gene for
1(II) where it is encoded by exon 2 and is conditionally
expressed in a developmentally regulated fashion
(17, 18, 19) . A quite different domain is found
in the analogous position in the other fibrillar collagen chains,
including
1(V),
1(XI), and
2(XI)
(20, 21, 22, 23, 24) . Here the
globular domain is much larger (>400 amino acids) and more closely
related to the amino-terminal noncollagenous domains of the
fibril-associated collagens, types IX, XII, and XIV
(25) . The
sequence contains only four cysteine residues and can be divided into a
weakly basic amino-terminal half containing the cysteine residues
(designated PARP-like after the pro-
2(XI) cleaved domain
(23, 24) ) and a very acidic half adjacent to the minor
helix (see Fig. 1).
Figure 1:
cDNA clones encoding rat
pro-1(XI) amino-terminal domain. Overlapping clones were obtained
for the amino-terminal domain of
1(XI) from a rat chondrosarcoma
cDNA library. Position of clones is compared to general structure of
the published amino propeptide; + indicates a slightly basic
domain; - - - indicates a strongly acidic region. The numerical scale
represents base pairs of DNA, with 0 at the start of translation.
Variations in the very acidic region are indicated in Fig. 2. The
5`-most clone (R8E) contains
300 bp of 5`-untranslated sequence.
R1-1 and R6D extend into the major triple helix-coding
( TH) region. Signal peptide ( sp), minor helix
( mh), telopeptide ( tp).
In cartilage, the structure of this domain
in type XI collagen has been of interest because unlike type II
collagen, proteolytic processing of the amino-terminal domain is very
slow and even after processing is completed, some portion of the domain
is retained
(26) . The site(s) of proteolytic cleavage have not
been identified. For the pro-1(XI) chain, it is likely that some
molecules with an intact amino-terminal domain are incorporated into
the growing fibril.
(
)
modulates fibrillogensis
(28, 29) . In vivo, failure to remove the amino
propeptide of type I collagen can result in derangement of the tightly
packed cylindrical fibril as seen in dermatosporaxis
(28, 30) . The low abundance of type XI collagen, the
size of the amino propeptides of type XI collagen, their persistence in
the tissue, and the location of the triple helix in the interior of the
cartilage fibril suggest that the role of this molecule in fibril
formation is more regulatory than structural. Such a role was proposed
recently for type V collagen, see also Ref. 21.
1(XI)
amino propeptide was originally cloned and sequenced from a
noncartilaginous source
(22) . Because the type XI amino
propeptide may help regulate formation of collagen fibrils in
cartilage, we have cloned and sequenced this domain from a
cartilaginous source, rat chondrosarcoma
(31) , and looked for
structural features specific to this tissue. Our results show that
alternative splicing at two sites within the acidic sequence generates
multiple forms of the amino-terminal domain. The additional isoforms
generated by alternative splicing are quite different from the original
noncartilaginous form and, in rat, are more prevalent in cartilage.
cDNA Cloning and Sequencing
cDNA clones encoding
the amino-terminal domain of rat pro-1(XI) were obtained by serial
screening of an RCS cDNA library. The library was generated from Swarm
rat chondrosarcoma poly(A)
RNA (prepared as described
previously
(32) ), primed for first strand synthesis using both
random hexamers and oligo(dT). A modified Gubler and Hoffman
(33) method of cDNA construction was used employing kit reagents
(Amersham). Double stranded cDNA with EcoRI linkers was
ligated to
gt11 arms and phage were packaged with commercial
extracts (Stratagene). The initial screen utilized a probe obtained by
reverse transcriptase-PCR amplification of a fragment of the rat
pro-
1(XI) carboxyl propeptide
(34) from RCS RNA. Primers
for amplification were 5`-GGAGAAGTCATACAGCCATTACCT-3` and
5`-CCAAGAAAACAAGCTGGACCAACTTC-3`, and the amplified fragment was cloned
and sequenced. DNA fragments were labeled for use as probes with
P by random primer extension (Multiprime, Amersham). The
5`-most clone was identified by sequencing of subclones and a new probe
from the 5` end prepared by PCR amplification template DNA with primers
5`-TGAGTCCTTGAGGGCCCCC-3` and TAGCCCTGAGTCCTTGAGG-3`. A second round of
screening produced clone R1-1 (Fig. 1) identified as the
5`-most by sequencing of the purified phage. A third screen was
performed in the same way using a probe generated by PCR with primers
5`-GTGTGGAGAAGAAAACTGTGACAATG and 5`-GTCACAGTCGGGACTGTAATGGTC-3` and
R1-1 template DNA. Phage containing the 5`-most cDNA were
identified as those yielding the largest products of amplification
between the downstream primer used to make the probe and a primer from
either side of the
gt11 cloning site. Overlapping clones spanning
the amino-terminal domain of rat pro-
1(XI) (see Fig. 1) were
subcloned into pBluescript (Stratagene) and sequenced on both strands.
