THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1932 by The American Soeiety for Biochemistry and Molecular Biology, Inc.

Vol. 267, No. 10, Issue of April 5, pp. 69834990,1392 Prrnted m U.S.A.

Binding Specificity of a Baby Hamster Kidney Lectin for H Type I and I1 Chains, Polylactosamine Glycans, and Appropriately Glycosylated Forms of Laminin and Fibronectin”

(Received for publication, December 4, 1991)

Sachiko Sat0 and R. Colin Hughes$ From the National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 I A A , United Kingdom

The carbohydrate binding specificity of M,. = 30,000 lectin (CBPSO) from baby hamster kidney (BHK) cells has been studied by inhibition of binding of the radio- labeled lectin to asialofetuin-Sepharose using model oligosaccharides and glycopeptides. CBP30 binds type I or I1 GaW(1+3(4))GlcNAc chains but not Gal(Bl+ 3)GalNAc. The inhibitory potency of straight chain polylactosamine structures or complex-type branched glycans is increased in proportion to the number of Gal(@1+3(4)) units present. Fucosylation or sialylation of terminal galactose residues or further substitution by (arl+3)-linked galactose or N-acetylgalactosamine does not affect binding whereas substitution of the penultimate N-acetylglucosamine residue drastically reduces binding. Thus, blood group A, H type I or H type I1 structures, shows high affinity whereas Le”, Le”, and Leb structures bind poorly. CBP30 binds to murine Engelbreth-Holm-Swarm (EHS) tumor laminin and human amniotic fluid fibronectin but not human plasma fibronectin. Binding involves polylactosamine glycans as well as tri- and tetraantennary complex- type glycans present in EHS laminin and amniotic fluid fibronectin but absent in plasma fibronectin. Proteo- lytic fragments of EHS laminin (ElX/Nd, P1, E8, and E3) bind CBP30, but only fragment E8 supports at- tachment and spreading of BHK cells. BHK cell adhe- sion to EHS laminin or fragment E8 was not disturbed by CBP3O-specific antibodies, but at relatively high concentrations (46 pglml) CBP3O inhibited spreading and partially attachment of cells on laminin.

Endogenous lectins binding to @-galactosides are ubiquitous in mammalian tissues (1). Extensive amino acid sequencing data indicate that these lectins represent a large family of related molecules, presumably with unique carbohydrate bind- ing properties and multifarious roles in cellular interactions (2, 3). However, these roles remain unclarified. Recent data suggest that members of the family have cytostatic or growth promoting properties (4, 5 ) whereas others have been sug- gested to assist in intracellular packaging and secretion of selected glycoconjugates (1). The fact that many of the @- galactoside-binding lectins are developmentally regulated, as are putative carbohydrate epitopes with affinity for the lec- tins, suggests other important roles in cellular interactions which dictate positional information during development (1, 6). Considerable interest in the lectins was stimulated by

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be sent. Fax: 081-906-4477.

several reports that certain members of the family bind to the basement membrane component, laminin (7-9). The proto- type laminin used in most of these studies was derived from the murine Engelbreth-Holm-Swarm (EHS)’ tumor (10). Re- cent reviews (11,12) list these lectins as laminin receptors on the basis of their binding affinities, but functional data relat- ing to any actual role in biologically significant cell interac- tions with laminin are very limited. However, the findings are of interest in view of the known properties of laminin isoforms in such phenomena as cell-substratum adhesion, growth pro- motion, and morphogenesis (lo), for example in kidney (13, 14).

The baby hamster kidney cell line BHK2l C13, established (15) from tissue of newborn animals, is a useful source of putative markers of kidney development. The exact tissue origin of the cells is not clear although their syntheses and secretion of the hamster equivalent of Tamm-Horsfall glyco- protein or uromodulin suggest a tubular epithelial phenotype (16). Previous work has shown that BHK cells contain at least four @-galactoside-binding proteins of M, = 14,500, 16,000, 19,000, and 30,000 (17). The M, = 30,000 carbohy- drate-binding protein CBP30 has been purified to homoge- neity by affinity chromatography of Triton X-100 extracts on asialofetuin-Sepharose and elution with lactose (16). CBP30 appears to be a monomeric protein that migrates with M, = 30,000 in SDS-polyacrylamide gel electrophoresis under re- ducing conditions and elutes from a calibrated gel filtration column under nondissociating conditions with M , = 30,000. CBP30 expression is developmentally regulated in kidney; it is expressed at highest levels in late embryonic kidney and immediately postpartum but is lost rapidly over 14 days post- natally and is undetectable in adult kidney. By contrast, other tissues such as intestine and colon contain similar levels of CBP30 from the embryo into the adult tissue. In the polarized epithelia expressing it, CBP3O is located at the apical surface and in embryonic kidney is present in the distal convoluted tubules at sites that in human kidney express the Tamm- Horsfall glycoprotein (16).

