Isolation, Cloning, and Sequence Analysis of the Integrin Subunit
10, a
1-associated Collagen Binding Integrin Expressed on
Chondrocytes*
Lisbet
Camper
§,
Ulf
Hellman¶, and
Evy
Lundgren-Åkerlund
From the
Department of Cell and Molecular Biology,
Section for Cell and Matrix Biology, Lund University, S-221 00 Lund and the ¶ Ludwig Institute for Cancer Research,
S-75124 Uppsala, Sweden
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ABSTRACT |
We have found that chondrocytes express a novel
collagen type II-binding integrin, a new member of the
1-integrin
family. The integrin
subunit, which has a
Mr of 160 kDa reduced, was isolated from bovine
chondrocytes by collagen type II affinity purification. The human
homologue was obtained by screening a human chondrocyte library with a
bovine cDNA probe. Cloning and cDNA sequence analysis of the
human integrin
subunit designated
10 show that it shares the
general structure of other integrin
subunits. The predicted amino
acid sequence consists of a 1167-amino acid mature protein, including a
signal peptide (22 amino acids), a long extracellular domain (1098 amino acids), a transmembrane domain (25 amino acids), and a short
cytoplasmic domain (22 amino acids). The extracellular part contains a
7-fold repeated sequence, an I-domain (199 amino acids) and three
putative divalent cation-binding sites. The deduced amino acid sequence
of
10 is 35% identical to the integrin subunit
2 and 37%
identical to the integrin subunit
1. Northern blot analysis shows a
single mRNA of 5.4 kilobases in chondrocytes. A peptide antibody
against the predicted sequence of the cytoplasmic domain of
10
immunoprecipitated two proteins with masses of 125 and 160 kDa from
chondrocyte lysates under reducing conditions. The peptide antibody
specifically stained chondrocytes in tissue sections of human articular
cartilage, showing that
10
1 is expressed in cartilage tissue.
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INTRODUCTION |
The integrins are a large family of transmembrane glycoproteins
that mediate cell-cell and cell-matrix interactions (1-5). All known
members of this superfamily are noncovalently associated heterodimers
composed of an
and a
subunit. At present, 8
-(
1-
8) (See Ref. 6 and references therein) and 16
subunits (
1-
9,
v,
M,
L,
X,
IIb,
E, and
D) have been characterized
(6-21), and these subunits associate to generate more than 20 different integrins. The
1 subunit has been shown to associate with
10 different
subunits,
1-
9 and
v and to mediate
interactions with extracellular matrix proteins such as collagens,
laminins, and fibronectin. The major collagen binding integrins are
1
1 and
2
1 (22-25). The integrins
3
1 and
9
1
have also been reported to interact with collagen (26, 27), although
this interaction is not well understood (28). The extracellular
N-terminal regions of the
and
integrin subunits are important
in the binding of ligands (29, 30). The N-terminal region of the
subunits is composed of a 7-fold repeated sequence (12, 31) containing FG and GAP consensus sequences. The repeats are predicted to fold into
a
-propeller domain (32), with the last three or four repeats
containing putative divalent cation binding sites. The
-integrin
subunits
1,
2,
D,
E,
L,
M, and
X contain an ~200
amino acid inserted domain, the I-domain (A-domain), that shows
similarity to sequences in von Willebrand factor, cartilage matrix
protein, and complement factors C2 and B (33, 34). The I-domain is
localized between the second and third FG-GAP repeats; it contains a
metal ion-dependent
adhesion site (MIDAS), and it is involved in
binding of ligands (35-38).
Chondrocytes, the only type of cells in cartilage, express a number of
different integrins including
1
1,
2
1,
3
1,
5
1,
6
1,
v
3, and
v
5 (39-41). We have shown that
1
1
and
2
1 mediate chondrocyte interactions with collagen type II
(25), which is one of the major components in cartilage. We have also shown that
2
1 is a receptor for the cartilage matrix protein chondroadherin (42). In the present study we have isolated a novel
collagen type II binding integrin,
10
1, from bovine articular chondrocytes. Cloning and sequence analysis of the human homologue is
described, and expression of
10 on chondrocytes is examined.
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MATERIALS AND METHODS |
Antibodies--
A polyclonal antiserum was generated against the
10 cytoplasmic domain peptide CKKIPEEEKREEKLE. Peptide synthesis and
conjugation to keyhole limpet hemocyanin, injection of rabbits and
affinity purification were performed by Innovagen AB (Lund, Sweden).
