Glycosaminoglycan chains from α 5 β 1 integrin are involved in fibronectin-dependent cell migrationDedicated to the memory of Professor Carl P. Dietrich

Glycosaminoglycan chains from a5b1 integrin areinvolved in fibronectin-dependent cell migration1

Celia R.C. Franco, Edvaldo S. Trindade, Hugo A.O. Rocha, Rafael Bertoni daSilveira, Katia Sabrina Paludo, Roger Chammas, Silvio S. Veiga, Helena B. Nader,and Carl P. Dietrich

Abstract: a5b1 integrin from both wild-type CHO cells (CHO-K1) and deficient in proteoglycan biosynthesis (CHO-745)is post-translationally modified by glycosaminoglycan chains. We demonstrated this using [35S]sulfate metabolic labelingof the cells, enzymatic degradation, immunoprecipitation reaction with monoclonal antibody, fluorescence microscopy, andflow cytometry. The a5b1 integrin heterodimer is a hybrid proteoglycan containing both chondroitin and heparan sulfatechains. Xyloside inhibition of sulfate incorporation into a5b1 integrin also supports that integrin is a proteoglycan. Also,cells grown with xyloside adhered on fibronectin with no alteration in a5b1 integrin expression. However, haptotactic mo-tility on fibronectin declined in cells grown with xyloside or chlorate as compared with controls. Thus, a5b1 integrin is aproteoglycan and the glycosaminoglycan chains of the integrin influence cell motility on fibronectin. Similar glycosylationof a5b1 integrin was observed in other normal and malignant cells, suggesting that this modification is conserved and im-portant in the function of this integrin. Therefore, these glycosaminoglycan chains of a5b1 integrin are involved in cellularmigration on fibronectin.

Key words: integrin, glycosaminoglycan, migration on fibronectin, adhesion on fibronectin, proteoglycan.

Resume : L’integrine a5b1 des cellules CHO sauvages (CHO-K1) et deficientes en biosynthese de proteoglycanes (CHO-745) est modifiee au niveau post-traductionnel par des chaınes de glycosaminoglycanes. Nous l’avons demontre a l’aided’un marquage metabolique des cellules au [35S]sulfate, par degradation enzymatique, par immunoprecipitation a l’aided’un anticorps monoclonal, par microscopie en fluorescence et par cytometrie de flux. L’heterodimere a5b1 est un protero-glycane hybride contenant des chaınes de chondroıtine et d’heparane sulfate. L’inhibition par le xyloside de l’incorporationde sulfate dans l’integrine a5b1 appuie aussi l’hypothese que l’integrine soit un proteoglycane. Les cellules cultivees avecdu xyloside adherent aussi sur la fibronectine, sans que l’expression de a5b1 soit modifiee. Cependant, la motilite haptotac-tique sur la fibronectine diminuait chez les cellules cultivees en presence de xyloside ou de chlorate, comparativement auxcontroles. Ainsi, l’integrine a5b1 est un proteoglycane et les chaınes de glycosaminaglycanes de l’integrine influencent lamotilite cellulaire sur la fibronectine. Une glycosylation similaire de l’integrine a5b1 a ete observee chez d’autres types decellules normales ou malignes, suggerant que cette modification est conservee et qu’elle est importante a la fonction decette integrine. Consequemment, ces chaınes de glycosaminaglycanes de l’integrine a5b1 sont impliquees dans la migrationcellulaire sur la fibronectine.

Mots-cles : integrine, glycosaminoglycane, migration sur fibronectine, adhesion sur fibronectine, proteoglycane.

[Traduit par la Redaction]

Introduction

Extracellular matrices (ECM) comprise glycoprotein mac-romolecules that interact with each other in the intercellularspace. Cell–ECM interactions have been implicated in cell

adhesion, migration, growth, and differentiation and therebyplay an important physiological role in tissue architectureand integrity as well as pathological phenomena (Dietrich1984; Lopes et al. 2006a; Sampaio and Nader 2006; Chanet al. 2007, Nemeth et al. 2007). Cell–ECM interactions are

Received 7 January 2009. Revision received 31 March 2009. Accepted 29 April 2009. Published on the NRC Research Press Web site atbcb.nrc.ca on 24 July 2009.

C.R.C. Franco and E.S. Trindade. Departamento de Bioquımica, Universidade Federal de Sao Paulo, Rua Tres de Maio, 100 - CEP04044-020, Sao Paulo, SP, Brazil; Departamento de Biologia Celular, Universidade Federal de Parana, Curitiba, PR, Brazil.H.A.O. Rocha. Departamento de Bioquımica, Universidade Federal de Sao Paulo, Rua Tres de Maio, 100 - CEP 04044-020, Sao Paulo,SP, Brazil; Departamento de Bioquımica, Universidade Federal de Rio Grande do Norte, Natal, RN, Brazil.R. Bertoni da Silveira, H.B. Nader,2 and C.P. Dietrich. Departamento de Bioquımica, Universidade Federal de Sao Paulo, Rua Tres deMaio, 100 - CEP 04044-020, Sao Paulo, SP, Brazil.K.S. Paludo and S.S. Veiga. Departamento de Biologia Celular, Universidade Federal de Parana, Curitiba, PR, Brazil.R. Chammas. Laboratorio de Oncologia Experimental, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP, Brazil.

