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Post-translational Modifications of α5β1 Integrin by Glycosaminoglycan Chains
1997; Elsevier BV; Volume: 272; Issue: 19 Linguagem: Inglês
10.1074/jbc.272.19.12529
ISSN1083-351X
AutoresSílvio Sanches Veiga, Maria Carolina Elias, Waldemiro Gremski, Marimélia Porcionatto, Roseli da Silva, Helena B. Nader, Helena Brentani,
Tópico(s)Proteoglycans and glycosaminoglycans research
ResumoCell-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 α5β1 integrin. Using [125I]iodine cell surface labeling or metabolic radiolabeling with sodium [35S]sulfate, we identified α5β1 integrin as the only sulfated integrin among β1 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 α5β1 integrin sulfation was evidenced by its resistance to treatments with high ionic, chaotrophic, and denaturing agents such as 4 m NaCl, 4 mMgCl2, 8 m urea, and 6 m guanidine HCl. Based on deglycosylation procedures as chemical β-elimination, proteinase K digestion, and susceptibility to glycosaminoglycan lyases (chondroitinase ABC and heparitinases I and II), it was demonstrated that the α5β1 heterodimer and α5 and β1 integrin subunits were proteoglycans. The importance of α5β1sulfation was strengthened by the finding that this molecule is also sulfated in MG-63 (human osteosarcoma) and HCT-8 (human colon adenocarcinoma) cells. 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 α5β1 integrin. Using [125I]iodine cell surface labeling or metabolic radiolabeling with sodium [35S]sulfate, we identified α5β1 integrin as the only sulfated integrin among β1 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 α5β1 integrin sulfation was evidenced by its resistance to treatments with high ionic, chaotrophic, and denaturing agents such as 4 m NaCl, 4 mMgCl2, 8 m urea, and 6 m guanidine HCl. Based on deglycosylation procedures as chemical β-elimination, proteinase K digestion, and susceptibility to glycosaminoglycan lyases (chondroitinase ABC and heparitinases I and II), it was demonstrated that the α5β1 heterodimer and α5 and β1 integrin subunits were proteoglycans. The importance of α5β1sulfation was strengthened by the finding that this molecule is also sulfated in MG-63 (human osteosarcoma) and HCT-8 (human colon adenocarcinoma) cells. Proteoglycans are complex molecules formed by a core protein to which one or more glycosaminoglycan (GAG) 1The abbreviations used are: GAG, glycosaminoglycan chains; RGD, peptide Arg-Gly-Asp; PAGE, polyacrylamide gel electrophoresis; ECM, extracellular matrix. 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 asN- and O-glycosylation increase the complexity of these molecules (for review see Refs. 1Jackson R.L. Bush S.J. Cardin A.D. Physiol. Rev. 1991; 2: 481-485Crossref Scopus (961) Google Scholar and 2Kjellén L. Lindahl U. Annu. Rev. Biochem. 1991; 60: 443-475Crossref PubMed Scopus (1676) Google Scholar). The biological functions of proteoglycans are numerous. They have been involved in several biological effects (1Jackson R.L. Bush S.J. Cardin A.D. Physiol. Rev. 1991; 2: 481-485Crossref Scopus (961) Google Scholar, 3Gallagher J.T. Curr. Opin. Cell Biol. 1989; 1: 1201-1213Crossref PubMed Scopus (212) Google Scholar, 4Wight T.N. Heinegard D.K. Hascall V.C. Hay E.D. Cell Biology of Extracellular Matrix. 2nd Ed. Plenum Press, New York1991: 45-78Crossref Google Scholar, 5Bernfield M. Kokenyesi R. Kato M. Hinkes M.T. Spring J. Gallo R.L. Lose E.J. Annu. Rev. Cell Biol. 1992; 8: 365-393Crossref PubMed Scopus (967) Google Scholar), such as extracellular matrix (ECM) assembly (6Yurchenco P. Schittny J. FASEB J. 1990; 4: 1577-1590Crossref PubMed Scopus (788) Google Scholar) and cell surface-ECM receptors for growth factors and hormones (2Kjellén L. Lindahl U. Annu. Rev. Biochem. 1991; 60: 443-475Crossref PubMed Scopus (1676) Google Scholar, 5Bernfield M. Kokenyesi R. Kato M. Hinkes M.T. Spring J. Gallo R.L. Lose E.J. Annu. Rev. Cell Biol. 1992; 8: 365-393Crossref PubMed Scopus (967) Google Scholar, 7Yanagishita M. Hascall V.C. J. Biol. Chem. 1992; 267: 9451-9454Abstract Full Text PDF PubMed Google Scholar) or have had a role in biological processes such as cell-cell recognition (8Dietrich C.P. Armelin H.A. Biochem. Biophys. Res. Commun. 