(Received for publication, November 26, 1996, and in revised form, February 21, 1997)
From the Department of Molecular Biology and Biochemistry, Okayama University Medical School, Okayama 700, Japan
Type XIX collagen is a poorly characterized
member of the fibril-associated collagens with an interrupted triple
helices (FACIT) class of collagen molecules. As a first step toward
elucidating its function, we have isolated full size cDNA clones
from the mouse 1(XIX) collagen gene (Col19a1) and
established its pattern of expression in the developing embryo and
adult organism. Col19a1 transcripts can be detected as
early as 11 days of gestation and in all embryonic tissues, except the
liver, of an 18-day postcoitum mouse. In contrast, only a few adult
tissues, brain, eye, and testis, seem to accumulate Col19a1
mRNA. Col19a1 transcripts are at least 10 times more
abundant in adult than fetal brain and significantly less in adult than
fetal muscle and skin. Consistent with the RNA data, polyclonal
antibodies for
1(XIX) collagen reacted with a 150-kDa protein in the
neutral salt extraction of adult mouse brain tissues. We therefore
propose that type XIX collagen plays a distinct role from the other
FACIT molecules, particularly in the assembly of embryonic matrices and
in the maintenance of specific adult tissues.
Collagenous networks play a critical role in the morphogenesis of the embryo and in the maintenance of tissue architecture of adult tissues. The 19 genetically distinct types of collagen molecules are expressed in developmental and tissue-specific manners (1-4). The collagen superfamily can be classified into several subgroups according to the structural features of its members, the supramolecular aggregates that they form, and the structure of the gene that the proteins are encoded. One of the most interesting and less characterized collagen subgroups is the one of the so-called FACIT1 (fibril-associated collagens with interrupted triple helices) which includes types IX, XII, XIV, XVI, and XIX collagen. Available evidence suggests that FACIT may play an important role in providing tissue-specific molecular links between collagen fibrils and other extracellular matrix aggregates (5, 6). With the exception of types IX and XII, virtually nothing is known about the expression pattern of the other FACITs. Without this information, no function has yet been postulated for types XIV, XVI, and XIX collagen.
Type XIX collagen was originally discovered through cDNA cloning of
RNA transcripts from the human rhabdomyosarcoma cell line RD (CCL 136)
(7). The predicted polypeptide was found to contain 1,142 amino acid
residues with a 23-residue signal peptide followed by the five
collagenous (COL) domains, interspersed and flanked with the six
non-collagenous (NC) domains (3, 4, 8). The coding region of the
1(XIX) mRNA is small compared with the length of the entire
transcript (10.4 kb), due to the presence of more than 5-kb long
3
-untranslated region. Additionally, an unusual number of splicing
events appeared to occur in the RD cell line. More interestingly, the
1(XIX) collagen gene (COL19A1) was located to human chromosome
6q12-q14, syntenic to the
1(IX) collagen (COL9A1) and
1(XII)
collagen genes (COL12A1) (7, 9). Type IX collagen is found in tissues
containing type II collagen, such as hyaline cartilage and the vitreous
body of the eye. On the other hand, type XII collagen is found in dense
connective tissues, such as tendons and ligaments, where type I is the
major collagenous component. Aside from the rhabdomyosarcoma cell line, there is currently no information about the tissue distribution of type
XIX collagen and, consequently, about its possible function.
The present study was designed to fill this gap of knowledge and to
generate suitable reagents to eventually establish type XIX collagen
function using the gene targeting approach in mouse embryonic stem
cells. Accordingly, we isolated overlapping cDNA clones coding for
the entire mouse 1(XIX) chain; we examined the pattern of mRNA
accumulation in the tissues of both the developing and adult mice; and
we identified the
1(XIX) protein in adult brain tissues.
