(CANCER RESEARCH 48, 4743-4748, September 1, 1988]

Glycosylation-dependent Collagen-binding Activities of Two MembraneGlycoproteins in MDAY-D2 Tumor Cells1

Suzanne Laferté2and James W. Dennis3

Division of Cancer and Cell Biology, Mount Sinai Hospital Research Institute, Toronto, Ontario M5G 1X5 [J. W. DJ, and Department of Medical Genetics, Universityof Toronto, Toronto, Ontario [J. W. D.J, Canada

ABSTRACT

Two highly glycosylated membrane sialoglycoproteins designated P2Aand P2B were isolated from the lymphoreticular tumor cell line calledMDAY-D2 and shown to be structurally similar to the lymphocyteglycoprotein called leukosialin and to lysosome-associated membraneglycoprotein termed I .AMP-1, respectively. The loss of sialic acid andpolylactosamine sequences in glycosylation mutants of MDAY-D2 has

been correlated previously with enhanced cell adhesion on extracellularmatrix proteins. Since these two glycoproteins bear the majority of thesialylated oligosaccharides found in membrane fractions of MDAY-D2,

they were tested for binding activity on extracellular matrix proteins.Both isolated glycoproteins bound to immobilized collagen type I withaffinities that were dependent on their glycosylation. Enzymatic removalof sialic acid, polylactosamine, or complete asparagine-linked chainsfrom purified P2B enhanced its binding to collagen, laminin, and fibro-

nectin. In contrast, P2A bound specifically to collagen type I and theinteraction required the presence of sialic acid residues which weresensitive to neuraminidase digestion but not to endoglycosidase F. Theresults suggest that oncodevelopmental regulation of oligosaccharideexpression on P2A and P2B glycoproteins may modulate their bindingto extracellular matrix glycoproteins.

INTRODUCTION

Malignant transformation is often accompanied by changesin oligosaccharide expression (1,2), and some of these changesmay be associated with altered cell adhesion to the ECM4 (3,

4) and with metastatic potential of the tumor cells (5, 6).Control of cell proliferation (3, 4) and certain differentiatedfunctions (7, 8) are regulated in part by specific contacts withECM and loss of these contacts following malignant transformation may contribute to tumor cell proliferation, cell motility,and metastasis (4, 9).

Studies on the class 1 glycosylation mutants of the highlymetastatic tumor cell line MDAY-D2 suggested that decreasedexpression of sialylated glycoconjugates may be related to theobserved increase in cell attachment on collagen-, fibronectin-,and laminin-coated surfaces (10-12). The class 1 mutants synthesize glycoproteins and glycolipids which are deficient insialic acid and galactose apparently due to a defect in transportof the sugar donor UDP-galactose into the golgi apparatus (13).Class 1 tumor cells were shown to be tumorigenic in syngeneicmice but nonmetastatic from a s.c. site. Suppression of the

Received 3/25/88; revised 5/27/88; accepted 6/6/88.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' This work was supported by the National Cancer Institute of Canada.2Recipient of a Postdoctoral Fellowship of the National Cancer Institute of

Canada. Present address: Department of Biochemistry, A3 Health SciencesBuilding, University of Saskatoon, Saskatoon, Saskatchewan S7N OWO,Canada.

3Research Scholar of the National Cancer Institute of Canada. To whom

requests for reprints should be addressed, at Division of Cancer and Cell Biology,Mount Sinai Hospital Research Institute, 600 University Avenue, Toronto,Ontario M5G IXS, Canada.