1978; 84: 794-801Crossref PubMed Scopus (31) Google Scholar) and control of cell growth (9Porcionatto M.A. Pinto C.R.M. Dietrich C.P. Nader H.B. Braz. J. Med. Biol. Res. 1994; 27: 2185-2190PubMed Google Scholar). The fact that several ECM proteins, such as fibronectin (10Ruoslahti E. Annu. Rev. Cell Biol. 1988; 4: 229-255Crossref PubMed Scopus (552) Google Scholar), laminin (11Skubitz A.P.N. McCarthy J.B. Charonis A.S. Furcht L.T. J. Biol. Chem. 1988; 263: 4861-4868Abstract Full Text PDF PubMed Google Scholar), thrombospondin (12Sun X. Mosher D.F. Rapraeger A. J. Biol. Chem. 1989; 264: 2885-2889Abstract Full Text PDF PubMed Google Scholar), vitronectin (13Lane D.A. Flynn A.M. Pejler G. Lindahl U. Choay J. Preissner K. J. Biol. Chem. 1987; 262: 16343-16348Abstract Full Text PDF PubMed Google Scholar), type IV collagen (14LeBaron R.G. Höök A. Esko J.D. Gay S. Höök M. J. Biol. Chem. 1989; 264: 7950-7956Abstract Full Text PDF PubMed Google Scholar), and tenascin (15Marton L.S. Gulcher J.R. Stefansson K. J. Biol. Chem. 1989; 264: 13145-13149Abstract Full Text PDF PubMed Google Scholar), 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 (5Bernfield M. Kokenyesi R. Kato M. Hinkes M.T. Spring J. Gallo R.L. Lose E.J. Annu. Rev. Cell Biol. 1992; 8: 365-393Crossref PubMed Scopus (967) Google Scholar), and tenascin (16Salmivirta M. Elenius K. Vainio S. Hofer U. Chiquet-Ehresmann R. Thesleff I. Jalkanen M. J. Biol. Chem. 1991; 266: 7733-7739Abstract Full Text PDF PubMed Google Scholar); the heparan sulfate proteoglycan of Schwann cells binds laminin (17Carey D.J. Crumbling D.M. Stahl R.C. Evans D.M. J. Biol Chem. 1990; 265: 20627-20633Abstract Full Text PDF PubMed Google Scholar); a cell surface chondroitin sulfate proteoglycan is apparently involved in cell adhesion to laminin (18Elias M.C.Q.B. Veiga S.S. Gremski W. Brentani R.R. Braz. J. Med. Biol. Res. 1996; 29: 1247-1249PubMed Google Scholar); and a cell surface phosphatidyl inositol-anchored heparan sulfate proteoglycan mediates melanoma cell adhesion to fibronectin (19Drake S. Klein D.J. Mickelson D.J. Oegema T.R. Furcht L.T. McCarthy J.B. J. Cell Biol. 1992; 117: 1331-1342Crossref PubMed Scopus (68) Google Scholar). Strong corroboration for these proteoglycan-ECM interactions comes from the presence of a heparan sulfate proteoglycan that co-localizes with β1 integrins as a widespread component of focal adhesion (20Woods A. Couchman J.R. Mol. Biol. Cell. 1994; 5: 183-192Crossref PubMed Scopus (278) Google Scholar). 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 (21McCarthy J.B. Chelberg M.K. Mickelson D.J. Furcht L.T. Biochemistry. 1988; 27: 1380-1388Crossref PubMed Scopus (109) Google Scholar, 22Ruoslahti E. J. Biol. Chem. 1989; 264: 13369-13372Abstract Full Text PDF PubMed Google Scholar, 23Haugen D.K. McCarthy J.B. Skubitz A.P.N. Furcht L.T. Letournean P.C. J. Cell Biol. 1990; 106: 1365-1373Google Scholar). 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 (24LeBaron R.G. Esko J.D. Woods A. Johanson S. Höök M. J. Cell Biol. 1988; 106: 945-952Crossref PubMed Scopus (107) Google Scholar, 25Couchman J.R. Austria R. Woods A. Hughes R.C. J. Cell Physiol. 1988; 136: 226-236Crossref PubMed Scopus (21) Google Scholar). The best studied receptors for fibronectin that bear adhesiveness and focal adhesion plaque formation are integrins that are α/β heterodimers widely expressed by almost all animal cells (26Albelda S.M. Buck C.A. FASEB J. 1990; 4: 2868-2880Crossref PubMed Scopus (1635) Google Scholar, 27Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9011) Google Scholar). 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 (28Akiyama S.K. Yamada S.S. Yamada K.M. J. Biol. Chem. 1989; 264: 18011-18018Abstract Full Text PDF PubMed Google Scholar, 29Zheng M. Fang H. Hakomori S. J. Biol. Chem. 1994; 269: 12325-12331Abstract Full Text PDF PubMed Google Scholar, 30Veiga S.S. Chammas R. Cella N. Brentani R.R. Int. J. Cancer. 1995; 61: 420-424Crossref PubMed Scopus (45) Google Scholar). 