Poly(A)+ RNA was isolated from 18 d.p.c.
mouse whole embryo tissue according to the standard protocol (10) and
was used as a template for cDNA synthesis. Double-stranded cDNA
was synthesized using random primers and then inserted into the gt10
vector using a commercial kit (Amersham Corp.). Three distinct mouse
embryonic cDNA libraries were screened under low stringency
conditions using probes specific for the human
1(XIX) collagen (4,
7). Hybridizations carried out at 55 °C overnight in a mixture
containing 5 × SSC (1 × SSC; 0.15 M NaCl, 0.015 M sodium citrate, pH 6.8), 1%
N-laurylsarcosine, 50 µg/ml salmon sperm DNA, and
32P-labeled probes after prehybridization which is the same
solution without probes for 1 h. Isolation and purification of the
positive clones were as described previously (7). Sequencing was
carried out according to the dideoxy chain termination methods using an ABI 373S automatic sequencer. After sequencing validation of positive clones, a 0.6-kb gap in the
1(XIX) coding sequence was resolved by
amplifying an aliquot of reverse-transcribed embryonic cDNA using
the polymerase chain technique (RT-PCR). Twenty microliters of reverse
transcription reaction mixture (50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol) containing 2 µg of total RNA from
18 d.p.c. mouse embryo, 0.25 mM dNTP, 2 units of
RNasin (Toyobo, Osaka), 400 ng of random hexamer, and 100 units of
Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.) was incubated at 37 °C for 1 h, heated to 70 °C for 10 min, and quick-chilled on ice, and diluted with 80 µl of water. To get appopriate RCR products, the nested PCR was performed. First PCR
reaction was performed using 2 µl of cDNA solution in 20 µl of
mixture containing 0.8 units of Tth DNA polymerase (Toyobo) using
primer 1 (forward, 5
-GGAACTTAAAGACACATGCC-3
, Fig. 2) and primer 2 (reverse, 5
-ATGTCTCCCAAGAGAGGAAG-3
, Fig. 2), under the following
condition: 94 °C for 1 min, 60 °C for 1 min, and 70 °C for 3 min for 35 cycles. Five microliters of the first PCR amplification
product was used for the second PCR reaction with primer 3 (forward,
5
-CCTCAATGGACAAGATGGTT-3
, Fig. 2) and primer 4 (reverse,
5
-TTCTACCAGGGATTCCTACAC-3
, Fig. 2) under same condition. PCR products
were subcloned in TA vectors (Invitrogen). The sequence was confirmed
with both strands of two clones.
Northern Blot and RT-PCR Analysis
For Northern blotting
analysis, approximately 3 or 5 µg of poly(A)+ RNA was
electrophoresed in 0.8% agarose gel under denaturing conditions,
blotted onto Hybond N nylon filter (Amersham Corp.), and to hybridized
species-specific probes (10). Sources of RNA included 18 d.p.c.
mouse whole embryo, 5-week-old mouse brain, human rhabdomyosarcoma cell
line (RD), human glioma cell line (U251MG), human infant skin
fibroblast culture cells, and human adult skin fibroblast culture
cells. The following mouse tissues and cultured cells were instead used
for RT-PCR amplification: 11, 12, and 14 d.p.c. mouse whole embryo;
different tissues from 18 d.p.c. mouse embryo and 5-week-old
mouse; the human rhabdomyosarcoma cell lines RD, A204 and KYM1; the
human glioma cell lines U251MG, U373MG, and A172; the human
neuroblastoma cell lines TGW and NB1; and skin fibroblasts from mouse
embryo and mouse adult and human infant and human adult. Single strand
cDNA was synthesized as mentioned above. PCR was performed with 35 cycles for 1(XIX) or 30 cycles for
actin using 0.8 units of Tth
DNA polymerase at 94 °C for 1 min, at 60 °C for 2 min, and at
70 °C for 3 min. The nucleotide sequences for the primers used in
these reactions are as follows:
1(XIX), 5
-AACTGCCAGCAGCAATGTTG-3
(forward) and 5
-CAATCTTCTGGATTACATCT-3
(reverse);
actin,
5
-AAGAGAGGTATCCTGACCCT-3
(forward) and 5
- TACATGGCTGGGGTGTTGAA-3
(reverse).
