Type XXIII Collagen, a New Transmembrane Collagen Identified in Metastatic Tumor Cells*
Jacqueline Banyard,
Lere Bao
and
Bruce R. Zetter
From the
Program in Vascular Biology and Department of Surgery, Children's
Hospital/Harvard Medical School, Boston, Massachusetts 02115
Received for publication, October 17, 2002
, and in revised form, March 14, 2003.
 |
ABSTRACT
|
---|
We have identified a transmembrane collagen, collagen XXIII, in rat
prostate carcinoma cells. Differential display of mRNA expression in prostate
carcinoma sublines with varying metastatic potential revealed overexpression
of this transcript in the metastatic AT6.1 subline. cDNA cloning identified a
2733-bp transcript from AT6.1 RNA, encoding a protein of 532 amino acids,
together with a 3067-bp human homologue, resulting in a 540-amino acid
protein. Collagen XXIII is predicted to be a type II membrane protein
consisting of an amino-terminal cytoplasmic domain, a transmembrane region,
and three collagenous domains flanked by short noncollagenous domains.
Collagen XXIII is a new member of the transmembrane collagen family, showing
structural homology with the transmembrane collagens XIII and XXV. We present
evidence that collagen XXIII is expressed as a
75-kDa protein at the cell
surface and that it can be cleaved by furin protease activity. Cleavage
results in a
60-kDa soluble protein that forms a multimeric complex and
exhibits a low affinity interaction with heparin.
 |
INTRODUCTION
|
---|
The extracellular environment consists of a complex mix of matrix
macromolecules together with sequestered growth factors. Each of these
components can interact with cells to influence their function and behavior.
Proteolytic cleavage of these molecules can release soluble growth factors and
matrix fragments that also possess biological activity. The collagen
superfamily represents the most abundant group of extracellular matrix
macromolecules, and it is becoming clear that this family encompasses a
structurally and functionally diverse group of proteins
(1). Collagens can be divided
into two major groups: fibrillar and nonfibrillar, with a key characteristic
of both being the presence of a repeating Gly-Xaa-Yaa, where Xaa and Yaa are
frequently proline and hydroxyproline, respectively. A subfamily of the
nonfibrillar collagens is the transmembrane collagens. This group currently
consists of three members, collagens XIII and XVII and the recently identified
collagen XXV, each containing a single-pass hydrophobic transmembrane domain.
Both collagens XIII and XVII show widespread distribution in epithelia
(2,
3), whereas collagen XXV is
specifically overexpressed in neurons
(4). Functionally both collagen
XIII and XVII have been co-localized in cell adhesions, collagen XVII has been
co-localized in hemidesmosomes
(5), collagen XIII has been
co-localized in focal contacts
(2), and their extracellular
domains have been shown to support cell adhesion
(6,
7,
8). We report here the cloning
of a new transmembrane collagen overexpressed in rat prostate adenocarcinoma
cells and characterize its cellular localization, its cleavage, and the
properties of its soluble ectodomain.
 |
EXPERIMENTAL PROCEDURES
|
---|
Cell CultureThe Dunning rat R-3327 prostate carcinoma
sublines, AT2.1, AT3.1, and AT6.1, provided by J. Isaacs (Johns Hopkins
University) were maintained as previously described
(9). K562 and LoVo cells were
purchased from ATCC and grown in recommended culture media. Transfection was
performed in AT2.1 cells, unless otherwise stated, using LipofectAMINE 2000
(Invitrogen). The furin inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethylketone
(CMK),1 was purchased
from Bachem.
Differential DisplaymRNA differential display PCR was
performed on AT2.1, AT3.1, and AT6.1 cells as described previously
(10).
cDNA Cloning and Construct PreparationAn oligo(dT)-primed
cDNA library was constructed in the
gt10 vector (Amersham
Biosciences), using poly(A)+ RNA obtained from AT6.1 cells. The
library was screened using a 32P-labeled cDNA fragment, identified
from differential display, as a probe. Inserts from positive clones were
subcloned into pBluescript II SK+/- and subjected to DNA
sequencing. Partial rat and human collagen XXIII cDNAs were extended using a
Marathon cDNA amplification kit (Clontech) with AT6.1 mRNA and Marathon-ready
human brain (cerebellum) cDNA (Clontech), respectively. Further extension of
the 5' sequence was performed using a SMART RACE cDNA amplification kit
(Clontech), according to the manufacturer's instructions, using human heart
poly(A)+ RNA and brain (cerebellum) poly(A)+ RNA
(Clontech). Inverse RACE on AT6.1 RNA was performed as described
(11), using ThermoScript
(Invitrogen), 10% GC-Melt (Clontech), and 5% Me2SO, reverse
transcribing with primer R1 (5'-GCCCCGCACGCCGCAACAGTTCG-3').
Circularized DNA was amplified by PCR using nested reverse primer R2
(5'-CAGCGCCGCCACTCGGCCATGCAAG-3') and forward primer F1
(5'-GCGAACTGTTGCGGCGTGCG-3'). The genomic sequence was amplified
from adapter-ligated, restriction enzyme-digested rat genomic DNA from the Rat
Genome Walker kit (Clontech) with Advantage-GC Genomic PCR reaction mix
(Clontech) with adapter and gene-specific PCR primers. Amplified PCR products
were inserted into pCR2.1-TOPO (Invitrogen) and plasmid DNA-sequenced.
