From the
An analysis was performed of differential splicing of primary
transcripts in the noncollagenous variable region located in the amino
terminus of the pro-
In many tissues, such as cornea or vitreous humor, the diameter
of each collagen fibril is carefully regulated
(1) . However,
the mechanism by which this is controlled is still unknown. Numerous
studies have shown that most, if not all, collagen fibrils contain a
small proportion of one or more members of the type V/XI family of
collagens
(2, 3, 4, 5, 6) .
Originally, it was thought that type XI collagen is specific for
cartilaginous tissues
(3) . However, other studies have
demonstrated the presence of transcripts for both the pro-
Initially, a
function for the type V/XI collagen family was suggested from in
vitro experiments which demonstrated that type V collagen will
limit the diameter of collagen fibrils formed from type I collagen
(15, 16) . Similar experiments indicating an involvement
of type XI in the control of fibril diameter were reported for
cartilage collagens in which type II, type IX, and type XI collagen
were all found to be required for the assembly of fibrils of constant
diameter
(17) . Separate evidence for an involvement of the type
V/XI collagen family in influencing fibril diameter was obtained from
an analysis of the cho/cho mouse which exhibits an autosomal
recessive chondrodysplasia in which thick collagen fibrils are observed
by electron microscopy within the cartilage matrix
(18, 19) . Recent analyses show that the cho mutation is located in the mouse
A model was presented recently for the location of
type V on the surface of a fibril of type I collagen present in the
chicken embryonic cornea
(22) . It was proposed that the major
collagen triple helix of type V collagen lies buried within the fibril
(as was also proposed earlier
(4, 23) ). However, the
amino terminus of each molecule, including a second short collagen
triple helix, was proposed to project away from the surface of the
fibril. Thus, the noncollagenous amino terminus of each molecule could
potentially interact with other connective tissue macromolecules such
as proteoglycans, and therefore be involved in maintaining the overall
organization of the matrix. The nucleotide-derived amino acid sequence
of the amino terminus of the human and chicken
Chicken gizzard, heart, kidney, skeletal muscle,
brain, skin, lung, liver, and tendon were all dissected from 17-day
embryos and used to isolate RNA without dissociation into individual
cells.
Total RNA was isolated from 17-day chick
chondrocytes and other chick tissues using RNAzol
Two
overlapping genomic clones were obtained (
For the
isolation of chicken genomic
For the isolation of a human genomic
To obtain biotinylated probes for Northern
hybridization, unidirectional PCR was performed using the antisense
primer VS5 for IIA and VC2 for IIB and a mixture of dNTPS in which dUTP
was biotinylated (Millipore, Bedford, MA). Each template (0.2 mg) was
primed with 5
Previously, we reported
(28) the molecular cloning of
human PARP from hyaline cartilage and the subsequent biosynthesis by
RT-PCR of a 1.86-kilobase pair product which bridged from PARP to the
published nucleotide sequence of the human pro-
These results suggested that differential splicing
potentially may occur within the variable region of the pro-
The remarkable feature of these
results is the difference in predicted amino acid composition of exon
IIB when compared to exons IIA, III, and IV. Exon IIB is highly basic
with a calculated pI of 10.6, whereas IIA, III, and IV are all highly
acidic with calculated pI values of 3.2, 3.8, and 3.7, respectively. To
understand the potential significance of these splicing events,
experiments were first performed in which RT-PCR was carried out on 20
µg of total RNA isolated from 17-day chicken embryo tendon, skin,
skeletal muscle, lung, liver, kidney, heart, gizzard, cartilage, and
brain in which the primers PL1 and PL2 were used which span the
variable region (see Fig. 6for location of primers). The results
of this experiment are shown in Fig. 9. Some variability can be noted
in the products of the RT-PCR reaction in different tissues, but in all
tissues transcripts of the pro-
Several interesting examples of differential splicing exist
in the connective tissue field
(40) . For example, for type II
collagen an additional exon is present in the aminopropeptide which is
present in precursor cells for cartilage and in noncartilage cells
during development, but is apparently absent in mature cartilage
(41, 42, 43, 44, 45) . The
present analyses show that a region of the pro-
Analyses of the biosynthesis of
the pro-
The potential presence of a basic sequence (exon IIB of
the
The nucleotide
sequences reported in this paper have been submitted to the
GenBank
We thank Pauline M. Mayne, Brian Wood, and Jeanne
Holloway for their assistance in the preparation of this manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
1(XI) and pro-
2(XI) collagen chains. The
results for the pro-
2(XI) chain showed that human cartilage or
fibroblasts in culture contain transcripts in which a single highly
acidic exon encoding for 21 amino acids is present or absent. For the
chicken pro-
1(XI) chain a more complex pattern of alternative
splicing was detected with six possible variants. Of special interest
was the alternative use of two exons (called IIA and IIB) in which IIA
encodes for 39 amino acids and is highly acidic (estimated pI =
3.2), whereas IIB encodes for 49 amino acids and is highly basic
(estimated pI = 10.6). A similar alternative use of exon IIA or
exon IIB was also observed for human chondrocytes. Northern blotting
with probes specific for IIA or IIB showed that both exons are present
in transcripts from cartilage but exon IIB is preferentially utilized
in transcripts from tendon. Present results suggest that both the
pro-
1(XI) and pro-
2(XI) chains of type XI collagen undergo
limited processing in vivo and that the noncollagenous
variable region is initially retained on the surface of the fibrils.