4 The abbreviations used are: ECM, extracellular matrix; DOC, deoxycholate;WGA, wheat germ agglutinin; L-PHA, leukoagglutinin; GRGDPTL, Gly-Arg-Gly-Asp-Pro-Thr-Leu; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gelelectrophoresis; BSA, bovine serum albumin; ELISA, enzyme-linked immuno-sorbent assay; NCAM, neuron adhesion molecule; Gal, galactosyl; GalNAc, N-acetylgalactosamine; GlcNAc, /V-acetyl-glucosamine; Neu NAc, A'-acetylneuramicacid; RDG, Arg-Asp-Gly; GRGDSPK, Gly-Arg-Gly-Asp-Ser-Pro-Lys.

recessive class 1 mutation by somatic cell fusion with lymphoidcells restored the metastatic and nonadhesive phenotype in thehybrid cells (10). Similarly, partial revertants of the class 1mutation had mixtures of mutant and wild type oligosaccharidestructures as well as intermediate organ colonization potentialand intermediate cell-adhesive properties on ECM glycoproteins in vitro (11). Finally neuraminidase treatment of wild typeMDAY-D2 cells enhanced cell adhesion on collagen, laminin,and fibronectin suggesting that sialylation of certain glycoconjugates may be directly related to the nonadherent phenotypeofMDAY-D2cells(10).

The major sialylated glycoproteins in MDAY-D2 cells canbe detected by 125I-WGA staining of membrane glycoproteinsseparated by SDS-PAGE and appear to have molecular weightsof 80,000-160,000 (11, 13). The glycoproteins also stainedwith 125I-L-PHA indicating the presence of /91—6 branched

complex type oligosaccharides. Digestion of membrane preparations with neuraminidase and endo-ß-galactosidase reducedthe size heterogeneity and suggested that only a few glycoproteins possessing L-PHA reactive oligosaccharides were presentin MDAY-D2 cells. In an effort to identify these glycoproteinsand their possible role in the malignant and/or adhesive phe-notypes of MDAY-D2 cells, two major sialoglycoprotein werepurified from MDAY-D2 cells and structurally characterized.5

In this report, the glycoproteins designated P2A and P2B werecompared before and after glycosidase digestion for bindingactivity on immobilized ECM glycoproteins. P2A bound specifically to collagen type I and this activity appeared to bedependent on sialylation of C>linked oligosaccharides. P2Bbound weakly to collagen fibronectin and laminin and this wasenhanced by removal of sialic acid and polylactosamine presenton TV-linkedoligosaccharides.

MATERIALS AND METHODS

Purification of P2A and P2B. The glycoproteins were purified from50 ml of packed MDAY-D2 cells as described previously.5 Briefly, thepelleted cells were extracted at 4°Cfor 2 h with constant stirring in

200 ml of 50 HIMTris-HCl (pH 8.0)-0.15 M NaCl (buffer A) containing1 mM EOT A, 1% Triton X-100, and 2 mM phenylmethylsulfonylfluoride. The extract was centrifuged at 15,000 x g for 30 min and thesupernatant was diluted 4-fold with buffer A. The extract was appliedto a 25-ml column of WGA-Sepharose (4 mg lectin/ml Sepharose),preequilibrated in buffer A containing 0.2% Triton. The column waswashed with 10 volumes of 10 mM Tris-HCl, pH 8.0, containing 0.4%sodium deoxycholate and 1 mM EDTA and eluted with the same buffercontaining 5% M-acetylglucosamine. P2A and P2B were detected as L-PHA-reactive glycoproteins in the eluted fractions.5

The L-PHA-positive fractions eluted from WGA-Sepharose werepooled, concentrated to 5 ml by ultrafiltration using an Amicon P-10membrane, and applied to a column (2.5 x 120 cm) of Bio-Gel A-5m(100-200 mesh). The column separated the material into two major L-PHA-reactive peaks which contained more than 90% of the appliedglycoproteins and the fractions were designated P2A and P2B. P2Aand P2B were each dialyzed against 20 mM NH4HCO3-0.01% SDS,

5S. Lafertéand J. W. Dennis. Purification of two glycoproteins expressing01-6 branched Asn-linked oligosaccharides from metastatic tumor cells, submittedfor publication.