Amino-terminal truncated rat collagen XXIII(del 148)Myc
corresponding to amino acids Ala49Lys532, was
obtained by PCR using LA Taq (Panvera, WI) with forward primer
5'-GCCTTGCATGGCCGAGTGGCGGCG-3' and reverse primer
5'-CGCTCTAGACTACAGATCTTCTTCAGAAATAAGTTTTTGTTCCTTATGCCAGCAACCAGGCACAGGCA-3'
and inserted into pcDNA4/HisMaxC (Invitrogen) in frame with an amino-terminal
His tag. Full-length (wild type) rat collagen XXIII-Myc was constructed by
overlap extension PCR. Exon 1 sequence was amplified by PCR from rat genomic
DNA, as determined by genome walking, to overlap cloned cDNA sequence by a
primer length. Fragments were annealed, extended using Advantage-GC Genomic
polymerase, and amplified by PCR. The soluble cleaved form of human collagen
XXIII, corresponding to Glu111Lys540, was
obtained using forward (5'-GGGGTACCGAAGCTCCATCCGAATGTGTCTGC-3')
and reverse (5'-CCGCTCGAGGCTTATGCCAGCAGCCAGGCACAG-3') primers and
inserted into pSecTag2HygroB (Invitrogen) with an amino-terminal Ig
secretion signal and carboxyl-terminal His and Myc tags.
DNA was sequenced using the Sequenase 2.0 DNA sequencing kit (U.S.
Biochemical Corp.) or submitted to the Dana Farber Molecular Biology Core
Facility (Boston, MA) for DNA sequencing. Sequence data were compiled using
GeneToolLite Multi-Align (BioTools, Inc., Alberta, Canada) and peptides
aligned using GCG PileUp, PEP comparison matrix. Amino acid and nucleotide
distances were compared using the Jotun Hein method.
RT-PCR AnalysisTotal RNA was isolated from tissue culture
cells using a RNeasy miniprep kit (Qiagen), treated with 25 units of RQ1 DNase
(Promega), phenol-chloroform extracted, and ethanol-precipitated. 2 µg of
total RNA was reverse transcribed using 200 units of Superscript II
(Invitrogen) and 500 ng of oligo(dT)1218 at 45 °C.
Serial dilutions of cDNA were amplified by PCR using 18 cycles with the rat
glyceraldehyde-3-phosphate dehydrogenase primers (forward primer,
5'-TGAAGGTCGGTGTCAACGGATTTGGC-3'; reverse primer,
5'-CATGTAGGCCATGAGGTCCACCAC-3'). PCR bands were analyzed by
National Institutes of Health Scion Image (Scion Corporation), and cDNA
concentration was equalized accordingly such that housekeeping gene
amplification was equivalent (data not shown). Equalized cDNA was amplified by
PCR using LA Taq using primers designed to span multiple exons (rat
collagen XXIII forward primer, 5'-GACTCGACGGTTTCCCTGGACCCA-3';
reverse primer, 5'-GATACAAGTTCTGAACAGGAGGCC-3'). PCR was performed
using a PTC-200 Peltier Thermal Cycler (MJ Research) as follows: 94 °C for
3 min; 38 cycles of 94 °C for 30 s, 65 °C for 30 s, and 72 °C for
1 min; followed by a 72 °C 5-min elongation step. Housekeeping genes were
amplified using the same PCR conditions for 21 cycles. RNA analysis of
collagen XXIII expression in normal tissues was performed on a rat multiple
tissue cDNA panel (Clontech) using the PCR conditions listed above and primers
(forward primer, 5'-GGCCCTCACGGTCTGCCTGGACC-3'; reverse primer,
5'-TTGGGGGCTATGCCCGAGGATCACT-3').
Preparation of Collagen XXIII AntiseraRat AT6.1 cDNA
encoding the carboxyl-terminal 108 amino acids of collagen XXIII was cloned
into pGEX3X vector (Amersham Biosciences). Protein was expressed in
Escherichia coli DH5
(Invitrogen) and purified over a
glutathione-Sepharose column (Amersham Biosciences). New Zealand White rabbits
were immunized with 100 µg of collagen XXIII glutathione
S-transferase fusion protein emulsified with complete Freund's
adjuvant. The bleeds were screened by dot blot against the glutathione
S-transferase fusion protein. Positive antiserum from rabbit 22225B,
hereafter known as 6.1B, was used for subsequent analysis.
Protein PreparationThe cells were collected in RIPA or
M-PER extraction reagent (Pierce) supplemented with Complete EDTA-free
protease inhibitors (Roche Applied Science). For serum-free conditioned medium
analysis, the cells and debris were removed by centrifugation at 220 x
g for 5 min, followed by 0.2-µm filtration. For conditioned medium
analysis following transfection of wild type collagen XXIII-Myc, protein was
concentrated using PAGEprep slurry (Pierce) according to the manufacturer's
instructions and eluted in 100 mM sodium citrate, 50 mM
Tris, pH 7.5, and 1% SDS. For heparin and ion exchange binding studies,
conditioned medium from collagen
XXIII(Glu111Lys540) transfected cells was
analyzed without concentration. Heparin-agarose bead slurry (Pierce) was
washed with heparin equilibration buffer (10 mM Tris, 50
mM NaCl, pH 7.0), incubated with filtered conditioned medium,
aliquoted into spin columns, washed in heparin equilibration buffer, and
eluted with increasing concentration of NaCl in heparin equilibration buffer.
