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• W2000738994 abstract "The tetraspanin CD151 forms a stoichiometric complex with integrin α3β1 and regulates its endocytosis. We observed that down-regulation of CD151 in various epithelial cell lines changed glycosylation of α3β1. In contrast, glycosylation of other transmembrane proteins, including those associated with CD151 (e.g. α6β1, CD82, CD63, and emmprin/CD147) was not affected. The detailed analysis has shown that depletion of CD151 resulted in the reduction of Fucα1–2Gal and bisecting GlcNAc-β(1→4) linkage on N-glycans of the α3 integrin subunit. The modulatory activity of CD151 toward α3β1 was specific, because stable knockdown of three other tetraspanins (i.e. CD9, CD63, and CD81) did not affect glycosylation of the integrin. Analysis of α3 glycosylation in CD151-depleted breast cancer cells with reconstituted expression of various CD151 mutants has shown that a direct contact with integrin is required but not sufficient for the modulatory activity of the tetraspanin toward α3β1. We also found that glycosylation of CD151 is also critical; Asn159 → Gln mutation in the large extracellular loop did not affect interactions of CD151 with other tetraspanins or α3β1 but negated its modulatory function. Changes in the glycosylation pattern of α3β1 observed in CD151-depleted cells correlated with a dramatic decrease in cell migration toward laminin-332. Migration toward fibronectin or static adhesion of cells to extracellular matrix ligands was not affected. Importantly, reconstituted expression of the wild-type CD151 but not glycosylation-deficient mutant restored the migratory potential of the cells. These results demonstrate that CD151 plays an important role in post-translation modification of α3β1 integrin and strongly suggest that changes in integrin glycosylation are critical for the promigratory activity of this tetraspanin. The tetraspanin CD151 forms a stoichiometric complex with integrin α3β1 and regulates its endocytosis. We observed that down-regulation of CD151 in various epithelial cell lines changed glycosylation of α3β1. In contrast, glycosylation of other transmembrane proteins, including those associated with CD151 (e.g. α6β1, CD82, CD63, and emmprin/CD147) was not affected. The detailed analysis has shown that depletion of CD151 resulted in the reduction of Fucα1–2Gal and bisecting GlcNAc-β(1→4) linkage on N-glycans of the α3 integrin subunit. The modulatory activity of CD151 toward α3β1 was specific, because stable knockdown of three other tetraspanins (i.e. CD9, CD63, and CD81) did not affect glycosylation of the integrin. Analysis of α3 glycosylation in CD151-depleted breast cancer cells with reconstituted expression of various CD151 mutants has shown that a direct contact with integrin is required but not sufficient for the modulatory activity of the tetraspanin toward α3β1. We also found that glycosylation of CD151 is also critical; Asn159 → Gln mutation in the large extracellular loop did not affect interactions of CD151 with other tetraspanins or α3β1 but negated its modulatory function. Changes in the glycosylation pattern of α3β1 observed in CD151-depleted cells correlated with a dramatic decrease in cell migration toward laminin-332. Migration toward fibronectin or static adhesion of cells to extracellular matrix ligands was not affected. Importantly, reconstituted expression of the wild-type CD151 but not glycosylation-deficient mutant restored the migratory potential of the cells. These results demonstrate that CD151 plays an important role in post-translation modification of α3β1 integrin and strongly suggest that changes in integrin glycosylation are critical for the promigratory activity of this tetraspanin. Transmembrane