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lyophilized, resuspended in 2 ml SDS-sample buffer (0.03 M Tris-HCl,pH 6.8-5% 2-mercaptoethanol-5% glycerol-1.2% SDS), and subjectedto preparative SDS-PAGE. A vertical strip of acrylamide was removedfrom each gel (0.35 x 16 cm), cut into 0.5-cm lengths, and extractedovernight in buffer A containing 0.1 % SDS, and the extracted proteinwas counted for radioactivity. Regions of the gel corresponding to theglycoproteins were pooled and purified P2A and P2B isolated byelectroelution. The sample was dialyzed into 10 mM Tris, pH 7.4-0.15mM NaCl-0.4% DOC and concentrated to 3 ml by ultrafiltration.Protein determinations were carried out with BCA reagent (Pierce)using BSA as the standard.

Binding of '"I-labeled P2A and P2B to Collagen-Sepharose 4B.Twenty-five ^g of P2A and P2B were radioiodinated with 250 ¿iCiofNa'25I (Amersham) and lodogen beads (Pierce) for 10 min at 20°C,

followed by desalting on a Bio-Gel P-10 column developed in 10 mMTris-HCl, pH 7.4-0.15 M NaCl-0.4% DOC. The specific activity was6-12 x 106cpm/ng and the labeled glycoproteins were made 0.1 % BSAand stored at 4°C.

CNBr-activated Sepharose 4B (Pharmacia) was coupled accordingto manufacturer's instructions with rat tail collagen type I, collagen

type IV (Sigma), human plasma fibronectin, or the chemically synthesized peptide GRGDSPK. Collagens were dissolved in 0.1 M aceticacid and then dialyzed into coupling buffer 0.1 M NaHCO3, pH 8.O.Proteins were coupled at a ratio of 10 mg protein/g Sepharose with a70-80% efficiency as determined by Aâ„¢before and after coupling. Thecoupled Sepharose was equilibrated with 10 mM Tris, pH 7.4-1 mMCaCli-1 mM MnClz-0.1% Triton X-100 and 50-^1 aliquots were incubated with P2A or P2B, 2 x 10s cpm, on ice for 2 h. The beads were

washed 5 times with the Tris buffer and eluted with buffer containing0.2 M NaCl at 4°Cfor 30 min. Eluted samples were diluted with 2 x

SDS-PAGE sample buffer and separated on a 7.5% polyacrylamide gelunder denaturing conditions which were dried and exposed to X-ray

film.Glycosidase Digestions. Aliquots of iodinated P2A and P2B (5-10

ng) were incubated at 37°Covernight with 0.2 unit endoglycosidase F

from Flavobacterium meningosepticum (Boehringer) in 20 ^1 of 10 mMTris-HCl, pH 7.4-0.4% DOC for 16 h. For neuraminidase and endo-/3-galactosidase digestion, aliquots of P2A and P2B in the same bufferwere diluted with an equal volume of 50 mM sodium acetate, pH 5.0,and incubated at 37°Covernight with either 5 mU of endo-/3-galacto-sidase (Escherichia freundi; Miles) or 50 mU neuraminidase (Clostri-

dium perfringens; Sigma type X). The glycosidase digestions produceda characteristic shift in the molecular weights of P2A and P2B on SDS-PAGE. Neuraminidase and endoglycosidase F digestions under theseconditions eliminated WGA and L-PHA reactivity, respectively, forboth purified P2A and P2B and for crude membrane preparations ofthe glycoproteins. Undigested samples were incubated in buffer alone.

P2A and P2B Binding to Collagen-coated ELISA Plates. ELISA plateswere coated overnight at 4°Cwith 2.5 ^g/well of rat tail type I collagen

or type IV prepared by the pepsin method (Sigma). The wells wereblocked for 30 min at 20°Cwith 1% BSA in 10 mM Tris-HCl (pH 7.5)-1 mM MgClz-l mM CaCh and then washed in the same buffer. I2SI-

Labeled P2A or P2B preincubated with either buffer, neuraminidase,glycopeptidase F, or endo-/3-galactosidase was diluted in the Tris buffercontaining 1% BSA, such that 10s cpm were added to each well.

Following incubation at room temperature for 4 h, wells were washed5 times with Tris buffer, the wells were cut out, and the radioactivecontent of each well was measured in a gamma counter.