1Dedicated to the memory of Professor Carl P. Dietrich.2Corresponding author (e-mail: [email protected]).

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Biochem. Cell Biol. 87: 677–686 (2009) doi:10.1139/O09-047 Published by NRC Research Press

mediated by integrins, heterodimeric integral plasma mem-brane proteins that are widely expressed in most animalcells. In addition to cell adhesion, integrins also regulate cy-toskeletal organization, transmembrane signal transduction,cell growth, and Ca2+ and H+ concentration in the cytoplasmand modulate phosphoinositide metabolism (Yamada andMiyamoto 1995; Laukaitis et al. 2001; Humphries et al.2006; Zaidel-Bar et al. 2007).

Fibronectin is one of the most studied ECM molecules,serving as a prototype for adhesive molecules, due to thepresence of the tripeptide RGD (highly conserved cell adhe-sion determinant) in its sequence. Fibronectin is involved ina number of important RGD-dependent regulatory processesthat include cell growth, cellular differentiation during em-bryogenesis and morphogenesis, cell adhesion and migra-tion, wound healing, and angiogenesis (Ruoslahti andPierschbacher 1987; Reyes-Reyes et al. 2006).

However, fibronectin–cell interactions that influence cel-lular anchoring, traction, and migration are dependent ondifferent peptides. The tripeptide RGD in domain 10 is themajor binding site for a5b1 integrin (Ruoslahti and Piersch-bacher 1987; Plow et al. 2000). Fibronectin–cell interactionsare also dependent on fibronectin–glycosaminoglycan(GAG) binding domains. Proteoglycan (PG) deficient cellshave defective focal adhesion contact formation in responseto fibronectin (LeBaron et al. 1988).

Several integrins that share the b1 subunit are receptorsfor fibronectin. These include a3b1, a4b1, avb1, and a5b1 in-tegrin, the classic fibronectin receptor (Plow et al. 2000).Cell surface expression of a5b1 integrin is required for mo-tility and locomotion on fibronectin substrate, discovered us-ing a5b1 integrin deficient CHO cells and transfection ofthese cells with a5 subunit cDNA (Laukaitis et al. 2001).

PGs have long been recognized as essential and criticalfor numerous biological functions. They comprise a familyof cell surface receptors or ECM molecules with the abilityto bind growth factors, hormones, ECM constituents, andcell surface molecules. Based on such abilities, PGs arethought to be involved in a variety of physiological andpathophysiological processes such as ECM assembly andproteolysis, axon guidance, embryonic development, inflam-matory and immune response, tumor formation and metasta-sis, among others (Dietrich 1984; Bernfield et al. 1999;Lopes et al. 2006a; Sampaio and Nader 2006).

Although a5b1 integrin has been implicated in several as-pects of cell motility, the molecular mechanisms involved incell locomotion are still unclear. How cells coordinatestrong adhesion to fibronectin substrate, the development oftension and a coordinated protrusion of the plasma mem-brane with release, and the formation of new adhesions ispoorly understood, especially how a5b1 integrin influencescell motility on fibronectin. Here, we approached this prob-lem and demonstrated that as a part-time PG, the GAGchains of a5b1 integrin play an essential role in the controlof haptotactic motility of cells on fibronectin.

Materials and methods

Reagents and antibodiesFibronectin was purified from fresh human plasma (ob-

tained from Hospital Sao Paulo, UNIFESP, SP, Brazil) by

affinity chromatography on gelatin–Sepharose (GE Health-care Life Sciences) following Engvall and Ruoslahti (1977).We used monoclonal antibody NKI-SAM-1 (MAB anti-a5)that recognizes the a5 integrin subunit from Chemicon Inter-national Inc. (Temecula, California), fluorescein-conjugatedanti-mouse IgG antibody from Jackson ImmunoresearchLaboratories, Inc. (West Grove, Pennsylvania), and DAPI(4’,6-diamidino-2-phenylindole, dihydrochloride) from Mo-lecular Probes, Inc. (Eugene, Oregon).

