Post-translational Modifications of alpha 5beta 1 Integrin by Glycosaminoglycan Chains
THE alpha 5beta 1 INTEGRIN IS A FACULTATIVE PROTEOGLYCAN*

(Received for publication, October 2, 1996, and in revised form, December 23, 1996)

Silvio S. Veiga Dagger §, Maria Carolina Q. B. Elias Dagger , Waldemiro Gremski Dagger §, Marimelia A. Porcionatto par , Roseli da Silva par , Helena B. Nader par and Ricardo R. Brentani Dagger

From the Dagger  Ludwig Institute for Cancer Research, R. Prof. Antonio Prudente, 109, 4 A, 01509-010, São Paulo, SP, Brazil, the § Department of Cell Biology, Federal University of Parana, Curitiba, Centro Politécnico, Seter de Ciências Biológicas, Jardim des Americas, 81531-990, Curitiba, PR, and the par  Department of Biochemistry, Federal University of São Paulo, Universidade Federal de São Paulo, Escola Paulista de Medicina, rea 3 de maio, 100 4 andar, 04044-020, São Paulo, SP, Brazil

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Cell-fibronectin interactions, mediated through several different receptors, have been implicated in a wide variety of cellular properties. Among the cell surface receptors for fibronectin, integrins are the best characterized, particularly the prototype alpha 5beta 1 integrin. Using [125I]iodine cell surface labeling or metabolic radiolabeling with sodium [35S]sulfate, we identified alpha 5beta 1 integrin as the only sulfated integrin among beta 1 integrin heterodimers expressed by the human melanoma cell line Mel-85. This facultative sulfation was confirmed not only by immunoprecipitation reactions using specific monoclonal antibodies but also by fibronectin affinity chromatography, two-dimensional electrophoresis, and chemical reduction. The covalent nature of alpha 5beta 1 integrin sulfation was evidenced by its resistance to treatments with high ionic, chaotrophic, and denaturing agents such as 4 M NaCl, 4 M MgCl2, 8 M urea, and 6 M guanidine HCl. Based on deglycosylation procedures as chemical beta -elimination, proteinase K digestion, and susceptibility to glycosaminoglycan lyases (chondroitinase ABC and heparitinases I and II), it was demonstrated that the alpha 5beta 1 heterodimer and alpha 5 and beta 1 integrin subunits were proteoglycans. The importance of alpha 5beta 1 sulfation was strengthened by the finding that this molecule is also sulfated in MG-63 (human osteosarcoma) and HCT-8 (human colon adenocarcinoma) cells.


INTRODUCTION

Proteoglycans are complex molecules formed by a core protein to which one or more glycosaminoglycan (GAG)1 chains are linked. This basic definition, although true, hides the molecular complexity shown by these molecules. They encompass an exceptionally large range of structures involving different core proteins, different classes of GAGs, and different numbers and lengths of individual GAG chains. Other post-translation modifications such as N- and O-glycosylation increase the complexity of these molecules (for review see Refs. 1 and 2).

The biological functions of proteoglycans are numerous. They have been involved in several biological effects (1, 3-5), such as extracellular matrix (ECM) assembly (6) and cell surface-ECM receptors for growth factors and hormones (2, 5, 7) or have had a role in biological processes such as cell-cell recognition (8) and control of cell growth (9). The fact that several ECM proteins, such as fibronectin (10), laminin (11), thrombospondin (12), vitronectin (13), type IV collagen (14), and tenascin (15), have GAG binding sites adds credence to the postulated multiple roles of proteoglycans. Supporting the idea of proteoglycans as ECM receptors, syndecan type I binds fibronectin, thrombospondin, collagens (5), and tenascin (16); the heparan sulfate proteoglycan of Schwann cells binds laminin (17); a cell surface chondroitin sulfate proteoglycan is apparently involved in cell adhesion to laminin (18); and a cell surface phosphatidyl inositol-anchored heparan sulfate proteoglycan mediates melanoma cell adhesion to fibronectin (19). Strong corroboration for these proteoglycan-ECM interactions comes from the presence of a heparan sulfate proteoglycan that co-localizes with beta 1 integrins as a widespread component of focal adhesion (20).

Among the several ECM molecules that bind proteoglycans, the role of fibronectin should be emphasized not only because of its GAG binding domains but also because of the adhesive properties conferred to this molecule by these domains together with the RGD cell-binding fragment (21-23). Cells devoid of proteoglycans or bearing proteoglycans with altered GAG chains have a reduced capacity of adhesion to fibronectin and have a defective focal adhesion plaque formation in response to this molecule (24, 25).