RESULTS

P2A and P2B Glycoproteins. The purified glycoproteins P2Aand P2B were distinct from one another as indicted by aminoacid sequencing of the amino ends. Secondly, endoglycosidaseF digestion of the purified glycoproteins indicated differentcontents of N- and CMinked oligosaccharides and this wasconfirmed by lectin binding and monosaccharide composition(Table 1). Finally, rabbit polyclonal antiserum raised againstP2B did not recognize purified P2A.5

Based on these analysis, P2A appeared to be structurally

Table 1 Characteristics ofP2A and P2BSummary of glycoprotein characterization.' Estimates of N- and O-linked

content are based on glycosidase digestions and monosaccharide composition.Amino acid compositions for P2A and P2B were compared to an average of 204proteins.

P2A P2B

Migration on SDS-PAGE(A/,)NativeNeuraminidaseNeuraminidase + endo-

rf-galactosidaseEndoglycosidase F

W-Linked content (%)0-Linked content (%)Amino acid composition

Lectin bindingTissue distribution by

Western blotting

90,000-120,000120,000-140,000100,000-120,000

65,000-75,000

15-2520-30

High, Ser, Thr, ProLow, Ile, LysWGA, L-PHA, PNA"

ND*

90,000-150,00090,000-150,00090,000-95,000

40,000

50-60<5

High, Ser, GlyLow, Ile, LysWGA, L-PHAWidely distributed

" Peanut agglutinin (PNA) bound only following neuraminidase digestion.WGA reacts with a2—3 sialylated structures (38) and L-PHA binds to /31-6-branched complex type oligosaccharides (39).

* ND, not determined.

similar to human (14,15) and rat leukosialin (16,17), a lymph-oid cell surface glycoprotein with a high content of O-linkedoligosaccharides. Amino end sequence showed that 4 of thefirst 10 residues were identical between P2A and rat leukosialin(17). The molecular weights of these glycoproteins are 100,000-130,000, by SDS-PAGE and show an apparent increase inmolecular weight of 20,000-30,000 following release of sialicacid from O-linked oligosaccharides with neuraminidase (Table1). The asialo forms of the glycoproteins bound peanut agglutinin indicating the presence of unsubstituted Gal-GlcNAc onserine/threonine residues (18). The rat form of leukosialin lacksyV-linked oligosaccharides (17), the human form has 1 or 2chains (15) and mouse P2A also has several jV-linked oligosaccharides.5

P2B shared amino end sequence homology with murinelysosomal membrane glycoprotein designated LAMP-1 (15 ofthe first 20 amino acids). Both P2B and LAMP-1 have beenshown to have unusually high contents of jV-linked oligosaccha-ride, in the order of 50% as suggested by the decrease in theirapparent molecular weights following glycosidase digestions(Table 1) (19).5

Binding of P2A and P2B to ECM Glycoproteins. Two assayswere used to measure binding of 125I-labeled P2A and P2B to

ECM proteins in the presence of low NaCl. The iodinatedglycoproteins were incubated with Sepharose beads coupledwith fibronectin or collagen types I or IV and the boundradioactivity was eluted with 0.2 M NaCl and visualized following SDS-PAGE. This procedure allowed a qualitative assessment of the bound radioactivity and confirmation of the glycosidase digestions. The second assay measured direct bindingof the radiolabeled glycoproteins to ELISA plates coated withtitered amounts of ECM proteins.

P2A bound specifically to collagen type I-Sepharose and notto fibronectin and collagen type IV. In contrast P2B boundweakly to collagen type I but did not appear to bind to fibronectin or collagen type IV (Fig. 1). Reapplication of unboundP2A and P2B to fresh collagen type I-Sepharose showed littleadditional binding indicating that the amount of affinity matrixused in the assay was saturating (Fig. \li). Binding of P2A andP2B to ELISA plates coated with ECM glycoproteins wasdependent on the ECM concentration and confirmed the specificities observed for binding to ligand-coupled Sepharose (Fig.2).