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 (31Conforti G.L. Zanetti A. Pasquali-Ronchetti I. Quaglino D. Neyroz P. Dejana E. J. Biol. Chem. 1990; 265: 4011-4019Abstract Full Text PDF PubMed Google Scholar), by divalent metal binding (32Grzesiak J.J. Davis G.E. Kirchhofer D. Pierschbacher M.D. J. Cell Biol. 1992; 117: 1109-1117Crossref PubMed Scopus (127) Google Scholar), 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 (27Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9011) Google Scholar,33Shaw L.M. Kessier J.M. Mercurio A.M. J. Cell Biol. 1990; 110: 2167-2174Crossref PubMed Scopus (209) Google Scholar). In the present study, we characterize α5β1integrin 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. Human fibronectin was purified from fresh plasma (obtained from Hospital A. C. Camargo, São Paulo, Brazil) by gelatin affinity chromatography as described (34Engvall E. Ruoslahti E. Int. J. Cancer. 1977; 20: 1-5Crossref PubMed Scopus (1429) Google Scholar). Monoclonal antibody A-1A5 that recognizes the β1 integrin subunit (35Hemler M.E. Jacobson J.G. Strominger J.L. J. Biol. Chem. 1985; 260: 15246-15252Abstract Full Text PDF PubMed Google Scholar) and B-5G10 that reacts with the α4 integrin subunit (36Hemler M.E. Huang C. Takada Y. Schwarz L. Strominger J.L. Clabby M.L. J. Biol. Chem. 1987; 262: 11478-11485Abstract Full Text PDF PubMed Google Scholar) were provided by Dr. Martin E. Hemler (Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA). Monoclonal antibody II-F5, which specifically recognizes the α3 integrin subunit (37Weitzman J.B. Pasqualini R. Takada Y. Hemler M.E. J. Biol. Chem. 1993; 268: 8651-8657Abstract Full Text PDF PubMed Google Scholar), was a gift from Dr. Renata Pasqualini (The Burnham Institute, San Diego, CA). Monoclonal antibodies against α2 integrin subunit CLB-thromb/4 and α5integrin 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 α7 integrin subunit (38Song W.K. Wang W. Sato H. Bielser D.A. Kauffman S.J. J. Cell Sci. 1993; 106: 1139-1152Crossref PubMed Google Scholar) 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 α5β1 integrin molecule (RB3847) that in immunoblotting reacts only with the β1 integrin subunit 2K. Yamada, personal communication. 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. 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. Cell surface expression of β1 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 (39Morrison M. Methods Enzymol. 1980; 70: 214-220Crossref PubMed Scopus (91) Google Scholar). 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 mmphenylmethanesulfonyl 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 (40Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207159) Google Scholar) followed by electrotransference onto nitrocellulose membranes (41Towbin H. Staehilin T. Gorden G. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44915) Google Scholar) 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-α5integrin antibody. The [35S]sulfate-labeled immunoprecipitates obtained with anti-α5 integrin monoclonal antibody were incubated with 4 m NaCl, 4m MgCl2, 6 m guanidine HCl, and 8m urea for 2 h at 37 °C. Mixtures were then boiled for 5 min, and α5β1 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 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 (30Veiga S.S. Chammas R. Cella N. Brentani R.R. Int. J. Cancer. 1995; 61: 420-424Crossref PubMed Scopus (45) Google Scholar). SDS gel electrophoresis was performed as described (40Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207159) Google Scholar). 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 (41Towbin H. Staehilin T. Gorden G. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44915) Google Scholar). Molecular mass markers (myosin, 205 kDa; β-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 (42O' Farrel P.Z. Goodman P. O'Farrel P.H. Cell. 1977; 12: 1133-1142Abstract Full Text PDF PubMed Scopus (2583) Google Scholar) 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 (43Dietrich C.P. Dietrich S.M.C. Anal. Biochem. 1976; 70: 645-647Crossref PubMed Scopus (251) Google Scholar). 