Isolation, Cloning, and Sequence Analysis of the Integrin Subunit alpha 10, a beta 1-associated Collagen Binding Integrin Expressed on Chondrocytes*

Lisbet CamperDagger §, Ulf Hellman, and Evy Lundgren-ÅkerlundDagger

From the Dagger  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

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have found that chondrocytes express a novel collagen type II-binding integrin, a new member of the beta 1-integrin family. The integrin alpha  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 alpha  subunit designated alpha 10 show that it shares the general structure of other integrin alpha  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 alpha 10 is 35% identical to the integrin subunit alpha 2 and 37% identical to the integrin subunit alpha 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 alpha 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 alpha 10beta 1 is expressed in cartilage tissue.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha  and a beta  subunit. At present, 8 beta -(beta 1-beta 8) (See Ref. 6 and references therein) and 16 alpha  subunits (alpha 1-alpha 9, alpha v, alpha M, alpha L, alpha X, alpha IIb, alpha E, and alpha D) have been characterized (6-21), and these subunits associate to generate more than 20 different integrins. The beta 1 subunit has been shown to associate with 10 different alpha  subunits, alpha 1-alpha 9 and alpha v and to mediate interactions with extracellular matrix proteins such as collagens, laminins, and fibronectin. The major collagen binding integrins are alpha 1beta 1 and alpha 2beta 1 (22-25). The integrins alpha 3beta 1 and alpha 9beta 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 alpha  and beta  integrin subunits are important in the binding of ligands (29, 30). The N-terminal region of the alpha  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 beta -propeller domain (32), with the last three or four repeats containing putative divalent cation binding sites. The alpha -integrin subunits alpha 1, alpha 2, alpha D, alpha E, alpha L, alpha M, and alpha 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 alpha 1beta 1, alpha 2beta 1, alpha 3beta 1, alpha 5beta 1, alpha 6beta 1, alpha vbeta 3, and alpha vbeta 5 (39-41). We have shown that alpha 1beta 1 and alpha 2beta 1 mediate chondrocyte interactions with collagen type II (25), which is one of the major components in cartilage. We have also shown that alpha 2beta 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, alpha 10beta 1, from bovine articular chondrocytes. Cloning and sequence analysis of the human homologue is described, and expression of alpha 10 on chondrocytes is examined.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Antibodies-- A polyclonal antiserum was generated against the alpha 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 beta 1 (P4C10), alpha 2 (P1E6), and alpha 3 (P1B5) (unpurified ascites fluid) were from Life Technology Inc. The monoclonal antibody against human integrin subunit alpha 1 (TS2/7; hybridoma supernatant) was a kind gift from Timothy Springer, Boston Blood Center, Boston, MA (43). Polyclonal antibody (serum) against the rat beta 1-integrin subunit was kindly provided by Staffan Johansson, Uppsala, Sweden (44). Polyclonal antibodies (serum) against human integrin subunits alpha 2 (AB1936), alpha 3 (AB1920), and polyclonal antibody (serum) against rat integrin subunits alpha 1 (AB1934) were from Chemicon International Inc. (Temecula, CA). Polyclonal antibodies against the integrin subunit alpha 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 beta 1,alpha 1,alpha 2, or alpha 3 (unpurified ascites fluid, dilution 1/100), polyclonal antibody against the rat integrin subunit beta 1 (purified IgG, 50-100 µg/ml), polyclonal peptide antibodies against the integrin subunits alpha 1, alpha 2, alpha 3, and alpha 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 alpha 10 (10 µg/ml affinity-purified IgG) or beta 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 beta 1 antibody (20 µg/ml) in blocking buffer containing 1% dried milk. The beta 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 alpha 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 alpha 10, we designed the primer AAC TCG TCT TCC AGT GCC ATT CGT GGG (reverse; residues 1254-1280 in alpha 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 alpha 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 lambda 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 alpha 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 beta 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 alpha 9 or alpha 10 (5 µg/ml in blocking buffer). For control, the alpha 10 antibody was preincubated with the alpha 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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Identification and Isolation of the Chondrocyte alpha 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 alpha 1beta 1 and alpha 2beta 1, express an unidentified beta 1-related alpha  subunit (Fig. 1). This integrin subunit had an apparent molecular mass of approximately 160 kDa under reducing condition and was slightly larger than the alpha 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 beta 1-associated alpha  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 beta 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 beta 1(P4C10), alpha 1(TS2/7), alpha 2(P1E6),and alpha 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 alpha 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 alpha 10-integrin
Peptides were isolated by in-gel digestion with trypsin and sequenced by Edman degradation.