Monoclonal antibodies against human integrin subunit
1 (P4C10),
2
(P1E6), and
3 (P1B5) (unpurified ascites fluid) were from Life
Technology Inc. The monoclonal antibody against human integrin subunit
1 (TS2/7; hybridoma supernatant) was a kind gift from Timothy
Springer, Boston Blood Center, Boston, MA (43). Polyclonal antibody
(serum) against the rat
1-integrin subunit was kindly provided by
Staffan Johansson, Uppsala, Sweden (44). Polyclonal antibodies (serum) against human integrin subunits
2 (AB1936),
3 (AB1920), and polyclonal antibody (serum) against rat integrin subunits
1 (AB1934) were from Chemicon International Inc. (Temecula, CA). Polyclonal antibodies against the integrin subunit
9 (affinity-purified IgG)
were a kind gift from Dean Sheppard, University of California San
Francisco Lung Biology Center, San Fransisco, CA (6).
Cell Isolation and Culture--
Bovine chondrocytes were
isolated by digestion of articular cartilage from 4-6-month-old calves
with collagenase (CLS1; Worthington Biochemical Corp., Lakewood, NJ) as
described elsewhere (45). Briefly, cartilage slices were digested by
collagenase in Earle's balanced salt solution (Life Technologies,
Inc.) for 15-16 h at 37 °C. The tissue digest was filtered through
a 100-µm nylon filter, and the isolated cells were then washed three
times in Dulbecco's modified phosphate-buffered saline
(PBS),1 Life Technologies,
Inc.). Human chondrocytes from articular cartilage were isolated by
digestion with Pronase (Calbiochem) for 1 h followed by
collagenase (Boehringer Mannheim) for 15-18 h, as described by
Häuselmann et al. (46). The cells were filtered and
washed as described above. Human chondrocytes were cultured in
Dulbecco's minimum essential medium and F-12 (1:1) supplemented with
10% fetal calf serum, 25 µg/ml ascorbic acid, 50 IU of penicillin, and 50 µg/ml streptomycin (Life Technologies, Inc.). To harvest cells, the culture dish was washed three times with
Ca2+/Mg2+-free PBS, and the cells were
incubated with 0.5% trypsin and 1 mM EDTA (Life
Technologies, Inc.) in Ca2+/Mg2+-free PBS for 5 min. Detached cells were suspended in medium containing 10% fetal calf
serum or in PBS containing 1 mg/ml trypsin inhibitor (Sigma) and then
washed in PBS.
Coupling of Affinity Columns--
Collagen type II isolated from
nasal cartilage by pepsin digestion (47) was coupled to CNBr-Sepharose
4B (Pharmacia Biotech, Uppsala, Sweden) according to the published
procedure (25). A control column was produced by treating
CNBr-Sepharose 4B in a similar manner but in the absence of protein.
Bovine fibronectin (Sigma) was coupled to CNBr-Sepharose 4B according
to instructions from the manufacturer. After blocking, the
fibronectin-Sepharose was washed three times with PBS.
Affinity Purification and Immunoprecipitation of Chondrocyte
Membrane Proteins--
Human chondrocyte cell surface proteins were
125I-labeled and affinity-purified on collagen type
II-Sepharose according to the published procedure (25). Cell lysates or
affinity-purified samples were immunoprecipitated as described earlier
(42). The following antibodies were used in immunoprecipitation
experiments: monoclonal antibodies against the human integrin subunits
1,
1,
2, or
3 (unpurified ascites fluid, dilution 1/100),
polyclonal antibody against the rat integrin subunit
1 (purified
IgG, 50-100 µg/ml), polyclonal peptide antibodies against the
integrin subunits
1,
2,
3, and
10 (serum, dilution 1/100).
The immunoprecipitated proteins were separated by 4-12% SDS-PAGE and
visualized by image analysis using the BioImaging Analyzer Bas2000
(Fuji Photo Film Co., Tokyo, Japan).
Western Blot--
Human chondrocyte membrane proteins
immunoprecipitated with polyclonal antibodies against
10 (10 µg/ml
affinity-purified IgG) or
1 (100 µg/ml IgG) were separated by 8%
SDS-PAGE and transferred to a nitrocellulose membrane essentially as
described by Towbin et al. (48). The membrane was blocked
with 3% dried milk in 10 mM Tris-HCl, pH 7.4, 0.15 M NaCl, and 0.2% Tween (blocking buffer) and then
incubated with the
1 antibody (20 µg/ml) in blocking buffer
containing 1% dried milk. The
1 subunit was detected after
incubation with a secondary antibody conjugated with horseradish by
chemiluminescence using the ECL system (Amersham Pharmacia Biotech).