The best studied receptors for fibronectin that bear adhesiveness and focal adhesion plaque formation are integrins that are alpha /beta heterodimers widely expressed by almost all animal cells (26, 27). Integrins represent good examples of how post-translational modifications can alter the structure of a molecule, thus modulating its biological activity. Integrin glycosylations represent a kind of regulation by which a wide variety of these receptors have their specificity and affinity modulated in several cell lines (28-30). However, the versatility of cells to modulate the binding properties of integrins is not restricted to glycosylation. Integrin functions can be modulated by acylation of membrane lipid (31), by divalent metal binding (32), and, for the cytoplasmic domain, by tyrosine phosphorylation, which is the best understood example of this type of biological modification, especially in leukocytes and platelets (27, 33).

In the present study, we characterize alpha 5beta 1 integrin as a part-time proteoglycan containing both heparan and chondroitin sulfate, which per se could affect cell adhesion to both fibronectin RGD and GAG binding domains.


EXPERIMENTAL PROCEDURES

Reagents and Antibodies

Human fibronectin was purified from fresh plasma (obtained from Hospital A. C. Camargo, São Paulo, Brazil) by gelatin affinity chromatography as described (34). Monoclonal antibody A-1A5 that recognizes the beta 1 integrin subunit (35) and B-5G10 that reacts with the alpha 4 integrin subunit (36) were provided by Dr. Martin E. Hemler (Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA). Monoclonal antibody II-F5, which specifically recognizes the alpha 3 integrin subunit (37), was a gift from Dr. Renata Pasqualini (The Burnham Institute, San Diego, CA). Monoclonal antibodies against alpha 2 integrin subunit CLB-thromb/4 and alpha 5 integrin chain SAM-1 were purchased from the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service (Amsterdam, the Netherlands). Rabbit polyclonal antibody against the cytoplasmic domain of the alpha 7 integrin subunit (38) was a gift from Dr. Stephen J. Kaufman (Department of Cell and Structural Biology, University of Illinois, Urbana, IL), and rabbit polyclonal antiserum against the alpha 5beta 1 integrin molecule (RB3847) that in immunoblotting reacts only with the beta 1 integrin subunit2 was provided by Dr. Kenneth M. Yamada (National Institute for Dental Research, Bethesda, MD). Monoclonal antibody against chondroitin sulfate chains (CS-56) was purchased from Sigma.

Cell Culture

A human melanoma cell line (Mel-85) was provided by Dr. Stephan Carrel (Ludwig Institute for Cancer Research, Lausanne, Switzerland). A human osteosarcoma cell line (MG-63) was given by Dr. Eva Engvall (Burnham Institute, San Diego, CA), and a human colon adenocarcinoma cell line (HCT-8) was given by Dr. M. M. Brentani (Department of Oncology, School of Medicine, São Paulo University, Brazil). All cells were grown in RPMI 1640 medium (Sigma) supplemented with 10% fetal calf serum (Cultilab, Campinas, Brazil) and gentamicin (50 µg/ml) at 37 °C, 5% CO2 in humidified conditions. Cells were harvested using divalent cation-free phosphate-buffered saline containing 2 mM EDTA. For [35S]sulfate incorporation, cells were labeled in the presence of sodium [35S]sulfate (240 µCi/ml of medium) for 24 h.

Immunoprecipitation Reactions

Cell surface expression of beta 1 integrin heterodimers in Mel-85 cells was probed through immunoprecipitation reactions of cells that were surface labeled (using [125I]iodine) by the lactoperoxidase-H2O2 method as described previously (39). After washing, cells were solubilized by lysis buffer (50 mM Tris-HCl, pH 7.3, 1% Triton X-100, 50 mM NaCl, 5 mM CaCl2, 5 mM MgCl2, 1 mM phenylmethanesulfonyl fluoride, and 2 µg/ml of aprotinin) for 15 min at 4 °C. The extract was clarified by centrifugation for 10 min at 13,000 - g, and the supernatant was preincubated with either normal mouse or rabbit serum followed by precipitation with protein A-Sepharose (Sigma). Mel-85 extract (at the same mass of protein, 1 mg) was incubated respectively with antibodies against different integrin subunits (as shown above), and for B-5G10 (an IgG1 molecule), rabbit IgG was preincubated against mouse IgG followed by protein A-Sepharose. Affinity beads were washed with lysis buffer, and the immunoprecipitates were eluted by boiling for 5 min with Laemmli buffer.

[35S]Sulfate-labeled Mel-85 cell extracts were immunoprecipitated using the same mono- or polyclonal antibodies as above. Immunoprecipitates were analyzed by 7.5% SDS-PAGE (40) followed by electrotransference onto nitrocellulose membranes (41) and exposure to x-ray films (Kodak, Rochester, NY). The same procedure was used to study HCT-8 and MG-63 cell extracts with an anti-alpha 5 integrin antibody.

Ionic, Chaotropic, and Denaturating Experimental Conditions

The [35S]sulfate-labeled immunoprecipitates obtained with anti-alpha 5 integrin monoclonal antibody were incubated with 4 M NaCl, M MgCl2, 6 M guanidine HCl, and 8 M urea for 2 h at 37 °C. Mixtures were then boiled for 5 min, and alpha 5beta 1 integrin was separated from Sepharose beads by centrifugation for 1 min at 13,000 × g. Supernatants were dialyzed against water, concentrated in a speed vaccum concentrator, subjected to 7.5% SDS-PAGE under nonreducing conditions, and electrotransferred onto nitrocellulose membranes that were then exposed to x-ray films at room temperature for 10 days.