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 (44Dietrich C.P. Nader H.B. Straus A.H. Biochem. Biophys. Res. Commun. 1983; 111: 865-871Crossref PubMed Scopus (94) Google Scholar), dermatan sulfate from pig skin, and chondroitin sulfate from shark cartilage (Seikagaku, Kogyo Co., Tokyo, Japan). To study the specific pattern of glycosylation of α5 and β1 integrin subunits, [35S]sulfate-labeled Mel-85 cell lysate was immunoprecipitated using a monoclonal antibody against the α5 integrin subunit as already described, and the precipitate was submitted to a preparative 7.5% SDS-PAGE under nonreducing conditions using prestained β-galactosidase (116 kDa) that comigrates with the β1 integrin subunit as a standard. Autoradiography of separated α5 and β1 subunits was done in identical conditions as above and used as a guide. Gel pieces were then cut off in the positions of separated α5 and β1 subunits, and proteins were excised and eluted from the gel by incubation in 50 mmTris-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 α5 and β1 integrin subunits were then submitted to β-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 β1 integrin subunit but not with the denatured α5 integrin subunit) and a monoclonal antibody against chondroitin sulfate chains (CS-56) were performed as described previously (30Veiga S.S. Chammas R. Cella N. Brentani R.R. Int. J. Cancer. 1995; 61: 420-424Crossref PubMed Scopus (45) Google Scholar). The GAG chains from [35S]sulfate-labeled α5β1 integrin, purified by immunoprecipitation using a monoclonal antibody against the α5 integrin subunit, were liberated by digestion of the protein core using excess proteinase-K (50 μg; Sigma) at 58 °C overnight or by a β-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. β-Eliminated materials were submitted to digestion with chondroitinase ABC from Proteous vulgaris(Seikagaku, Kogyo Co, Tokyo, Japan), heparitinases fromFlavobacterium heparinum (45Nader H.B. Porcionatto M.A. Moraes C.T. Dietrich C.P. J. Biol. Chem. 1990; 265: 6807-6813Google Scholar), or all enzymes and analyzed by agarose gel electrophoresis. 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 shownin Fig. 1, Mel-85 cells in culture efficiently incorporate [35S]sulfate. The cell lysate was submitted to immunoprecipitation with a monoclonal antibody against the β1 integrin subunit, and a [35S]sulfated αβ1 integrin molecule dimer was detected. This suggests that β1 integrin is a substrate for post-translational sulfation. After the demonstration that αβ1dimer integrin is a sulfated molecule, our next experimental procedure was to identify the α subunit complementing the β1subunit 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.2 A) the presence of β1, α2, α3, α4, α5, α7, and probably α1 subunit, a 200-kDa signal (Fig. 2 A, lane 1) that could be precipitated with the β1 subunit. Neither cell flow cytometry nor immunoprecipitation showed detectable levels of the α6 integrin subunit in Mel-85 cells (data not shown). Interestingly, Fig. 2 B shows that only α5 and the corresponding β1 subunit are sulfated. These findings suggested that α5β1 integrin is a facultative sulfated β1 integrin molecule because none of the other β1 integrin molecules incorporated [35S]sulfate. It is known that after reduction of the disulfide bonds by β-mercaptoethanol (chemical reduction), the α5 integrin subunit comigrates with the β1 subunit (36Hemler M.E. Huang C. Takada Y. Schwarz L. Strominger J.L. Clabby M.L. J. Biol. Chem. 1987; 262: 11478-11485Abstract Full Text PDF PubMed Google Scholar). The immunoprecipitates obtained using monoclonal antibodies to β1 and α5 integrin subunits or a polyclonal antibody against the α5β1 integrin dimer from a [35S]sulfate-labeled Mel-85 cell lysate were then subject to chemical reduction. As shown in Fig. 2 C, after chemical reduction the immunoprecipitates reveal just one band in the gel, confirming that α5β1 integrin is a sulfated molecule. It is also known that the α5β1 integrin has fibronectin as the only ECM ligand (27Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9011) Google Scholar, 46Ruoslahti E. Pierschbacher M.D. Science. 