Affinity Purification of the Integrin Subunit
10 on Collagen
Type II-Sepharose--
Freshly isolated bovine chondrocytes (2500 × 106) were lysed in 6 ml of 1% Triton X-100, 100 µg/ml
aprotinin, 2 µg/ml leupeptin, 2 µg/ml pepstatin A, 1 mM
phenylmethylsulfonyl fluoride, 1 mM MnCl2, 1 mM MgCl2, and 10 mM Tris-HCl, pH
7.4, for 1 h on ice. The lysate was centrifuged for 30 min at
10,000 rpm, and the pellet was discarded. Collagen type II-Sepharose (4 ml) and the fibronectin-Sepharose (2 ml) were equilibrated with at
least 20 volumes of 0.1% Triton X-100, 1 mM
phenylmethylsulfonyl fluoride, 1 mM MnCl2, 1 mM MgCl2, and 10 mM Tris-HCl, pH
7.4 (equilibration buffer). The entire cell lysate was passed over the
fibonectin-Sepharose twice, and the flow through was then incubated
with the collagen-Sepharose end over end for 3 h. The columns were
washed (15 gel volumes) with the equilibration buffer containing 75 mM NaCl, and bound proteins were eluted with 20 mM EDTA, 0.1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and 10 mM Tris-HCl, pH
7.4.
Isolation of Internal Peptides by In-gel Digestion and Peptide
Sequencing--
Affinity-purified proteins were concentrated by
precipitation using the methanol/chloroform protocol (49). After
reduction/alkylation with dithiothreitol/iodoacetamide (50), the
precipitated proteins were subjected to SDS-PAGE on a 4-12%
polyacrylamide gel, and protein bands were visualized by Coomassie
staining. The 160-kDa protein band was excised from the gel and
prepared for in-gel digestion (51). Briefly, the gel slice was washed
extensively to remove SDS and the dye, and after complete drying,
protease was forced into the gel by rehydration with a solution of
modified trypsin (Promega, Madison, WI) in 0.2 M
NH4HCO3 buffer. After an overnight incubation,
peptides were extracted and then isolated by narrow bore reversed phase
liquid chromatography on a µRPC C2/C18 stainless steel 2.1/10 column
operated in a SMART System (Amersham). Several peptides were analyzed
by Edman degradation in a Perkin-Elmer Applied Biosystem Model 476 sequencer operated according to the manufacturer's instructions.
mRNA Purification and cDNA Synthesis--
mRNA from
bovine or human chondrocytes were isolated using a
QuickPrep® Micro mRNA purification kit (Pharmacia).
cDNA was synthesized at 42 °C for 1 h using the
SuperscriptTM II RNase H
Reverse
Transcriptase cDNA Synthesis system (Life Technologies, Inc.)
random DNA hexamers and oligo(dT) (Promega, Madison, WI).
PCR Amplification--
PCR reactions were performed in 50-µl
reaction volumes and contained 1 × Taq polymerase
buffer (Life Technologies, Inc.), 1.5 mM MgCl2,
1 µM of each primer, 0.025 units/µl Taq
polymerase, 1 µl of DNA template (bovine chondrocyte cDNA), and
0.1 mM each of dATP, dGTP, dCTP, and dTTP (Boehringer
Mannheim). PCR samples were heated to 94 °C for 5 min in a
thermocycler and then subjected to 35 cycles consisting of 30 s at
94 °C (denaturation), 30 s at 48 or 52 °C (annealing) and 3 min at 72 °C (extension). The PCR products were re-amplified using 1 µl of each product for an additional 35 cycles. Amplified DNA was
analyzed by 1% agarose gel electrophoresis. Small DNA fragments were
analyzed using 4% MethaPhoreTM-agarose (FMC BioProducts,
Rockland, ME).
The degenerate primers GAY AAY ACI GCI CAR AC (DNTAQT, forward)
and TIA TIS WRT GRT GIG GYT (EPHHSI, reverse) were used in PCR to
amplify the nucleotide sequence corresponding to the bovine peptide 1 (Table I). A 900 base pair PCR fragment was then amplified from bovine
cDNA using an internal specific primer TCA GCC TAC ATT CAG TAT (SAYIQY, forward) corresponding to the cloned nucleotide sequence of
peptide 1 together with the degenerate primer
ICK RTCCCA RTG ICC IGG (PGHWDR, reverse) corresponding to the
bovine peptide 2 (Table I). Mixed bases were used in positions that
were 2-fold degenerate, and inosines were used in positions that were
3- or 4-fold degenerate.