Fibronectin Affinity Cromatography

Fibronectin-affinity chromatography of [35S]sulfate-labeled Mel-85 cell extract was performed using purified human plasma fibronectin coupled to CNBr-activated Sepharose (Pharmacia Biotech Inc.) as detailed elsewhere (30).

Gel Electrophoresis, Purification of alpha 5 and beta 1 Integrin Subunits, and Immunoblotting

SDS gel electrophoresis was performed as described (40). Samples under reducing or nonreducing conditions were analyzed on 5 or 7.5% polyacrylamide gels, and proteins were transferred overnight to nitrocellulose filters as described (41). Molecular mass markers (myosin, 205 kDa; beta -galactosidase, 116 kDa; and phosphorylase b, 98 kDa; albumin, 67 kDa) were purchased from Sigma. For two-dimensional electrophoresis, samples were separated in the first dimension by isoelectric focusing (42) using an ampholyte gradient (pH 4.0-6.5, six parts and pH 3.0-10.0, four parts, Pharmacia) followed by 7.5% SDS-PAGE in nonreducing conditions.

Glycosaminoglycan analysis was performed using agarose gel electrophoresis in 0.05 M 1,3-diaminopropane acetate buffer pH 9.0 (Aldrich). After the electrophoretic run, compounds were precipitated in the gel using 0.1% Cetavlon for 2 h at room temperature (43). After drying, the gel was stained with toluidine blue and exposed to x-ray films (X-Omat, Kodak) for 10 days at room temperature. GAG standards used were heparan sulfate from bovine pancreas (44), dermatan sulfate from pig skin, and chondroitin sulfate from shark cartilage (Seikagaku, Kogyo Co., Tokyo, Japan).

To study the specific pattern of glycosylation of alpha 5 and beta 1 integrin subunits, [35S]sulfate-labeled Mel-85 cell lysate was immunoprecipitated using a monoclonal antibody against the alpha 5 integrin subunit as already described, and the precipitate was submitted to a preparative 7.5% SDS-PAGE under nonreducing conditions using prestained beta -galactosidase (116 kDa) that comigrates with the beta 1 integrin subunit as a standard. Autoradiography of separated alpha 5 and beta 1 subunits was done in identical conditions as above and used as a guide. Gel pieces were then cut off in the positions of separated alpha 5 and beta 1 subunits, and proteins were excised and eluted from the gel by incubation in 50 mM Tris-HCl, pH 7.3, containing 0.1% Triton X-100 overnight at 4 °C. The mixtures were then filtered through 0.45-µm filters (Nalgene, Rochester, NY) to remove polyacrylamide, and the solutions containing extracted proteins were dialyzed against water and concentrated 20-fold. Purified alpha 5 and beta 1 integrin subunits were then submitted to beta -elimination reaction to release free GAG chains, which were incubated with chondroitinase ABC, heparitinases I and II, or a mixture of these enzymes (see below), and the digests were analyzed by agarose gels.

Immunoblotting reactions using Rb3847 (a rabbit polyclonal antibody that only reacts with the beta 1 integrin subunit but not with the denatured alpha 5 integrin subunit) and a monoclonal antibody against chondroitin sulfate chains (CS-56) were performed as described previously (30).

Chemical beta -Elimination and Enzyme Digestions

The GAG chains from [35S]sulfate-labeled alpha 5beta 1 integrin, purified by immunoprecipitation using a monoclonal antibody against the alpha 5 integrin subunit, were liberated by digestion of the protein core using excess proteinase-K (50 µg; Sigma) at 58 °C overnight or by a beta -elimination reaction (treatment overnight at 37 °C with 0.1 M NaOH in the presence of 2 M NaBH4; Sigma). The products obtained were analyzed by agarose gel electrophoresis. beta -Eliminated materials were submitted to digestion with chondroitinase ABC from Proteous vulgaris (Seikagaku, Kogyo Co, Tokyo, Japan), heparitinases from Flavobacterium heparinum (45), or all enzymes and analyzed by agarose gel electrophoresis.


RESULTS

Sodium [35S] Sulfate Labeling of alpha beta 1 Dimer Integrin

Because integrins are substrates for several different post-translational modifications, we decided to determine whether they could function as substrates for sulfation. We decided to address this question using [35S]sulfate labeling of the cells, immunoprecipitation, and blotting experiments. As shown in Fig. 1, Mel-85 cells in culture efficiently incorporate [35S]sulfate. The cell lysate was submitted to immunoprecipitation with a monoclonal antibody against the beta 1 integrin subunit, and a [35S]sulfated alpha beta 1 integrin molecule dimer was detected. This suggests that beta 1 integrin is a substrate for post-translational sulfation.