proteins from the tetraspanin superfamily are assembled in microdomains (referred to as tetraspanin-enriched microdomains (TERM) 2The abbreviations used are:TERMtetraspanin-enriched microdomain(s)DMEMDulbecco's modified Eagle's mediumAbantibodymAbmonoclonal antibody ) which also incorporate a number of tetraspanin-interacting receptors (e.g. integrins and receptor tyrosine kinases) (1Hemler M.E. Nat. Rev. Mol. Cell. Biol. 2005; 6: 801-811Crossref PubMed Scopus (1025) Google Scholar). It has been shown that tetraspanins regulate the activity of the associated receptors via various mechanisms involving ligand binding, clustering, and trafficking (1Hemler M.E. Nat. Rev. Mol. Cell. Biol. 2005; 6: 801-811Crossref PubMed Scopus (1025) Google Scholar, 2Levy S. Shoham T. Physiology. 2005; 20: 218-224Crossref PubMed Scopus (195) Google Scholar, 3Berditchevski F. Odintsova E. Traffic. 2007; 8: 89-96Crossref PubMed Scopus (232) Google Scholar). tetraspanin-enriched microdomain(s) Dulbecco's modified Eagle's medium antibody monoclonal antibody N-Linked glycosylation is one of the most common and diverse modifications of transmembrane proteins. The role of glycosylation in regulation of protein stability, folding, and dimerization and trafficking to and from the plasma membrane as well as between intracellular organelles is documented in numerous reports (4Helenius A. Aebi M. Science. 2001; 291: 2364-2369Crossref PubMed Scopus (1998) Google Scholar, 5Mitra N. Sinha S. Ramya T.N. Surolia A. Trends Biochem. Sci. 2006; 31: 156-163Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 6Xu J. He J. Castleberry A.M. Balasubramanian S. Lau A.G. Hall R.A. J. Biol. Chem. 2003; 278: 10770-10777Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 7Kohno T. Wada A. Igarashi Y. FASEB J. 2002; 16: 983-992Crossref PubMed Scopus (65) Google Scholar, 8Isaji T. Sato Y. Zhao Y. Miyoshi E. Wada Y. Taniguchi N. Gu J. J. Biol. Chem. 2006; 281: 33258-33267Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Furthermore, earlier studies have shown that glycosylation of a number of transmembrane proteins, which would be later identified as tetraspanin-associated partners, regulate their functions. Glycosylation of intercellular adhesion molecule 1 and components of major histocompatibility class I complex is important for interactions of the proteins with their respective receptors (9Shen L. Kane K.P. J. Exp. Med. 1995; 181: 1773-1783Crossref PubMed Scopus (15) Google Scholar, 10Diamond M.S. Staunton D.E. Marlin S.D. Springer T.A. Cell. 1991; 65: 961-971Abstract Full Text PDF PubMed Scopus (654) Google Scholar). Trafficking of CD4 to the cell surface was impaired in cells treated with tunicamycin (11Konig R. Ashwell G. Hanover J.A. J. Biol. Chem. 1988; 263: 9502-9507Abstract Full Text PDF PubMed Google Scholar). More recently, it was found that glycosylation of H,K-ATPase β subunit, a partner for tetraspanin CD63, regulates internalization and subsequent degradation of the protein (12Vagin O. Turdikulova S. Sachs G. J. Biol. Chem. 2004; 279: 39026-39034Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). A number of reports have described that surface expression, conformation, ligand binding, dimerization, and endocytosis of epidermal growth factor receptor are regulated by glycosylation (13Fernandes H. Cohen S. Bishayee S. J. Biol. Chem. 2001; 276: 5375-5383Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 14Tsuda T. Ikeda Y. Taniguchi N. J. Biol. Chem. 2000; 275: 21988-21994Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 15Wang X. Gu J. Ihara H. Miyoshi E. Honke K. Taniguchi N. J. Biol. Chem. 2006; 281: 2572-2577Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). The N-linked glycan on ErbB3 prevents spontaneous