Plasmids were prepared using the alkaline lysis method
(35) and
purified using DNA Purification Resin (Promega).
DNA Sequencing
Sequencing was performed by the
dideoxy chain termination method
(36) using either Sequenase
DNA polymerase (U. S. Biochemical Corp.) or sequencing grade Taq DNA
polymerase and thermal cycle DNA sequencing protocol (fmol, Promega).
In some cases, sequences were obtained by automated DNA sequencing
using an ABI-373A sequencer (Perkin Elmer). Sequence alignments were
performed using programs provided by the Genetics Computer Group
(37) .
Cloning of Genomic DNA
A rat genomic DNA library
(Clontech) was screened with the ApaI/ EcoRI
restriction fragment from clone R1-1 (Fig. 1). Positive
plaques were purified and phage DNA grown and prepared as described
(35) . EcoRI fragments were screened for exons by
Southern hybridization analysis with oligonucleotides from C1, V1b, V2,
and C3. Exon-containing fragments were subcloned into pBluescript and
sequenced.
Northern Analysis
A probe for V1b was prepared by
PCR amplification of cloned template DNA between primers
5`-AAAAGAAATCCAATTACACAAAGA-3` and 5`-GACCCCTAGTTTGGCTTTG-3`. Rat
chondrosarcoma poly(A)RNA was probed by Northern
analysis as described previously
(11) .
RNA Preparation
RNA was extracted from specified
tissues of 17-day fetal rat, from a fetal rat skin cell line (FR)
(ATCC), and from IRC cells (provided by Walter E. Horton Jr.
(38) ), by the method of Chomczynski
(39) .
PCR Analysis of Alternatively Spliced
Domains
Primer pairs for PCR bracketing variable region V1 were
5`-CCAAGGCAGCATATGACTACTGTG-3` and 5`-GCCGAGGAGACTCAGTCTGG-3` (C1-C2),
bracketing variable region V2 were 5`-CCAGACTGAGTCTCCTCGGC-3` and
5`-CATTCCGGGTTCAACTACAGC-3` (C2-C3), and bracketing both V1 and V2 were
5`-CCAAGGCAGCATATGACTACTGTG-3` and 5`-CATTCCGGGTTCAACTACAGC-3` (C1-C3).
Primer pairs for glyceraldehyde-3-phosphate dehydrogenase,
5`-GTCAACGGATTTGGCCGTATTGG-3` and 5`-AAAGTTGTCATGGATGACCTTGGCC-3`
(40) , were used as a control for PCR. Annealing temperatures
were calculated by the PRIMER program (kindly provided by Drs. S.
Lincoln, M. Daly, and E. Lander, Massachusetts Institute of Technology,
Center for Genome Research). PCR was performed according to established
protocols
(41) using 1.5 m
M MgClin the
amplification buffer and 30 cycles of amplification. As a negative
control, reverse transcriptase was omitted. PCR products were analyzed
by electrophoresis on 3.5% Nusieve 3:1 agarose gels (FMC) and staining
with ethidium bromide. Individual amplification products were
identified by direct sequencing of gel bands (fmol, Promega) after
removal of agarose (PCR clean up resin, Promega). To analyze PCR
products without heteroduplex formation, amplification products were
end labeled with [
-
P]ATP (ICN) and
polynucleotide kinase (Promega) and electrophoresed on alkaline
denaturing gels
(35) . Electrophoretic markers, FC
174/ HaeIII digest (Promega), were labeled in the same way.
1(XI) and some of the 5`-untranslated
region (Fig. 1). The sequence from the signal peptide to the start of
the major triple-helical domain is presented in Fig. 2. This
corresponds to the sequence of clone R6D. In general, the rat
amino-terminal domain was very similar to the reported human sequence.
The sequence 5` of the central pair of cysteine residues at nucleotides
703 and 724 of the rat sequence showed 87% nucleic acid sequence
identity, translating to 93% amino acid similarity. The region of the
minor helix and amino telopeptide (nucleotides 1276 to 1615 of the rat
sequence) showed 87% identity at the nucleic acid level and 98%
similarity at the amino acid level. Between these two regions of high
homology lies an area of variability. Two major differences between rat
and human were observed in this variable region. Variable region 1
(V1), starting at nucleotide 775 ( shaded area in Fig. 2)
was unrelated to the human sequence. Variable region 2 (V2) is the
region 93 bases downstream, between 1021 and 1275 ( second shaded
area in Fig. 2) which was missing in clones R8E, R6B, and
R1-1.