Fig. 1. There is a sulfated alpha beta 1 integrin in Mel-85 cells. 35S-Labeled Mel-85 cell extract (lane 1) was immunoprecipitated by a specific monoclonal antibody to the beta 1 integrin subunit (lanes 3 and 5). As a negative control, the same extract was precipitated by normal mouse serum (lanes 2 and 4). Immunoprecipitates were separated by 7.5% SDS-PAGE, transferred onto a nitrocellulose membrane that was exposed to an x-ray film (lanes 2 and 3), or exposed to a rabbit polyclonal antiserum against the beta 1 integrin subunit (lanes 4 and 5). Open arrow, pre-beta 1 integrin subunit; closed arrow, beta 1 integrin subunit; arrowhead, alpha  subunit. Molecular mass markers are on the left.
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alpha 5beta 1 Is the Only Sulfated Integrin in Mel-85 Cells

After the demonstration that alpha beta 1 dimer integrin is a sulfated molecule, our next experimental procedure was to identify the alpha  subunit complementing the beta 1 subunit in this particular integrin heterodimer. To investigate this, Mel-85 cells were surface radiolabeled with [125I]iodine by the lactoperoxidase method or metabolically labeled with [35S]sulfate. Both cell lysates were immunoprecipitated with antibodies against different integrin subunits. We can see in the [125I]iodine-labeled immunoprecipitates (Fig. 2A) the presence of beta 1, alpha 2, alpha 3, alpha 4, alpha 5, alpha 7, and probably alpha 1 subunit, a 200-kDa signal (Fig. 2A, lane 1) that could be precipitated with the beta 1 subunit. Neither cell flow cytometry nor immunoprecipitation showed detectable levels of the alpha 6 integrin subunit in Mel-85 cells (data not shown). Interestingly, Fig. 2B shows that only alpha 5 and the corresponding beta 1 subunit are sulfated. These findings suggested that alpha 5beta 1 integrin is a facultative sulfated beta 1 integrin molecule because none of the other beta 1 integrin molecules incorporated [35S]sulfate.


Fig. 2. The alpha 5beta 1 integrin is a facultative sulfated molecule. Mel-85 cell extracts of cell surface [125I]iodinated (A) or metabolically labeled with sodium [35S]sulfate (B) were immunoprecipitated with monoclonal antibodies to specific integrin subunits. beta 1 subunit (lanes 1), alpha 2 subunit (lanes 2), alpha 3 subunit (lanes 3), alpha 4 subunit (lanes 4), alpha 5 subunit (lanes 5), and a polyclonal antibody against the alpha 7 subunit (lanes 6). Immunoprecipitates were separated by 7.5% SDS-PAGE and transferred onto nitrocellulose membranes that were exposed to x-ray films. C, detergent extracts from Mel-85 cells metabolically labeled with [35S]sulfate were immunoprecipitated by monoclonal antibodies against the alpha 5 integrin subunit (lane 1), beta 1 integrin subunit (lane 3) and by a rabbit polyclonal antibody against alpha 5beta 1 integrin (lane 5) or normal mouse serum (lanes 2 and 4) and normal rabbit serum (lane 6) as negative controls. The immunoprecipitates were reduced by a beta -mercaptoethanol containing buffer, separated by 7.5% SDS-PAGE, and electrotransferred to a nitrocellulose membrane that was exposed to an x-ray film. D, cell lysate from [35S]sulfate-labeled Mel-85 cells were chromatographed on a bovine serum albumin-Sepharose column (lanes 1 and 3) or a fibronectin-Sepharose column (lanes 2 and 4). EDTA-eluted materials were separated by 7.5% SDS-PAGE and transferred onto a nitrocellulose membrane that was exposed to an x-ray film (lanes 1 and 2), or the membrane was treated with a specific rabbit polyclonal antiserum against the beta 1 integrin subunit (lanes 3 and 4). The arrow represents the beta 1 integrin subunit, and the arrowhead represents the alpha 5 integrin subunit. Molecular mass markers are on the left.
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It is known that after reduction of the disulfide bonds by beta -mercaptoethanol (chemical reduction), the alpha 5 integrin subunit comigrates with the beta 1 subunit (36). The immunoprecipitates obtained using monoclonal antibodies to beta 1 and alpha 5 integrin subunits or a polyclonal antibody against the alpha 5beta 1 integrin dimer from a [35S]sulfate-labeled Mel-85 cell lysate were then subject to chemical reduction. As shown in Fig. 2C, after chemical reduction the immunoprecipitates reveal just one band in the gel, confirming that alpha 5beta 1 integrin is a sulfated molecule. It is also known that the alpha 5beta 1 integrin has fibronectin as the only ECM ligand (27, 46). Fig. 2D shows that after elution from a fibronectin affinity chromatography alpha 5beta 1 integrin is detected as a sulfated molecule.

alpha 5beta 1 Integrin Is the Only Sulfated Molecule; No Other Sulfated Molecule Co-precipitates with Integrin