For competitive RT-PCR, 1(XIX) collagen and
actin cDNA
clones were modified using restriction enzymes to delete 51 bp in the
former and 61 bp in the latter. These modified clones were then used as
competitors in RT-PCR amplifications of various mouse samples. To
determine the optimal condition, a series of RT-PCR reactions
containing 2-fold serial dilutions of competitor (ranging from 1 pg to
1 fg/µl for
1(XIX), and from 50 pg to 1.6 pg/µl for
actin)
were first carried out (11). PCR was then performed described above
except that 5 more cycles were added to each reaction. Aliquots of each
amplification were electrophoresed in 2% or 2.5% agarose gels (Sigma)
containing 0.5 µg/ml ethidium bromide; gels were photographed with
Polaroid film (Polaroid type 667); photographs were quantitatively
scanned using the NIH image software.
A recombinant
1(XIX) NC1-COL1-NC2 peptide was made by expressing a cDNA
fragment subcloned into EcoRI and PstI sites of
pMALc2 (New England BioLabs Inc.) expression plasmid. Production of the MBP (maltose binding protein)-
1(XIX)COL fusion proteins was induced by the addition of isopropyl-
-D-thiogalactopyranoside
(final concentration of 0.3 mM) to the bacterial cultures
incubated at 37 °C for 2 h. The cells were lysed by lysozyme
treatment, and the fusion proteins were purified by affinity
chromatography on an amylase column as describe by the manufacturer
(New England Biolabs Inc.). After incubation of the fusion protein with
Xa-specific peptidase, the
1(XIX)COL recombinant protein was
separated on 12.5% SDS-PAGE gel and isolated in a gel slice after
Coomassie Brilliant Blue R (Merck, Darmstadt, Germany) staining (12). The gel slices containing approximately 500 µg of protein in total were washed with phosphate-buffered saline, cut into the small pieces,
emulsified with the equal volume of complete Freund's adjuvant
(Difco), and then injected subcutaneously into a rabbit. Booster
injections (200 µg) were given with incomplete Freund's adjuvant
(Difco) 3 weeks after the first injection. The titers of antisera were
measured with enzyme-linked immunosorbent assay (13).
All steps were carried out at 4 °C or on ice (14). Brains, dissected from 40 5-week-old mice, were homogenized in 500 ml of cold isotonic buffer (0.25 M sucrose, 10 mM HEPES, pH 7.0, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 mM N-ethylmaleimide), and then stirred for 60 min. The homogenate was centrifuged for 30 min at 15,000 rpm. The pellet was suspended in 500 ml of extraction buffer (1 M NaCl, 50 mM Tris-HCl, pH 7.5, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 mM N-ethylmaleimide) and incubated for 48 h with stirring. The suspension was centrifuged at 35,000 rpm for 30 min, and the solubilized proteins were precipitated with 4 M NaCl of final concentration. The precipitate was collected by centrifugation at 25,000 rpm for 30 min, and then the pellet was suspended in 0.5 M acetic acid. The insoluble material was removed by centrifugation at 35,000 rpm for 30 min. The supernatant was dialyzed against 5 mM acetic acid and lyophilized. The sample was resolved with 200 µl of 50 mM acetic acid and stored at 4 °C.
Western BlottingProteins were separated on 6.0% SDS-PAGE
in the presence or absence of 2-mercaptoethanol (12). An aliquot of the
sample was also digested with bacterial collagenase before loading onto the gel. The sample for digestion was first neutralized with 0.5 M NaOH and then incubated with bacterial collagenase
solution (250 units/ml Wakojunyaku Form III; 50 mM
Tris-HCl, pH 7.5, 0.2 M NaCl, 5 mM
CaCl2) containing 5.5 mM CaCl2 for
5 h at 37 °C. Samples were transferred onto polyvinylidene
difluoride membranes electrophoretically (15), and the gel was stained
with Coomassie Brilliant Blue R. The transferred sample was allowed to
react with anti-1(XIX)COL antibodies, followed by incubation with
the peroxidase-conjugated secondary antibodies against anti-rabbit IgG.