For ion exchange, conditioned medium was diluted in binding buffer (25
mM Tris/HCl, pH 8.0, <25 mM salt), added to
buffer-equilibrated columns, washed, and eluted in 1 M NaCl.
Immunoblot AnalysisProtein sample concentrations were
equalized by BCA Assay (Pierce) and separated by SDS-PAGE in the presence
(reducing conditions) or absence (nonreducing conditions) of 5%
-mercaptoethanol. The proteins were transferred to Immobilon-P
polyvinylidene difluoride membrane (Millipore) using Towbin buffer.
Immunoblotting was performed using rabbit polyclonal anti-XXIII antibody 6.1B
or 9E10 anti-Myc antibody (Santa Cruz), followed by horseradish
peroxidase-conjugated secondary antibodies (Amersham Biosciences) and detected
by an enhanced chemiluminescence detection system (PerkinElmer Life
Sciences).
ImmunofluorescenceTransfected cells on 10 µg/ml
fibronectin-coated cover slips were fixed using 4% paraformaldehyde (Electron
Microscopy Sciences) in PBS, and either washed in PBS or PBS containing 0.1%
Triton X-100 for membrane permeabilization. Nonspecific staining was blocked
by incubation in 10% horse serum in PBS. The cells were incubated in 6.1B
rabbit antibody followed by donkey anti-rabbit Texas Red-conjugated secondary
antibody (Amersham Biosciences). The coverslips were mounted in Vectashield
(Vector Laboratories) and examined under fluorescence illumination.
 |
RESULTS
|
---|
Identification and Cloning of a Novel Collagen Expressed in Metastatic
Prostate Adenocarcinoma CellsSublines of the Dunning rat R-3327
rat prostate adenocarcinoma (9)
were analyzed for differences in mRNA expression by differential display
analysis. An mRNA transcript was identified that was highly overexpressed in
the metastatic subline AT6.1 (Fig.
1A). The cloned cDNA fragment was used to screen an AT6.1
cDNA library, isolating a 1412-bp partial cDNA. Sequence analysis revealed a
novel mRNA containing an 108-amino acid open reading frame containing the
repeating amino acid motif [Gly-Xaa-Yaa]n, where Xaa and
Yaa were frequently proline and hydroxyproline, characteristic of the collagen
superfamily of proteins (1).
The partial mRNA sequence was extended by both the 5' and 3' RACE
methods (12). Sequence
obtained via multiple 5' RACE reactions suggested an incomplete
transcript, because of an additional collagenous coding sequence upstream of
potential start sites. Consequently PCR genomic walking
(13) was used to amplify
additional 5' sequence, revealing a potential translation start site
upstream of the known exon sequence. Inverse RACE
(11) confirmed this sequence
in rat AT6.1 mRNA and further determined a potential transcriptional start
site 128 bp upstream of the translation start site
(Fig. 1B). Thus we
identified a 2733-bp transcript from AT6.1 RNA. BLAST
(14) comparison of the new
collagen nucleotide sequence against available data base information revealed
an expressed sequence tag fragment from human retina with homology. Northern
blot and RT-PCR was used to identify human tissues and cell lines expressing
this new collagen, revealing expression in human heart and brain and in the
human leukemia cell line, K562 (data not shown). RNA from these sources was
used to amplify the human homologue using RACE and PCR, identifying a 3067-bp
transcript. BLAST searching revealed identity with the carboxyl-terminal end
of an incomplete human cDNA from testis, DKFZp434K0621 (accession number
AL137461
[GenBank]
), and most recently to the complete Mus musculus collagen
1 type XXIII (accession number AF410792
[GenBank]
). Thus we have identified and
cloned both the human and rat type XXIII collagen
1-chains. At the
nucleotide level, human and rat collagen
1(XXIII) show 76% identity.
Rat and mouse collagen
1(XXIII) predictably show even greater cDNA
identity, at 91%. Considering the high identity between human, rat, and mouse
cDNAs, and the longer 5' sequences identified in human and mouse, we
note the possibility of additional transcriptional start sites in the rat
collagen XXIII gene, in addition to the site we have identified by Inverse
RACE. Certainly multiple transcriptional start sites have been identified in
the structurally similar collagen XIII
(15). BLAST searching the
National Institutes of Health genome data base located the
1(XXIII)
gene on Homo sapiens chromosome 5q35 (NT_023132.9) and M.
musculus chromosome 11B1 + 2 (NW_000039.1), hereafter referred to simply
as collagen XXIII, in the absence of known alternate
-chains. Across
the open reading frames human and rat collagen XXIII show 65% nucleotide
identity, translated into 532- and 540-amino acid proteins, respectively, with
91% identity (Fig. 2).
Structurally, collagen XXIII consists of a long amino-terminal noncollagenous
(NC) domain, NC-1, containing a short cytoplasmic region and a putative
membrane spanning domain, followed by three collagenous (COL1COL3)
domains in the extracellular region of the protein that are interrupted by
short noncollagenous domains (NC2NC4), as shown in the schematic
(Fig. 3). The structural
organization of human and rat collagen XXIII is similar to the published
transmembrane collagens XIII
(16) and collagen XXV
(4). At the amino acid level
collagens XIII and XXV show 54 and 56% identity with collagen XXIII,
respectively, although this homology exists primarily across the collagenous
domains. However, in all three structurally similar transmembrane collagens,
types XXIII, XIII, and XXV, there is high identity across the 20-amino acid
carboxyl-terminal noncollagenous domain
(Fig. 4). In addition, the
COL-1 domain of collagen XXIII contains a conserved consensus binding motif
for the
5
1 integrin, RGD, and multiple
copies of a KGD motif reportedly used for integrin-mediated cell adhesion by
collagen type XVII (8).