heterodimerization and activation of the receptor (16Yokoe S. Takahashi M. Asahi M. Lee S.H. Li W. Osumi D. Miyoshi E. Taniguchi N. Cancer Res. 2007; 67: 1935-1942Crossref PubMed Scopus (38) Google Scholar). There is a substantial body of evidence indicating that glycosylation of integrins plays a critical role in their function. Early experiments have shown that differential glycosylation of β2 integrin subunit is critical for its pairing with either αL or αM (17Dahms N.M. Hart G.W. J. Biol. Chem. 1986; 261: 13186-13196Abstract Full Text PDF PubMed Google Scholar). Altered glycosylation of β1 integrins correlated with differences in attachment of cells to fibronectin and laminin (18Oz O.K. Campbell A. Tao T.W. Int. J. Cancer. 1989; 44: 343-347Crossref PubMed Scopus (43) Google Scholar). Similarly, binding of α5β1 integrin to its ligand was decreased in cells treated with 1-deoxymannojirimycin, a compound that inhibits conversion of the high mannose to hybrid and complex glycosylated species of the protein (19Akiyama S.K. Yamada S.S. Yamada K.M. J. Biol. Chem. 1989; 264: 18011-18018Abstract Full Text PDF PubMed Google Scholar). Subsequent work from various laboratories has extended these observations to other integrins, including α6β1 (20Chammas R. Veiga S.S. Travassos L.R. Brentani R.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1795-1799Crossref PubMed Scopus (70) Google Scholar), αv integrins (21Lehmann M. El B.A. Abadie B. Martin J.M. Marvaldi J. J. Cell. Biochem. 1996; 61: 266-277Crossref PubMed Scopus (15) Google Scholar), and α3β1 (22Zhao Y. Itoh S. Wang X. Isaji T. Miyoshi E. Kariya Y. Miyazaki K. Kawasaki N. Taniguchi N. Gu J. J. Biol. Chem. 2006; 281: 38343-38350Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 23Zhao Y. Nakagawa T. Itoh S. Inamori K. Isaji T. Kariya Y. Kondo A. Miyoshi E. Miyazaki K. Kawasaki N. Taniguchi N. Gu J. J. Biol. Chem. 2006; 281: 32122-32130Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 24Pochec E. Litynska A. Bubka M. Amoresano A. Casbarra A. Eur. J. Cell Biol. 2006; 85: 47-57Crossref PubMed Scopus (31) Google Scholar, 25Pochec E. Litynska A. Amoresano A. Casbarra A. Biochim. Biophys. Acta. 2003; 1643: 113-123Crossref PubMed Scopus (108) Google Scholar). In addition, it has been proposed that glycosylation-dependent interactions of integrins with their ligands involve gangliosides (26Hakomori S. Handa K. FEBS Lett. 2002; 531: 88-92Crossref PubMed Scopus (118) Google Scholar, 27Zheng M. Tsuruoka T. Tsuji T. Hakomori S. Biochem. Biophys. Res. Commun. 1992; 186: 1397-1402Crossref PubMed Scopus (17) Google Scholar). Together, α3 and β1 integrin chains have 27 potential N-linked glycosylation sites: 14 on the α3 subunit and 13 on the β1 subunit. Detailed analyses of N-glycans by mass spectrometry have revealed significant diversity of oligosaccharides that decorate α3β1 integrin purified from various cell types (22Zhao Y. Itoh S. Wang X. Isaji T. Miyoshi E. Kariya Y. Miyazaki K. Kawasaki N. Taniguchi N. Gu J. J. Biol. Chem. 2006; 281: 38343-38350Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 23Zhao Y. Nakagawa T. Itoh S. Inamori K. Isaji T. Kariya Y. Kondo A. Miyoshi E. Miyazaki K. Kawasaki N. Taniguchi N. Gu J. J. Biol. Chem. 2006; 281: 32122-32130Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 25Pochec E. Litynska A. Amoresano A. Casbarra A. Biochim. Biophys. Acta. 2003; 1643: 113-123Crossref PubMed Scopus (108) Google Scholar, 28Litynska A. Pochec E. Hoja-Lukowicz D. Kremser E. Laidler P. Amoresano A. Monti C. Acta Biochim. Pol. 2002; 49: 491-500Crossref PubMed Scopus (26) Google Scholar, 29Litynska A. Przybylo M. Ksiazek D. Laidler P. Acta Biochim. Pol. 2000; 47: 427-434Crossref PubMed Scopus (27) Google Scholar). Importantly, specific changes in