The signals were detected by enhanced chemiluminescence Western blotting detection reagents (Amersham Corp.).
To identify the
best source for 1(XIX) collagen, we initially performed RT-PCR
amplifications using RNA from a variety of embryonic and adult mouse
tissues and a couple of sets of primers derived from the human
sequence. The most convincing results of this initial survey were
obtained using embryonic tissues. Sequencing of these amplification
products showed that the clones were the mouse counterparts of the
human COL19A1 gene. Based on these data, we constructed and screened a
cDNA library from 18 d.p.c. mouse whole embryo. Five
overlapping clones were isolated and, upon sequencing, found to cover
all but 0.6 kb of the region coding for the mouse
1(XIX) chain; this
last gap was finally covered by RT-PCR amplification (Fig.
1).
The deduced amino acid sequence of the mouse 1(XIX) chain shows 82%
identity to the human counterpart with the highest level of sequence
conservation at the carboxyl termini than the amino termini. All of the
potentially important structural-functional features previously noted
in the human
1(XIX) chain are also conserved in the mouse
polypeptide (Fig. 2) The major difference between the
two mammalian polypeptides is the total number of 1,136 amino acids in
mouse versus 1,142 amino acids in human. The differences are
due to the deletion of three amino acids in each NC6 and NC5 domain and
the substitution of cysteinyl residues for serinyl residues in the
signal peptide of the mouse chain. Additional structural differences
include the NC5 and NC3 domains, which are shorter in the mouse
compared with the human chain, and the relative position of small
imperfection in the COL5 domain. These few differences notwithstanding,
the primary structure of the
1(XIX) collagen chains is remarkably
conserved between the two mammalian species.
To define the pattern of expression of Col19a1,2 we surveyed tissues from various embryonic stages and 5-week-old mouse tissues. The transcripts were readily detectable by RT-PCR analysis in the whole embryo at 11, 12, and 14 d.p.c. (data not shown). Positive tissues of 18 d.p.c. embryo include limbs, vertebrae, heart, brain, tail, kidneys, calvaria, lung, muscle, skin, and intestine (Table I). A very different pattern was observed in adult tissues where Col19a1 expression was only seen in the cerebrum, cerebellum, eyes, and testis, and perhaps in the aorta, lungs, and kidneys (Table I). Based on these data, we used the same approach to identify cultured cells that accumulate COL19A1 (and Col19a1) transcripts. This second survey included human rhabdomyosarcoma cell lines (RD, A204, and KYM1), human glioma cell lines (U251MG, U373MG, and A172), human neuroblastoma cell lines (TGW and NB1), human normal skin fibroblasts (infant and adult), and mouse normal skin fibroblasts (18 d.p.c. embryo and adult) (Table II). Among malignant cell lines, only RD (rhabdomyosarcoma) and U251MG (glioma) showed significant COL19A1 mRNA accumulation. Detectable amounts were also noted in human neonatal fibroblasts, mouse embryo fibroblasts, and to a much lesser extent, in adult fibroblasts.
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To examine whether Col19a1 gene expression may change during
development, we compared transcript levels in the muscle, skin, brain,
and liver of 18 d.p.c. embryos and 5-week-old mice using the
quantitative technique of competitive RT-PCR (Fig. 3).
Relative quantitation of amplification from the endogenous transcript
and the competitor plasmid revealed that 1 µl of cDNA from
muscle, skin, and brain of 18 d.p.c. embryo and 5-week-old mouse
contained 52.3, 7.1, 24.5, 0.7, 0.4, and 149.3 fg, respectively. When
normalized to the actin values, the relative amounts of
Col19a1 transcripts were significantly higher in embryonic
muscle and skin than in the adult counterparts, whereas transcript
levels in the brain were approximately 10 times more in adult animals
than embryos (Table III).