View larger version (53K):
[in this window]
[in a new window]
|
FIG. 1. RNA analysis reveals a transcript up-regulated in metastatic AT6.1 rat
prostate carcinoma cells. A, differential display PCR of Dunning
Rat R-3327 prostate carcinoma cells demonstrates an up-regulated transcript in
metastatic AT6.1 cells, as marked with an arrowhead. B, inverse RACE
was used to identify a potential transcriptional start site for collagen XXIII
in AT6.1 RNA. RNA was reverse transcribed using a gene-specific primer to
collagen XXIII, circularized, and amplified by PCR. Lane 1, forward
(F1) and reverse (R2) primers; lane 2, forward primer only; lane
3, reverse primer only. The DNA size in nucleotides is indicated.
|
|

View larger version (59K):
[in this window]
[in a new window]
|
FIG. 2. Amino acid sequences of human and rat collagen XXIII. The predicted
amino acid sequences of human (upper rows) and rat (lower
rows) collagen XXIII are shown. Collagenous domains are boxed,
and a putative transmembrane region, as predicted by THHMM
(17), is shaded. The
potential furin protease cleavage site is underlined. The RGD motif
is underlined with a dashed line.
|
|

View larger version (7K):
[in this window]
[in a new window]
|
FIG. 3. Schematic representation of Collagen XXIII structure. The sequence
predicted domain structure of collagen XXIII is shown. Collagenous domains
(COL) are white boxes. Noncollagenous domains (NC)
are black boxes. The transmembrane domain is indicated with
cross-hatching. The amino acid positions (human collagen XXIII) are
listed across the top. The positions of cysteine residues are marked
(C). The potential cleavage site is marked (X).
|
|

View larger version (13K):
[in this window]
[in a new window]
|
FIG. 4. Alignment of noncollagenous NC-4 domains of transmembrane collagens
XXIII, XIII, and XXV. The carboxyl-terminal 20 amino acid noncollagenous
NC-4 domains of transmembrane collagens XXIII, XIII, and XXV were aligned.
Identical amino acids are in black boxes, and similar amino acids in
a gray box.
|
|
Collagen XXIII Is a Type II Transmembrane ProteinSequence
analysis revealed the presence of a group of hydrophobic amino acids close to
the amino terminus of the collagen XXIII protein. Multiple sequence analysis
algorithms, including TMHMM2.0
(17) and TMPred
(18), predict this region to
be a transmembrane helix, in accord with the structural homology with
transmembrane collagens XIII and XVII. To investigate the cellular
localization of collagen XXIII, a truncation mutant, rat collagen XXIII(del
148)Myc, was constructed by deletion of the putative cytoplasmic and
transmembrane domains. The length of the putative transmembrane helix is
variable between prediction programs. Because the ectodomain sequence is
important for collagen XIII chain association
(19), a minimal deletion was
made. AT2.1 prostate carcinoma cells were transfected with either wild type
rat collagen XXIII-Myc or the amino-terminal truncation mutant, rat collagen
XXIII(del148). Cellular localization was determined by
immunofluorescence staining using antibody 6.1B, which recognizes the carboxyl
terminus of rat collagen XXIII. Fig.
5 demonstrates that wild type collagen XXIII could be detected
either with or without cell permeabilization. In contrast, collagen
XXIII(del148) was detected only following permeabilization of the
cells, indicating an intracellular localization for the amino-terminal
truncation mutant. This demonstrates that the carboxyl terminus of wild type
collagen XXIII is present on the cell surface and that the amino-terminal
cytoplasmic and transmembrane domains are required for cell surface
targeting.

View larger version (13K):
[in this window]
[in a new window]
|
FIG. 5. Collagen XXIII is a type II transmembrane protein. Overexpressed
wild type collagen XXIII-Myc (A and C) and an amino-terminal
truncation mutant, collagen XXIII(del 148)Myc (B and
D) were detected by immunofluorescence in AT2.1 cells using the
carboxyl-terminal 6.1B antibody under Triton X-100 permeabilized (A
and B) and nonpermeabilized (C and D) conditions.
This experiment reveals that amino-terminal deletion of cytoplasmic and
transmembrane domains results in loss of cell surface localization of collagen
XXIII. The scale bar indicates 100 µm.
|
|
Collagen XXIII Expression Is Up-regulated in Metastatic Tumor
CellsWe first identified collagen XXIII in the highly metastatic
AT6.1 subline from the Dunning Rat R-3327 prostate adenocarcinoma model. To
investigate its expression pattern, RT-PCR analysis was performed on
additional sublines in this rat prostate cancer series.
Fig. 6A shows that
nonmetastatic NbE cells and poorly metastatic AT2.1 cells did not express
collagen XXIII, whereas highly metastatic AT6.1 cells show high levels of
expression, and further revealed that an additional metastatic subline,
MatLyLu, expressed low levels of collagen XXIII. We have examined collagen
XXIII expression in normal rat tissues by RT-PCR analysis and found expression
in brain and lung (data not shown).

View larger version (26K):
[in this window]
[in a new window]
|
FIG. 6. Expression pattern of collagen XXIII in prostate carcinoma cells.