Cloning and Sequencing of the Human Integrin alpha -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 lambda ZapII cDNA library to obtain the human integrin alpha -subunit homologue. Two overlapping clones, hc1 and hc2 (Fig. 3), were isolated, subcloned, and sequenced. These clones contained <FR><NU>2</NU><DE>3</DE></FR> of the nucleotide sequence, including the 3' end of the cDNA. A third clone (Race1; Fig. 3), which contained the 5'end of the alpha 10 cDNA, was obtained using the RACE technique. From these three overlapping clones of alpha 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 alpha -subunit family, and the subunit was named alpha 10.


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Fig. 3.   Schematic map of the sequencing strategy. The overlapping alpha 10 clones hc1 and hc2 were obtained by screening a human articular chondrocyte library with a bovine alpha 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 alpha 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.

Comparison of alpha 10 Integrin Subunit with Other alpha  Subunits-- Analysis of alpha 10 with known alpha  subunits showed that its structure follows the conserved pattern of integrin alpha  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 alpha 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 alpha 10 with the general structure of integrin alpha  subunits. The conserved repeats 1-7 are indicated with boxes. An I-domain is found in some alpha -integrin subunits. Other alpha  integrin subunits are posttranslationally cleaved near the transmembrane domain (TM). The integrin subunit alpha 10 contains the seven conserved repeats and an I-domain located between repeat two and three.

In contrast to most alpha -integrin subunits, the cytoplasmic domain of alpha 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 alpha 10 showed the highest identity to the collagen-binding integrin subunits alpha 1 (37%) and alpha 2 (35%). The similarity of integrin alpha  subunits are shown in Fig. 6.

                              
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Table II
Comparison of the cytoplasmic tails of I-domain-containing integrin alpha  subunits
The underlined sequence in alpha 10 represents the peptide that was used for antibody production.


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Fig. 6.   Sequence similarity between integrin alpha  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 alpha -integrin subunits, one subfamily that contains I-domains (alpha 1, alpha 2, alpha M, alpha X, alpha L, and alpha 10), one subfamily that is cleaved (alpha 3, alpha 5, alpha 6, alpha 7, alpha 8, alpha nu , and alpha IIb), and one subfamily that neither contains I-domains nor is cleaved (alpha 4, alpha 9).

Expression of the alpha 10 Integrin Subunit on Chondrocytes-- Northern blot analysis of mRNA from bovine chondrocytes showed that a human alpha 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 beta 1 was used. This cDNA probe hybridized a mRNA band of approximately 3.5 kilobases on the same filter. Translation of the alpha 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 alpha 10 and beta 1 mRNA. Bovine chondrocyte mRNA was hybridized with 32P-labeled cDNA probes corresponding to the integrin subunits alpha 10 or beta 1. The alpha 10 probe hybridized to an mRNA of 5.4 kilobases, and the beta 1-probe hybridized to an mRNA of 3.5 kilobases on the same filter.

To study expression of alpha 10 at the protein level, 125I-labeled membrane proteins from human chondrocytes were immunoprecipitated with polyclonal antibodies against the integrin subunits beta 1, alpha 1, alpha 2, alpha 3, and alpha 10 (Fig. 8). A polyclonal peptide antibody raised against the cytoplasmic domain of alpha 10 precipitated two protein bands with molecular masses of approximately 160 and 125 kDa under reducing conditions. The alpha 10-associated beta -chain migrated as the beta 1 integrin subunit both under reducing and nonreducing conditions (Figs. 8, a and b). To verify that the alpha 10-associated beta -chain indeed is beta 1, chondrocyte lysates were immunoprecipitated with antibodies against alpha 10 or beta 1 followed by Western blot using antibodies against the beta 1 subunit (Fig. 8c). These results clearly demonstrated that alpha 10 is a member of the beta 1-integrin family.