1987; 238: 491-497Crossref PubMed Scopus (3878) Google Scholar). Fig. 2 D shows that after elution from a fibronectin affinity chromatography α5β1integrin is detected as a sulfated molecule. Data in the literature describe proteoglycans as α5β1 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 (19Drake S. Klein D.J. Mickelson D.J. Oegema T.R. Furcht L.T. McCarthy J.B. J. Cell Biol. 1992; 117: 1331-1342Crossref PubMed Scopus (68) Google Scholar, 22Ruoslahti E. J. Biol. Chem. 1989; 264: 13369-13372Abstract Full Text PDF PubMed Google Scholar, 47Woods A. Couchman J.R. J. Cell Sci. 1992; 101: 227-290Google Scholar, 48Damsky C.H. Werb Z. Curr. Opin. Cell Biol. 1992; 4: 772-781Crossref PubMed Scopus (487) Google Scholar). We cannot therefore discard the possibility of a physical association between a third molecule that comigrates with αβ 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 α5 and β1 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-β1antibody or anti-α5 antibody show only a sulfated signal of α5β1 dimer. To corroborate the findings described above and demonstrate that the sulfate groups in α5β1 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 mNaCl) and chaotrophic agents (6 m guanidine HCl, 4m 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. 3 C). These results show that the α5β1 integrin bears covalently linked sulfate groups. Proteoglycans represent the best characterized sulfated molecules containing serine-linked sulfated GAG chains as a result of post-translational modifications of the protein core (2Kjellén L. Lindahl U. Annu. Rev. Biochem. 1991; 60: 443-475Crossref PubMed Scopus (1676) Google Scholar, 7Yanagishita M. Hascall V.C. J. Biol. Chem. 1992; 267: 9451-9454Abstract Full Text PDF PubMed Google Scholar,10Ruoslahti E. Annu. Rev. Cell Biol. 1988; 4: 229-255Crossref PubMed Scopus (552) Google Scholar). To determine the site of sulfate substitution in the α5β1 integrin dimer, [35S]sulfate-labeled Mel-85 cell lysate was immunoprecipitated using antibody against the α5 subunit and subjected to β-elimination. This procedure cleaves GAG chains from the protein core. As shown in Fig. 4 A(lane 2), α5β1 integrin after β-elimination showed two sulfated bands that comigrate electrophoretically with chondroitin and heparan sulfate standards. An identical result was obtained when [35S]sulfated α5β1 integrin was submitted to proteinase-K digestion (a serine protease of broad specificity) (Fig. 4 A,lane 3). These experiments suggest that α5β1 integrin is a hybrid chondroitin/heparan sulfate proteoglycan. This was further investigated by degradation of the β-eliminated material from α5β1 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. 4 B), demonstrating that α5β1 integrin is in fact a hybrid chondroitin/heparan sulfate proteoglycan. Because in Mel-85 cells α5 and β1 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, α5β1integrin 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. 5 A). 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 β1 integrin subunit (Fig. 5 B). Interestingly, we can see that only the 116-kDa β1integrin subunit, which corresponds to the completely glycosylated form, has chondroitin sulfate chains. In contrast, the pre-β1 integrin chain (100 kDa), which corresponds to a high mannose structure, has no chondroitin sulfate chains. Pre-β1 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-β1 integrin should be expected (49Lohmander L.S. Hascall V.C. Yanagishita M. Kuettner K.E. Kimura J.H. Arch. Biochem. Biophys. 1986; 250: 211-227Crossref PubMed Scopus (27) Google Scholar). This finding was also substantiated by results shown in Fig. 1 in which the pre-β1 integrin subunit, although coprecipitated, is not sulfated. These results demonstrate that the integrin dimer α5β1 is a proteoglycan and that in Mel-85 cells both integrin subunits have covalently linked chondrotin sulfate chains. As a second approach we have used successive immunoprecipitation reactions to isolate α5β1 integrin from a35S-labeled Mel-85 cell lysate. The purified α5β1 integrin was submitted to preparative polyacrylamide gel electrophoresis. Using an autoradiogram of the gel and prestained molecular mass standards as guides, separated α5 and β1 subunits were removed from the gel and subjected to β-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. 