Differential splicing in the variable region may potentially influence
the interaction of collagen fibrils with other molecules of the
extracellular matrix and more specifically with sulfated
glycosaminoglycan chains or with hyaluronan. Such interactions may play
a key role in establishing both the organization of the collagen
fibrils within the extracellular matrix and in limiting the diameter of
collagen fibrils.
1(XI)
chain
(7, 8, 9) and pro-
2(XI) chain
(10, 11) in a variety of cells in culture and in
different tissues during development. In addition, adult bovine bone
was shown to contain a significant amount of the
1(XI) chain in
addition to the
1(V) chain
(12) . More recent results show
that a molecule of chain organization
1(XI)
2(V)
is the major collagen synthesized by the rhabdomyosarcoma cell line
A204
(13) . In addition, the isolation of
1(XI) and
2(V) chains, but not
2(XI) chains, from bovine vitreous
(6) , further supports the growing evidence that there is a
single type V/XI family of collagens with a variety of different chain
organizations
(14) . It appears likely that all of the collagen
molecules in this family will share a common function, and that
individual members of the family may be potentially involved in the
generation of tissue-specific collagen fibrils.
1(XI) collagen gene and that
these animals have only a limited ability to synthesize the
pro-
1(XI) chain
(20) . In addition, two Dutch families were
recently analyzed each with an inherited chondrodysplasia resembling
Stickler syndrome but without eye involvement. In both families
mutations in the
2(XI) chain were located showing the importance
of type XI collagen in the cartilagenous structures of man
(21) .
1(V) chain
(22, 24, 25) , the human
2(V) chain
(26) , the human and bovine
1(XI) chain
(8, 27) , and the human
2(XI) chain
(28) are all available. Analysis of these sequences shows that
the pro-
2(V) chain is closely related to the chains of type I, II,
and III collagen, whereas the pro-
1(V), pro-
1(XI), and
pro-
2(XI) chains are closely related to each other and share
several unique features
(14, 25, 28) . For the
pro-
2(XI) chain, a disulfide-bonded peptide called
proline/arginine-rich protein or PARP
(
)
is
released from the amino terminus during extracellular processing and is
subsequently retained in the cartilage matrix from which it can be
isolated in significant amounts
(29) . The pro-
1(V) and
pro-
1(XI) chains also have an amino-terminal domain which is
homologous in amino acid sequence and location of cysteine residues to
PARP (30-32; see Fig. 1). However, it is not known if there
is a specific cleavage and release of these PARP-like domains into the
matrix. For the pro-
1(V), pro-
1(XI), and pro-
2(XI)
chains there is a highly acidic amino acid sequence which follows the
basic PARP-like domains and is variable in length. This is the only
region of the molecule for which little or no homology can be
recognized in amino acid sequence when the
1(V),
1(XI), and
2(XI) chains are compared
(24, 25, 28) . We
previously called this sequence the variable region
(28) . We
have now performed a detailed analysis of transcripts containing the
variable region using RT-PCR and, with additional analyses of the
genomic organization, report alternative mRNA processing of the
pro-
1(XI) and pro-
2(XI) chains.