Cell cultureWild-type CHO cells (CHO-K1) and CHO cells are defi-

cient in PG biosynthesis (CHO-745) were obtained from Dr.Jeffrey D. Esko (Glycobiology Research and Training Cen-ter, UCSD, San Diego, California) (Esko et al. 1985). CHOcells transfected with cholecystokinin receptors (CHO-CCK)(Hadac et al. 1996) were donated by Dr. Carlos F. Toledo(UNIFESP, SP, Brazil). Additional cell lines used were en-dothelial cell (RAEC) and smooth muscle cell (RSMC) linesderived from rabbit aorta (Buonassisi and Venter 1976),RAEC transfected with EJ-ras oncogene (Lopes et al.2006b), and human umbilical vein endothelial cells (ECV-304) (Takahashi et al. 1990). Cells were grown in F-12 me-dium (Invitrogen, Carlsbad, California) supplemented with10% fetal calf serum (Cultilab, Campinas, SP, Brazil) withpenicillin (100 U/mL) and streptomycin (100 mg/mL) at37 8C at 2.5% relative humidity (RAEC, RSMC, ECV-304)or 5% CO2 (CHO cells). Cells were subcultured every weekwith pancreatin (Sigma-Aldrich Chemical Co., St. Louis,Missouri) and harvested using divalent cation free phos-phate-buffered saline (PBS) containing 2 mmol/L EDTA.For [35S]sulfate incorporation, cells were labeled with150 mCi/mL (1 mCi = 37 kBq) carrier-free [35S]sulfate(IPEN, Sao Paulo, Brazil) in F-12 medium for 18 h (Naderet al. 1987).

Immunoprecipitation reactionsCells were labeled with [35S]sulfate as described above

and, after incubation, the cells were removed and washedtwice with PBS and harvested with 2 mmol/L EDTA inPBS. The cells were washed three times with PBS and sus-pended in 500 mL of lysis buffer (50 mmol/L Tris–HCl(pH 7.4) containing 1% Triton X-100, 50 mmol/L NaCl,5 mmol/L CaCl2, 5 mmol/L MgCl2, 1 mmol/L PMSF (phe-nylmethanesulfonylfluoride), 2 mg/mL aprotinin, and 2 mg/mL leupeptin (protease inhibitors from Sigma-Aldrich)) for15 min. Protein was determined following the Bradford(1976) method. The lysates were then separated into 1 mgprotein aliquots that were then clarified by centrifugation(13 000g for 10 min). The supernatant was preincubatedwith mouse serum followed by precipitation with 20 mL ofprotein A-Sepharose (GE Healthcare) for 1 h at 4 8C. Cellextracts (normalized for protein content) were incubatedwith MAB anti-a5 (2 mg/mL) (mouse, IgG) for 2 h at 4 8Cfollowed by protein A-Sepharose beads for 1 h at 4 8C.Agarose beads were washed in lysis buffer and immunocom-plexes were then solubilized by adding SDS–PAGE bufferunder nonreducing conditions for 5 min at 100 8C and thenanalyzed with linear-gradient 3%–20% (w/v) SDS–PAGE(Pinhal et al. 2001). Membranes were exposed to multipur-pose films at room temperature for 15–30 days. Radioactiv-

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ity was quantified (counts per minute) using a radioscanner(Cyclone Storage Phosphor System by Perkin-Elmer LifeSciences/Wallac-Oy, Turku, Finland). Molecular massmarkers included myosin (205 kDa), Escherichia coli b-gal-actosidase (116 kDa), phosphorylase B (98 kDa), bovine se-rum albumin (BSA) (67 kDa), ovalbumin (44 kDa), andcarbonic anhydrase (29 kDa), all from Sigma-Aldrich.

Agarose gel electrophoresisPGs and GAGs were analyzed by agarose gel electropho-

resis in 0.05 mol/L 1,3-diaminopropane acetate bufferpH 9.0. Following electrophoresis, compounds in gel wereprecipitated with 0.1% Cetavlon for 2 h at room temperature(Dietrich and Dietrich 1976). After drying, the gel wasstained with toluidine blue and exposed on multipurposefilm for 15–30 days. Radioactivity was measured (countsper minute) using a radioscanner. GAG standards are hep-aran sulfate (HS) from bovine pancreas (Dietrich et al.1983), dermatan sulfate (DS) from pig skin, and chondroitinsulfate (CS) from shark cartilage (Seikagaku, Kogyo Co.,Tokyo, Japan).

Flow cytometric analysisCHO-K1 and CHO-745 were grown in F-12 medium (see

above) followed by cell removal with 2 mmol/L EDTA inPBS for 10 min. Cells (1 � 106) were suspended andwashed three times with PBS containing 1% BSA. Cell sus-pensions were then incubated with MAB anti-a5 (2 mg/mLin PBS plus 1% BSA) for 1 h at 4 8C, washed with PBS,and incubated with the secondary antibody (fluorescein iso-thiocyanate conjugated goat anti-mouse IgG) for 30 min at4 8C followed by three washes with FACS solution as rec-ommended by the manufacturer. Events (n = 10 000) wereanalyzed with a FACScan flow cytometer (Becton Dickin-son Imunocytometry System, San Jose, California).