glycosylation of α3β1 observed in tumor cells correlated with their migratory and invasive potential (25Pochec E. Litynska A. Amoresano A. Casbarra A. Biochim. Biophys. Acta. 2003; 1643: 113-123Crossref PubMed Scopus (108) Google Scholar, 29Litynska A. Przybylo M. Ksiazek D. Laidler P. Acta Biochim. Pol. 2000; 47: 427-434Crossref PubMed Scopus (27) Google Scholar). Recent studies have demonstrated that tetraspanin CD81 regulates glycosylation of its proximal partners CD19 and EWI-2 (30Shoham T. Rajapaksa R. Kuo C.C. Haimovich J. Levy S. Mol. Cell. Biol. 2006; 26: 1373-1385Crossref PubMed Scopus (77) Google Scholar, 31Stipp C.S. Kolesnikova T.V. Hemler M.E. J. Cell Biol. 2003; 163: 1167-1177Crossref PubMed Scopus (72) Google Scholar). In the case of CD19, the region responsible for this activity was mapped to the predicted N-terminal cytoplasmic portion of the protein (30Shoham T. Rajapaksa R. Kuo C.C. Haimovich J. Levy S. Mol. Cell. Biol. 2006; 26: 1373-1385Crossref PubMed Scopus (77) Google Scholar). In addition, modulation in the expression levels of CD82/KAI-1 affected maturation and surface expression of β1 integrin subunit in lung carcinoma cells (32Jee B.K. Lee J.Y. Lim Y. Lee K.H. Jo Y.H. Biochem. Biophys. Res. Commun. 2007; 359: 703-708Crossref PubMed Scopus (35) Google Scholar). α chains of canonical laminin-binding integrins (i.e. α3β1, α6β1/β4, and α7β1) are post-translationally cleaved in Golgi by proprotein convertases to form light and heavy chains that are held together by a single disulfide bond and β subunit (33Belkin A.M. Stepp M.A. Microsc. Res. Technol. 2000; 51: 280-301Crossref PubMed Scopus (300) Google Scholar). It is thought that cleavage of α subunits is required for “inside-out” activation of integrin heterodimers (34van der F.A. Sonnenberg A. Cell Tissue Res. 2001; 305: 285-298Crossref PubMed Scopus (818) Google Scholar). There are two splice variants of the light chains described for α3 subunit, each having three potential N-linked glycosylation sites (35de Melker A.A. Sonnenberg A. BioEssays. 1999; 21: 499-509Crossref PubMed Scopus (108) Google Scholar). Tetraspanin CD151 forms stable and highly stoichiometric complexes with α3β1, α6β1/β4, and α7β1 integrin heterodimers (36Yauch R.L. Berditchevski F. Harler M.B. Reichner J. Hemler M.E. Mol. Biol. Cell. 1998; 9: 2751-2765Crossref PubMed Scopus (270) Google Scholar, 37Sterk L.M. Geuijen C.A. Oomen L.C. Calafat J. Janssen H. Sonnenberg A. J. Cell Biol. 2000; 149: 969-982Crossref PubMed Scopus (190) Google Scholar). Interactions with CD151 regulate ligand-binding and signaling properties of these integrins (38Berditchevski F. Odintsova E. Sawada S. Gilbert E. J. Biol. Chem. 2002; 277: 36991-37000Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 39Kazarov A.R. Yang X. Stipp C.S. Sehgal B. Hemler M.E. J. Cell Biol. 2002; 158: 1299-1309Crossref PubMed Scopus (142) Google Scholar, 40Lammerding J. Kazarov A.R. Huang H. Lee R.T. Hemler M.E. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7616-7621Crossref PubMed Scopus (141) Google Scholar, 41Lau L.M. Wee J.L. Wright M.D. Moseley G.W. Hogarth P.M. Ashman L.K. Jackson D.E. Blood. 2004; 104: 2368-2375Crossref PubMed Scopus (109) Google Scholar, 42Nishiuchi R. Sanzen N. Nada S. Sumida Y. Wada Y. Okada M. Takagi J. Hasegawa H. Sekiguchi K. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 1939-1944Crossref PubMed Scopus (133) Google Scholar, 43Sawada S. Yoshimoto M. Odintsova E. Hotchin N.A. Berditchevski F. J. Biol. Chem. 2003; 278: 26323-26326Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 44Takeda Y. Kazarov A.R. Butterfield C.E. Hopkins B.D. Benjamin L.E. Kaipainen A. Hemler M.E. Blood. 