RT-PCR analysis of collagen XXIII expression in Dunning rat prostate carcinoma
cell lines demonstrates up-regulation in AT6.1 and MatLyLu cells, relative to
the nonmetastatic and poorly metastatic NbE and AT2.1 cells. Upper
panel, collagen (col) XXIII; lower panel,
glyceraldehyde-3-phosphate dehydrogenase (G3PDH).
|
|
To investigate expression of collagen XXIII at the protein level, a rabbit
polyclonal antibody, antibody 6.1B, was raised to a glutathione
S-transferase fusion protein encoding the carboxyl-terminal 108 amino
acids of rat collagen XXIII. Fig.
7A demonstrates antigenic recognition of transfected rat
collagen XXIII(del 148)Myc by 6.1B antibody. Specificity was compared
with immunoblot using anti-Myc 9E10 monoclonal antibody, as shown in
Fig. 7B. Endogenous
collagen XXIII was detected in rat AT6.1 and MatLyLu metastatic prostate
carcinoma cells by immunoblot using 6.1B antibody, as a protein with a
molecular mass of
70,00075,000 Da. No collagen XXIII protein was
detectable in the nonmetastatic NbE cells or in the poorly metastatic AT2.1
subline (Fig. 7C),
confirming the expression pattern at the RNA level. These results suggest that
collagen XXIII expression is up-regulated in certain metastatic prostate
cancer cells relative to nonmetastatic or poorly metastatic prostate cancer
cells.

View larger version (51K):
[in this window]
[in a new window]
|
FIG. 7. Immunoblot analysis of collagen XXIII. Lysate was prepared from
AT2.1 Dunning rat prostate carcinoma cells transfected with empty vector
(lane 1) or collagen XXIII(del148)Myc (lane 2),
separated by 8% SDS-PAGE, and immunoblotted with rabbit polyclonal antibody
6.1B raised against the carboxyl terminus of collagen XXIII (A), or
9E10 monoclonal anti-Myc antibody (B). C, analysis of 100
µg of cell lysate separated by 6% SDS-PAGE and immunoblotted with 6.1B
antibody indicates expression of the 75-kDa collagen XXIII protein in AT6.1
cells and MatLyLu cells, marked by an arrowhead. Molecular mass
markers (kDa) are shown on the left.
|
|
Collagen XXIII Is Cleaved from the Cell Surface by Furin Protease
ActivityBecause most collagens are secreted proteins, we explored
whether collagen XXIII was retained on the cell surface by analyzing cell
lysates and cell-conditioned media of cells transfected with Myc-tagged wild
type rat collagen XXIII. Western blot analysis revealed the presence of a
70,00075,000-Da form on the cell surface along with a smaller,
60-kDa soluble form of the protein in the cell media
(Fig. 8, A and
B). Amino acid analysis of the extracellular domain of
collagen XXIII revealed potential sites for cleavage by furin, a protease that
requires the minimal basic amino acid motif Arg-Xaa-Xaa-Arg
(20). Collagen XXIII contains
a suitable basic motif,
94KLRTVR99 in rat
collagen XXIII and
105KIRTAR110 in human
collagen XXIII. Interestingly, these motifs show homology with the furin
cleavage sites reported in the transmembrane collagens XIII
(19), XVII
(3), and XXV
(4). To investigate the role of
furin proteases in collagen XXIII cleavage, wild type rat collagen XXIII-Myc
overexpressing cells were treated with a synthetic furin inhibitor,
decanoyl-RVKR-CMK. Fig. 8 (A and
B) demonstrates that cleavage of collagen XXIII was
reduced by CMK treatment. Equal loading of lysate protein is indicated by
-actin immunoblot (Fig.
8C). In addition, when wild type collagen XXIII-Myc was
overexpressed in LoVo cells, a human colon carcinoma cell line that lacks
endogenous furin activity
(21), full-length protein was
seen in the cell lysate, but no soluble cleavage product was detectable in the
medium fraction (Fig.
8D). Taken together, these data indicate a role for furin
protease cleavage in the processing of collagen XXIII.

View larger version (43K):
[in this window]
[in a new window]
|
FIG. 8. Collagen XXIII is cleaved from the cell surface by a furin protease.
A and B, immunoblot analysis of AT2.1 cell lysate
(A) and conditioned medium (B) from cells transfected with
wild type collagen XXIII-Myc, using anti-Myc antibody. -, control. +, 100
µM furin inhibitor decanoyl-Arg-Val-Lys-Arg-CMK. C,
equal protein lysate loading is indicated by immunoblot with anti- -actin
antibody. D, LoVo cells that lack furin protease activity were
transfected with wild type collagen XXIII-Myc. Cell lysate (lanes
LYS) and conditioned medium (lanes CM), controls (-), and 100
µM CMK (+). The molecular mass markers (kDa) are shown on the
left.
|
|
Multimer Formation and Heparin Binding of Soluble Collagen
XXIIICollagen XXIII is a new member of an increasingly
structurally diverse collagen superfamily, many members of which associate
with homotypic or heterotypic collagen polypeptide chains to form multimers.