To gain further insights into the biological roles of BHK CBP30 we have examined its carbodydrate binding specificity. Particular attention has been given to the affinity of CBP30 with the matrix proteins, laminin and fibronectin. The latter protein is abundantly expressed in the mesenchyme of kidney whereas laminin, present in basement membranes, has been shown to play crucial roles in kidney development and the establishment of the polarized epithelium (13,14). Our results show that CBP30 has high affinity for type I and I1 carbo-

’ The abbreviations used are: EHS, Englebreth-Holm-Swarm; CBP30, M, = 30,000 carbohydrate-binding protein; ConA, concana- valin A; BHK, baby hamster kidney; BSA, bovine serum albumin; SDS, sodium dodecyl sulfate.

6983

6984 BHK Cell Lectin

hydrate epitopes as well as multiple N-acetyllactosamine units, either in linear polyglycosyl chains or in highly branched complex-type glycans. CBP30 binds with high affin- ity to multiple sites within the laminin of murine EHS tumor and to amniotic fluid but not plasma-type fibronectin, con- sistent with the glycosylation patterns of these isoforms, and modulates at relatively high concentrations BHK cell inter- action with laminin substrata.

EXPERIMENTAL PROCEDURES

Reagents-Chemicals, enzymes, and other reagents were obtained from Sigma unless specified otherwise. Arthrobacter ureafaciens sial- idase was from Nacalai Tesque, Kyoto, Japan. Biotinylated Lycoper- sicon esculentum (tomato lectin), Datura stramonium agglutinin, and concanavalin A (ConA) were from Vector Laboratories, Burlingame, CA. Tetracarpidium conophorum agglutinin was purified as described (18) and biotinylated by a standard procedure (Enzo Biochemicals, New York). Endo @-galactosidase from Bacterwdes fragilis, Diplococ- cus pneumoniue @-galactosidase, green coffee bean a-galactosidase, Dactylium dendroides galactose oxidase, and human plasma fibronec- tin were from Boehringer Mannheim. Other preparations of human and bovine plasma fibronectin and human amniotic fluid fibronectin were obtained by gelatin-Sepharose affinity chromatography (19). Laminin was purified by Terry Butters (of this institute) from murine EHS tumor as described (20). Generous samples of EHS laminin- nidogen complex, elastase digestion fragments ElX/Nd, E3, and E8, and pepsin digestion fragment P1 were kindly supplied by Dr. Rupert Timpl, Martinsreid, Germany. Human IgE was from Calbiochem. N - Succinimidyl [2,3-3H]proprionate (519 mCi/mg) was from Amersham International.

Purification of CEP30-CBP30 was isolated from BHK21 C13 cell extracts as described previously (16). All steps were done at 4 "C. In brief, cells were homogenized with 75 mM Tris-HC1 buffer, pH 7.2, 50 mM CaCI2, 150 mM NaCI, 2 mM 8-mercaptoethanol (buffer A) supplemented with 1 mM phenylmethylsulfonyl fluoride and 1% Triton X-100, in a Dounce homogenizer. The extract was centrifuged at 3,000 X g for 15 min and at 100,000 X g for 2 h to remove the top lipid phase and pellet. The clear supernatant was applied to asialo- fetuin-Sepharose (5 mg of protein/ml, 15 x 1.3 cm) at 10 ml/h, and the column was washed thoroughly with buffer A with 1% Triton X- 100 followed by buffer A. CBP3O was eluted with 150 mM lactose in buffer A and dialyzed against 20 mM Tris-HC1, pH 7.2, supplemented with 2 mM mercaptoethanol (buffer B). Usually 100 pg of CBP3O was obtained from 7-10 ml of packed cells.

Preparation of 3H-Labeled CBP3O-CBP3O was dialyzed against 10 mM phosphate buffer, pH 7.5, and concentrated to 0.1-0.2 mg/ml by a SpeedVac concentrator (Savant). Concentrated CBP3O (250 pl) was added to an Eppendorf tube containing 0.5 mCi (5.45 nmol) of N- succinimidyl [2,3-3H]propionate, previously dried down under NI gas to remove the solvent, and incubated for 2 h at 20 'C (21). The reaction was stopped by adding 50 p1 of 1 M Tris-HC1, pH 7.5, and further incubation for 5 min. The mixture was applied to an asialo- fetuin-Sepharose column as described before. Tritium-labeled CBP30 was eluted with 150 mM lactose in buffer A, mixed with crystalline bovine serum albumin (0.5%), and dialyzed against buffer B. The specific activity was about 42 X 10' cpm/pg protein.