Sialic Acid on P2A Required for Binding to Collagen. Neither4744

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COLLAGEN-BINDING GLYCOPROTEINS

P2A P2B P2A P2B'C NME F' 'C N N,E F' C N N,E F C N N,E F

92-

68*

45*

Type ETCollagen- Sepharose

Fibronectin-sepharose

P2A P2BP2A P2B

B C N N.E—ii—F C

-\ °N N,EF «

M ir92-

68-*-

45-

30-

Typel Collagen-SepharoseFig. 1. Binding specificity of '''M-labeled P2A and P2B on collagen and

fibronectin coupled to Sepharose. Aliquots of P2A and P2B containing HP cpmwere predigested with nil (C), neuraminidase (N), neuraminidase plus endo-/3-galactosidase ( v, £),or endoglycosidase F (/') and then applied to collagen or

fibronectin coupled to Sepharose. After the Sepharose beads were washed, boundmaterial was eluted with 0.2 M NaCI and separated by SDS-PAGE. In the 4righthand lanes of B, unbound material was reapplied to fresh collagen-Sepharose(second elution) to assure that binding capacity of the collagen was not exceededwith these conditions. For P2A on collagen type I-Sepharose and glycosidase-digested P2B on fibronectin and collagen type I-Sepharose, binding ranged from10 to 15% of the applied counts, similar to that observed with the ELISA plates(Fig. 2).

removal of polylactosamine sequences by endo-ß-galactosidasedigestion nor complete removal of TV-linked oligosaccharidesby endoglycosidase F digestion altered P2A binding to collagen

type I (Fig. 2). Endoglycosidase F digestion removed TV-linkedoligosaccharides, as indicated by the increased mobility of collagen-bound P2A separated by SDS-PAGE (Fig. IA). Theendoglycosidase F-treated P2A retained its diffuse appearanceon SDS-PAGE due to the presence of sialylated O-linkedoligosaccharides.

Neuraminidase-treated P2A showed reduced binding to collagen-Sepharose and to collagen-coated ELISA plates (Figs,and 1 and 2). Since sialic acid associated with TV-linked oligosaccharides would be removed by endoglycosidase F digestion,the sialic acid residues on O-linked structures of P2A appear tobe required for collagen binding. Reduced and alkylated P2Ashowed a similar loss of collagen-binding activity suggestingthat the native conformation of the glycoprotein is required forbinding (Fig. 2).

TV-LinkedSialic Acid and Polylactosamine Reduce P2B Binding. Compared to P2A, P2B bound poorly to the ECM glyco-proteins. However, endoglycosidase F-digested P2B (i.e., M,40,000) showed increased affinity for collagens, laminin, andfibronectin (Figs. 1 and 2). Similarly, the removal of polylactosamine sequences or sialic acid from P2B also enhancedbinding to the ECM glycoproteins. Therefore, the bindingaffinity of P2B for these ECM glycoproteins appeared to bereduced by the presence of sialic acid residues which may belinked to polylactosamine sequences associated with TV-linkedoligosaccharides.

The considerable size heterogeneity observed for P2B separated by SDS-PAGE was reduced by endo-0-galactosidasedigestion (Fig. I).5 This suggested that subpopulations of puri

fied P2B differing in polylactosamine content might be distinguished by their binding affinities for ECM glycoproteins. Totest this hypothesis, purified P2B was applied to type I collagen-Sepharose and eluted in a stepwise manner with increasingconcentrations of NaCI. P2B could be partially eluted with 20mM NaCI, while endoglycosidase F-digested P2B remainedbound (Fig. 3). Raising the NaCI concentration to 50 mM wassufficient to elute most of the bound P2B, both untreated andendoglycosidase F digested. A comparison of untreated P2Beluted from the collagen-Sepharose with increasing salt concentrations indicated that a higher molecular weight subfraction ofP2B eluted first (Fig. 3). This supports the conclusions drawnfrom the studies using glycosidases and further suggests thatpolylactosamine sequences as well as sialylation may reduce thebinding affinity of P2B for collagen.