To investigate the association of collagen XXIII
-chains, the
truncation mutant collagen XXIII(del148)Myc was used, such that protein
would be retained in the cell lysate and not cleaved into the cell conditioned
medium. The cells were transfected with rat collagen XXIII(del148)Myc,
and cell lysate was analyzed by immunoblot under nonreducing and reducing
conditions. Fig. 9A
shows the formation of multimeric complexes, indicative of a dimer and trimer,
which were reduced to a monomer upon addition of the reducing agent
-mercaptoethanol. Because we have shown that the wild type protein can
be cleaved from the cell surface, we next determined whether the cleaved
collagen remains in this multimeric form. Conditioned medium from cells
overexpressing wild type collagen XXIII-Myc was collected and analyzed by
immunoblot under nonreducing and reducing conditions
(Fig. 9B). These data
indicate that furin-cleaved soluble collagen XXIII can exist as an
3(XXIII) homotrimer and also suggest that the amino acids required for
-chain association are beyond the site of furin cleavage. To further
investigate the function of soluble collagen XXIII, an expression plasmid was
constructed encoding the cleaved form of human collagen XXIII, amplified from
K562 human leukemia cell cDNA, in frame with a secretion signal sequence.
Serum-free conditioned medium from collagen
XXIII(Glu111Lys540)-transfected cells was
immunoblotted under nonreducing conditions, resulting in equivalent multimer
formation, validating
-chain association of this secreted protein (data
not shown).
Because a soluble collagen may be available for cell surface and/or
extracellular matrix binding, we next investigated the heparin binding
affinity of collagen XXIII. Conditioned medium from cells transfected with
secreted collagen XXIII(Glu111Lys540) was
incubated with heparin-agarose at pH 7.0
(Fig. 10A) and pH 8.6
(data not shown), and the fractions were eluted with increasing salt
concentration. Secreted collagen
XXIII(Glu111Lys540) bound heparin with weak
affinity, with elution beginning at 200 mM NaCl. We also determined
that collagen XXIII(Glu111Lys540) bound to anion
exchange columns of weak and strong affinity at pH 8.0
(Fig. 10B),
indicating a net negative charge.

View larger version (37K):
[in this window]
[in a new window]
|
FIG. 10. Heparin binding of collagen XXIII. A, conditioned medium
from cells transfected with secreted collagen
XXIII(Glu111Lys540) was incubated with
heparin-agarose beads. The fractions were eluted in increasing concentrations
of NaCl in equilibration buffer, separated by SDS-PAGE, and immunoblotted
using anti-Myc antibody. B, secreted collagen
XXIII(Glu111Lys540) in conditioned medium was
diluted in binding buffer, added to weak (diethylamine; lanes 1 and
2) or strong affinity (quaternary ammonium; lanes 3 and
4) basic anion exchange columns, washed in binding buffer, and eluted
in buffer containing 1 M NaCl. Column flow-through (lanes
1 and 3) and eluate (lanes 2 and 4) were
separated by SDS-PAGE and immunoblotted with anti-Myc antibody.
|
|
 |
DISCUSSION
|
---|
We have identified a new transmembrane collagen, collagen XXIII, from rat
and human RNA. This collagen is the fourth in the subgroup of nonfibrillar
type II transmembrane collagens and displays similarities in domain structure
with collagens XIII and XXV. It is expressed in normal human heart and retina
as well as in metastatic prostate cancer cells. Although sequence identity is
low across collagens XXIII, XIII, and XXV, their 20-amino acid NC-4
carboxyl-terminal noncollagenous domains show significant identity. We
hypothesize that this motif may be involved in functional interaction with
cell surface or extracellular matrix proteins. Such an interaction might occur
either between the integral membrane "precursors" of these
transmembrane collagens or following cleavage to their soluble forms. It is
thus possible to envisage a bifunctional signaling process, in a similar
manner as has been shown for soluble and membrane bound forms of neuropilin
interacting with the vascular endothelial growth factor (VEGF) receptor
(22). It should be noted that
the cytoplasmic domains of the structurally similar collagens XXIII, XIII, and
XXV display minimal sequence identity. That the NC-4 noncollagenous domain is
not critical for
-chain association is suggested by trimer formation of
truncated collagen XIII, where both the COL-3 and NC-4 domains were deleted
(19).
Independent of this conserved noncollagenous domain, there are several
amino acid motifs present in collagen XXIII that may contribute toward cell
interaction. We report that collagen XXIII contains a species conserved RGD
motif, the binding site for
5
1 integrin,
and three copies of a KGD motif, which contributes to cell adhesion and
migration on collagen XVII via weak
5
1 and
v
1 integrin subunit interaction
(8). In addition, of the
traditional collagen receptors,
1
1 but not
2
1 mediated interaction with native
collagen XIII (7). These
reports suggest the possibility of similar integrin receptor-mediated
interactions with collagen XXIII. These interactions may be dependent on
collagen chain association and folding, because the peptide motifs RGD and KGD
lie within collagenous domains and could be hidden within a collagen triple
helix. Such cryptic motifs are exposed upon proteolytic cleavage or unfolding
via denaturation, as shown for collagen types I, V, VI, and XVII
(8,
23,
24,
25). Conversely, other peptide
motifs may depend on triple helix conformation for ligand presentation to
integrin receptors, as observed for the collagen recognition motif GFOGER
(26). Clearly the state of
collagen folding could influence cellular interaction and function. We have
shown that recombinantly expressed forms of collagen XXIII formed multimers
suggestive of a dimer and trimer under nonreducing conditions. Although the
trimeric complex might be predicted to contain nucleated triple helical
domains characteristic of collagens, it has been observed that expression of
other recombinant transmembrane collagens also results in dimeric structures.