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Fig. 8.   Immunoprecipitation of the alpha 10 integrin subunit from human chondrocytes. Triton X-100 lysates of 125I-labeled human chondrocytes were immunoprecipitated with polyclonal antibodies against the integrin subunits beta 1, alpha 1, alpha 2, alpha 3, or alpha 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 alpha 10 or beta 1 were separated by SDS-PAGE (8%) under nonreducing conditions and analyzed by Western blot using the polyclonal beta 1 antibody and chemiluminescent detection. C, control.

Expression of alpha 10 in cartilage was examined by immunostaining of human articular cartilage from the trochlear groove with the polyclonal alpha 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 alpha 10 peptide. A control antibody against the alpha 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 alpha 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 alpha 10 peptide (B). A control antibody recognizing the alpha 9 integrin subunit did not bind to the chondrocytes (C).

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The present study demonstrated that human chondrocytes express a novel, collagen type II-binding integrin in the beta 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 alpha 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 beta 1 antibody immunoprecipitated an unknown collagen-binding integrin alpha  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 alpha 10beta 1. However, this will be investigated in cell adhesion experiments using cells transfected with the alpha 10 subunit.

The immunoprecipitation experiments showed that alpha 2 and alpha 10 integrin subunit have similar molecular masses under reducing conditions (Fig. 1). To avoid contamination of alpha 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 alpha 10 subunit (Fig. 4).

The deduced amino acid sequence of alpha 10 was found to share the general structure of the integrin alpha  subunits described in previously published reports (6-21). The large extracellular N-terminal part of alpha 10 contains a 7-fold repeated sequence that was recently predicted to fold into a beta -propeller domain (32). The integrin subunit alpha 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 alpha -integrin subunits, the cytoplasmic domain of alpha 10 does not contain the conserved sequence KXGFF(R/K)R. The predicted amino acid sequence in alpha 10 is KLGFFAH. Several reports indicate that the integrin cytoplasmic domains are crucial in signal transduction (54) and that membrane-proximal regions of both alpha - and beta -integrin cytoplasmic domains are involved in modulating conformation and affinity state of integrins (55-57). It is suggested that the GFFKR motif in alpha -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 alpha 10 may have a key function in regulating the affinity between alpha 10beta 1 and collagen.

Integrin alpha  subunits are known to share an overall identity of 20-40% (61). Sequence analysis showed that the alpha 10 subunit is most closely related to the I domain-containing alpha  subunits (Fig. 6) with the highest identity to alpha 1 (37%) and alpha 2 (35%). The integrins alpha 1beta 1 and alpha 2beta 1 are known receptors for both collagens and laminins (24, 62, 63), and we have also recently demonstrated that alpha 2beta 1 interacts with the cartilage matrix protein chondroadherin (42). Since alpha 10beta 1 was isolated on a collagen type II-Sepharose, we know that collagen type II is a ligand for alpha 10beta 1. We have also shown by affinity purification experiments that alpha 10beta 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 alpha 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 beta 1-associated alpha  subunit that was affinity-purified on collagen type II-Sepharose. The 125-kDa protein was in Western blot recognized by an antibody to the beta 1 subunit. This, together with previous findings that alpha 1beta 1 and alpha 2beta 1 are present on isolated chondrocytes demonstrate that chondrocytes express at least three collagen-binding integrins in the beta 1 family (25). Further studies will answer the question whether these integrins have similar or different functions in cartilage.

Immunohistochemistry using the alpha 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 alpha 10 peptide completely abolished the staining. An antibody against the integrin subunit alpha 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 alpha 10beta 1. The alpha 10 subunit was isolated from bovine chondrocytes, and the human homologue was cloned and sequenced. Antibodies against the alpha 10-integrin subunit stained chondrocytes in tissue sections of articular cartilage, indicating that alpha 10beta 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 alpha 10beta 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.
    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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