2006; 109: 1524-1532Crossref PubMed Scopus (135) Google Scholar, 45Winterwood N.E. Varzavand A. Meland M.N. Ashman L.K. Stipp C.S. Mol. Biol. Cell. 2006; 17: 2707-2721Crossref PubMed Scopus (113) Google Scholar, 46Yamada M. Sumida Y. Fujibayashi A. Fukaguchi K. Sanzen N. Nishiuchi R. Sekiguchi K. FEBS J. 2008; 275: 3335-3351Crossref PubMed Scopus (37) Google Scholar, 47Yang X.H. Richardson A.L. Torres-Arzayus M.I. Zhou P. Sharma C. Kazarov A.R. Andzelm M.M. Strominger J.L. Brown M. Hemler M.E. Cancer Res. 2008; 68: 3204-3213Crossref PubMed Scopus (149) Google Scholar). Furthermore, CD151 regulates endocytosis of α3β1 integrin (45Winterwood N.E. Varzavand A. Meland M.N. Ashman L.K. Stipp C.S. Mol. Biol. Cell. 2006; 17: 2707-2721Crossref PubMed Scopus (113) Google Scholar, 48Hasegawa M. Furuya M. Kasuya Y. Nishiyama M. Sugiura T. Nikaido T. Momota Y. Ichinose M. Kimura S. Lab. Invest. 2007; 87: 882-892Crossref PubMed Scopus (52) Google Scholar, 49Liu L. He B. Liu W.M. Zhou D. Cox J.V. Zhang X.A. J. Biol. Chem. 2007; 282: 31631-31642Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). In this report, we describe a previously unknown role for CD151 as a modulator of α3β1 glycosylation. We have also established that the direct interaction of CD151 with α3β1 is necessary but not sufficient for the modulatory activity of the tetraspanin. Finally, we provide strong evidence that changes in integrin glycosylation are important for the α3β1-dependent promigratory function of CD151. The MDA-MB-231 and HeLa cell lines were purchased from the Cancer Research UK. Cell lines were maintained in DMEM (Invitrogen) supplemented with 10! fetal calf serum (PAA Laboratories). The mouse anti-CD81 and anti-CD82 mAbs (M38 and M104, respectively) were kindly provided by Dr. O. Yoshie. The anti-CD63 (6H1) and anti-CD151 (5C11 and 11B1G4) mouse mAbs and rabbit anti-CD151 polyclonal Ab were described previously (50Berditchevski F. Bazzoni G. Hemler M.E. J. Biol. Chem. 1995; 270: 17784-17790Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 51Berditchevski F. Chang S. Bodorova J. Hemler M.E. J. Biol. Chem. 1997; 272: 29174-29180Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar, 52Kishimoto, T., Kikutani, H., von dem Borne, A. E. G. K., Goyert, S. M., Mason, D., Miyasaka, M., Moretta, L., Okumure, K., Shaw, S., and Springer, T. A., eds) Garland Publishing, New YorkGoogle Scholar, 53Sincock P.M. Fitter S. Parton R.G. Berndt M. Gamble J.R. Ashman L.K. J. Cell Sci. 1999; 112: 833-844Crossref PubMed Google Scholar). The anti-CD9 mAb BU16 was from The Binding Site (Birmingham, UK). The anti-CD82 (TS82) mAbs were generously provided by Dr. E. Rubinstein (Villejuif, France). The anti-integrin mAbs used were A2-VIIC6 (anti-α2) (54Berditchevski F. Odintsova E. J. Cell Biol. 1999; 146: 477-492Crossref PubMed Scopus (258) Google Scholar), A3-IVA5 (anti-α3) (55Weitzman J.B. Pasqualini R. Takada Y. Hemler M.E. J. Biol. Chem. 1993; 268: 8651-8657Abstract Full Text PDF PubMed Google Scholar), P1D6 (anti-α5) (56Wayner E.A. Carter W.G. J. Cell Biol. 1987; 105: 1873-1884Crossref PubMed Scopus (540) Google Scholar), A6-ELE (anti-α6) (57Tachibana I. Bodorova J. Berditchevski F. Zutter M.M. Hemler M.E. J. Biol. Chem. 1997; 272: 29181-29189Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), TS2/16 (anti-β1) (58Hemler M.E. Sánchez-Madrid F. Flotte T.J. Krensky A.M. Burakoff S.J. Bhan A.K. Springer T.A. Strominger J.L. J. Immunol. 1984; 132: 3011-3018Crossref PubMed Google Scholar), G0H3 (anti-α6) (Chemicon International), and 3E1 (anti-β4) (Chemicon International). Rabbit polyclonal antibodies to α3 and α6 integrin subunits were gifts from Dr. F. Watt (Cambridge, UK) and Dr. A. Cress (Tucson, AZ). Laminin-332 was isolated from SCC25 cells as previously described (59Eble J.A. Wucherpfennig K.W. Gauthier L. Dersch P. Krukonis E. Isberg R.R. Hemler M.E. Biochemistry. 