Expression of recombinant collagen types XIII
(19,
27), XVII
(3), and XXV
(4) generates both dimers and
trimers. However, rotary shadowing electron microscopy of the structurally
similar collagen XIII has revealed the formation of rodlike structures
(27), consistent with triple
helix folding. This might imply that these collagen dimers represent a
processing intermediate, although dimeric collagen
XXV/CLAC100 is a major form detected in brain tissue from
patients with Alzheimer's disease
(4). We have not yet been able
to determine whether endogenous collagen XXIII shows the same distribution of
monomer and multimers because of expression levels and/or detection
sensitivity. However, collagen XXIII chain association will be an important
consideration in the functional analysis of this protein.
Collagens can also interact with other components of the extracellular
matrix, as demonstrated by collagen XXV binding to fibrillized
-amyloid
peptide, a key component of the characteristic senile plaques deposited in
Alzheimer's disease (4), and
collagen XIII interaction with fibronectin, nidogen-2, and heparin
(27). We report here low
affinity heparin binding of collagen XXIII. Indeed, collagen XXIII contains
clusters of basic amino acid residues that might mediate heparin interaction.
Heparan sulfate glycosaminoglycans exist both at the cell surface and as a
matrix component; thus any interaction may be relevant in the context of
either cell-matrix or cell-cell contact
(28), and we are currently
further defining this binding activity.
Our results demonstrate that cleavage of type XXIII collagen is a furin
convertase/paired basic amino acid-cleaving enzyme-mediated event. Many
transmembrane proteins are cleaved by this group of proteases, including
growth factors such as transforming growth factor-
1 and other proteases,
such as stromelysin 3 (matrix metalloprotease-11) and membrane type matrix
metalloprotease-1 (reviewed in Ref.
20). Furin cleavage of many
proteins is a required activation event, resulting in the release of a soluble
mature molecule from an inactive precursor. This raises the possibility that
soluble collagen XXIII represents the mature form of collagen XXIII.
Alternatively, membrane proteins can possess activities independent of the
cleaved protein, as demonstrated with soluble sHB-EGF and proHB-EGF
(29). Because furin protease
cleavage may control the rate of release of soluble collagen XXIII, it is
interesting to note that increased furin expression has been reported in lung
(30) and head and neck tumors
(31). Moreover, reduced
tumorigenicity resulted from inhibition of furin activity
(32). Because collagen XXIII
expression was up-regulated in more aggressive cell lines of the Dunning rat
prostate carcinoma model, concurrent furin activity level may be an important
factor regulating transmembrane collagen cleavage in the tumor environment. A
useful consequence of increased cleavage of a membrane protein up-regulated in
tumor cells is its potential use as a diagnostic or prognostic marker. We have
preliminary evidence correlating increased collagen XXIII immunohistochemical
staining with increasing tumor stages in human prostate cancer (data not
shown) and are further characterizing expression to explore this
possibility.
 |
FOOTNOTES
|
---|
The nucleotide sequence(s) reported in this paper has been submitted to
the GenBankTM/EBI Data Bank with accession number(s) AY158895
[GenBank]
and AY158896
[GenBank]
.
* This work was supported by Grant R01 CA 37393 from the National Institutes
of Health. The costs of publication of this article were defrayed in part by
the payment of page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact. 
Present address: Pintex Pharmaceuticals, 313 Pleasant St., Watertown, MA
02472. 
To whom correspondence should be addressed: Dept. of Surgery, Enders 1074,
Children's Hospital, 300 Longwood Ave., Boston, MA 02115. E-mail:
bruce.zetter{at}tch.harvard.edu.
1 The abbreviations used are: CMK, chloromethylketone; RACE, rapid
amplification of cDNA ends; COL, collagenous; NC, noncollagenous; RT, reverse
transcription; PBS, phosphate-buffered saline. 
 |
ACKNOWLEDGMENTS
|
---|
The Dunning rat prostate carcinoma variants were generously provided by J.
T. Isaacs (Johns Hopkins University). The NbE rat prostate cell line was
provided by M. R. Freeman (Children's Hospital, Boston, MA).
 |
REFERENCES
|
---|
- Myllyharju, J., and Kivirikko, K. I. (2001)
Ann. Med. 33,
7-21[Medline]
[Order article via Infotrieve]
- Peltonen, S., Hentula, M., Hagg, P., Yla-Outinen, H., Tuukkanen,
J., Lakkakorpi, J., Rehn, M., Pihlajaniemi, T., and Peltonen, J.
(1999) J. Invest. Dermatol.
113,
635-642[Abstract/Free Full Text]
- Schacke, H., Schumann, H., Hammami-Hauasli, N., Raghunath, M., and
Bruckner-Tuderman, L. (1998) J. Biol.
Chem. 273,
25937-25943[Abstract/Free Full Text]
- Hashimoto, T., Wakabayashi, T., Watanabe, A., Kowa, H., Hosoda, R.,
Nakamura, A., Kanazawa, I., Arai, T., Takio, K., Mann, D. M., and Iwatsubo, T.
(2002) EMBO J.
21,
1524-1534[Abstract/Free Full Text]
- Hopkinson, S. B., Findlay, K., deHart, G. W., and Jones, J. C.
(1998) J. Invest. Dermatol.
111,
1015-1022[Abstract]
- Hagg, P., Vaisanen, T., Tuomisto, A., Rehn, M., Tu, H., Huhtala,
P., Eskelinen, S., and Pihlajaniemi, T. (2001) Matrix
Biol. 19,
727-742[CrossRef][Medline]
[Order article via Infotrieve]
- Nykvist, P., Tu, H., Ivaska, J., Kapyla, J., Pihlajaniemi, T., and
Heino, J. (2000) J. Biol. Chem.