Figure 1:
Model for the
domain structure of the pro-2(XI), pro-
1(XI), pro-
1(V),
and pro-
2(V) chains. The order of the domains is as described
previously (28) with COL1 forming the major collagen triple helix and
COL2 forming the minor, amino-terminal triple helix. Note that there
are three noncollagenous domains (NC1
NC3) and that the NC3
domain is further subdivided into the signal peptide ( SP), the
PARP region, the variable region ( VR), and a short constant
region ( CR), the latter being highly homologous in primary
amino acid sequence for all three chains. Also note that the
pro-
2(V) chain does not contain a PARP-like domain, a variable
region, or a constant region.
Sources of Cells and Tissues
Human chondrocytes
were isolated from sternal cartilage obtained from an 8-year-old white
female during pectus excavatum surgery as described previously
(28) . Human skin fibroblasts were initially obtained from a
skin biopsy of a 34-year-old white female and were at the fifth
passage. The cells were grown in McCoy's modified 5a medium plus
10% fetal calf serum and 1% antibiotic antimycotic. Human gingival
fibroblasts were obtained as outgrowths from a normal adult male and
grown in minimal essential medium ( medium) plus 10% fetal calf
serum, 1%
L-glutamine, and 4% antibiotic/antimycotic mixture.
The cells were passaged with trypsin four to six times before being
utilized for RNA isolation. Human histiocytic lymphoma cells were
obtained from American Type Culture Collection (ATCC CRL 1593 U-937)
and were grown in Dulbecco's minimal essential medium containing
10% fetal calf serum and 1% antibiotic/antimycotic. Chicken
chondrocytes were obtained from the sterna of 17-day embryos and were
isolated as described previously
(33) . All culture medium and
supplements were from Life Technologies, Inc./BRL Life Technologies,
Gaithersburg, MD.
Isolation of RNA and First Strand cDNA
Synthesis
Total RNA from cells or tissues was obtained by the
single step acid-guanidine method
(34) and poly(A)-rich RNA
selected by oligo(dT)-cellulose chromatography
(35) . Synthesis
of first strand cDNA was based on a kit (Reverse Transcription System;
Promega Corp., Madison, WI) using either 5 µg of poly(A)-rich RNA
or 10-30 µg of total RNA in 50 µl of buffer. A mixture of
random primers (10 µl) was added and the mixture incubated at 65
°C (10 min) and rapidly cooled on ice (1 min). To the RNA/primer
mixture, 5 µl of 100 m
M methylmercuric hydroxide
(Alfa/Johnson Matthey, Ward Hill, MA) was added with incubation for 10
min at room temperature followed by 5 µl of 700 m
M
-mercaptoethanol and incubation for 5 min at room temperature. For
first strand cDNA synthesis, avian myeloblastosis virus reverse
transcriptase (Promega) was employed with incubation at 45 °C for
45 min followed by 55 °C for 15 min. Precipitation was performed
with absolute ethanol in the presence of 3
M Na-acetate, pH
5.2 (10:1 ratio), at -20 °C and the pellet washed with
ice-cold ethanol (80%).
B
according to the manufacturer's instructions (Biotecx
Laboratories, Inc., Houston, TX). Poly(A)-rich RNA was isolated with
streptavidin-paramagnetic particles using a kit (PolyATract mRNA
Isolation System; Promega). First strand cDNA was prepared from chicken
tissues as described above for human cells.
Polymerase Chain Reaction (PCR)
In order to
analyze the variable region of the human pro-2(XI) chain
(28) , PCR was performed with primers DE1
(5`-
CAGAAGGAGCTGGAATGCGAG
-3`) and PO2
(5`
CGGAATGGGAGCATGAGAGA3`
) using GeneAmp
PCR core reagents (Perkin Elmer, Norwalk, CT), and Taq I polymerase
(Promega). The cycle parameters were 2 min denaturation (94 °C),
1.5 min annealing (58 °C), and 3 min polymerization (72 °C) for
33 cycles with 5 min final elongation (75 °C) using a Programmable
Thermal Controller (MJ Research Inc., Watertown, MA). For the initial
PCR to analyze the variable region of the chicken pro-
2(XI),
pro-
1(XI), and pro-
1(V) chains, degenerate oligonucleotides
XB1 5`-GA(CT)GA(AG)GA(AG)GTITT(CT)GA(AG)GG-3` and XB2
5`-GG(TC)TCICC(TC)TT(TC)T(GC)(TGCA)CC(TC)TT-3` were designed from
shared amino acid sequences with substitution by inosine residues at
some sites in the 5` ends of the primers
(33) . Subsequently,
the variable region of the chicken pro-
1(XI) chain was analyzed by
RT-PCR with primers PL1 and PL2 located in exons I and V (see
Fig. 6
for location and sequence of primers).