Oligosaccharides-2'-Fucosyllactose, Fuc(a1+2)Gal((31+4)Glc; 3- fucosyllactose, Gal((31+4)[Fuc(al+3)]Glc and A hexasaccharide GalNAc(al+3)[Fuc(a1+2)]Gal((3l+3)GlcNAc((31+3)Gal((31+ 4)Glc were from BioCarb Chemicals. The following oligosaccharides were a kind gift from Dr. A. S. R. Donald, Research Sugars, Long Crendon, Aylesbury, U.K.: lacto-N-tetraose, LNT, Gal(@1+3)Glc- NAc((31+3)Ga1((31-+4)Glc; lacto-N-neotetraose, LNNT, Gal (@1+4)GlcNAc((31-~3)Ga1(@1+4)Glc; lacto-N-fucopentaose I, LNFPI, Fuc(a1+2)Gal(@l+3)GlcNAc(~1-+3)Gal((31+4)Glc; lacto- N-fucopentaose 11, LNFPII, Ga1(@1+3[Fuc(al+4)]GlcNAc((31+ 3)Gal(@l+4)Glc; lacto-N-fucopentaose 111, LNFPIII, Gal(@1+4) [Fuc(al+3)]ClcNAc((31+3)Gal((31+4)Glc; lacto-N-fucopentaose V, LNFPV, Gal((3l+3)GlcNAc(@l+3)Gal(@l+4)[Fuc(al+3)]Glc; lacto-N-difucohexaose I, LNDFHI, Fuc(al+2)Gal((31+B)[Fuc(al+ 4)]GlcNAc((31+3)Gal (@1+4)Glc; LS-tetrasaccharide a LSTa, Neu- NAc(a2+3)Ga1((31+3)GlcNAc(@1+3)Gal((31+4)Glc; LS-tetrasac- charide b, LSTb, Gal(@l+3)[NeuNAc(a2+6)]GlcNAc(@1+3)Gal (B14)Glc; LS-tetrasaccharide c, LSTc, NeuNAc(a24)Gal(@l+ 4)GlcNAc(@l+3)Gal(~l+4)Glc; p-lacto-N-hexaose, pLNH, Gal (@l~3)GlcNAc(@l+3)Gal(@l+4)GlcNAc((31+3)Gal(@l+4)Glc;p-

lacto-N-neohexaose, pLNnH,Gal((3l+4)GlcNAc((31+3)Gal((314) GlcNAc(@l +)Gal(@1+4)Glc; fucosyl-p-lacto-N-hexaose, Fp- LNHIV,Gal((31+3)GlcNAc((31+3)Gal((31+4)[Fuc(al+3)] GlcNAc((3l+3)Gal((31+4)Glc; lacto-N-hexaose LNH, Gal((31-4) GlcNAc(@1+6)[Gal((31+3)GlcNAc(@l~)]Gal(@1+4)Glc; 1acto-N- neohexaose, LNnH,Gal(@1+4)GlcNAc(@l~6)[Gal(@l~4)GlcNAc ((31+3)]Gal((314)Glc. Gal((31+3)GalNAc was from Toronto Re- search Chemicals Inc. Oligosaccharides 25 and 26 were obtained from human transferrin and bovine fetuin, respectively by Pronase (Cal- biochem) digestion. Gal(al+3)Gal(~l+4)GlcNAc was from Chem- biomed Ltd., Canada. Oligosaccharide 27 was obtained from the acidic glycopeptide fraction after Pronase digestion of human plasma a1 acid glycoprotein. Oligosaccharides 23 and 24 were gifts from Dr. G. Strecker (Universibi de Sciences et Techniques, Lille, France).

Hapten Inhibition Assay-To study the binding activity of CBP3O with various oligosaccharides we examined their ability to inhibit lectin binding to asialofetuin-Sepharose. The asialofetuin-Sepharose was diluted to 4 volumes of phosphate-buffered saline; 25 pl of the solution was aliquoted into microcentrifuge tubes (Laser Laboratory Systems Ltd., Southampton, U. K.) and centrifuged to remove the phosphate-buffered saline. Known amounts of the test oligosaccha- ride solution (20 pl) and 20 pl of [3H]CBP30 (4,000 cpm) were added and incubated for 1 h at toom temperature on an end-over-end rotor. The solution was centrifuged, and the aliquots of the supernatant were measured for unbound radioactivity. Control tubes contained BSA-Sepharose.

Enzyme-linked Immunoassay-The wells of Nunc-Immuno plates I were coated with a solution of glycoprotein in phosphate-buffered saline (10 pg/ml, 50 pl) by incubation overnight at 4 "C. The wells were washed three to five times with 20 mM Tris-HC1, pH 7.2, 0.1 M NaCl (buffer C) containing 0.1% Nonidet P-40, and free surface sites were blocked by further incubation at room temperature for 2 h with 150 pl of 10% crystalline bovine serum albumin in buffer C. All of the following steps were done at 4 "C. The wells were washed as before, and then solutions of CBP3O (10 pg/ml, 50 pl) were added and incubated for 16 h. Bound lectin was assayed by sequential addition to the wells of rabbit polyclonal antibody raised against CBP3O (50 pl, 1:1,000 dilution in buffer C), anti-rabbit IgG-peroxidase conjugate (Sigma, 50 p1, 1:5,000 dilution in buffer C). Incubation times were carried out for 8 and 16 h, respectively, and the wells were washed as before after each step. Finally, 0.045% o-phenylenediamine, 0.012% H2O2 citrate phosphate buffer, pH 5.0, was added and the color developed over 30 min and was read at 498 nm in a Titertek Multiskan apparatus (Labsystems UK Ltd., Basingstoke, U. K.)