Binding to GRGDSPK-Sepharose. Since many of the cellattachment receptors recognize RGD-containing peptide sequences present in glycoproteins such as fibronectin and vitro-nectin (20-22), P2A and P2B were compared for their abilityto bind to an immobilized GRGDSPK. Similar amounts ofendoglycosidase F-digested 125I-labeled P2B bound to collagen

type I and GRGDPTL-Sepharose 4B while P2A did not bindto the peptide (Table 2). The same result was obtained using apeptide-Sepharose column coupled with a 5 times higher pep-tide:Sepharose ratio. Approximately 25% of applied P2B, bothendoglycosidase F-digested and untreated samples, bound tothe column, while <1% of the applied P2A was retained on thecolumn. Since removal of TV-linkedoligosaccharides from P2Bdid not appear to be required for binding to the RGD peptide,it is possible that the conformational context of the peptidesequence in ECM glycoproteins may also influence P2B accessor binding.

P2B differed in peptide size from that of both the a or ßsubunits of the fibronectin or vitronectin receptors (20, 21),LFA-1 or Hb/IIIa, the latter found on lymphocytes and platelets, respectively (22). Since P2B appears to be structurally

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Fig. 2. Binding of '"I-labeled P2A andP2B to ELISA plates coated with ECM gly-coproteins. The wells of ELISA plates (Falcon)were coated with serially diluted collagen typesI or IV, fibronectin, or laminin overnight at4'C. The wells were then blocked with 1%BSA in 10 mM Tris-HCl, pH 7.4, for 30 minand then washed with the same buffer plus0.1% Triton X-100. Aliquots of 125I-labeledP2A and P2B (50 nl, 10s cpm) either mockdigested (A) or digested with glycosidases (B)was applied to duplicated wells for 4 h at 20°C;

then plates were washed 5 times and radioactivity was determined in a gamma counter.Data are the mean of 2 experiments and replicates showed less than 10% variation. Thebinding data in B were obtained on wells coatedwith 2.5 MSof ECM glycoprotein. Control, C\glycopeptidase F, F: endo-0-galactosidase,,i; neuraminidase, N; reduced and alkylated,RIA.

9-

8-

I 4-1

3-

2-

1 -

To

B

•Collagen I di£Collagen IV ¡S3o Fibronectin•Laminin

—I—2.5 1.25 0.62 0.31

Ligandfcg/well)

—i—0.15 0.07 C F ß N N«/3R/A

9-1

8-

7-

a.

6-

5-

4-

•Collagen lA Collagen IVo Fibronectin•Laminin

Biv

ES3nnJ.1|jija

<r\i•M\W«3'f;g?iIm

?'WSÌ¡1is1ia'£'*g1&1''*l§11pg.%Hiij%i1ifli1

2.5 1.25 0.62 0.31 0.15 0.07

Ligand (//g/well)

related to lysosomal associated membrane proteins, RGD recognition by these glycoproteins may play a role in traffickingof intracellular vesicles or movement of proteins containingRGD sequences into or out of specific vesicular compartments.These activities might also be modulated by oligosaccharideprocessing on P2B.

DISCUSSION

Some of the commonly observed changes in oligosaccharidestructures following malignant transformation or tumor progression include increased sialylation of available galoctosyland GalNAc residues (5), increased branching of complex typeasparagine-linked oligosaccharides (6,23), increased expressionof unsubstituted GalNAc and Gal|81-3GalNAc presumably onCMinked (24, 25), and neoexpression of embryonic and bloodgroup antigens (2). Studies on the glycosylation mutants ofMDAY-D2 have suggested that loss of sialic acid and galactoseor decreased branching of complex type asparagine-linked oli

gosaccharides are directly related to loss of metastatic potential(6, 26). To begin to understand how oligosaccharide expressionmay affect malignant tumor cell growth, membrane glycoproteins that had previously been identified on SDS-PAGE as themajor L-PHA/WGA-reactive species were purified from metastatic MDAY-D2 lymphoma cells and structurally characterized.5