Figure 6:
Nucleic acid sequence of the variable
region of the chicken pro-1(XI) chain with a comparison to the
human pro-
1(XI) chain. Location of several primers (sense and
antisense) is shown by underlining the nucleotides.
Intron/exon boundaries are shown by
. The nucleic acid sequence
and derived peptide sequence for human I, IIA, III, IV, and V was taken
from Ref. 27. The nucleic acid sequence of the previously undescribed
human exon IIB is also included in the figure. Primers XB1 and XB2,
location of the degenerate primers used initially to amplify the
differentially spliced variants. Primers PL1 and PL2 were used to
investigate the differentially spliced variants specifically of the
chicken pro-
1(XI) chain. Primers VS4 and VS5 were used to amplify
specifically exon IIA and primers VC1 and VC2 were used to amplify exon
IIB.
Screening of Genomic Libraries
For isolation of a
human genomic 2(XI) clone, a human leukocyte genomic library
constructed in EMBL 3A vector (Clontech Laboratories, Inc., Palo Alto,
CA) was used. For screening, a 162-bp fragment was generated by PCR on
the NZ2 clone derived from human chondrocyte RNA
(28) using
primers LA5 =
5`-
TCAAGACTTCACAGGCCACA
-3` and LA6 =
5`-
TTCCAACACTGCAGGCTCTC
-3`. The 162-bp
fragment was extracted from agarose gel using a QIAEX agarose gel
extraction kit (Qiagen Inc., Chatsford, CA). For screening, the 162-bp
fragment was labeled using a random primed DNA labeling kit (Boehringer
Mannheim). The specific activity of labeling was ≅1
10
cpm/µg DNA. The labeling reaction was performed with
3000 mCi/mmol [
-
P]dCTP (DuPont NEN).
HGCol18 and
HGCol9). For sequencing, the whole insert was first removed by
digestion with HindIII or EcoRI/ SacI, and
then subcloned from the
HGCol18 clone into pBluescript II SK+
plasmid. The constructed plasmids were called p18H15 and p18RS12 and
used for DNA sequencing and analysis of genomic structure.
1(XI) clones a chicken genomic
library in
FIX II vector (Stratagene) was screened with 558- and
528-bp fragments generated by PCR on plasmid DNAs containing exon IIB
(called pXB28) or exon IIA (called pXB45) using primers PL1 and PL2
(see Fig. 6). Fragments were labeled using a random primed DNA
labeling kit (Boehringer Mannheim). Two overlapping genomic clones
(
CHCol5 and
CHCol13) were obtained. The whole inserts from
both
clones were subcloned into pBluescript II using the
NotI restriction site.
1(XI) clone containing exon IIB, a PCR procedure was conducted
using total genomic DNA from human leukocytes (Clontech Laboratories,
Inc., Palo Alto, CA) with primers RX6
(5`-
TGCACCAGAGGATATAATCG
-3`) located in
exon IIA and HT4 located in exon III
(5`-
AACATGCCTAGGAGCTTCTG
-3`) as described
in Ref. 27. A PCR fragment (955 bp) was subcloned into PCR
II vector using the TA cloning system (Invitrogen Corp., San
Diego, CA) for DNA sequencing. To isolate cDNA for human IIB exon,
RT-PCR was performed on human chondrocyte RNA with primers LA1
(5`-
AGGAACCTCAGATAGATAG
-3`) located in exon
I, and VC5 (5`-
AAATCATCAACGATGTTTGC
-3`)
located in exon III
(27) . Two PCR products were obtained which
after subcloning into the PCR
II vector and DNA sequencing
were shown to contain exon IIA or exon IIB.