SDS-Gel Electrophoresis and Western Blotting-Proteins were heated in Laemmli sample buffer a t 100 'C for 5 min and applied to 6% polyacrylamide gels. After electrophoresis the proteins were trans- ferred to nitrocellulose filters of 0.45-pm porosity (Schleicher & Schuell) in the presence of 20 mM Tris-HC1, 192 mM glycine, 0.1% SDS, 20% methanol at pH 8.3 using a Sartoblot I1 semidry electro- blotter (Sartorius, Belmont, Surrey, U. K.). Proteins were located by staining with 0.5% Ponceau S solution (BDH, Poole, U. K.). The filters were blocked with 10% bovine serum albumin in phosphate- buffered saline at 4 "C overnight or at 37 'C for 2 h. The following steps were done at 4 'C. Filters were incubated overnight with CBP3O (1 pg/ml) in buffer C, washed with buffer C containing 0.5% Tween 20 (5 times for 10 min), and bound lectin was detected by sequential addition of rabbit antibody raised against CBP3O (1:200 dilution in buffer C) and anti-rabbit IgG-peroxidase conjugate (1:1,000 dilution in buffer C).

In Situ Enzyme Treatment of Laminin on Nitrocellulose Filters- After transfer to nitrocellulose and blocking with 10% BSA, the filter was incubated at 37 "C overnight as follows: a-galactosidase, 5 units/ ml in 0.15 M citrate phosphate buffer, pH 6.5, with 0.5% BSA and 1 mg/ml y-galactolanolactone; endo-(3-galactosidase, 0.12 unit/ml in 50 mM acetate buffer, pH 5.8, with 2 mg/ml BSA; sialidase, 0.125 unit/ ml in 50 mM acetate buffer, pH 5.0; galactose oxidase, 6.25 units/ml in 0.1 M potassium phosphate buffer, pH 6.0. Controls were incubated with the same buffers without enzymes.

Dot-Blot Assays-Various amounts (0.01-0.1 pg in 1 pl) of glyco- proteins were spotted on a 0.45-pm nitrocellulose filter. The filter was blocked with 10% bovine serum albumin in phosphate-buffered saline overnight at 4 "C, incubated with biotinylated lectin (2 pg/ml) in 10 mM Tris HCl, pH 7.5, 0.15 M NaCl, 0.1% Tween 20 (supple- mented with 1 mM MgC12, MnC12, and CaCL for T. conophorum agglutinin and ConA only) at room temperature for 2 h, washed, and treated with streptavidin peroxidase (1:1,000 dilution) a t room tem- perature for 1 h. After washing, the filter was developed as before.

BHK Cell Lectin 6985

The intensity of staining was estimated visually. Adhesion Assays-Twenty-four-well plates (Cel-Cult, Sterilin, Fel-

tham, U. K. or Costar, High Wycombe, U. K.) were coated with solutions of laminin, laminin fragments, fibronectin, or crystalline bovine serum albumin a t 37 "C for 1 h or overnight a t 2 "C. Routinely, 10 pg/ml solutions were used except in dose-response experiments when up to 100 pg/ml concentrations were examined. The wells were rinsed twice with phosphate-buffered saline (0.5 ml) before use. Subconfluent monolayer cultures of BHK cells were trypsinized as described (22, 23), washed twice in serum-free Eagle's medium, and diluted to approximately 5 X 10' cells/ml in serum-free Eagle's medium. Aliquots (0.25 ml) were added to the coated wells with or without polyclonal rabbit antibodies against CBP30 or BHK plasma membrane glycoproteins (100-300 pg of purified IgG/ml of final concentration). The specificities of the antibodies have been described fully (16,22). In other experiments the cell suspensions contained 10 or 45 pg/ml of purified CBP3O. After incubation a t 37 "C for 2 h the nonadherent cells were removed by gently rinsing each well once with phosphate-buffered saline (0.5 ml), and adherent cells were fixed at room temperature for 30 min with 1% glutaraldehyde in phosphate- buffered saline and stained with 0.1% crystal violet in H20 for 1 h at room temperature (24). In some experiments, cells were allowed to attach and spread maximally over 2-3 h a t 37 "C, nonadherent cells were removed as before, and 0.25-ml antibody solutions (100-300 pg/ ml) in serum-free Eagle's medium were added. After further incuba- tion at 37 "C for 5-8 h the wells were rinsed as before, and adherent cells were fixed and stained. Control wells received no antibody. To quantitate cell attachment, 0.25 ml of 0.2% Triton X-100 was added to each well and the absorbance measured at 540 nm in an enzyme- linked immunosorbent assay reader. Alternatively, attached cells were counted using a calibrated graticule in the microscope.