Data in the literature describe proteoglycans as alpha 5beta 1 integrin-associated molecules that complement the requirements involved in cell adhesion to fibronectin. In addition, in melanoma cells a heparan sulfate proteoglycan of 150/175 kDa has been described to bind fibronectin (19, 22, 47, 48). We cannot therefore discard the possibility of a physical association between a third molecule that comigrates with alpha beta integrin subunits, masking the sulfated signals in the autoradiograms. To rule out this possibility the same [35S]sulfate-labeled Mel-85 cell extract was again immunoprecipitated by specific monoclonal antibodies to alpha 5 and beta 1 integrin subunits and now submitted to a two-dimensional electrophoresis (Fig. 3, A and B). We can observe that the immunoprecipitation reactions using either anti-beta 1 antibody or anti-alpha 5 antibody show only a sulfated signal of alpha 5beta 1 dimer.


Fig. 3. No other sulfated molecule co-precipitates with alpha 5beta 1 integrin. Sulfate groups in alpha 5beta 1 integrin are covalently linked. Cell extracts from [35S]sulfate-labeled Mel-85 cells were immunoprecipitated by monoclonal antibodies against the beta 1 integrin subunit (A) or the alpha 5 integrin subunit (B); the precipitates were separated by two-dimensional electrophoresis (isoelectric focusing and 7.5% SDS-PAGE) and electrotransferred onto nitrocellulose membranes that were exposed to x-ray film. The arrows represents the beta 1 integrin subunit, and the arrowheads represents the alpha 5 subunit. pH gradient is shown on the top. C, immunoprecipitates obtained using monoclonal antibodies against alpha 5 integrin subunit from a Mel-85 cell extract labeled as described above were incubated for 2 h in the presence of 4 M NaCl (lane 1), 4 M MgCl2 (lane 2), 8 M urea (lane 3), and 6 M guanidine HCl (lane 4). After elution and dialysis, precipitates were separated by 7.5% SDS-PAGE and transferred onto nitrocellulose strips that were exposed to x-ray films. Molecular mass standards are on the left.
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To corroborate the findings described above and demonstrate that the sulfate groups in alpha 5beta 1 integrin are covalently linked and not adsorbed to this molecule or to the beads during immunoprecipitation, this integrin was immunoprecipitated from a [35S]sulfate-labeled Mel-85 cell. After washing with phosphate-buffered saline, the beads were submitted to different conditions of elution such as high ionic strength (4 M NaCl) and chaotrophic agents (6 M guanidine HCl, 4 M MgCl2, 8 M urea). After boiling, the precipitates were dialyzed against water, submitted to 7.5% SDS-PAGE, and transferred onto nitrocellulose filters, which were then exposed to an x-ray film (Fig. 3C). These results show that the alpha 5beta 1 integrin bears covalently linked sulfate groups.

alpha 5beta 1 Integrin Is a Hybrid Proteoglycan

Proteoglycans represent the best characterized sulfated molecules containing serine-linked sulfated GAG chains as a result of post-translational modifications of the protein core (2, 7, 10). To determine the site of sulfate substitution in the alpha 5beta 1 integrin dimer, [35S]sulfate-labeled Mel-85 cell lysate was immunoprecipitated using antibody against the alpha 5 subunit and subjected to beta -elimination. This procedure cleaves GAG chains from the protein core. As shown in Fig. 4A (lane 2), alpha 5beta 1 integrin after beta -elimination showed two sulfated bands that comigrate electrophoretically with chondroitin and heparan sulfate standards. An identical result was obtained when [35S]sulfated alpha 5beta 1 integrin was submitted to proteinase-K digestion (a serine protease of broad specificity) (Fig. 4A, lane 3). These experiments suggest that alpha 5beta 1 integrin is a hybrid chondroitin/heparan sulfate proteoglycan. This was further investigated by degradation of the beta -eliminated material from alpha 5beta 1 integrin with specific enzymes that degrade chondroitin sulfate (chondroitinase ABC) and heparan sulfate (heparitinases type I and II). We can see that under these conditions, both sulfated bands were completely degraded by the specific enzymes (Fig. 4B), demonstrating that alpha 5beta 1 integrin is in fact a hybrid chondroitin/heparan sulfate proteoglycan.