1998; 37: 10945-10955Crossref PubMed Scopus (105) Google Scholar). Biotinylated lectins were purchased from Vector Laboratories. To generate stable MDA-MB-231/CD151(–) and HeLa/CD151(–) cell lines, transfections were carried out using Fugene6 (Roche Applied Science) for HeLa or GeneJammer (Stratagene) for MDA-MB-231 cells. Transfected cells were selected and maintained in DMEM containing 0.5–1.0 μg/ml puromycin. CD151 knockdown was confirmed by flow cytometry analyses (COULTER Epics XL). CD151-positive and -negative populations were selected by cell sorting (BDFACSVantage SE) and confirmed by flow cytometry and Western blotting. MDA-MB-231/CD9(–), MDA-MB-231/CD63(–), and MDA-MB-231/CD81(–) cell lines were established using the same approach. To generate MDA-MB-231/rec series (MDA-MB-231/CD151(–) cells with the reconstituted expression of the CD151 wild-type or CD151 mutants) pZeoSV-based constructs were introduced into the cells using GeneJammer and selected in growth medium containing 100–300 μg/ml Zeocin. Various reconstituted CD151 cell lines were sorted to obtain a pool of cells expressing CD151 at levels similar to that of the control MDA-MB-231. Plasmids Expressing Short Hairpin RNA—pSuperior-based constructs for specific targeting of tetraspanins were generated using a standard protocol. The constructs targeted the following sequences: CD151, 5′-AGTACCTGCTGTTTACCTACA (45Winterwood N.E. Varzavand A. Meland M.N. Ashman L.K. Stipp C.S. Mol. Biol. Cell. 2006; 17: 2707-2721Crossref PubMed Scopus (113) Google Scholar); CD81, 5′-ATCTGGAGCTGGGAGACAA (60Mazzocca A. Sciammetta S.C. Carloni V. Cosmi L. Annunziato F. Harada T. Abrignani S. Pinzani M. J. Biol. Chem. 2005; 280: 11329-11339Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar); CD63, 5′-GGTTTTTCAATTAAACGGA. pSuper-CD9 (kind gift from Dr. E. Rubinstein (Villejuif, France)) targets the following sequence: 5′-ACCTTCACCGTGAAGTCCT (61Barreiro O. Yanez-Mo M. Sala-Valdes M. Gutierrez-Lopez M.D. Ovalle S. Higginbottom A. Monk P.N. Cabanas C. Sanchez-Madrid F. Blood. 2005; 105: 2852-2861Crossref PubMed Scopus (178) Google Scholar). CD151 Constructs—The original CD151ΔC, CD151palm(–)/CD151Cys8, and SW6 mutants have been described earlier (38Berditchevski F. Odintsova E. Sawada S. Gilbert E. J. Biol. Chem. 2002; 277: 36991-37000Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 43Sawada S. Yoshimoto M. Odintsova E. Hotchin N.A. Berditchevski F. J. Biol. Chem. 2003; 278: 26323-26326Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 62Berditchevski F. Gilbert E. Griffiths M.R. Fitter S. Ashman L.K. Jenner S.J. J. Biol. Chem. 2001; 276: 41165-41174Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). CD151-QRD mutant was generated as described by others (39Kazarov A.R. Yang X. Stipp C.S. Sehgal B. Hemler M.E. J. Cell Biol. 2002; 158: 1299-1309Crossref PubMed Scopus (142) Google Scholar). CD151glyco(–) and 63-N-151 mutants were generated using a standard PCR approach; in CD151glyco(–) mutant, a predicted glycosylation site (Asn159) was substituted to glutamine, and in 63-N-151 mutant, the N-terminal cytoplasmic sequence of CD151 was substituted to a corresponding region of CD63. Semiconfluent cells were detached using Cell Dissociation Buffer (Invitrogen), incubated with saturating concentrations of primary mouse mAb for 1 h on ice, washed twice in phosphate-buffered saline, and then labeled with fluorescein isothiocyanate-conjugated goat anti-mouse IgG for 1 h at 4 °C. Surface labeling was analyzed by flow cytometry using COULTER Epics XL. For sorting, cells were prepared as above except that all the solutions were sterilized by filtration. Cells were lysed overnight at 4 °C in 1! Triton X-100/phosphate-buffered saline