Generation of Specific Probes for Exon IIA or Exon
IIB
PCR was performed on plasmid DNAs pXB28 and pXB45 which
contain the exon sequences I-IIA-III-IV-V and I-IIB-III-IV-V with sets
of primers VS4 (5`-TACACACCAGAAGATTTTAT-3`) and VS5
(5`-CTCTGTTTGTGCTATTGTCT-3`) designed to amplify specifically exon IIA
and VC1 (5`-AAAAAGAAAGCCATGGTCAA-3`) and VC2
(5`-CCCTAGTTTGTCTCTTGTTG-3`) designed to amplify specifically exon IIB
(see Fig. 6for location of primers). The PCR products were
subcloned into PCRII vector and the correct sequences
were confirmed.
10
M of antisense
primer in the presence of 0.5 units of Taq I polymerase (Promega) in
reaction buffer with 2.5 m
M MgCl
with cycle
parameters of denaturation (95 °C, 2 min), annealing (54 °C, 1
min), polymerization (72 °C, 3 min) for 40 cycles with a final
elongation (75 °C, 5 min). Biotinylated single stranded probes were
precipitated with absolute ethanol in the presence of 3
M
NaAc, and boiled prior to hybridization.
Northern Hybridization
Total RNA (20 µg/lane)
was isolated from 17-day embryonic chicken brain, kidney, gizzard,
heart, skeletal muscle, liver, lung, skin, tendon and isolated
chondrocytes before being run on a 1% agarose-formamide gel. Transfer
was to maximum strength NYTRAN membrane (Schleicher and Schuell, Keene,
NH) prehybridized in 5 SSC (1
SCC = 0.15
M NaCl, 0.015
M sodium citrate, pH 7.0), 5
Denhardt's solution, 0.8% SDS with 100 µg/ml herring sperm
DNA (Promega) at 65 °C for 2 h. After washing, membranes were
hybridized with single stranded biotinylated exon IIA or exon IIB
probes in the same solution as above at 65 °C. Filters were washed
twice for 30 min with 2
SSC, 0.1 SDS at 65 °C and once for
15 min in 0.2
SSC, 0.1% SDS at 65 °C. Biotinylated signals
were detected with a PolarPlex Chemiluminescent kit (Millipore).
DNA Sequencing
All sequencing was performed on
both strands with Sequenase version 2.0 DNA sequencing kit (U.S.
Biochemical Corp.). All plasmid templates for sequencing were isolated
using the Wizardminipreps DNA purification system
(Promega).
2(XI) chain
(36) . The PCR product was cloned and the complete nucleotide
sequence determined. Analysis of this sequence showed that it included
a variable region which was found to be much shorter than the variable
region of the pro-
1(XI) and pro-
1(V) chains (Fig. 1). During
these experiments, it was found on nucleotide sequencing of individual
DNA molecules cloned from the RT-PCR reaction that a short stretch of
63 nucleotides was missing in some clones. The nucleotide-derived amino
acid sequence of this stretch was found to be highly acidic
(DPTPGEEEEILESSLLPPLEE) and was located in the variable region of the
human pro-
2(XI) chain (nucleotides 646-708; Ref. 28). This
initial observation suggested that differential splicing of the primary
transcript may occur in the variable region. Accordingly, RT-PCR
reactions were performed with 20-21-mer oligonucleotides which
directly span the variable region and using RNA preparations obtained
from a variety of human cells in culture. Fig. 2 compares the products
of RT-PCR reactions performed for human chondrocytes ( lane 1)
and for human skin fibroblasts ( lane 2). In both cases, two
major products were observed which were cloned into the PCR
II vector using the TA cloning protocol and shown to differ by
the expected 63 nucleotides. The same two products were also obtained
in varying proportions for human gingival fibroblasts in culture and
for a line of human histiocytic lymphoma cells. Small amounts of other
products were sometimes observed by RT-PCR (for example, a band of
approximately 7-800 nucleotides for human skin fibroblasts;
Fig. 2
, lane 2) but were not investigated further. It is
possible that additional alternatively spliced forms of the
pro-
2(XI) chain may also be synthesized during the RT-PCR reaction
since two additional exons have recently been identified in transcripts
from several mouse tissues but not from mouse cartilage
(37, 38) .
Figure 2:
RT-PCR reaction performed with primers
that span the variable region of the human pro-2(XI) chain. The
sense primer (DE1) was located in the PARP domain and the antisense
primer (PO2) was located largely in the NC2 domain as described
previously (28). Lane 1, human chondrocytes from sternal
cartilage. Lane 2, human skin fibroblasts. The location of
standard oligonucleotides of different sizes is
shown.