To obtain cDNA that encoded the 5' end of
10, we designed the
primer AAC TCG TCT TCC AGT GCC ATT CGT GGG (reverse; residues 1254-1280 in
10 cDNA) and used it for rapid amplification of the cDNA 5' end (RACE) as described in the MarathonTM
cDNA amplification kit (CLONTECH INC., Palo
Alto, CA).
Cloning and Sequencing of cDNA--
PCR fragments were
isolated and purified from agarose gels using Jet Sorb DNA extraction
kit (Genomed Inc. Research Triangle Park, CA). Purified fragments were
then cloned with the pCR ScriptTM Sk(+) kit (Stratagene, La
Jolla, CA). Selected plasmids were purified from liquid cultures using
QIAGEN plasmid midi preparation kit (QIAGEN Inc. Valencia, CA) and
sequenced by ABI 373A sequencer using ABI PrismTMDye
Terminator Cycle Sequencing Core kit (Perkin-Elmer) together with T3,
T7, and internal specific primers.
Library Screening--
The cloned 900-base pair PCR fragment
corresponding to bovine
10-integrin was digoxigenin-labeled
according to the DIG DNA labeling kit (Boehringer Mannheim) and used as
a probe for screening of a human articular chondrocyte
ZapII
cDNA library (provided by Michael Bayliss, The Royal Veterinary
Basic Sciences, London, UK) (52). Positive clones containing the
pBluescript SK+ plasmid with the cDNA insert were
rescued from the ZAP vector by in vivo excision as described
in the ZAP-cDNA® synthesis kit (Stratagene). Selected
plasmids were purified and sequenced as described earlier using T3, T7,
and internal specific primers.
Northern Blot Analysis--
Bovine chondrocyte mRNA was
purified using a QuickPrep®Micro mRNA purification kit
(Amersham), separated on a 1% agarose formaldehyde gel, transferred to
nylon membranes, and immobilized by UV cross-linking. cDNA probes
were 32P-labeled with Random Primed DNA labeling kit
(Boehringer Mannheim). Filters were prehybridized for 2-4 h at
42 °C in 5× SSE (20 × SSC, 3M NaCl, 0.3 M
trisodium citrate·2H2O, pH adjusted to 7.0 with 1 M HCl), 5× Denhardt's solution, 0.1% SDS, 50 µg/ml
salmon sperm DNA, and 50% formamide and then hybridized overnight at 42 °C with the same solution containing the specific probe
(0.5-1 × 106 cpm/ml). Specifically bound cDNA
probes were analyzed using the phosphoimaging system (Fuji). Filters
were stripped by washing in 0.1% SDS for 1 h at 80 °C before
reprobing. The
10-integrin cDNA probe was isolated from the
race1-containing plasmid using the restriction enzymes BamHI
(Life Technologies, Inc.) and NcoI (Boehringer Mannheim).
The rat
1-integrin cDNA probe was a kind gift from Staffan
Johansson, Uppsala, Sweden (25).
Tissue Staining--
Human cartilage from the trochlear groove,
obtained during surgery, was provided by Anders Lindahl, Sahlgrenska
University Hospital, Gothenburg, Sweden. Frozen sections of cartilage
tissue were fixed in acetone at
18 °C for 5 min, washed in PBS,
and then treated with 2 mg/ml hyaluronidase (Sigma) in PBS, pH 5.0, for
15 min at 37 °C. After washing with PBS, sections were blocked for
15 min at room temperature in 0.1% H2O2 in PBS
to remove endogenous peroxidase activity. Sections were then washed in
PBS, blocked with 0.5% casein and 0.05% thimerosal in PBS (blocking
buffer) for 15 min at room temperature, and then incubated overnight at 4 °C with the affinity-purified antibodies against the integrin subunits
9 or
10 (5 µg/ml in blocking buffer). For control, the
10 antibody was preincubated with the
10 peptide (0.1 mg/ml) for
30 min at 4 °C. After washing in PBS, sections were incubated with
biotinylated goat anti-rabbit secondary antibody (Vector Laboratories
Inc; diluted 1:200 in blocking buffer) at room temperature for 60 min.