Immunofluorescence microscopyCHO cells were grown on glass cover slips (&500 cells

each) for 7 days followed by fixation with 2% formaldehydein PBS for 30 min at 4 8C and incubated with 0.1 mol/Lglycine for 3 min. Cells were sequentially blocked withPBS containing 1% BSA for 1 h at room temperature. Integ-rin a5 was detected with MAB anti-a5 (2 mg/mL). Followingthree washes with PBS and blocking with PBS containing1% BSA for 30 min at room temperature, cells were thenincubated with secondary antibody fluorescein isothiocya-nate conjugate at room temperature for 40 min. Cell stainingwas then analyzed using an Eclipse E600 fluorescence mi-croscope (Nikon Instruments Inc., Melville, New York).

GAG chain analysisFree GAG chains from a5 and b1 integrin subunits were

obtained after overnight protein core digestion at 58 8Cwith maxatase (alkaline protease from Sporobacillus, 4 mg/mL) (Biocon do Brasil, Rio de Janeiro, Brazil). Digestionproducts were analyzed by agarose gel electrophoresis (de-scribed above). Samples were treated with chondroitinaseABC from Proteus vulgaris (Seikagaku Kogyo Co, Tokyo,Japan) and heparitinases I and II from Flavobacterium hep-arinum (Nader et al. 1990) and analyzed by agarose gelelectrophoresis.

Cell culture with chlorate and xylosidePostconfluent cultures of CHO cells were incubated with

either 75 mmol/L sodium chlorate (Merck, Rio de Janeiro,Brazil) or 300 mmol/L b-D-phenyl xyloside (Sigma-Aldrich)for 18, 24, and 48 h and 4, 8, 10, and 15 days, respectively.GAG chains were labeled by cell exposure to 150 mCi/mL[35S]sulfate in F-12 medium for the final 18 h. The mediumwas then removed and cells were washed twice with F-12medium and then harvested with 2 mmol/L EDTA in PBSfor 10 min. Cell viability was determined with the TrypanBlue exclusion method.

Adhesion assayCells were first incubated for 30 min at room temperature

with MAB anti-a5 at different concentrations. Tissue cultureplates (24 wells) (Costar by Corning Inc. Life Sciences,Lowell, Massachusetts) were coated for 2 h with fibronectin(10 mg/mL in PBS). Nonadhesive substrate was prepared bycoating the wells with 1% BSA for 60 min at 37oC. Plateswere washed with PBS and blocked with 1% BSA in PBSfor 1 h. Cells (treated with the antibody or not) were washedthree times, suspended (5 � 105 cells in 0.5 mL of F-12 me-dium), and allowed to attach to the substrate for 1 h at 37oCunder 5% atmospheric CO2. Following incubation, unat-tached cells were removed by washing with PBS. Attachedcells were fixed in methanol for 20 min, stained with 0.8%crystal violet (Sigma-Aldrich) dissolved in 20% ethanol, andwashed five times with PBS. The dye was eluted with 50%ethanol in 0.1 mol/L sodium citrate pH 4.2 and the opticaldensity measured at 540 nm.

Motility assayTo examine the involvement of a5b1 integrin on cell mo-

tility, cells were incubated for 30 min at room temperaturewith MAB anti-a5 at different concentrations. Haptotacticmotility experiments were carried out using transwell motil-ity chambers (8 mm pore size, Corning). An insoluble solid-phase gradient was established by floating the polycarbonatefilters in a solution containing fibronectin (10 mg/mL in PBSfor 40 min at 37 8C). The filters were then blocked with 1%BSA (1 h, 37 8C). Cells (1 � 106) were suspended in100 mL of F-12 medium and added to the upper transwellchamber and then incubated for 5 h at 37 8C in a humid at-mosphere containing 5% CO2. Following incubation, cellson the upper surface were removed using a cotton swab.Membranes were fixed with 2% formaldehyde for 10 minand stained for 10 min with 1% toluidine blue in Borax.The dye was eluted by adding SDS–PAGE buffer and cellmotility, measured as a function of migrating cells, was ex-pressed as optical density values at 630 nm. Experimentswere repeated three times.

Results

Different cell lines exhibit sulfated a5b1 integrinWe previously demonstrated that a5b1 integrin is post-

translationally modified by sulfate groups using a humanmelanoma cell line (Mel-85), a human osteosarcoma cellline (MG-63), and a human colon adenocarcinoma cell line(HCT-8). a5b1 integrin was confirmed as a part-time PGcompared with other b1 integrins expressed by Mel-85 cells

Franco et al. 679


(Veiga et al. 1997). To better understand this post-transla-tional modification and the biological significance thereof,we tested whether the sulfation of the a5b1 integrin wasalso expressed by either transformed or nontransformed(‘‘normal’’) cell lines. Six cell lines (CHO-K1, CHO-CCK,RSMC, ECV-304, RAEC, and RAEC-EJ-ras) were metabol-ically labeled with [35S]sulfate and the lysates immunopreci-pitated using a monoclonal antibody that recognizes the a5integrin subunit (MAB anti-a5) and analyzed by SDS–PAGE. The results confirmed that sulfate groups are incor-porated into the a5b1 integrin subunits in both normal andmalignant cells (Fig. 1), suggesting that this modification isconserved, transformation independent, and likely related tothe function of this integrin. Also, the proportion of a5b1 in-tegrin sulfated subunits varies among the different cell lines(Table 1).