RESULTS

The CBP3O samples used in this study were purified from Triton X-100 extracts of BHK cells by affinity chromatogra- phy on asialofetuin-Sepharose and elution with lactose. On SDS-polyacrylamide gel electrophoresis each sample gave a homogeneous M, = 30,000 component under reducing condi- tions. After radiolabeling as described under "Experimental Procedures" at least 90% of the labeled protein was bound to asialofetuin-Sepharose in the microassay, and binding was inhibited in a dose-dependent manner by lactose. The con- centration of lactose required for 50% inhibition of binding ID,, was 500 PM, and complete inhibition was obtained above 10 mM lactose.

Hapten Inhibition of CBP30 Binding to Asialofetuin-Seph- arose-Table I shows the concentrations of various oligosac- charides and glycopeptides required to inhibit [3H]CBP30 binding to asialofetuin-Sepharose by 50%. In each case bind- ing was blocked completely at the highest concentrations tested except for compounds 3,6, and 13.

Lactose and N-acetyllactosamine had similar inhibitory activities, indicating that the N-acetyl group plays a trivial role in CBP30 binding: However, Gal(@l+3)GalNAc had greatly reduced activity, indicating a preference for Gal(@l+ 4) linked to a gluco- rather than galacto-sugar. Substitution of the galactose by residues at C2 (compound 4) or C3 (com- pound 5) enhanced activity, indicating that these hydroxyls are not essential for activity. CBP30 binds equally to type I (compound 7) or type I1 (compound 8) structures, and fuco- sylation on C2 of galactose does not affect activity (compound 9). Similarly, sialylation at C3 or C6 of galactose (compounds 15 and 16) had no deleterious effect. Substitution at C2 and C3 of galactose as in compound 14 increased activity about 3- fold, perhaps because of a nonreducing GalNAc-linked ( d + 3) to Gal; an ((u1+3) galactose substituent also increased 3- 4-fold the activity of N-acetyllactosamine (compounds 5 and 2, respectively). Interestingly, substitution of the penultimate GlcNAc residue of either type I or type I1 chains as in compounds 10 and 11, respectively, reduced activity 3-%fold compared with the unsubstituted chains (compounds 7 and 8,

TABLE I Inhibition of binding of CBP30 to asialofetuin-Sepharose by various

oligosaccharides Oligosaccharide IDm

P M

1 G a l ( p l 4 ) G l c 500 2 Gal(pl4)GlcNAc 250 3 Gal(@l+3)GalNAc 1,600 4 Fuc(cul+2)Gal(~l4)Glc 360 5 Gal(c~1+3)Gal(pl4)GlcNAc 70 6 GlcNAc(pl4)GlcNAc(Pl-+i)GlcNAc >10,000 7 Gal(~l-+3)GlcNAc(~l+3)Gal(~l4)Glc 67 8 Gal(~l4)GlcNAc(~l+3)Gal(~l4)Glc 64 9 Fuc(al-+2)Gal(~l-3)GlcNAc(~l+3)Gal 44