buffer containing inhibitors (2 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin). The lysate was centrifuged at 12,000 × g for 10 min to remove insoluble material. Equal amounts of protein lysates were resolved by 12! SDS-PAGE under reducing and nonreducing conditions, transferred onto nitrocellulose membrane, and incubated with appropriate primary Ab. Protein bands were visualized after subsequent incubations with appropriate horseradish peroxidase-conjugated secondary Ab and Chemiluminescence Reagent Plus (PerkinElmer Life Sciences). Cell lysates were prepared as above (except when the analysis of tetraspanin-tetraspanin association was performed) and precleared by incubation for 3 h at 4 °C with agarose beads conjugated with goat anti-mouse antibodies (mIgG-beads; Sigma). Immune complexes were collected using appropriate mAbs prebound to the mIgG-beads and washed four times with the immunoprecipitation buffer. The complexes were eluted from the beads with Laemmli sample buffer. Proteins were resolved by SDS-PAGE, transferred to the nitrocellulose membrane, and developed with the appropriate Ab. For the analysis of α3β1-tetraspanin (other than CD151) and tetraspanin-tetraspanin interactions, cell lysis and immunoprecipitation was carried out in 1! Brij 96 in the presence of aforementioned inhibitors. Migration was analyzed using a standard Boyden Chamber protocol. In brief, 1–2 × 105 cells were detached using Cell Dissociation Solution (Invitrogen) and suspended in 500 μl of serum-free DMEM. Cells were subsequently added into the inner compartment of Nunc's tissue culture inserts with polycarbonate membranes (8-μm pores), the bottom sides of which were coated with 2 μg/ml Laminin-332 or 10 μg/ml fibronectin (Sigma). Cells were allowed to migrate toward serum-free DMEM supplemented with 10 ng/ml epidermal growth factor for 8 h. Nonmigrated cells were removed, and nuclei of migrating cells were stained with 4′,6-diamidino-2-phenylindole. Membranes were mounted on glass slides and analyzed using a Nikon Eclipse E600 microscope. Up to seven random fields per membrane were photographed and scored using ImageJ nuclear/cell counter program. Each of the experiments was done in quadruplicates, and 2–3 independent experiments were carried out for each cell line. Protein lysate (∼100 μg) was boiled for 10 min in the presence of 0.5! SDS before deglycosylation. The denatured lysate or immunoprecipitated complexes eluted in 0.2× Laemmli buffer were deglycosylated overnight at 37 °C with either peptide:N-glycanase F in Buffer G7 or endoglycosidase H in Buffer G5 (New England Biolabs). Samples were then resolved by 12! SDS-PAGE, transferred onto nitrocellulose membrane, and detected with Abs for various proteins. Deglycosylation was performed in both reducing (in the presence of 40 mm dithiothreitol) and nonreducing conditions. A standard static adhesion assay (30–35 min) was carried out as previously described (55Weitzman J.B. Pasqualini R. Takada Y. Hemler M.E. J. Biol. Chem. 1993; 268: 8651-8657Abstract Full Text PDF PubMed Google Scholar); 2,7-bis-(2-carboxyethyl)-5-(and-6)carboxyfluorescein-labeled cells aliquoted into 96-well plates precoated overnight with various concentrations of laminin-332 or fibronectin. CD151 Regulates Glycosylation of α3β1 Integrin—The tetraspanin CD151 forms stoichiometric complexes with various laminin-binding integrins, including α3β1, α6β1, α6β4, and α7β1. To investigate whether CD151 affects biosynthetic processing of integrins, we established two epithelial cell lines in which expression of the tetraspanin was decreased, using a specific short hairpin RNA construct (45Winterwood N.E. Varzavand A. Meland M.N. Ashman L.K. Stipp C.S. Mol. Biol. Cell. 2006; 17: 2707-2721Crossref