Expression and sulfation of a5b1 integrin on CHO wild-type and mutant cells

To study the relevance of sulfation of the integrin subu-nits, we used CHO cells deficient in PG biosynthesis (CHO-745) and wild-type cells (CHO-K1) (Esko et al. 1985, 1987).Mutant cells express GAGs at approximately 5% of the ex-pression of wild-type cells (Fig. 2A). However, both celltypes synthesize the same proportions of CS and HS. Cellsurface a5b1 integrin was examined by flow cytometric anal-ysis and immunofluorescence microscopy using MAB anti-a5 (Figs. 2B and 2C). Both mutant and wild-type CHO cellsshowed similar levels of a5b1 integrin expression at the cellsurface. Metabolically, [35S]sulfate labeled lysates from bothcells, immunoprecipitated with MAB anti-a5 and analyzedby SDS–PAGE, showed that a5b1 integrin dimers from bothcells are sulfated macromolecules (Fig. 2D). Note that incor-poration of [35S]sulfate in the integrin subunits is similar forboth cell lines and contrasts with synthesis of sulfatedGAGs, which is reduced in the mutant type (Fig. 2A).

a5b1 integrin heterodimers from both wild-type andvariant CHO cells are PGs

[35S]sulfate labeled CHO cell lysates, immunoprecipitatedwith MAB-anti a5 and digested by protease followed byagarose gel electrophoresis, show two sulfated bands thatcomigrate electrophoretically with CS and HS standards(Fig. 3A). CS and HS covalently linked to a5b1 integrin forthe mutant cells are approximately 45% of those of the wild

Fig. 1. Sulfation of a5b1 integrin is a phenomenon displayed by different cell lines. Lysates normalized for protein content from [35S]sulfate-labeled CHO-K1, CCK, RSMC, ECV-304, RAEC, and RAEC EJ-ras cells were immunoprecipitated using MAB anti-a5. Immunoprecipi-tates were separated by SDS–PAGE under nonreducing conditions and transferred to a nitrocellulose membrane that was exposed to multi-purpose film for 30 days. Phase-contrast micrographs. Bar = 55 mm.

Table 1. Relative proportion of sulfated a5b1 integrin sub-units in different cell lines.

Cell line % of a5 subunit % of b1 subunitCHO-K1 59.14 40.86CCK 72.96 27.04RSMC 76.61 23.39ECV-304 37.96 62.04RAEC 37.47 62.53RAEC-EJ-ras 24.82 75.18



type. This result, along with degradation with chondroitinaseand heparitinases (Figs. 3B and 3C), indicates that the mu-tant cell line is preferentially synthesizing the GAG chainsthat are linked to the integrin. Also, immunoprecipitateda5b1 integrin, treated with chondroitinase or heparitinasesplus chondroitinase, resulted in total degradation after GAGlyases (data not shown), thereby also indicating that a5b1 in-tegrin from CHO cells is a PG.

Effect of xyloside on sulfate incorporation into a5b1integrins from CHO cells

Cell lysates immunoprecipitated with MAB anti-a5 and

subjected to electrophoresis on SDS–PAGE resulted in a70% reduction of sulfate incorporation into the a chain ofthe integrin and a 95% reduction in the b chain when CHOwild-type cells were grown with xyloside (data not shown),which inhibits PG synthesis (Fritz and Esko 2001). Thus,this molecule is indeed a PG.

a5b1 integrin expression and adhesion to fibronectin ofCHO cells exposed to xyloside

CHO wild-type cells were grown in the presence or ab-sence of xylosides and were then tested for a5b1 integrin ex-pression with MAB anti-a5 and adhesion to fibronectin.

Fig. 2. Both wild-type and mutant CHO cells express similar levels of sulfated a5b1 integrin. (A) Lysates from [35S]sulfate-labeled CHO-K1and CHO-745 cells normalized for protein content were digested by maxatase and subjected to agarose gel electrophoresis. After drying, thegel was exposed to multipurpose film for 5 days. The migration of standard CS, DS, and HS is shown on the left part of the figure. (B)Equal numbers of CHO-745 and CHO-K1 were treated with MAB anti-a5 followed by staining with fluorescein-conjugated anti-mouse IgGand analyzed by flow cytometry. (C) Immunofluorescence of CHO-K1 and CHO-745 cells adhered to glass cover slips and stained withMAB anti-a5 and fluorescein-labeled secondary antibody. (D) [35S]sulfate-labeled lysates normalized for protein content from CHO-K1 andCHO-745 cells were immunoprecipitated by MAB anti-a5, separated by SDS–PAGE under nonreducing conditions, transferred onto a nitro-cellulose membrane, and exposed to multipurpose film for 30 days. The positions of the a5 and b1 integrin chains are shown on the left ofthe figure. Bar = 12.6 mm.