10 Gal(~l+3)[Fuc(al4)]GlcNAc(~l+3)Gal 600

11 Gal(pl4)[Fuc(cul+3)]GlcNAc(~l+3)Gal 190

12 Gal(~l+3)GlcNAc(@1+3)Gal(~l4)[Fuc(a1+3)] 140

13 Fuc(al+2)Gal(@1+3)[Fuc(al4)]GlcNAc 1,800

14 Fuc(al+2)[GalNAc(al+3)]Gal(~l-+3) 15

15 NeuNAc(a2+3)Gal(pl+3)GlcNAc 35

16 N e u N A c ( a 2 4 ) G a l ( ~ 1 4 ) G l c N A c 52

17 Gal(pl+3))NeuNAc(a2~6)]GlcNAc 210

18 Gal(pl+3)GlcNAc(@l+3)Gal 15

19 Gal(pl4)GlcNAc(pl+3)Gal 26

20 Gal(pl-+3)GlcNAc(~l+3)Gal(pl4)[Fuc(al+3)] 60

Gal(@l4)GlcNAc(pl 62

( 0 1 4 ) G l c

( p l 4 ) G l c

( p l 4 ) G l c

Glc

(p1-*3)Gal(p14)Glc

G l c N A c ( ~ l + 3 ) G a l ( ~ l 4 ) G l c

(pl"t3)Gal(pl4)Glc

(p1-+3)Gal(p1-+4)Glc

(p1+3)Gal(pI4)Glc

(p l4)GlcN.4c(p l~3)Gal (p l4)Glc

( p l 4 ) G l c N A c ( ~ l ~ 3 ) G a l ( ~ l ~ 4 ) G l c

GlcNAc(pl-+3)Gal(pl4)Glc

21 6, 3) Ga l (p l4 )Glc Gal(pl+3)GlcNAc(p17 Gal(f314)GlcNAc(pl

22 6)Gal(pl-+i)Glc Gal(pl4)GlcNAc(pl Gal(pl+4)GlcNAc(~l ,,

/T 3,

6) 23 Man(&

30

/I 2, -6) Gal(pl4)GlcNAc(@l Man(pl+4) GlcNAc

41

Gal(~l4GlcNAc(@l-+2Man(al Gal (p l4 )GlcNAc(p l+2)Man(a l~

2 3)

6) 24 M a n ( p l 4 ) GlcNAc

44 Gal(pl4)GlcNAc(B1"+4) r 3 )

G a l ( p l 4 ) G l c N A c ( p l ~ 2 ) Man(cu1

25 Asialo transferrin glycopeptide 80 26 Asialo fetuin glycopeptide 35 27 Asialo a, acid glycoprotein glycopeptide 16

respectively). Substitution of the GlcNAc residue with sialic acid (compound 17) also reduced activity 3-4-fold. More sur- prisingly, distal fucosylation of the type I chain affected binding to CBP30 (compound 12), indicating a cumulative effect of each Gal(@l~3[4])GlcNAc[Glc] sequence in com- pound 7. This indication that CBP30 binding appears to be enhanced markedly in oligosaccharides containing multiple copies of Gal(@l+4)GlcNAc[Glc] or Gal(@l+3)GlcNAc[Glc] sequences was confirmed using longer oligosaccharides. Oli- gosaccharides 18 and 19 containing three reactive disaccha-

6986 BHK Cell Lectin

rides were 4-5-fold more active than compounds 7 and 8 containing only two disaccharide units. Again, distal GlcNAc substitution reduced by about %fold the activity (compare compounds 20 and 18). The topology of the reactive disaccha- ride units plays some role in CBP3O interactions. For example, compounds 21 and 22, which contain three such units, were somewhat less active than the straight chain compounds 18 and 19 carrying the same number of units. CBP3O also shows relatively high affinity for branched complex-type glycans. The isomeric triantennary glycans 23 and 24 showed identical activity that was similar to compounds 21 and 22 as expected from the same number of reactive disaccharides in each case. Finally, the results obtained with desialylated glycopeptides from transferrin, fetuin, and a, acid glycoprotein show an increasing activity, as expected from the known glycan com- positions of these glycoproteins. Transferrin contains bian- tennary-type glycans, fetuin contains triantennary glycans similar to those in compound 24, and a1 acid glycoprotein carries mainly tetraantennary type glycans.

To determine which of the disaccharide units of multiple copy oligosaccharides plays the major role in CBP30 binding, p-LNnH (compound 19) was treated with @-galactosidase to remove the terminal G a l ( P 1 4 ) residue. As shown in Fig. 1, the agalacto derivative was less active than the parent com- pound. Surprisingly, the oligosaccharide was also less active than oligosaccharide 8, suggesting that substitution of galac- tose by a GlcNAc(@1+3) residue affected binding although a similar substitution by sialic acid as in compound 15 had no effect.

Binding of CBP30 to Laminin and Fibronectin-Murine EHS tumor laminin contains three polypeptide chains: A, B1, and B2, with M, = 400,000,200,000, and 180,000, respectively (10,20,25). In the native molecule the subunits assemble into a cross with globular domains at the ends of the long arm and each of the three short arms. Nidogen, a polypeptide of M, = 148,000, forms a noncovalent interaction at the center of the cruciform laminin structure. In SDS-polyacrylamide gel elec- trophoresis under reducing conditions the A chain is separated from the two B chains that migrate together (Fig. 2A, track 1). CBP30 binds to both A and B subunits (Fig. 2B, track 1 ). The intensity of the labeling of the A chain was variable compared with B chain labeling, probably because of varia- bility in the efficiency of electrophoretic transfer of the large A polypeptide. To identify the nature of the carbohydrates recognized on the laminin subunits by CBP30, laminin was treated with various glycosidases prior to exposure to the lectin. As Fig. 2C shows, removal of the numerous terminal a-galactosyl residues (26, 27) from murine EHS laminin had

pLNnH

Agalactosyl pLNnH

LNnT

l - . - . . . . . . I 0 2 0 40 6 0 80 100

% Bfndlng

FIG. 1. Inhibition of CBP30 binding to asialofetuin-sepha- rose by LNnT, pLNnH, and agalactosyl pLNnH. LNnT (oligo- saccharide 8, Table I), pLNnH (oligosaccharide 191, and P-galacto- side-treated pLNnH (50 p ~ ) were added to 3H-labeled CBP30, and binding to asialofetuin Sepharose was measured. Controls contained no sugar hapten.