5 C). The result obtained from this last set of experiments support the concept that α5β1 integrin is a proteoglycan. It shows that both subunits contain chondroitin sulfate and heparan sulfate, thereby demonstrating that α5β1 integrin is a hybrid chondroitin/heparan sulfate proteoglycan. 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 α5β1 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 α5 integrin subunit and analyzed by SDS-PAGE followed by electroblotting onto nitrocellulose. A polyclonal antibody against the β1 integrin subunit (Fig.6 A) was used to detect the integrin. We can see that both cells display the [35S]sulfate incorporation into the α5 integrin subunit. The results indicate that GAG substitution of α5β1integrin has been conserved, suggesting its biological significance. However, in the case of the MG-63 cells, only the α5subunit was labeled; the β1 subunit found in MG-63 cells did not contain sulfate. To corroborate the results described in Fig.6 A and provide more evidence that the glycosylation of integrin α5β1 integrin as a proteoglycan is maintained in different tissues, we submitted [35S]sulfate-labeled α5β1integrin obtained from HCT-8 and MG-63 cell extracts to a β-elimination reaction. Products were analyzed by agarose gel electrophoresis. As shown in Fig. 6 B, we can see that heparan and chondroitin sulfates are present in α5β1 integrins of endodermic and mesodermic origins, as observed for neuro-ectodermic cells. The results not only confirm the conservative proteoglycan nature of α5β1 integrin from different origins but also indicate a similar glycosylation pattern. Working with the human melanoma cell line Mel-85, we have described α5β1 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 α5β1 integrin as the only sulfated integrin compared with other α(s)β1 heterodimers present in Mel-85 cells. Sulfation of α5β1integrin was confirmed not only by immunological methods but also by fibronectin affinity chromatography, two-dimensional electrophoresis, and reduction of disulfide bonds of the α5β1 heterodimer leading to comigration of both α5 and β1 integrin subunits, characteristic of this integrin as described (36Hemler M.E. Huang C. Takada Y. Schwarz L. Strominger J.L. Clabby M.L. J. Biol. Chem. 1987; 262: 11478-11485Abstract Full Text PDF PubMed Google Scholar). Based on different procedures such as chemical deglycosylation by β-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 α5β1 as the only sulfated integrin. Why are β1 subunits not sulfated in other αβ1 heterodimers? The existence of alternative splicing for the β1 integrin subunit as described (50Altruda F. Cervella P. Tarone G. Botta C. Balzac F. Stefanuto G. Silengo L. Gene ( Amst. ). 1990; 95: 261-266Crossref PubMed Scopus (84) Google Scholar) (reviewed in Ref. 27Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9011) Google Scholar) could explain in part such differences. However, because glycosylation of cell surface proteoglycans is restricted to extracellular domains (2Kjellén L. Lindahl U. Annu. Rev. Biochem. 1991; 60: 443-475Crossref PubMed Scopus (1676) Google Scholar) and the β1 integrin subunit has only alternatively spliced cytoplasmic domains (27Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9011) Google Scholar) this mechanism does not explain our findings. Oligomerization of αβ integrin heterodimers is an event that occurs during transit through the endoplasmic reticulum (28Akiyama S.K. Yamada S.S. Yamada K.M. J. Biol. Chem. 1989; 264: 18011-18018Abstract Full Text PDF PubMed Google Scholar) and precedes glycosaminoglycan biosynthesis, which occurs during transit through the Golgi (2Kjellén L. Lindahl U. Annu. Rev. Biochem. 