Fig. 4. The alpha 5beta 1 integrin is a hybrid proteoglycan. A, lysates from Mel-85 cells labeled with sodium [35S]sulfate were immunoprecipitated with a monoclonal antibody to the alpha 5 integrin subunit. The precipitates were submitted to beta -elimination reaction (lane 2) or digested with proteinase-K (lane 3), and the obtained materials were submitted to agarose gel electrophoresis in 0.05 M 1.3 diaminopropane acetate buffer, pH 9.0. GAGs were precipitated in the gel with 0.1% cetavlon. After drying and staining, the gel was exposed to x-ray film. Lane 1 represents glycosaminoglycan standards. CS, chondroitin sulfate; DS, dermatan sulfate; HS, heparan sulfate. B, 35S-labeled alpha 5beta 1 integrin obtained as above was submitted to beta -elimination reaction, and the obtained GAG chains were digested with chondroitinase ABC (lane 3), heparitinases I and II (lane 4), or a mixture of the enzymes (lane 5). Lane 2 represents beta -eliminated material before enzymic digestion, and lane 1 represents glycosaminoglycan standards as above. The incubation mixtures were analyzed by agarose gel electrophoresis and were dried and exposed to x-ray film.
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Both alpha 5 and beta 1 Integrin Subunits Have Chondroitin and Heparan Sulfate Chains

Because in Mel-85 cells alpha 5 and beta 1 subunits of integrin contain sulfate, our next goal was to determine the specific pattern of glycosylation, that is, to which subunit chondroitin and heparan sulfates are linked. Two complementary approaches based on immunologic specificities were used. Fist, alpha 5beta 1 integrin was immunoprecipitated from a 35S-labeled Mel-85 cell extract as described above. After separation by polyacrylamide gel electrophoresis, the immunoprecipitate was exposed to an x-ray film, blotted, and reacted with a monoclonal antibody specific for chondroitin sulfate chains (Fig. 5A). We can see that both integrin subunits bear chondroitin sulfate chains. Also, Mel-85 cell lysate was immunoprecipitated with a anti-chondroitin sulfate monoclonal antibody and blotted with a polyclonal antiserum against the beta 1 integrin subunit (Fig. 5B). Interestingly, we can see that only the 116-kDa beta 1 integrin subunit, which corresponds to the completely glycosylated form, has chondroitin sulfate chains. In contrast, the pre-beta 1 integrin chain (100 kDa), which corresponds to a high mannose structure, has no chondroitin sulfate chains. Pre-beta 1 integrin shows the glycosylation profile of a protein that has not crossed the Golgi. Because synthesis of GAG occurs in the Golgi, the absence of chondroitin sulfate in the pre-beta 1 integrin should be expected (49). This finding was also substantiated by results shown in Fig. 1 in which the pre-beta 1 integrin subunit, although coprecipitated, is not sulfated. These results demonstrate that the integrin dimer alpha 5beta 1 is a proteoglycan and that in Mel-85 cells both integrin subunits have covalently linked chondrotin sulfate chains.


Fig. 5. Both alpha 5 and beta 1 integrin subunits have chondroitin and heparan sulfate. A, alpha 5beta 1 integrin from [35S]sulfate-labeled Mel-85 cell lysate was immunoprecipitated using a monoclonal antibody to the alpha 5 integrin subunit. The immunoprecipitates were separated by electrophoresis in 7.5% SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was exposed to x-ray film (lane 1) and reacted with a monoclonal antibody against chondroitin sulfate chains (lane 2) or normal mouse serum (lane 3). The arrow shows the position of beta 1 integrin subunit, and the arrowhead shows the alpha 5 integrin subunit. B, Mel-85 cell extract was submitted to an immunoprecipitation reaction using normal mouse serum as a negative control (lane 2) or monoclonal antibody against chondroitin sulfate chains (lane 3). In lane 1 Mel-85 lysate before immunoprecipitation is shown. Immunoprecipitates were separated by 7.5% SDS-PAGE, electrotransferred onto nitrocellulose membranes, and immunoblotted with a rabbit polyclonal antiserum against the beta 1 integrin subunit. The open arrow depicts the position of pre-beta 1 integrin subunit, and the closed arrow depicts complex glycosylated beta 1 integrin subunits. Molecular mass markers are on the left. C, [35S]sulfate-labeled Mel-85 cell lysate was depleted of alpha 5beta 1 integrin by successive immunoprecipitation reactions using an anti-alpha 5 integrin subunit monoclonal antibody. The immunoprecipitates were electrophoresed in preparative 7.5% SDS-PAGE under nonreducing conditions using prestained molecular mass standards. Separated alpha 5 and beta 1 integrin subunits were excised and eluted from polyacrylamide gels and beta -eliminated as described under "Experimental Procedures." beta -Eliminated materials from alpha 5 or beta 1 subunits (lane 2) were treated with chondroitinase ABC (lane 3), heparitinases I and II (lane 4), and a mixture of the enzymes (lane 5) and electrophoresed in an agarose gel as described in the legend to Fig. 4. Lane 1 shows standard glycosaminoglycan. CS, chondroitin sulfate; DS, dermatan sulfate; HS, heparan sulfate.
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As a second approach we have used successive immunoprecipitation reactions to isolate alpha 5beta 1 integrin from a 35S-labeled Mel-85 cell lysate. The purified alpha 5beta 1 integrin was submitted to preparative polyacrylamide gel electrophoresis. Using an autoradiogram of the gel and prestained molecular mass standards as guides, separated alpha 5 and beta 1 subunits were removed from the gel and subjected to beta -elimination to obtain GAG free chains. These chains were analyzed by agarose gel electrophoresis before and after treatment with chondroitinase ABC, heparitinases, and a mixture of these enzymes (Fig. 5C). The result obtained from this last set of experiments support the concept that alpha 5beta 1 integrin is a proteoglycan. It shows that both subunits contain chondroitin sulfate and heparan sulfate, thereby demonstrating that alpha 5beta 1 integrin is a hybrid chondroitin/heparan sulfate proteoglycan.