Genomic clones coding for the variable
region of the pro-2(XI) chain were obtained by screening a human
leukocyte library (EMBL 3A) with a 162-bp fragment prepared by PCR
using nested primers (LA5 and LA6) on the NZ2 plasmid as a template so
that the 63-bp sequence was included. Two overlapping
genomic
clones were obtained (called
HGCol18 and
HGCol9) and the
whole inserts from each clone were subcloned into plasmid pBluescript
II SK+ vector for DNA sequencing. The genomic organization was
determined by DNA sequencing reactions performed with primers
synthesized from the known cDNA sequence and by PCR reactions using
primers which spanned one or more exons. Fig. 3 shows that the 63-bp
sequence arises from a single exon which is located between two large
introns. In addition, the location of five additional exons was
determined for the human pro-
2(XI) chain (Fig. 3) and a short
stretch of the nucleotide sequence at each intron/exon boundary is
shown in Fig. 4. All introns showed canonical sequences for intron/exon
junctions.
1(V)
and pro-
1(XI) collagen chains. Examination of the published
sequences for the pro-
1(V), pro-
1(XI), and pro-
2(XI)
chains showed the presence of two short stretches of amino acids which
span the variable region and are highly conserved for all three chains.
One sequence DEEVFEG is located in the ``PARP-like'' domain
of chicken pro-
1(V) (amino acid positions 210-216; Ref. 22),
human pro-
1(V) (amino acid positions 213-219; Ref. 25),
human pro-
1(XI) (amino acid positions 212-218; Ref. 27), and
human pro-
2(XI) (nucleotide positions 436-456; Ref. 28). A
second sequence in the constant region of NC3 (Fig. 1) contains a
short sequence, KGQKGEP, for chicken pro-
1(V) (positions
448-454; Ref. 22), human pro-
1(V) (positions 454-458;
Ref. 25), and human pro-
1(XI) (positions 425-431; Ref. 27)
with one conservative change to KGEKGEP for human pro-
2(XI)
(28) . This suggested that it may be possible to amplify the
variable region of these three chains of the type V/XI family by a
single RT-PCR reaction using degenerate oligonucleotides designed from
these amino acid sequences. Fig. 5 A shows that numerous bands
were obtained when an RT-PCR reaction was performed on 17-day embryonic
chicken cartilage RNA using a sense primer
5`-GA(CT)GA(AG)GA(AG)GTITT(CT)GA(AG)GG-3` and an antisense primer
5`-GG(TC)TCICC(TC)TT(TC)T(GC)(TGCA)CC(TC)TT-3` in which some positions
were substituted with inosine residues
(33, 39) . Each
band was cloned into the PCR
II vector and the complete
nucleotide sequence determined for several of these clones. The results
showed that there are at least six different products from the RT-PCR
reaction for the chicken pro-
1(XI) chain (Fig. 5 B),
suggesting a complex pattern of alternative splicing. However, only one
PCR product was obtained for the pro-
1(V) chain for which the
sequence agreed with the previously published nucleotide sequence
(22) . For reasons that are unclear, the pro-
2(XI) chain
has not so far been detected in the products of the RT-PCR reaction.
This may be a consequence of designing the degenerate primers by
including the sequence of the human pro-
2(XI) chain rather than
the chicken pro-
2(XI) chain for which the sequence is not
available. In addition, several products were obtained during the PCR
reactions that were found on nucleotide sequencing to arise from
priming at additional locations ( e.g. the band of low mobility
in Fig. 5 A) and were not investigated further.
Figure 5:
A, agarose gel electrophoresis showing the
separation of the products of an RT-PCR reaction performed with RNA
from two different preparations of chick chondrocytes using degenerate
nucleotides which span the variable region of the pro-1(V),
pro-
1(XI), and pro-
2(XI) chains. Each band is designated by
its molecular weight and was identified after cloning into the
PCR
II vector and nucleic acid sequencing (band 735
= pro-
1(V); all other bands were found to be derived from
the pro-
1(XI) chain; 683 = I-IIB-III-IV-V; 653 =
I-IIA-III-IV-V; 536 = I-III-IV-V, 437 = I-IIB-III-V; 407
= I-IIA-III-V; 290 = I-III-V. B, diagram showing
the organization of six possible variants obtained for the chicken
pro-
1(XI) chain after RT-PCR performed across the variable region.