1991; 60: 443-475Crossref PubMed Scopus (1676) Google Scholar). Perhaps the best explanation for the part-time proteoglycan nature of α5β1 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 αβ1heterodimers. This conformational hypothesis is consistent with the lack of a consensus sequence for proteoglycan biosynthesis initiation (1Jackson R.L. Bush S.J. Cardin A.D. Physiol. Rev. 1991; 2: 481-485Crossref Scopus (961) Google Scholar, 2Kjellén L. Lindahl U. Annu. Rev. Biochem. 1991; 60: 443-475Crossref PubMed Scopus (1676) Google Scholar) 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 (51Mann D.M. Yamaguchi Y. Bourdon M.A. Ruoslahti E. J. Biol. Chem. 1990; 265: 5317-5323Abstract Full Text PDF PubMed Google Scholar). Considering the findings described above, it is possible to assume that the same conformational folding of α5β1 that makes this integrin capable of recognizing the RGD peptide only in fibronectin among several other ECM molecules (52Hautanen A. Gailit J. Mann D.M. Ruoslahti E. J. Biol. Chem. 1989; 264: 1437-1442Abstract Full Text PDF PubMed Google Scholar) 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 α6β1 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 α6β1 integrin binding site (53Mercurio A.M. Trends Cell Biol. 1995; 5: 419-423Abstract Full Text PDF PubMed Scopus (200) Google Scholar). Furthermore, the structural relationship of the α5 chain with αIIb and αv (reviewed in Ref. 27Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9011) Google Scholar) could suggest other αβ integrin heterodimers as putative acceptors for GAG addition. Interestingly, Hayashi, Madri, and Yurchenco (54Hayashi K. Madri J.A. Yurchenco P.D. J. Cell Biol. 1992; 119: 945-959Crossref PubMed Scopus (164) Google Scholar) have shown that endothelial cell interaction with the basement membrane proteoglycan (perlecan) occurs between the core protein of perlecan and β1 and β3 integrins, an interaction partially RGD-independent and modulated by GAGs. The β1integrin heterodimer involved in this adhesion resembles the α5β1 molecule and the β3integrin, the αvβ3 vitronectin receptor (54Hayashi K. Madri J.A. Yurchenco P.D. J. Cell Biol. 1992; 119: 945-959Crossref PubMed Scopus (164) Google Scholar). The present study suggests for the first time that integrins such as α5β1 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 (21McCarthy J.B. Chelberg M.K. Mickelson D.J. Furcht L.T. Biochemistry. 1988; 27: 1380-1388Crossref PubMed Scopus (109) Google Scholar, 23Haugen D.K. McCarthy J.B. Skubitz A.P.N. Furcht L.T. Letournean P.C. J. Cell Biol. 1990; 106: 1365-1373Google Scholar). Because our work describes α5β1 integrin as a part-time proteoglycan compared with other αβ1 dimers, we can postulate that the fibronectin-α5β1 integrin interaction, which occurs primarily through the RGD peptide in fibronectin, is complemented and stabilized by the secondary interactions of α5β1 chondroitin or heparan sulfate chains with the fibronectin heparin binding domains. The possibility that α5β1 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 (10Ruoslahti E. Annu. Rev. Cell Biol. 1988; 4: 229-255Crossref PubMed Scopus (552) Google Scholar) and that soluble proteoglycans can inhibit cell adhesion to fibronectin (reviewed in Ref. 22Ruoslahti E. J. Biol. Chem. 1989; 264: 13369-13372Abstract Full Text PDF PubMed Google Scholar) and by the existence of nonintegrin fibronectin receptors like CD44 (a chondroitin sulfate proteoglycan) and a heparan sulfate proteoglycan (19Drake S. Klein D.J. Mickelson D.J. Oegema T.R. Furcht L.T. McCarthy J.B. J. Cell Biol. 1992; 117: 1331-1342Crossref PubMed Scopus (68) Google Scholar, 48Damsky C.H. Werb Z. Curr. Opin. Cell Biol. 1992; 4: 772-781Crossref PubMed Scopus (487) Google Scholar) 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 (55Whittard J.D. Mould A.P. Craig S.E. Askari J.A. Humphries M.J. Mol. Biol. Cell. 1995; 6 (abstr.): 47Google Scholar). 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 α5β1 integrin by two binding sites in the integrin molecule that bind RGD peptide and GAG binding domains in fibronectin. 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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