Washed sections were then incubated with VECTASTAIN® ABC reagent
(Vector Laboratories, Inc. Burlingame, CA) for 1 h at room
temperature and washed, and the color was developed using 1 mg/ml
diaminobenzidine, 0.02% H2O2 and 0.1 M Tris-HCl, pH 7.2. Sections were rinsed in water for 5 min
followed by 75, 95, and 99.5% ethanol for 5 min each and then three
times in xylene for 3 min at room temperature. Samples were mounted in
Pertex (Histolab Products AB, Gothenburg, Sweden) and examined by light
microscopy.
 |
RESULTS |
Identification and Isolation of the Chondrocyte
10 Integrin
Subunit--
Affinity purification of 125I-labeled
membrane proteins from human chondrocytes on collagen type II-Sepharose
followed by immunoprecipitation showed that these cells, in addition to
1
1 and
2
1, express an unidentified
1-related
subunit
(Fig. 1). This integrin subunit had an
apparent molecular mass of approximately 160 kDa under reducing
condition and was slightly larger than the
2 integrin subunit. This
finding is in agreement with a previous study from our group showing
that bovine chondrocytes also express an unidentified collagen binding
1-associated
subunit of similar molecular mass (25). To isolate
this protein, we affinity-purified collagen type II-binding proteins
from bovine chondrocytes. The chondrocyte lysate was first applied to a
fibronectin-Sepharose precolumn, and the flow-through was then applied
to a collagen type II-Sepharose column. As shown in Fig.
2, a number of proteins were eluted from the affinity columns. A protein with molecular mass of approximately 160 kDa was specifically eluted with EDTA from the collagen column but
not from the fibronectin column. The molecular mass of this protein
corresponded with the molecular mass of the unidentified
1-related
integrin subunit (Fig. 1). The 160-kDa protein band was excised from
the SDS-PAGE gel and digested with trypsin, and several of the isolated
peptides were analyzed. Table I shows the
amino acid sequence of six individual peptides.

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Fig. 1.
Affinity purification and immunoprecipitation
of collagen type II-binding integrins from Triton X-100 lysate of
125I-labeled human chondrocytes. The lanes
show immunoprecipitation of integrins using monoclonal antibodies
against the integrin subunits 1(P4C10), 1(TS2/7), 2(P1E6),and
3(P1B5). The proteins eluted by EDTA from the collagen type
II-Sepharose are shown in lane E. The proteins were
separated by SDS-PAGE (4-12%) under nonreducing conditions and
visualized using phosphoimaging.>
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Fig. 2.
Affinity purification of the 10 integrin
subunit on collagen type II-Sepharose. A Triton X-100 lysate of
bovine chondrocytes (2.5 × 109 cells) was applied to
a fibronectin-Sepharose precolumn followed by a collagen type
II-Sepharose column. The lanes show EDTA-eluted proteins
from the fibronectin-Sepharose (A), flow-through from the
collagen type II-Sepharose column (B), and EDTA-eluted
proteins from the collagen type II-Sepharose (C). The eluted
proteins were precipitated by methanol/chloroform, separated by
SDS-PAGE (4-12%) under reducing conditions, and stained with
Coomassie Blue. The 160-kDa protein with affinity for collagen type II
is indicated with an arrow.
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Table I
Amino acid sequences of peptides from bovine 10-integrin
Peptides were isolated by in-gel digestion with trypsin and sequenced
by Edman degradation.
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Cloning and Sequencing of the Human Integrin
-Subunit
Homologue--
The nucleotide sequence corresponding to peptide 1 (Table I) was obtained by PCR amplification, cloning, and sequencing of bovine cDNA. From this nucleotide sequence an exact primer was designed and applied in PCR amplification with degenerate primers corresponding to peptides 2-6 (Table I). Primers corresponding to
peptides 1 and 2 amplified a 900-base pair PCR fragment from bovine
cDNA that was cloned, sequenced, and used for screening of a human
articular chondrocyte
ZapII cDNA library to obtain the human
integrin
-subunit homologue. Two overlapping clones, hc1 and hc2
(Fig. 3), were isolated, subcloned, and
sequenced. These clones contained
of the nucleotide sequence, including the 3' end of the cDNA. A third clone
(Race1; Fig. 3), which contained the 5'end of the
10
cDNA, was obtained using the RACE technique. From these three
overlapping clones of
10 cDNA, 3884 nucleotides were sequenced
(Fig. 4). The sequence contains a
3504-nucleotide open reading frame that is predicted to encode a 1167 amino acid mature protein. The predicted sequence included a signal
peptide (22 amino acids), a long extracellular domain (1098 amino
acids), a transmembrane domain (25 amino acids), and a short
cytoplasmic domain (22 amino acids). Sequence analysis of the 160-kDa
protein sequence showed that it was a member of the integrin
-subunit family, and the subunit was named
10.