Figure 2:
Sequence of cDNA encoding rat 1(XI)
amino-terminal domain. Sequence of rat
1(XI) amino-terminal domain
is shown in capital letters and numbered with respect
to the first base of the start codon. The rat cDNA sequence is compared
to that of human; only human DNA sequence different from that of rat is
shown in the line of text directly above the rat sequence. The
translation of the rat sequence is shown immediately below the
rat DNA sequence and differences in human sequence are indicated
above the human DNA sequence. Very few differences exist
between 1 and 774 and from 927 into the major triple helix, and most
result in conservative changes at the amino acid level. An additional
codon for serine exists between nucleotides 15 and 16 of the rat
sequence and there is an additional codon for proline between
nucleotides 738 and 739 of the rat sequence. Shaded area from
nucleotide 775 to 927 indicates subdomain 1 of the variable region V,
designated V1. The acidic human sequence is termed V1a and the basic
rat sequence is termed V1b. Shaded area from 1021 to 1275
indicates subdomain 2 of the variable region, designated V2. The
constant region between V1 and V2 is called C2, and the 5`- and
3`-constant regions flanking the variable domains are C1 and C2,
respectively. Dashes indicate an absence of nucleotide or
amino acid, dots indicate that the nucleotide is the same in
both human and rat sequences. Continuous minor helix is
underlined. The beginning of the major helix is indicated by
solid triangle. The four cysteine residues are
boxed.
Inspection of the rat and human deduced amino acid
sequence showed that the very acidic (theoretical pI of 3.34) human
sequence of 39 amino acids
(22) termed V1a was replaced in RCS
by a very basic (pI of 11.90) 51-amino acid sequence termed V1b.
Northern analysis of RCS poly(A)RNA (data not shown)
indicated that this sequence was well represented among RCS
pro-
1(XI) transcripts. In RCS, the replacement of V1a by V1b,
combined with the frequent absence of V2, which also encodes a very
acidic sequence, would be predicted to strikingly alter the biochemical
nature of this region of the amino-terminal domain. To examine the
basis for the differences between the rat and human sequences, a rat
genomic library was screened with a cDNA probe specific for this
region, the ApaI- EcoRI fragment of cDNA clone
R1-1 (Fig. 1). Two non-overlapping genomic clones were
obtained and exons were identified and analyzed by sequencing. Sequence
data revealed that the V1a domain was also present in the rat gene
(Fig. 3). V1a, V1b, C2 (constant region 2), and V2 each occur as
separate exons, in this order (Fig. 4). The intron-exon boundaries of
the four exons analyzed conform to the consensus splice acceptor and
donor sites
(42) and do not involve split codons.
1(XI) pointed to
alternative splicing of mRNA as the likely mechanism to generate this
diversity. The pattern of exon utilization in various tissues was
examined in cDNAs encoding these regions which were amplified by PCR
and analyzed by electrophoresis and sequencing. IRC cell, RCS, and
17-day fetal rat sternal cartilage and limb cartilage were used as
sources of chondrocyte RNA; placenta and a fetal rat skin cell line
were used as sources of noncartilaginous RNA. The V1 region was
amplified using primers within the C1 and C2 sequences (Fig.
5 A). Three products which correspond to the presence of V1b
(319 bp), the presence of V1a (282 bp), and to the absence of a V1 exon
(166 bp) were detected. The identity of these bands was verified by
sequencing. All three forms were observed in normal chondrocytes,
whereas RCS lacked V1a and IRC lacked V1b. The band just above 319 bp
in the IRC lane as well as the uppermost band in RCS are heteroduplexes
of the lower two bands. In placenta and skin fibroblasts, only V1a was
detected. In only one case was a transcript containing both V1a and V1b
observed. Amplification using primers within V1a and C2
(Fig. 5 C) resulted in a larger than expected band only
in RCS and this band proved to be V1a + V1b as identified by
sequencing. Because V1a is itself rarely expressed in RCS, PCR priming
within V1a selects for rare transcripts. Analysis of the region
including V2 by amplification between C2 and C3 is shown in
Fig. 5B. In all cartilage-derived sources, both splice
forms with (364 bp) and without (109 bp) V2 were present. The V2 domain
was relatively more abundant in IRC and less so in RCS when compared to
normal cartilage. In placenta and skin fibroblasts, only the form with
V2 was expressed.