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Xyloside-treated CHO-K1 cells have cell surface a5b1 integ-rin expression levels similar to those of control cells in theabsence of xyloside (data not shown), supporting that GAGchains of a5b1 integrin do not influence cellular traffickingof this integrin to the plasma membrane. CHO cells grownwith xyloside have levels of adhesion to fibronectin similarto those of controls (Fig. 4A), which depends on a5b1 integ-rin cell surface expression and hence suggests that GAGchains from a5b1 integrin are unnecessary for adhesion ofCHO cells to fibronectin. This agrees with a previous studythat demonstrated that CHO cells deficient in PG synthesisadhered to fibronectin as well as CHO wild-type cells. Onthe other hand, inhibition experiments using a monoclonalantibody that binds to a5 integrin showed that CHO cell ad-hesion on fibronectin is dependent on this integrin (Fig. 4B).

Motility on fibronectin of CHO cells exposed to xylosideIt has been shown that a5b1 integrin is required for migra-

tion of cells on fibronectin and for fibronectin matrix assem-bly (Laukaitis et al. 2001). Then, we tested whether GAGchains from the a5b1 integrin influence cell motility on fi-bronectin. Motility of mutant CHO cells decreases on fibro-nectin to approximately 53% of that observed in wild-typecells (data not show). Xyloside treatment markedly reducedthe motility of cells on fibronectin, showing that GAGchains from the a5b1 integrin are involved in fibronectincell motility (Fig. 5A). The xyloside treatment did not influ-ence cell viability (>96%). Also, using an anti-a5 MAB, cellmigration was inhibited (Fig. 5B), thus confirming that a5b1integrin from CHO cells determines at least in part cell mo-tility on fibronectin.

The incorporation of [35S]sulfate in both HS and CS de-creases in the cell extract after exposure to xyloside(Fig. 5C). Around day 2, there was an inhibition of approx-imately 50% of GAG synthesis (Fig. 5D) paralleled with asignificant decrease in cell motility (Fig. 5A).

Motility on fibronectin of CHO cells exposed to chlorateAfter cell exposure to chlorate (a potent inhibitor of sulfa-

tion (Veiga et al. 1997), like selenate (Dietrich et al. 1988)),cell motility on fibronectin declines by approximately 50%

(Fig. 6) along with a decrease in PG synthesis (data notshown). Cell viability with chlorate was similar to that ofcontrol cells.

Discussion

The integrin family of cell surface receptors exemplifieshow post-translational modifications may result in confor-mational changes in molecules, thereby influencing their bi-ological activity. Integrin glycosylation is known to becritical for integrin activity by regulating the specificity andaffinity of binding to ligand integrins (Akiyama et al. 1989;Chammas et al. 1991, 1993; Veiga et al. 1995). Integrins aremetalloproteins with three to five divalent cation bindingsites. Cations exert important effects on molecular structureand biological functions of the integrins by promoting or in-hibiting binding to these molecules (Plow et al. 2000). Tyro-sine and serine phosphorylation of cytoplasmic domains arepost-translational modifications by which cells can regulatethe activity of integrins by impairing their ability to bind cy-toskeletal molecules and to interact with ECM ligands(Martin et al. 2002). Membrane lipids seem also to modifyintegrins, thus interfering with their function (Conforti et al.1990). The diversity of post-translational modifications ofintegrins was further expanded by the description of a5b1 in-tegrin as a part-time PG containing both HS and CS cova-lently linked chains (Veiga et al. 1997). In this study, weevaluated the contributions of GAG chains of a5b1 integrinto adhesion dynamics on fibronectin by using distinct cellmodels. Initially, our data showed that all tested cell linesin culture when metabolically labeled with [35S]sulfateshowed sulfate residues in their a5b1 integrins. This con-firms that a5b1 integrin is a facultative PG molecule andthat this activity occurs in all cell models, since previousstudies used only transformed cell lines (Veiga et al. 1997).These results point to a biological significance for this post-translational modification based on similar and conservedphenomena for all cellular models studied.