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Fig. 3.
Schematic map of the sequencing
strategy. The overlapping 10 clones hc1 and hc2 were obtained
by screening a human articular chondrocyte library with a bovine 10
probe. The Race1 clone was obtained from human chondrocyte cDNA
using the RACE technique. Arrows indicate the direction and
extent of nucleotide sequencing. kb, kilobases.
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Fig. 4.
Nucleotide sequence and deduced amino acid
sequence of the human 10 integrin subunit. The amino acid
translation is under the first nucleotide of the corresponding codon.
The signal peptide cleavage site is marked with an arrow,
human homologues to bovine peptide sequences are underlined,
and the I-domain is boxed. Metal ion binding sites are
indicated with a dashed underline, potential
N-glycosylation sites are indicated by an
asterisk, and the putative transmembrane domain is
double underlined. The normally conserved cytoplasmic
sequence is indicated by a dot and dash underline. The
sequence data is available from GenBankTM under accession number
AF074015.
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Comparison of
10 Integrin Subunit with Other
Subunits--
Analysis of
10 with known
subunits showed that
its structure follows the conserved pattern of integrin
subunits
(Fig. 5). The extracellular domain
contains a 7-fold repeated sequence including FG and GAP consensus
sequences, three putative divalent cation binding sites
(DXD/NXD/NXXXD), and an I domain of
199 amino acids. The protein contains 10 potential N-linked
glycosylation sites (NX(T/S)). The calculated molecular mass
is 153 kDa if carbohydrate chains with an average molecular weight of
2.5 kDa are assumed to attach to all 10 putative glycosylation sites.
This is in agreement with the molecular mass of
10 as judged by
SDS-PAGE where the molecular mass was estimated to approximately 160 kDa.

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Fig. 5.
Diagrammatic comparison of 10 with the
general structure of integrin subunits. The conserved repeats
1-7 are indicated with boxes. An I-domain is found in some
-integrin subunits. Other integrin subunits are
posttranslationally cleaved near the transmembrane domain
(TM). The integrin subunit 10 contains the seven
conserved repeats and an I-domain located between repeat two and
three.
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In contrast to most
-integrin subunits, the cytoplasmic domain of
10 does not contain the conserved sequence KXGFF(R/K)R (Table II). Instead, the predicted amino
acid sequence is KLGFFAH. The deduced amino acid sequence of
10
showed the highest identity to the collagen-binding integrin subunits
1 (37%) and
2 (35%). The similarity of integrin
subunits
are shown in Fig. 6.
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Table II
Comparison of the cytoplasmic tails of I-domain-containing integrin subunits
The underlined sequence in 10 represents the peptide that was used
for antibody production.
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Fig. 6.
Sequence similarity between integrin subunits. The similarity tree was constructed using the GCG
software and the program "Pileup." Percent identities were
calculated using the Jotun Hein algorithm provided in the Lasergene
DNASTAR software. The similarity tree indicates three different
subfamilies of -integrin subunits, one subfamily that contains
I-domains ( 1, 2, M, X, L, and 10), one subfamily that
is cleaved ( 3, 5, 6, 7, 8,  , and IIb), and one
subfamily that neither contains I-domains nor is cleaved ( 4,
9).
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Expression of the
10 Integrin Subunit on
Chondrocytes--
Northern blot analysis of mRNA from bovine
chondrocytes showed that a human
10 cDNA probe hybridized with a
single mRNA of approximately 5.4 kilobases (Fig.
7). As a comparison, a cDNA probe
corresponding to the integrin subunit
1 was used. This cDNA
probe hybridized a mRNA band of approximately 3.5 kilobases on the
same filter. Translation of the
10 nucleotide sequence revealed an
open reading frame of 3504 nucleotides (Fig. 4), which indicates that
around 2000 nucleotides in the mRNA is not translated.

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Fig. 7.