Note the presence of either IIA or IIB but not both, and the presence
or absence of IV.
The
six different products of pro-1(XI) from the RT-PCR reaction
suggested that a complex pattern of splicing occurs in which there is
alternative usage of exon IIA or exon IIB and that exon IV may be
present or absent (see below for the genomic structure). A summary of
the chicken pro-
1(XI) and new human pro-
1(XI) sequences is
shown in Fig. 6 together with a comparison to the previously published
human pro-
1(XI) sequence
(27) . To examine the intron/exon
organization directly a chicken genomic library in the
FIX II
vector (Stratagene) was screened with fragments generated by a PCR
reaction that was performed on two different plasmids with primers PL1
and PL2 located in exons I and V (see Fig. 6) so that both IIA
and IIB were included in the probe. Two overlapping genomic clones were
obtained and the whole insert of each clone was subcloned from the
vector into pBluescript II SK+ for sequencing. The location
of the exons was determined from PCR reactions with primers designed
for specific exons and is shown in Fig. 7 A. The results showed
that the different products observed by RT-PCR arose from alternative
splicing of different exons such that either exon IIA or IIB is
utilized and exon IV is either present or absent. The sequence of I,
IIA, III, IV, and V corresponds to the previously published human cDNA
sequence for pro-
1(XI)
(27) . It was therefore of interest
to determine if exon IIB is also present in human genomic DNA. Total
human genomic DNA was used as a template for PCR with primers of known
sequence located in IIA and III. A PCR product of 955 bp was obtained
which was cloned and the presence of exon IIB was demonstrated by DNA
sequencing (Fig. 7 B). In addition, it was possible to
demonstrate the alternative splicing of exons IIA and IIB by RT-PCR on
human chondrocyte RNA with primers located in exon I and exon III so
that products were obtained that upon DNA sequencing were found to
contain exon IIA or IIB but not both. In Fig. 8 the nucleotide
sequences of the intron/exon boundaries of exons IIA, IIB, III, and IV
for chicken genomic DNA and exon IIB for human genomic DNA are shown.
In addition, the nucleotide and predicted protein sequences of human
exon IIB are shown in Fig. 6.
1(XI) chain could be detected.
However, these experiments involving RT-PCR do not take into
consideration differences in level of the initial mRNAs which may be
very low in some tissues. Accordingly, Northern analyses were performed
to assess the relative amount of expression specifically of exon IIA or
IIB with specific biotinylated probes prepared for these exons (Fig.
10). In each lane 20 µg of total RNA was probed. Both exon IIA and
exon IIB were easily detected in chicken chondrocytes with two bands of
6.3 and 6.6 kilobases as described previously
(8, 9, 27) . In most other tissues, in the
conditions of hybridization used, we were unable to detect a signal
although our previous RT-PCR analyses demonstrated the presence of low
levels of transcripts for the pro-
1(XI) chain (Fig. 9).
Interestingly, transcripts containing exon II were readily detected in
tendon suggesting that there is a high level of transcripts of the
pro-
1(XI) chain in this tissue. These transcripts were largely of
exon IIB.
Figure 9:
Agarose gel electrophoresis after RT-PCR
performed with RNA (20 µg) from a variety of tissues of 17-day
chick embryo using primers PL1 and PL2 (see Fig. 6 for location of
primers). Note that all tissues gave a similar profile of products.
Lane 1, embryonic chondrocytes; 2, gizzard;
3, heart; 4, kidney; 5, skeletal muscle;
6, tendon.
1(XI) and
pro-
2(XI) chains undergoes complex alternative splicing and
similar results to ours have recently been reported for the mouse
(46) and rat
(47) pro-
1(XI) chain and the mouse
(37, 38) pro-
2(XI) chain. It appears that either
acidic stretches of sequence are present or absent or, in the case of
the pro-
1(XI) chain, an acidic stretch is replaced by a very basic
stretch of amino acids. The physiological significance of these events
is at present unknown but these splicing events are likely to be
important since they are conserved between chicken and rat
(47) . We have evidence that in the embryonic tendon the basic
exon IIB is preferentially utilized (Fig. 10). In addition, this
same basic exon is preferentially utilized in the mRNA from the
transplantable rat chondrosarcoma
(47) . This would suggest that
the preferential expression of this exon may be important in
establishing the organization of the extracellular matrix in some
tissues.