Sulfate Incorporation in alpha 5 Integrins Is a Conserved Phenomenon

Because all experiments described so far were performed using the human melanoma cell line Mel-85, which has a neuro-ectodermic origin, we decided to investigate whether this alpha 5beta 1 integrin post-translational modification was also present in cell lines of endodermic (HCT-8 cells) and mesodermic (MG-63 cells) origin. Cells were labeled with [35S]sulfate, and lysates were immunoprecipitated with monoclonal antibodies against the alpha 5 integrin subunit and analyzed by SDS-PAGE followed by electroblotting onto nitrocellulose. A polyclonal antibody against the beta 1 integrin subunit (Fig. 6A) was used to detect the integrin. We can see that both cells display the [35S]sulfate incorporation into the alpha 5 integrin subunit. The results indicate that GAG substitution of alpha 5beta 1 integrin has been conserved, suggesting its biological significance. However, in the case of the MG-63 cells, only the alpha 5 subunit was labeled; the beta 1 subunit found in MG-63 cells did not contain sulfate. To corroborate the results described in Fig. 6A and provide more evidence that the glycosylation of integrin alpha 5beta 1 integrin as a proteoglycan is maintained in different tissues, we submitted [35S]sulfate-labeled alpha 5beta 1 integrin obtained from HCT-8 and MG-63 cell extracts to a beta -elimination reaction. Products were analyzed by agarose gel electrophoresis. As shown in Fig. 6B, we can see that heparan and chondroitin sulfates are present in alpha 5beta 1 integrins of endodermic and mesodermic origins, as observed for neuro-ectodermic cells. The results not only confirm the conservative proteoglycan nature of alpha 5beta 1 integrin from different origins but also indicate a similar glycosylation pattern.


Fig. 6. Sulfate incorporation in alpha 5 integrins is a conservative phenomenon. A, lysates from [35S]sulfate-labeled HCT-8 cells (lanes 1 and 2) and MG-63 cells (lanes 3 and 4) were immunoprecipitated using a anti-alpha 5 integrin monoclonal antibody. Following 7.5% SDS-PAGE separation and electrotransference onto nitrocellulose membrane, materials were exposed to x-ray films (lanes 1 and 3) or reacted with a rabbit polyclonal anti-beta 1 integrin subunit (lanes 2 and 4). The arrowhead points to the beta 1 integrin position, and the arrow shows the alpha 5 subunit. Molecular mass markers are on the left. B, alpha 5beta 1 integrin obtained from [35S]sulfate-labeled extracts of HCT-8 cells or MG-63 cells were subjected to beta -elimination to obtain the free GAG chains. These chains were subjected to agarose gel electrophoresis, and the gel was dried an exposed to an x-ray film as described in the legend to Fig. 4. Lane 1, standard glycosaminoglycans. CS, chondroitin sulfate; DS, dermatan sulfate; HS, heparan sulfate. Lane 2, glycosaminoglycans from alpha 5beta 1 integrin of HCT-8 cells. Lane 3, glycosaminoglycans from alpha 5beta 1 integrin of MG-63 cells.
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DISCUSSION

Working with the human melanoma cell line Mel-85, we have described alpha 5beta 1 integrin as a hybrid chondroitin/heparan sulfate proteoglycan. Based on immunoprecipitation reactions from cell lysates that were cell surface labeled with [125I]iodine or metabolically labeled with [35S]sulfate, we were able to detect alpha 5beta 1 integrin as the only sulfated integrin compared with other alpha (s)beta 1 heterodimers present in Mel-85 cells. Sulfation of alpha 5beta 1 integrin was confirmed not only by immunological methods but also by fibronectin affinity chromatography, two-dimensional electrophoresis, and reduction of disulfide bonds of the alpha 5beta 1 heterodimer leading to comigration of both alpha 5 and beta 1 integrin subunits, characteristic of this integrin as described (36). Based on different procedures such as chemical deglycosylation by beta -elimination, proteinase-K digestion, immunological methods, and susceptibility to chondroitinase ABC and heparitinases, we were able to confirm this integrin as a proteoglycan. These results raise the important question of which mechanisms determine alpha 5beta 1 as the only sulfated integrin. Why are beta 1 subunits not sulfated in other alpha beta 1 heterodimers? The existence of alternative splicing for the beta 1 integrin subunit as described (50) (reviewed in Ref. 27) could explain in part such differences. However, because glycosylation of cell surface proteoglycans is restricted to extracellular domains (2) and the beta 1 integrin subunit has only alternatively spliced cytoplasmic domains (27) this mechanism does not explain our findings. Oligomerization of alpha beta integrin heterodimers is an event that occurs during transit through the endoplasmic reticulum (28) and precedes glycosaminoglycan biosynthesis, which occurs during transit through the Golgi (2). Perhaps the best explanation for the part-time proteoglycan nature of alpha 5beta 1 integrin is that the conformation of the heterodimer exposes the serine residues that are acceptors for the GAG chains, which does not happen with other alpha beta 1 heterodimers. This conformational hypothesis is consistent with the lack of a consensus sequence for proteoglycan biosynthesis initiation (1, 2) and by the experiments performed with decorin, a proteoglycan in which the primary structure of the protein core surrounding the sugar acceptor serine residue can be changed without appreciable modification in the glycosaminoglycan (51). Considering the findings described above, it is possible to assume that the same conformational folding of alpha 5beta 1 that makes this integrin capable of recognizing the RGD peptide only in fibronectin among several other ECM molecules (52) is also responsible for a specific GAG synthesis that complements the molecular requirements involved in the interaction of this integrin with fibronectin.