Northern blot analysis of 10 and 1
mRNA. Bovine chondrocyte mRNA was hybridized with
32P-labeled cDNA probes corresponding to the integrin
subunits 10 or 1. The 10 probe hybridized to an mRNA of
5.4 kilobases, and the 1-probe hybridized to an mRNA of 3.5 kilobases on the same filter.
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To study expression of
10 at the protein level,
125I-labeled membrane proteins from human chondrocytes were
immunoprecipitated with polyclonal antibodies against the integrin
subunits
1,
1,
2,
3, and
10 (Fig.
8). A polyclonal peptide antibody raised against the cytoplasmic domain of
10 precipitated two protein bands
with molecular masses of approximately 160 and 125 kDa under reducing
conditions. The
10-associated
-chain migrated as the
1
integrin subunit both under reducing and nonreducing conditions (Figs.
8, a and b). To verify that the
10-associated
-chain indeed is
1, chondrocyte lysates were immunoprecipitated
with antibodies against
10 or
1 followed by Western blot using
antibodies against the
1 subunit (Fig. 8c). These results
clearly demonstrated that
10 is a member of the
1-integrin
family.

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Fig. 8.
Immunoprecipitation of the 10 integrin
subunit from human chondrocytes. Triton X-100 lysates of
125I-labeled human chondrocytes were immunoprecipitated
with polyclonal antibodies against the integrin subunits 1, 1,
2, 3, or 10. The immunoprecipitated proteins were separated by
SDS-PAGE (4-12%) under reducing (a) and nonreducing
conditions (b) and visualized using a phosphoimager
(c). Triton X-100 lysates of human chondrocytes
immunoprecipitated with 10 or 1 were separated by SDS-PAGE (8%)
under nonreducing conditions and analyzed by Western blot using the
polyclonal 1 antibody and chemiluminescent detection. C,
control.
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Expression of
10 in cartilage was examined by immunostaining of
human articular cartilage from the trochlear groove with the polyclonal
10 antibody. As shown in Fig. 9, this
antibody specifically stained the chondrocytes in the cartilage tissue sections. The staining was completely abolished when the antibody was
preincubated with the
10 peptide. A control antibody against the
9 integrin subunit did not stain chondrocytes in the tissue sections
(Fig. 9).

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Fig. 9.
Immunostaining of human articular
cartilage. An antibody raised against the cytoplasmic domain of
10 (see Table II) stained the chondrocytes in tissue sections of
human articular cartilage (A). The staining was depleted
when the antibody was preincubated with the 10 peptide
(B). A control antibody recognizing the 9 integrin
subunit did not bind to the chondrocytes (C).
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DISCUSSION |
The present study demonstrated that human chondrocytes express a
novel, collagen type II-binding integrin in the
1 family. We have,
in an earlier study, presented some evidence for that bovine
chondrocytes and human chondrosarcoma cells also express this integrin
(25). Because bovine chondrocytes are readily available in large
amounts, we used these cells in the isolation of the integrin subunit
10. As shown in Fig. 2, several proteins were eluted from the
columns in the affinity purification experiments. It was difficult to
interpret the protein pattern in the eluate because typical integrin
bands were not clearly distinguished on the SDS-PAGE gel. This may be
explained by partial protein degradation, although a mixture of
protease inhibitors were included in the lysate buffer. Based upon the
finding that the
1 antibody immunoprecipitated an unknown
collagen-binding integrin
subunit with a moleculare mass of 160 kDa
(Fig. 1), a protein with similar molecular mass that was specifically
eluted with EDTA from the collagen type II column was excised from the
gel and used for peptide sequencing. This 160-kDa protein was not
eluted from the fibronectin-Sepharose, indicating that fibronectin is
not a ligand for
10
1. However, this will be investigated in cell
adhesion experiments using cells transfected with the
10
subunit.
The immunoprecipitation experiments showed that
2 and
10 integrin
subunit have similar molecular masses under reducing conditions (Fig.
1). To avoid contamination of
2, the 160-kDa protein was excised
from the SDS-PAGE gel as a very narrow band. This was apparently
successful since human homologues to all six bovine peptides (Table I)
that were isolated from the 160-kDa protein were found in the predicted
amino acid sequence of human
10 subunit (Fig. 4).