A 1 2 3

180D

116D

C a-Galactosidase

Endo- p -galactosidase

Sialidase

Galactose oxidase

180t

B I 2 3

FIG. 2. CBP30 binding to laminin and fibronectin. A, pro- teins (10 pg) were transferred onto a nitrocellulose filter after SDS- polyacrylamide gel electrophoresis and stained with Ponceau S. Lanes 1 , 2, and 3 contained murine EHS laminin, human plasma, and amniotic fluid fibronectin, respectively. B, the filter from A was incubated with CBPBO (1 pglml) after blocking, and bound lectin was detected by anti-CBPBO antibody. C, effects of various enzyme treat- ments on laminin binding to CBPBO. Laminin (10 Kg), transferred after electrophoresis to a nitrocellulose filter, was incubated with the enzymes indicated. After treatment, the filters were incubated with CBPBO, and bound lectin was detected as in B. The migrations of proteins standards are indicated (Mr X

no effect on CBP3O binding, but a subsequent treatment with endo-@-galactosidase reduced the labeling. Treatment of lam- inin with sialidase followed by galactose oxidase almost com- pletely abolished binding as did inclusion in the CBP30 so- lution of 100 mM lactose (results not shown). These results show that CBP3O binding to the laminin subunits requires intact terminal or subterminal @-galactosyl residues, at least part of which are present in polylactosamine structures sen- sitive to endo-P-galactosidase. In addition, there is significant residual binding to endo-@-galactosidase-resistant structures, probably tri- and tetraantennary complex-type glycans as indicated by the carbohydrate binding specificity of CBP3O deduced from hapten inhibition studies (Table I).

The presence of polylactosamine structures, expected from carbohydrate sequencing data on EHS laminin (26, 27), was confirmed by the affinity for L. esculentum lectin and D. stramonium agglutinin in dot blot assays (Table 11). The former lectin has high specificity for polylactosamine struc- tures (28) as shown by the insignificant reactivity of asialo- fetuin and asialotransferrin (Table 11) containing predomi- nantly triantennary complex-type glycans with C2,4 outer branching and biantennary complex-type glycans respec- tively: D. stramonium agglutinin also binds triantennary and tetraantennary complex glycans, especially with C2,6 outer chain branching (29), whereas T. conophorum agglutinin (18) shows highest affinity for nonsialylated triantennary com- plex-type glycans with C2,4 outer chain branching as in fetuin, and weaker affinity for bi- and triantennary glycans lacking

BHK Cell Lectin 6987

TABLE I1 Dot-blot analysis of lectin binding to EHS laminin and human plasma and amniotic fluid fibronectin

Glycoproteins (0.01, 0.05, 0.1 pg) were applied to a nitrocellulose filter. The blocked filter was reacted with biotinylated lectins (2 pg/ml), and bound lectins were detected with streptoavidin-peroxidase. The extent of binding was estimated visually.

T. conophorum L. esculentum D. stramonium agglutinin agglutinin agglutinin

ConA ~

0.01 0.05 0.1 0.01 0.05 0.1 0.01 0.05 0.1 0.01 0.05 0.1

Laminin +++ +++ +++ +++ +++ +++ ++ +++ +++ +++ +++ +++ Fibronectin (plasma) - ++ ++ - - - + ++ +++ Fibronectin (plasma)-sialidase - + ++ - - - - - - 2 + ++ Fibronectin (amniotic fluid) + ++ +++ - + ++ - + + + + Fibronectin (amniotic fluid)-sialidase + ++ ++ - +. ++ - + + + + Fetuin-sialidase + +++ +++ - - + - + ++ - + + Transferrin-sialidase - + ++ - - - - + +

- - -

- -

- - -

this branching pattern (18). Some of these structures were also shown to be present in laminin by dot blotting (Table 11), consistent with sequencing data (26, 27). Finally, the binding of ConA by laminin (Table 11) suggests the presence of biantennary complex-type glycans (26, 27) although these would not be expected to show high affinity for CBP30 (see Table I).

Plasma fibronectin obtained from adult animals contains glycans of the biantennary complex-type (30), and, as ex- pected, CBP30 showed no detectable affinity for this compo- nent (Fig. 2B, track 2). However, it is known that fibronectin isolated from fetal sources differs substantially in glycosyla- tion to fibronectin prepared from adult tissues (31, 32). Hu- man amniotic fluid fibronectin contains about 12% of its carbohydrate in the form of triantennary glycans (31), and the strong reaction obtained with D. stramonium agglutinin as well as the somewhat enhanced reaction with T. conopho- rum agglutinin compared with plasma fibronectin confirms this result (Table 11). As shown (Fig. 2B, track 3) CBP30 reacted strongly with human amniotic fluid fibronectin. Sur- prisingly, we found significant reaction of this fibronectin with L. esculentum lectin (Table 11), indicating that polylac- tosamine chains, not detected in the sequencing study (31), may be present contributing to the affinity of this fibronectin for CBP3O.