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To identify the protein in a tissue that expresses the 1(XIX)
mRNA, we utilized specific polyclonal antibodies (Fig.
4) in a Western blotting analysis of mouse adult brain
tissue. The antibodies were raised in rabbits against an
1(XIX)
recombinant peptide. As shown Fig. 5C, the
antibodies reacted with peptides of 150 and 145 kDa in size from a
fraction of neutral salt extraction of mouse adult brain. Consistent
with their identity, the 150- and 145-kDa bands were digested with
bacterial collagenase (Fig. 5C, lane 2). Under nonreducing
conditions, the reacting peptide migrated at about 400 kDa which
probably represents the trimeric form of type XIX collagen.
The above results suggest that type XIX collagen is an embryonic FACIT, since its expression is mostly confined to embryonic tissues and only a few adult organ systems. High Col19a1 expression in a couple of tumor lines indirectly supports the idea of a mostly embryonic gene product. We postulate that the uniqueness of this FACIT molecule extends to the kind of supermolecular aggregate it is associated with. We note that Col19a1 expression in the embryo does not follow the pattern of either of the major fibrillar collagens (types I or II) for it includes both cartilagenous and non-cartilagenous tissues. Even more intriguing is the expression of type XIX in the adult aorta, testis, and brain. The last organ, in particular, raises provoking new ideas. For example, type XIX collagen may be produced by glial cells to serve as a connecting bridge of the proteoglycan network or as an anchor for the surrounding cells. Irrespective of the alternatives, the evidence strongly supports the original prediction of Gordon and Olsen (16) for the existence of FACIT molecules that may provide highly specific properties to selected organ systems and/or at specific developmental stages.
Alternative Splicing of the COL19A1 TranscriptWe have
previously reported an unusual number of alternatively spliced COL19A1
products in the rhabdomyosarcoma cell line RD (4). Here we examined
whether or not alternatively spliced products appear to be tissue-
and/or stage-specific. Northern analysis of mRNA from 18 d.p.c. whole embryo yielded a single band with 5 (Fig.
6A, lane 3) and 3
(lane 4)
cDNA fragment as probes. Transcripts from mouse adult brain also
showed a single band with more strong signal (Fig. 6B, lane
3) than that from 18 d.p.c. mouse embryo (Fig. 6B, lane
2) when 5
probe was used. Likewise, mRNA from infant skin
fibroblasts showed only one hybridizing species (Fig. 6C, lane
3). In contrast, mRNA from glioma cell line (U251MG) yielded
multiple bands (Fig. 6C, lane 2).
Alternative splicing is a widely used means to generate protein
isoforms or even functionally distinct products from the same transcript. Alternative splicing has been reported to occur for several
different collagen types (17-24), although its functional significance
is understood only for a few of them. In the case of the 1(XIX)
chain, the results are apparently contradictory. Unlike two tumor
lines, multiple transcripts have not been seen in the present survey of
normal tissues and cultured cells. We therefore conclude that the
alternatively spliced COL19A1 transcripts seen in the tumor cells are
probably aberrant products of no physiological significance and without
counterparts in the normal organism. It is however unclear the reason
for and the mechanism behind this strange phenomenon in cancer
cells.
In summary, this study has provided new and important information about the possible role of type XIX collagen. We believe the expression data are consistent with the idea that type XIX collagen is somehow involved in the assembly of specialized structures of the developing embryo and/or the function of particular organ systems in the adult organism. Irrespective of the hypothesis, it is safe to predict that type XIX collagen will eventually prove to be unique among other FACIT members. The information and reagents produced by the present study provide the means to address this question using the powerful technique of gene targeting in the mouse.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB000636[GenBank].
We are indebted to Dr. F. Ramirez for the review and critical comments of the manuscript. We also thank M. Kohmoto and K. Frith for typing the manuscript.