The deduced amino acid sequence of
10 was found to share the general
structure of the integrin
subunits described in previously published reports (6-21). The large extracellular N-terminal part of
10 contains a 7-fold repeated sequence that was recently predicted to fold into a
-propeller domain (32). The integrin subunit
10
contains three putative divalent cation binding sites
(DXD/NXD/NXXXD) (53), a single
spanning transmembrane domain, and a short cytoplasmic domain. In
contrast to most
-integrin subunits, the cytoplasmic domain of
10
does not contain the conserved sequence KXGFF(R/K)R. The
predicted amino acid sequence in
10 is KLGFFAH. Several reports indicate that the integrin cytoplasmic domains are crucial in signal
transduction (54) and that membrane-proximal regions of both
- and
-integrin cytoplasmic domains are involved in modulating
conformation and affinity state of integrins (55-57). It is suggested
that the GFFKR motif in
-chains are important for association of
integrin subunits and for transport of the integrin to the plasma
membrane (58). The KXGFFKR domain has been shown to interact
with the intracellular protein calreticulin (59), and interestingly,
calreticulin-null embryonic stem cells are deficient in
integrin-mediated cell adhesion (60). It is, in this context, tempting
to speculate that the sequence KLGFFAH in
10 may have a key function
in regulating the affinity between
10
1 and collagen.
Integrin
subunits are known to share an overall identity of
20-40% (61). Sequence analysis showed that the
10 subunit is most
closely related to the I domain-containing
subunits (Fig. 6) with
the highest identity to
1 (37%) and
2 (35%). The integrins
1
1 and
2
1 are known receptors for both collagens and
laminins (24, 62, 63), and we have also recently demonstrated that
2
1 interacts with the cartilage matrix protein chondroadherin (42). Since
10
1 was isolated on a collagen type II-Sepharose, we
know that collagen type II is a ligand for
10
1. We have also shown by affinity purification experiments that
10
1 interacts with collagen type I (data not shown), but it remains to be seen whether laminin or chondroadherin are also ligands for this
integrin.
The peptide antibody that we raised against the cytoplasmic domain of
10 immunoprecipitated two proteins from human chondrocytes with
molecular masses of approximately 125 and 160 kDa. The molecular mass
of 160 kDa correlates with the unidentified
1-associated
subunit
that was affinity-purified on collagen type II-Sepharose. The 125-kDa
protein was in Western blot recognized by an antibody to the
1
subunit. This, together with previous findings that
1
1 and
2
1 are present on isolated chondrocytes demonstrate that
chondrocytes express at least three collagen-binding integrins in the
1 family (25). Further studies will answer the question whether
these integrins have similar or different functions in cartilage.
Immunohistochemistry using the
10 antibody showed staining of the
chondrocytes in tissue sections of human articular cartilage. The
antibody staining was clearly specific because preincubation of the
antibody with the
10 peptide completely abolished the staining. An
antibody against the integrin subunit
9 did not stain the
chondrocytes (6). This integrin is a receptor for tenascin C (64) and
is not known to be present in cartilage.
Taken together, we have isolated and characterized a novel collagen
type II-binding integrin designated
10
1. The
10 subunit was
isolated from bovine chondrocytes, and the human homologue was cloned
and sequenced. Antibodies against the
10-integrin subunit stained
chondrocytes in tissue sections of articular cartilage, indicating that
10
1 indeed is expressed in cartilage. Further investigations
including ligand interactions, tissue distribution, signal
transduction, and knockout mutation will demonstrate the function of
the integrin
10
1.
 |
ACKNOWLEDGEMENTS |
We are most grateful to Dick Heinegård,
Anders Aspberg, Danny Tuckwell, and Martin Humphries for valuable
discussions and to Michael Bayliss for providing the human chondrocyte
library.
 |
FOOTNOTES |
*
The work was supported by grants from the Swedish Medical
Research Council, Anna-Greta Crafoord's Stiftelse, Crafoord's
stiftelser, Gustav V's 80-års fond, Greta och Johan Kock's stiftele,
Kungliga fysiografiska sällskapets stiftelse,
Riksföreningen mot reumatism, Magnus Bergvall's Stiftelse,
Thelma Zoe'ga's fond, and Alfred Österlund's Stiftelse.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF074015.
§
Dept. of Cell and Molecular Biology, Section for Cell and Matrix
Biology, Lund University, P.O. Box 94, S-221 00 Lund, Sweden. Tel.:
+46-46-222-3126; Fax: 46-46-222-3128; E-mail: lisbet.camper{at}medkem.lu.se.
The abbreviations used are:
PBS, phoshate-buffered saline; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; RACE (Race), rapid amplification of the
cDNA end.
 |
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