Since previous studies on the class 1 glycosylation mutantsof MDAY-D2 also suggested that loss of sialic acid and/orpolylactosamine may be associated with increased cell adhesionon ECM glycoproteins (10-12), purified P2A and P2B weretested for ECM-binding activity. P2A bound specifically tocollagen type I and P2B bound weakly to collagen type I. Thesialic acid on P2A and P2B had opposing effects on the bindingactivities of the two purified glycoproteins. Sialylated O-linkedon P2A appeared to be required for collagen binding and lossof sialylated polylactosamine containing ./V-linked structures onP2B enhanced its binding on a number of ECM substratum.MDAY-D2 cells were previously shown to be nonadherent on

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20mMNaCI 50mM NaCI 100mMNaCI 200mM NaCI'P2A P2B1 P2A P2ÃŒT̂P2A P2ÃŒT'P2Ä P2ËT'C F C F' "C F C F C F C F C~T 'C~T

92-»-

68-»-

45-»-

Fig. 3. Elution of '"I-labeled P2A and P2B from collagen type I-Sepharose

with increasing concentrations of NaCI. Sample of P2A and P2B were incubatedalone (C) or with endoglycosidase F (/•')and applied to collagen-Sepharose. After

washing, the glycoproteins were eluted from the matrix by sequential 30-minincubations with 20, 50, 100, and 200 HIM NaCI in 10 mM Tris-HCl, pH 7.4-0.4% DOC. The eluted material was separated by SDS-PAGE and the dried gelwas exposed to X-ray film.

Table 2 Binding ofP2A and P2B to RGD peptidelodinated P2A and P2B (2 x 10s cpm) were added to 40 ti\ of Sepharose 4B

coupled with collagen type I, 10 mg/ml of GRGDSPK peptide, or Tris and leftat 20°Cwith periodic mixing for 4 h. The beads were washed and bound cpm

were measured in a gamma counter. P2B was predigested with glycopeptidase Fand P2A was untreated.

Sepharose 4BcoupledwithCollagen

GRGDPTLTrisP2A1

1,680 ±1,141°

2,071 ±2992,434 ±213P2B1

5,208 ±1,22612,398 ±1,7952,270 ±2,067

a Mean ±SD.

fibronectin, laminiti, and collagen type IV but could attach tocollagen type I which may be due to the presence of P2A (11,12). Loss of sialic acid and galactose in the glycoconjugates ofthe class 1 mutants was associated with enhanced cell adhesionon fibronectin, laminiti, and collagen type IV but little changeon type I. Based on the binding characteristics of purified P2Aand P2B, the cellular attachment phenotypes of MDAY-D2and the class 1 mutants are exactly as expected.

Developmental!}' regulated changes in oligosaccharide proc

essing on fibronectin (27) and NCAM (28) have been shown toalter or modulate their binding affinities. Enzymatic removalof polylactosamine from the TV-linked oligosaccharides of pla-cental fibronectin enhanced binding to gelatin making its binding profile similar to that of adult fibronectin which lackspolylactosamine (27). Interestingly, increased polylactosaminecontent in asparagine-linked oligosaccharides has been associated with increased malignancy in human thyroid carcinomas(29). Polylactosamine sequences in TV-linked structures of theBW5147 lymphoma were shown to be preferentially associated

with the ßl—6-linked antenna (30). Therefore increasedexpression of polylactosamine content in TV-linked structuresmay be related in part to increased branching at the trimannosylcore of complex type oligosaccharides which may in turn reducethe binding affinity of P2B for ECM glycoproteins.