Figure 5:
Analysis of exon usage in the V1 and V2
region. PCR amplification reactions were carried out across the
variable region of 1(XI) amino-terminal domain. A, three
different products are observed for the V1 domain, with V1a, with V1b
or minus either form of V1. B, two different products are
observed for the V2 domain, using PCR primers within C2 and C3. The
identity of these products was determined to be either with or without
the V2 exon. C, amplification using primers within V1a and C2
shows a 146-bp product in all tissues, but in addition, a larger
product in RCS (297 bp). This band consists of V1a and V1b expressed in
the same transcript. Tissues assayed were sterna ( STA), limb
( LMB), rat chondrosarcoma ( RCS), immortalized rat
chondrocytes ( IRC), placenta ( PLA), and a fetal rat
fibroblast cell line ( FB). Size markers are shown on the
left. PCR products were identified by sequencing gel bands.
Identity and sizes are shown on the
right.
The possibility of coordinate expression of exons
from the two domains was examined by PCR amplification across both V1
and V2 using primers in C1 and C3 (Fig. 6 A). A combination of
three possibilities with respect to V1 and two for V2 should yield six
possible products for this amplification. Six products of the expected
sizes were observed as well as additional bands of sizes not predicted
from this combination of exons. A simpler picture was obtained when the
PCR products were end-labeled and separated on a denaturing alkaline
gel (Fig. 6 B), indicating that part of the apparent
complexity could be explained by heteroduplex formation between the
various products of amplification in cases where more than one product
was formed. Only the products expected for combinations of V1 and V2
are present. V0, the splice form lacking both V1 and V2, is well
represented in all four chondrocyte-derived RNAs. The form V1a alone is
present in IRC but only barely detectable in sterna and limb. V1b was
present in normal chondrocytes and RCS but was not observed in IRC. V2
alone and V1a + V2 were found in limb, sterna, and IRC but not in
RCS. V1b + V2 was present in low amounts in limb, sterna, and RCS
but was absent from IRC. Placenta and skin fibroblasts show only V1a
+ V2, the form originally cloned and sequenced
(22) .
Figure 6:
Coordinate expression of exons of V1 and
V2. mRNA structure across the entire variable domain was analyzed by
amplifying cDNA using PCR primers within C1 and C3. A,
ethidium bromide-staining pattern of amplification products.
B, autoradiographic image of identical PCR products after end
labeling and separating on an alkaline gel. Tissues assayed are as
described in the legend to Fig. 5. The two bands below +V1a
+V2 in lane FB were not identified. Size markers are
shown on the left. Identified products and sizes are shown on
the right.
The examination of the pattern of alternative splicing of the
variable region was extended to RNA samples from several other
noncartilaginous tissues of fetal rat (Fig. 7). Amplification of
glyceraldehyde-3-phosphate dehydrogenase was performed on an identical
aliquot as a control for the quantity and quality of the RNA to permit
a rough estimation of the relative amount of pro-1(XI) transcript
in each tissue. Brain, calvaria, skeletal muscle, and skin showed
significant expression of pro-
1(XI) mRNA.. In each case, the
splice form containing V1a + V2 was the prominent species. The
splice form lacking both V1 and V2, V0, was also present in calvaria
and skeletal muscle as was a low level of +V1b (not visible on
this gel). The other tissues, heart, liver, kidney, and lung showed
barely detectable levels of pro-
1(XI) transcripts and then only
the V1a + V2 splice form was visible. Amplification of sternal RNA
revealed the same complex pattern of alternative splicing identified
above. In all of the PCR amplification experiments presented, no
products were amplified when reverse transcriptase was omitted.
1(II) gene
product can be modulated by alternative splicing of exon 2 which
encodes the cysteine-rich domain located between the minor helix and
the signal peptide in the amino propeptide
(17, 18, 19) . In the absence of direct
evidence, functional significance is inferred from the pattern and
location of the alternative spliced exons within the mRNA and protein,
specific tissue distribution, or specific spatial and temporal pattern
of expression during development.
1(XI) chain undergoes a complex pattern
of alternative exon usage involving two closely associated variable
regions, V1 and V2, located between the PARP-like region and the minor
helix of the amino-terminal domain. This region, identified from a
noncartilaginous source, is highly acidic with a predicted pI of 3.34
(22) . Transcripts from cartilage, analyzed by PCR, indicate
that these two variable regions can generate six forms of the
amino-terminal domain. These forms arise because in V1 either the V1a
exon, or the V1b exon or both can be spliced out while the V2 exon can
be either present or absent. These splice isoforms are also observed in
RNA from human and chick chondrocyte.