Since the a5b1 integrin dimer is the classical fibronectinreceptor and it mediates adhesion and migration of differentcells to this ECM molecule (Plow et al. 2000), we evaluated

Fig. 3. The a5b1 integrins from wild-type and mutant CHO cells are hybrid CS–HS PGs. (A) Lysates from CHO-K1 and CHO-745 labeledwith [35S]sulfate, normalized for their protein content, were immunoprecipitated by MAB anti-a5. The precipitates were digested with max-atase and submitted to electrophoresis on agarose gel that was dried and exposed to multipurpose film for 30 days. The positions of GAGstandards are shown on the left of the figure. (B and C) [35S]sulfate-labeled a5b1 integrin from CHO-K1 and CHO-745 cells, respectively,obtained after immunoprecipitation as described above, was digested with maxatase and the obtained GAG chains were incubated withchondroitinase ABC (ABC), heparitinases I and II (I + II), or no enzyme added (control). The incubation mixtures were applied to agarosegel electrophoresis. After drying, the gel was exposed to multipurpose film for 30 days. The positions of standard GAGs chains are shownon the left of the figures.



whether the GAG chains present in the integrin would inter-fere with either function of this particular integrin. To testthis, we studied CHO wild-type and mutant cells for PG bio-synthesis. Metabolically radiolabeling with [35S]sulfateshowed that mutant CHO cells incorporate much less sulfateon their GAGs than do wild-type cells (&5% of that of thewild-type cells) as described previously (Esko et al. 1987).Flow cytometric and immunofluorescence analysis withMAB anti-a5 showed similar a5b1 expression levels for bothwild-type and mutant CHO cells. Yet a5b1 integrin was sul-fated in both mutant and wild-type cells. Protease and GAGlyases digestions of the immunoprecipitated a5b1 integrinconfirmed this integrin as a PG. Mutant CHO cells, whilehaving a marked reduction in PG biosynthesis, still showsignificant glycosylation of their a5b1 with GAG chains.Not only does this demonstrates that GAG chains are incor-porated into a5b1 integrin but it also suggests that this post-

translational modification of a5b1 integrin cooperates in cel-lular events. Further evidence for GAG chains linked to a5b1integrin heterodimers come from metabolic [35S]sulfate ra-diolabeling in the presence of xyloside, an inhibitor of PGbiosynthesis. Inhibition of sulfate incorporation is clear ina5b1 integrin from CHO wild-type cells in the presence ofxyloside, which confirms the notion that a5b1 integrin is aPG (data not shown).

Which biological functions do the GAG chains in the a5b1integrin have in cell interactions with fibronectin? In a firstseries of experiments, we addressed the adhesive propertiesof wild-type CHO cells on fibronectin following their expo-sure to xylosides, comparing them with a control condition(sham treatment). a5b1 integrin cell surface expression(Fig. 4), cell viability, and adhesion to fibronectin were sim-ilar in both conditions. This suggests that GAG chains froma5b1 integrin in wild-type CHO cells are not required tomaintain adhesion on fibronectin, nor are they necessary forassembly of the a5b1 heterodimer or for transit through thethe Golgi apparatus and to the plasma membrane (Akiyamaet al. 1989). This last explanation is in agreement with ex-periments described by Akiyama et al. (1989) supportingthat assembly of ab integrin heterodimers occurs duringtransit through the endoplasmic reticulum and precedesGAG biosynthesis and PG formation, which occur mainlyduring transit through the Golgi apparatus (Esko and Lin-dahl 2001).

In an interesting way, the mutant cell, which produces 5%of GAGs when compared with the wild-type cell and, fromthese, approximately 50% are demonstrated to be covalentlylinked to integrin a5b1, had its motility reduced on fibronec-tin (53% compared with the wild-type cell) (Fig. 5). How-ever, our results clearly indicate that wild-type CHO cellsincubated with xyloside had decreased motility on fibronec-tin, exhibiting a residual motility of approximately 32% ofcontrols (without xyloside) in haptotactic experiments(Fig. 5). This suggests an important role for GAG chains ofthe a5b1 integrin during CHO cell motility towards fibronec-tin. Motility experiments of CHO cells on fibronectin withchlorate (an inhibitor of sulfation (Veiga et al. 1997)) furthersupport this possibility (Fig. 6).

The data presented here show for the first time in the lit-erature the role of GAG chains present in a5b1 integrin.However, further work is needed to elucidate the structureand functions of these GAG chains in fibronectin–cell inter-actions. The coreceptor hypothesis is reasonable and predictsthat GAG chains cooperate with the primary RGD peptide(Ruoslahti and Pierschbacher 1987) or secondary synergisticpeptides on fibronectin that bind a5b1 integrin (Plow et al.2000). This complements affinity interactions that are neces-sary for fibronectin–cell adhesion and migration. Previousstudies implicated the involvement of GAG binding sites offibronectin in cell adhesion to this molecule (Mercurius andMorla 2001). The involvement in fibronectin cell adhesionof a cell surface phosphatidylinositol-anchored PG (Bern-field et al. 1999) and the association of a fibronectin bindingcell surface PG with a5b1 integrin during fibronectin–celladhesion (Plow et al. 2000) support the synergism amongRGD and other secondary fibronectin adhesive peptideswith fibronectin GAG binding peptides during the processesof cell adhesion and migration on fibronectin.