The possibility that other integrins can also be sulfated is not ruled out by the present study because we could not detect alpha 6beta 1 integrin in Mel-85 cells. This is an integrin that binds laminin, a molecule with a GAG binding domain spatially close to the E8 domain corresponding to the alpha 6beta 1 integrin binding site (53). Furthermore, the structural relationship of the alpha 5 chain with alpha IIb and alpha v (reviewed in Ref. 27) could suggest other alpha beta integrin heterodimers as putative acceptors for GAG addition. Interestingly, Hayashi, Madri, and Yurchenco (54) have shown that endothelial cell interaction with the basement membrane proteoglycan (perlecan) occurs between the core protein of perlecan and beta 1 and beta 3 integrins, an interaction partially RGD-independent and modulated by GAGs. The beta 1 integrin heterodimer involved in this adhesion resembles the alpha 5beta 1 molecule and the beta 3 integrin, the alpha vbeta 3 vitronectin receptor (54).

The present study suggests for the first time that integrins such as alpha 5beta 1 may have two extracellular binding sites that play a role in fibronectin binding. Previous studies have implicated a specific involvement of the heparin binding site of fibronectin with cell adhesion. These data were based on the fact that the purified fibronectin fragment containing only the heparin binding domain without the RGD peptide can promote adhesion in several different cell models (21, 23). Because our work describes alpha 5beta 1 integrin as a part-time proteoglycan compared with other alpha beta 1 dimers, we can postulate that the fibronectin-alpha 5beta 1 integrin interaction, which occurs primarily through the RGD peptide in fibronectin, is complemented and stabilized by the secondary interactions of alpha 5beta 1 chondroitin or heparan sulfate chains with the fibronectin heparin binding domains. The possibility that alpha 5beta 1 integrin, an integrin that binds only fibronectin, has chondroitin and heparan sulfate chains interacting with the fibronectin heparin binding domains is suggested by the facts that during ECM assembly the fibronectin heparin binding domain can also bind chondroitin sulfate or dermatan sulfate proteoglycans (10) and that soluble proteoglycans can inhibit cell adhesion to fibronectin (reviewed in Ref. 22) and by the existence of nonintegrin fibronectin receptors like CD44 (a chondroitin sulfate proteoglycan) and a heparan sulfate proteoglycan (19, 48) as well as by the recent finding that monoclonal antibodies raised against the fibronectin heparin binding domain (Hep II/IIICS) inhibit cell adhesion and also partially inhibit integrin binding to fibronectin (55). A model is postulated in which the RGD and heparin binding sites in fibronectin, although linearly separated, are spatially close due to fibronectin folding. It is thus possible to assume that cell surface proteoglycans and integrin cooperativity during cell adhesion can really be achieved in the case of alpha 5beta 1 integrin by two binding sites in the integrin molecule that bind RGD peptide and GAG binding domains in fibronectin.


FOOTNOTES

*   This work was supported by grants from Conselho Nacional de Pesquisas and Fundação de Amparo à Pesquisa do estado de São Paulo.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.
   To whom correspondence should be addressed: Ludwig Inst. for Cancer Research, R. Prof. Antonio Prudente, 109, 4 A, cep: 01509-010, São Paulo, SP, Brazil.
1   The abbreviations used are: GAG, glycosaminoglycan chains; RGD, peptide Arg-Gly-Asp; PAGE, polyacrylamide gel electrophoresis; ECM, extracellular matrix.
2   K. Yamada, personal communication.

ACKNOWLEDGEMENTS

We thank Drs. E. Engvall, K. M. Yamada, M. E. Hemler, M. M. Brentani, R. R. Pasqualini, S. Carrel, and S. J. Kaufman for the gifts of reagents described under "Experimental Procedures." C. P. Dietrich, V. Buonassisi, and P. Colburn are gratefully acknowledged for revision of the manuscript.


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