Figure 10:
Northern blot prepared with probes
specific for exon IIA ( Panel A) or exon IIB ( Panel B)
with RNA preparations from a variety of tissues of 17-day chick
embryos. Lane 1, tendon; 2, skin; 3,
skeletal muscle; 4, lung; 5, liver; 6,
kidney; 7, heart; 8, gizzard; 9,
chondrocytes (preparation 1); 10, chondrocytes (preparation
2); 11, brain. Note that both IIA and IIB can be detected in
chondrocyte preparations but only IIB can be detected in tendon. In the
conditions of Northern hybridization, transcripts for IIA or IIB could
not be detected in other tissues.
Previously, it was reported that some of the pro-1(V)
chains in chicken tendon, but not other tissues, are different and were
designated pro-
1`(V)
(48) . It now appears from the results
described in Fig. 10that the pro-
1`(V) chain is likely to
be the pro-
1(XI) chain, and this would agree with other
observations that the pro-
1(XI) chain has a widespread
distribution in tissues
(9) . Although the presence of
transcripts for the pro-
1(XI) chain have not been described
previously in tendon, recent results show considerable expression of
transcripts for the
1(II) chain in the compressed region of the
adult bovine tendon
(49) .
1(XI) and pro-
2(XI) chains
(50, 51) ,
as well as the pro-
1(V) chain
(52) , all indicate that
extensive lengths of each amino-terminal peptide are retained even when
these chains are incorporated into fibrils. It would appear that the
short collagenous domain called COL2 (Fig. 1) is always retained
and in a recent model for type V collagen this domain is proposed to
project out from the surface of the fibril
(22) . This appears
to occur despite the presence of a potential aminopropeptide cleavage
site in the NC2 domain between COL2 and COL1 for the pro-
1(XI) and
pro-
1(V) chains
(24, 25, 27) . Thus, at
least part of the noncollagenous sequences may be located on the
surface of the fibrils. For the pro-
2(XI) chain there must be
cleavage prior to the variable region to give rise to the disulfide
bonded fragment called PARP
(29) . If this is the only cleavage
site for the pro-
2(XI) chain then the variable region would be
retained on the surface of the fibrils. It is not known if similar
PARP-like fragments are formed from the pro-
1(XI) and
pro-
1(V) chains, although these sequences are homologous including
the location of the cysteine bridges
(30, 31) . However,
a peptide antibody prepared to an upstream sequence of the
pro-
1(XI) chain between the PARP-like domain and the variably
spliced region was found by electron microscopy to recognize the thin
fibrils, but not the thicker fibrils, of the epiphyseal cartilage of
fetal calf and newborn human
(53) . Thus, the variable region of
pro-
1(XI) and pro-
2(XI) may be located on the surface of
fibrils. In contrast, the NH
-terminal sequence was recently
determined for the tissue form of the pro-
1(V) chain
(54) .
It was found to be located downstream of comparable regions where
differential splicing occurs in the variable region of the
pro-
1(XI) and pro-
2(XI) chains. This agrees with another
study which shows using peptide-derived polyclonal antibodies that
there is likely to be extensive processing of the tissue form of the
pro-
1(V) chain but not of the pro-
2(V) chain
(48) .
Thus, the pro-
1(V) chain appears to be more extensively processed
than the pro-
1(XI) and pro-
2(XI) chains so that the variable
region is largely missing from the fully processed form of the
molecule.
1(XI) chain) on the surface of the collagen fibrils of
cartilage and other tissues may be important for the interaction of
collagen fibrils with the glycosaminoglycan chains of proteoglycans or
with hyaluronan. We speculate that such interactions may be of
considerable importance in establishing the three-dimensional
organization of the matrix of cartilage and the vitreous humor.
However, it is also possible that the presence of chains of the type
V/XI family of collagens close to the fibril surface may play a role in
determining the diameter of each fibril by preventing the addition of
further molecules to its surface.
/EMBL Data Bank with accession numbers L38955 for
chicken pro-
1(XI) and L38956 for human pro-
1(XI), exon IIB.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.