Fig. 4. Xyloside treatment of CHO cells does not alter a5b1 integrinexpression at the cell surface and adhesion to fibronectin. (A) Timecourse adhesion of CHO-K1 on fibronectin-coated multiwell plates(10 mg/mL) cultivated in the presence of 300 mmol/L xyloside. Re-sults are means ± SE of three independent experiments performedin triplicate (ANOVA, P > 0.05). (B) Adhesion experiments to fi-bronectin (under the same conditions described above) except thatCHO-K1 cells were incubated with MAB anti-a5. BSA showsbackground values of cell adhesion to BSA. Results are means ±SE of four independent experiments performed in triplicate. Com-parisons were tested by one-way ANOVA followed by a Tukey testwith a significance level of P < 0.05 (asterisk).

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Control of cell motility to an ECM is a complex processand requires adhesion to and detachment from a coated sub-strate at the cell front, thereby providing the traction neces-sary for cell migration (Laukaitis et al. 2001). The level ofa5b1 expression at the cell surface controls cell adhesionand motility towards fibronectin (Plow et al. 2000). How-ever, we still are unable to fully explain the effects of GAGchains of the a5b1 integrin on fibronectin cell motility. Areasonable hypothesis is that GAG chains covalently linkedto a5b1 integrin could be necessary to organize and controlcell detachment from fibronectin, which is critically impor-tant for cell locomotion towards ECM substrates. The pres-ence of large negatively charged and hydrophilic residues,such as the GAG chains, in the a5b1 integrin could regulatethe exposure of integrin binding sites to their respective li-gands on fibronectin, thereby controlling cell motility onthis molecule.

Even when expressed by the same cell type, syndecansand glypicans (two cell surface HSPGs) can exert distinctfunctions, either as adhesive or as antiadhesive molecules(Couchman 2003). Syndecans increase cell adhesion and in-

Fig. 5. Motility on fibronectin of wild-type CHO cells exposed to xyloside. (A) A similar experiment as described in Fig. 4 was done exceptthat CHO-K1 cells were cultured either in the absence (time 0) or the presence of 300 mmol/L xyloside in a time course experiment. Mean ±SD values are shown for three independent experiments performed in triplicate. (B) Motility experiments on fibronectin of CHO-K1 (asdescribed above) incubated with different concentrations of MAB anti-a5. Results are means ± SE. One-way ANOVA tested for differencesamong treatments followed by a Tukey test; differences are considered significant at #P < 0.05 and *P < 0.01. (C) Cellular GAG synthesisin CHO wild-type cells exposed to xyloside. Lysates from [35S]sulfate-labeled CHO wild-type cells, grown in the presence of 300 mmol/Lxyloside in a time course experiment, normalized for their protein content were digested by maxatase and submitted to agarose gel electro-phoresis. After drying, the gel was exposed to multipurpose film for 15 days. The migration of the standard is shown on the left of thefigure. The control represents cells maintained in the absence of xyloside. (D) Quantification of the experiment shown in Fig. 5C expressedas a percentage of the control.

Fig. 6. Inhibitory activity of chlorate on the motility of wild-typeCHO cells on fibronectin. The experiments of motility on fibronec-tin were performed as described in Fig. 5 except that CHO-K1 cellswere treated with 75 mmol/L sodium chlorate for 16 h and thenprocessed as described. Mean ± SD values are shown for three in-dependent experiments performed in triplicate.



hibit cell invasion onto ECM-coated substrata (Woods 2001;Couchman 2003), while the expression of glypican-1 on thecell surface does not increase cell binding to collagen andfails to increase cellular invasiveness. The lack of GAGsdoes not affect initial attachment and subsequent spreadingof CHO cells on fibronectin (LeBaron et al. 1988). PG func-tions in cell–matrix and cell–cell adhesion and interactionsare dependent on GAG chains, the protein core, or both(Dietrich 1984; Bernfield et al. 1999; Porcionatto et al.1999; Woods 2001). It is unclear how a5b1 GAG chains in-terfere with cell migration on fibronectin. We suggest thatGAG chains from the integrin directly cause transient bind-ing of the a5b1 integrin to fibronectin. This is critical for celldisassembly to migrating substrates, during cell migration,or in some instances GAG chains participating in the modu-lation of the interaction between a5b1 integrin and cytoskele-tal components (which initiates cell release and precedesmigration). This suggestion is supported by the notion thatcells with a defective metabolism for biosynthesis of PGhave a defective focal adhesion plaque formation in re-sponse to fibronectin (LeBaron et al. 1988).

In summary, our results clearly indicate that a5b1 integrinis post-translationally modified by GAG chains in different‘‘normal’’ and malignant cell lines. Furthermore, we havedemonstrated that GAG chains in a5b1 integrin are requiredfor the control of cell locomotion on fibronectin.

AcknowledgementThe authors thank Dr. James J. Roper for help with the

English.

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Glycosaminoglycan chains from α 5 β 1 integrin are involved in fibronectin-dependent cell migrationDedicated to the memory of Professor Carl P. Dietrich