Splicing of these
exons is coordinated since V1a and V1b are typically expressed in a
mutually exclusive fashion; furthermore, V1b expression is usually
linked to the absence of V2, V1a to the presence of V2. The V1b exon
encodes a very basic amino acid sequence, predicted pI of 11.9, while
V1a and V2 are quite acidic. The combinations of these exons observed
would produce four prevalent versions of the variable segment of the
amino-terminal domain: a basic form, V1b + C2, strongly acidic
forms, V1a + C2 + V2 or C2 + V2, and a smaller less
acidic form consisting of C2 alone (Fig. 8), designated Vo. The V0
isoform would most closely resemble the structure of the
2(XI)
amino-terminal domain
(23) . Since C2 is also an acidic sequence
and is constitutively expressed, the differences of net charge
described among the isoforms are a matter of degree. While it is
simplistic to categorize sequences based solely on net charge, the
differences are striking enough to be noted.
1(XI) gene
(48) . Northern analysis of
poly(A)
RNA with a probe including the carboxyl
propeptide coding region showed that chick brain, skin, calvaria,
heart, and muscle express transcripts of the
1(XI) collagen gene.
Examination of
1(XI) mRNA isoforms in several different rat embryo
tissues by PCR showed a similar distribution with the exception of
heart. While it is difficult to assess abundance by PCR, use of a
standard transcript amplified in parallel provides some basis for
comparison. Several other tissues showed negligible levels of
1(XI) transcripts while yielding comparable levels of
amplification of transcripts of the housekeeping gene,
glyceraldehyde-3-phosphate dehydrogenase. The correlation of these PCR
results with the results from Northern analysis suggests that detection
of
1(XI) transcripts in these noncartilaginous tissues is not a
trivial result of the extreme sensitivity of PCR. These same tissues
also express low levels (relative to cartilage) of
1(II)
(49) and
2(XI) mRNA
(50) . Expression of
1(XI)
in these noncartilaginous tissues also shows a degree of specificity.
The acidic form, V1a + C2 + V2, is prominent in the type
XI-positive tissues, consistent with the splice form of the
amino-terminal domain initially cloned and sequenced in human placenta
and other sources
(22) . Rat placenta also expresses this form
primarily. Skin and calvaria may also express small amounts of V1b
+ C2 and V0. Northern analysis using total RNA, conditions
selective for more abundant transcripts, indicates that the
1(XI)
gene is transcribed more extensively only in cartilage and tendon
(58) . Surprisingly, splice forms with V1b but not V1a were
detected in tendon.
1(XI) synthesizes all of these forms or whether splice patterns
correlate with degree of differentiation or some other aspect of
chondrocyte biology. In this regard, it is interesting to note that the
two transformed chondrocytes examined, RCS and IRC, each express a
subset of the isoforms. IRC cells synthesize only the Vo and the acidic
forms, while RCS cells synthesize predominantly Vo and the basic form.
This could reflect some developmental or inherent property of
chondrocytes or it may just reflect an aberration resulting from
transformation. At the protein level, we have previously observed
multiple electrophoretic forms of pro-
1(XI) synthesized by
metabolically labeled chick sternal chondrocytes
(26) and IRC
cells
(11) . It is likely that these may represent different
splicing isoforms, although this has yet to be demonstrated.
1(XI) amino-terminal domain. In some cases the clusters are
separated by four or six amino acids in which case they fit the
putative hyaluronan binding motif, B(X7)B, of Yang et al. (51) , where B represents a basic amino acid and X denotes any non-acidic amino acid.
1(XI) chain,
essentially the same regions of the
2 (XI)
and
3(XI) ( i.e.
1(II)) undergo alternative splicing
events. The sequence of the variable region of
1(XI) is unrelated
to the analogous region of
1(V) while the remainder of the two
chains is very homologous. The sequence of this region of
1(V)
also shows significant species or tissue-dependent variation
(20, 21) , although no evidence of alternative exon
usage in this region has been reported.
1(XI) amino-terminal domain
(26) whose large size
would not likely be accommodated within the fibril; limitation of
cartilage fibril diameter when fibrils were reconstituted with the
matrix form of type XI
(53) ; demonstration by immunoelectron
microscopy of the PARP-like domain at the surface of thin but not thick
fibrils in cartilage growth plates
(54) ; and finally, failure
of the homozygous recessive chondrodystrophic mouse ( CHO) to
synthesize the
1(XI) chain
(55) , resulting in cartilage
which contains unusually large fibrils and fails to retain
proteogylcans effectively
(56, 57) .
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.