PubMed Scopus (113) Google Scholar); MDA-MB-231/CD151(–) and HeLa/CD151(–) lines express CD151 at levels of <5! of the corresponding parental cells (Fig. 1A). Flow cytometry and Western blotting have shown that down-regulation of CD151 did not affect surface and total levels of α3β1 and α6 integrins (α7β1 is not expressed in epithelial cells). Interestingly, we consistently observed that the protein band corresponding to the α3 integrin subunit runs slower in SDS-PAGE. This was particularly evident when we compared the positions of the α3 light chains. Light chains of α3 resolved as closely spaced three bands with the top, slower migrating band being more prominent in MDA-MB-231/CD151(–) than MDA-MB-231/CD151(+) cells (Fig. 1B). Conversely, the intensity of the lowest band was higher in the CD151-positive cells. The observed differences can be explained by one of the following: 1) differences in the furin-dependent cleavage; 2) differences in splicing; 3) differences in glycosylation. To distinguish between these possibilities, we compared the mobility of α3 light chains in SDS-PAGE after the treatment of protein lysates with peptide:N-glycanase. Since peptide:N-glycanase completely removes N-linked glycans from the protein backbone, this treatment would negate both qualitative and quantitative differences in glycosylation of the integrin in CD151-positive and CD151-negative cells. Indeed, we observed comparable patterns of the α3 light chains in the peptide:N-glycanase-treated lysates from the MDA-MB-231 and HeLa pairs (Fig. 2A, lanes 3 and 4 and lanes 9 and 10). To examine differences in the integrin glycosylation in more detail, we analyzed mobility of the α3 light chains after treatment with endoglycosidase H, which specifically cleaves high mannose and hybrid forms of N-linked glycans. Although all three major glycoforms of the α3 light chain were sensitive to the endoglycosidase H treatment, the products of the digestion run slower in SDS-PAGE than completely deglycosylated species (Fig. 2A). These results indicated that the light chain of the α3 integrin subunit, which has three putative glycosylation sites, is modified by both complex and hybrid/high mannose oligosaccharides. Importantly, depletion of CD151 changed relative abundance of various endoglycosidase H-resistant glycoforms in MDA-MB-231 and HeLa cells (Fig. 2A, lanes 5 and 6 and lanes 7 and 8). To gain further insight into the role of CD151 in maturation of α3β1, we analyzed glycosylation of the integrin in cells treated with swainsonine and deoxymannojirimycin. These chemicals block activities of trimming glycosidases and, thereby, inhibit processing of N-linked oligosaccharides to complex (swainsonine) and hybrid/complex forms (deoxymannojirimycin). Although less pronounced, differences between CD151(+) and CD151(–) cells were still visible after treatment with either swainsonine or deoxymannojirimycin (Fig. 2B). Taken together, these results demonstrated that CD151 influences glycosylation of α3β1 at the relatively early stages of the maturation process.FIGURE 2Down-regulation of CD151 changes glycosylation of α3 integrin subunit. A, cells were lysed in Triton X-100, and proteins were deglycosylated overnight with either peptide:N-glycanase F (PNG; lanes 3, 4, 9, and 10) or endoglycosidase H (EnH; lanes 5–8). Samples" @default.
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• W2000738994 date "2008-12-01" @default.
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• W2000738994 title "Tetraspanin CD151 Regulates Glycosylation of α3β1 Integrin" @default.
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• W2000738994 doi "https://doi.org/10.1074/jbc.m806394200" @default.
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