Similarly, polysialic acid sequences in NCAM isolated fromembryonic tissue reduces homotypic binding compared toNCAM from adult tissues which have less polysialic acid (28).Homotypic binding of NCAM appears to be mediated throughprotein domains and polysialic acid on the molecule reducesthe affinity of the interaction possibly by altering protein conformation. The binding of P2B to ECM proteins appears to bemodulated in a similar manner with sialic acid and polylactosamine sequences on asparagine-linked carbohydrates reducingbinding to collagen, laminin, and fibronectin. The similaritiesof these systems suggest that binding affinities of several adhesion-mediating glycoproteins may be modulated together bychanges in the activities of glycosyltransferases in the processing pathway for asparagine-linked oligosaccharides. Sinceoligosaccharide processing and expression of embryonic carbohydrate antigens are temporally and spatially regulated during embryogenesis (2), carbohydrate-dependent changes in theactivity or specificity of adhesion glycoproteins may influencecell motility, proliferation, and differentiation. These are alsothe parameters that change as a result of tumor progressionand appear to directly affect metastatic potential (31 ).

Since distinct «2—6and al—3 sialyltransferases act on O-and TV-linkedoligosaccharides (32), the sialylation of structureson P2B and P2A that modulate their affinity for collagen maybe controlled independently. Consequently, a shift in the distribution of sialic acids between O- and TV-linked structures mayradically change the adhesion properties of the cells. For example, attachment variants of the Esb lymphoma were foundto have unchanged levels of total sialic acid; however, lectin-binding studies suggested that the distribution of sialic acid onO- and TV-linked structures may be altered and could account

for the change in attachment and metastasis phenotypes (33).The sialylation of peanut agglutinin- and soybean agglutin-reactive oligosaccharides (i.e., Gal-GalNAc and GalNAc, respectively) has been shown to be directly related to the invasiveand metastatic potential of BW5147 lymphoma sublines (34).The metastatic sublines with sialylated O-linked structures weregenerated by fusing non melasi at ic BW5147 cells with activated"1-cells. Segregants that lost sialic acid on the O-Iinked oligo

saccharides also lost invasive and metastatic potential. Similarly, the acquisition of sialylated peanut agglutinin-reactiveoligosaccharides on M, 90,000-130,000 glycoproteins in B16

melanoma cells maintained in suspension culture has beenassociated with increased metastatic potential (35). These experimental models and our results suggest that an increasedcontent of sialylated O-linked structures may affect the metastatic potential of tumor cells by altering the activity of adhesion-mediating glycoproteins such as P2A.

Stimulation of lymphocytes results in an increase inNeuNAc«2-3Gal sialyltransferase and a decrease inNeuNAca2-6GalNAc transferase activities concordant with

changes in oligosaccharide expression on leukosialin (36). Itremains to be determined whether stimulation-related changesin oligosaccharide expression on leukosialin affect cellular interactions with ECM glycoproteins. Interestingly, leukosialinand the immunologically distinct GPlb have been shown to bedeficient on lymphocytes and platelets, respectively, from individuals with Wiscott-Aldrich syndrome (37). GPlb is structurally similar to leukosialin and P2A, showing a high contentof O-linked oligosaccharides, and appears to be involved in

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COLLAGEN-BINDING GLYCOPROTEINS

platelet adhesion to Von Willebrand factor. Since neuramini-dase-treated normal lymphocytes showed a loss of gpLllS/leukosialin and the appearance of a larger band in SDS-PAGEsimilar to a species observed in the Wiscott-Aldrich patients, itwas suggested that a glycosylation defect may explain thedysfunction of two distinct glycoproteins in these patients (37).Based on our observations with P2A, a defect in glycosylationleading to incomplete sialylation of O-linked oligosaccharidesmay inhibit binding activity of the sialoglycoprotein(s) and thisin turn may affect lymphocyte function.

In conclusion, the glycosylation state of two membrane glycoproteins, P2A and P2B, directly affected their binding affinityfor ECM glycoproteins. This suggests that the oncodevelop-mental state or cellular program of oligosaccharide processingmay regulate the binding affinity of adhesion-mediating proteins such as P2A and P2B and influence cellular processesduring development and tumor cell metastasis.

ACKNOWLEDGMENTS

The authors would like to thank Astrid Eberhart and Lynda Woodcock for their secretarial assistance.

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1988;48:4743-4748. Cancer Res   Suzanne Laferté and James W. Dennis  Membrane Glycoproteins in MDAY-D2 Tumor CellsGlycosylation-dependent Collagen-binding Activities of Two

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