275,
8255-8261[Abstract/Free Full Text]
- Nykvist, P., Tasanen, K., Viitasalo, T., Kapyla, J., Jokinen, J.,
Bruckner-Tuderman, L., and Heino, J. (2001) J. Biol.
Chem. 276,
38673-38679[Abstract/Free Full Text]
- Isaacs, J. T., Isaacs, W. B., Feitz, W. F., and Scheres, J.
(1986) Prostate
9, 261-281[Medline]
[Order article via Infotrieve]
- Bao, L., Loda, M., Janmey, P. A., Stewart, R., Anand-Apte, B., and
Zetter, B. R. (1996) Nat. Med.
2,
1322-1328[Medline]
[Order article via Infotrieve]
- Eyal, Y., Neumann, H., Or, E., and Frydman, A. (1999)
BioTechniques 27,
656-658[Medline]
[Order article via Infotrieve]
- Frohman, M. A., Dush, M. K., and Martin, G. R. (1988)
Proc. Natl. Acad. Sci. U. S. A.
85,
8998-9002[Abstract]
- Siebert, P. D., Chenchik, A., Kellogg, D. E., Lukyanov, K. A., and
Lukyanov, S. A. (1995) Nucleic Acids Res.
23,
1087-1088[Medline]
[Order article via Infotrieve]
- Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D.
J. (1990) J. Mol. Biol.
215,
403-410[CrossRef][Medline]
[Order article via Infotrieve]
- Kvist, A. P., Latvanlehto, A., Sund, M., Horelli-Kuitunen, N.,
Rehn, M., Palotie, A., Beier, D., and Pihlajaniemi, T. (1999)
Matrix Biol. 18,
261-274[CrossRef][Medline]
[Order article via Infotrieve]
- Hagg, P., Rehn, M., Huhtala, P., Vaisanen, T., Tamminen, M., and
Pihlajaniemi, T. (1998) J. Biol. Chem.
273,
15590-15597[Abstract/Free Full Text]
- Sonnhammer, E. L., von Heijne, G., and Krogh, A.
(1998) Proc. Int. Conf. Intell. Syst. Mol.
Biol. 6,
175-182[Medline]
[Order article via Infotrieve]
- Hofmann, K., and Stoffel, W. (1993) Biol.
Chem. Hoppe-Seyler 374,
166
- Snellman, A., Tu, H., Vaisanen, T., Kvist, A. P., Huhtala, P., and
Pihlajaniemi, T. (2000) EMBO J.
19,
5051-5059[Abstract/Free Full Text]
- Nakayama, K. (1997) Biochem.
J. 327,
625-635[Medline]
[Order article via Infotrieve]
- Takahashi, S., Kasai, K., Hatsuzawa, K., Kitamura, N., Misumi, Y.,
Ikehara, Y., Murakami, K., and Nakayama, K. (1993)
Biochem. Biophys. Res. Commun.
195,
1019-1026[CrossRef][Medline]
[Order article via Infotrieve]
- Gagnon, M. L., Bielenberg, D. R., Gechtman, Z., Miao, H. Q.,
Takashima, S., Soker, S., and Klagsbrun, M. (2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
2573-2578[Abstract/Free Full Text]
- Davis, G. E. (1992) Biochem. Biophys. Res.
Commun. 182,
1025-1031[Medline]
[Order article via Infotrieve]
- Ruggiero, F., Champliaud, M. F., Garrone, R., and Aumailley, M.
(1994) Exp. Cell Res.
210,
215-223[CrossRef][Medline]
[Order article via Infotrieve]
- Pfaff, M., Aumailley, M., Specks, U., Knolle, J., Zerwes, H. G.,
and Timpl, R. (1993) Exp. Cell Res.
206,
167-176[CrossRef][Medline]
[Order article via Infotrieve]
- Knight, C. G., Morton, L. F., Peachey, A. R., Tuckwell, D. S.,
Farndale, R. W., and Barnes, M. J. (2000) J. Biol.
Chem. 275,
35-40[Abstract/Free Full Text]
- Tu, H., Sasaki, T., Snellman, A., Gohring, W., Pirila, P., Timpl,
R., and Pihlajaniemi, T. (2002) J. Biol.
Chem. 277,
23092-23099[Abstract/Free Full Text]
- Park, P. W., Reizes, O., and Bernfield, M. (2000)
J. Biol. Chem. 275,
29923-29926[Free Full Text]
- Miyoshi, E., Higashiyama, S., Nakagawa, T., Hayashi, N., and
Taniguchi, N. (1997) J. Biol. Chem.
272,
14349-14355[Abstract/Free Full Text]
- Mbikay, M., Sirois, F., Yao, J., Seidah, N. G., and Chretien, M.
(1997) Br. J. Cancer
75,
1509-1514[Medline]
[Order article via Infotrieve]
- Bassi, D. E., Mahloogi, H., Al-Saleem, L., Lopez De Cicco, R.,
Ridge, J. A., and Klein-Szanto, A. J. (2001) Mol.
Carcinog. 31,
224-232[CrossRef][Medline]
[Order article via Infotrieve]
- Bassi, D. E., Lopez De Cicco, R., Mahloogi, H., Zucker, S., Thomas,
G., and Klein-Szanto, A. J. (2001) Proc. Natl. Acad.
Sci. U. S. A. 98,
10326-10331[Abstract/Free Full Text]