Binding of CBP30 to Laminin Domains-Laminin is var- iously reported (26, 27) to contain from 13 to 15% to 25 to 30% carbohydrate distributed throughout the molecule (27). T o determine to which of the many glycosylation sites of EHS laminin CBP30 binds, we examined the reaction of the lectin with well characterized proteolytic fragments (25). Pepsin digestion fragment P1 contains three rod-like segments orig- inating from the central domains of the A, B1, and B2 chains; the elastase fragment E1X originates from but extends the same portion of the native laminin and carries nidogen in a noncovalent complex; elastase fragment E8 derives from the long arm; elastase fragment E3 derives from the heparin binding domain at the end of the long arm of native laminin. SDS-polyacrylamide gel electrophoresis under reducing con- ditions showed that all of these proteolytic fragments pro- duced discrete polypeptide components (Fig. 3A).

CBP3O binding was first screened by Western blotting of approximately equimolar (Fig. 3B) or equal weights (Fig. 3C) of the fragments and intact laminin, included in each experi- ment as a control. The results show clearly that CBP30 was bound to each laminin fragment. On a molar basis, the great- est binding was obtained for the largest fragment ElXNd that presumably accounts for the largest proportion of the total carbohydrate of native laminin (27). Fragments E8 and P1 bound to similar extents and fragment E3 somewhat less.

A LN 'r$" PI E8 E3

180 D

116D

84 D

58 D

48.5D

"

B LN

1

BOD fl I t

116D

C LN

180D

116D

E I X Nd E8 PI E3

180D 116D 84 D

I 5RD - .. 116D 48.50

04 D

E I X Nd E8 PI E3

180D

1160

84D

1160 84D

48.5D 58D

FIG. 3. CBP30 binding to laminin and laminin proteolytic fragments. A , Coomassie Brilliant Blue staining of EHS laminin ( L N ) and laminin fragments (3 pg) subjected to 7.5% SDS-polyacryl- amide gel electrophoresis under reducing conditions. B, equimolar amounts of laminin and laminin fragments (1.25 pmol) were trans- ferred onto nitrocellulose filters after SDS-polyacrylamide gel elec- trophoresis and incubated with CBP30 (1 pglml). Bound CBP3O was detected as described in Fig. 2. C , equal amounts of fragments (10 pg) were transferred onto nitrocellulose filters and incubated with CBP30 (1 pglml).

In solid state immune assay CBP30 was also found to bind to each proteolytic fragment (Fig. 4). Furthermore, the frag- ments as well as intact laminin were found to be effective inhibitors of CBP30 binding to asialofetuin-Sepharose; the IDs0 was a t least 15 times less than asialofetuin itself (Table 111), indicating a high affinity of CBP30 for native EHS laminin and laminin domains.

BHK Cell Adhesion on Laminin and Fibronectin-BHK cells attached on EHS laminin to similar extent (Fig. 5) and with similar kinetics (results not shown) as on fibronectin. Human plasma or amniotic fluid fibronectin was equally active. Maximal attachment required a coating solution of approximately 10 pg/ml protein, and substrata prepared by coating with less than 1 pg/ml solutions were nonadhesive. The cells attached on laminin fragment E8 in a manner similar to that on intact laminin whereas fragments ElXNd and P1 were totally inactive. Fragment E3 promoted cell attachment to a small but significant extent. The cells on fibronectin, laminin, and fragment E8 were well spread but on fragment E3 appeared with rounded morphology (Fig. 6). Attachment to laminin or fragment E8 was Ca2+-dependent (Fig. 7). Polyclonal antibodies to CBP3O a t concentrations up to 300 pg/ml failed to block cell attachment to laminin or fragment E8 (Fig. 7) and did not induce cell rounding when

6988 BHK Ce

-0 .4 E W

U m

'0.3 a, 0 C m 0

a

e 2 0.2

--C E I X

+ € 8 * € 3

0.1 .1 1 1 0 1 0 0

Laminin fragment concentration (, pglml ) FIG. 4. Binding of CBPBO to immobilized laminin frag-

ments on microtiter wells. Microtiter wells were coated with various concentrations of EHS laminin or laminin fragments (50 pl) and blocked with 10% BSA. Wells were incubated with CBP30 (10 pg/ml, 50 pl), and bound lectin was detected by addition of anti- CBP30 antibody and anti-rabbit IgG-peroxidase.

TABLE I11 Inhibition effects of EHS laminin and laminin fragments on CBP30

binding to asialofetuin-Sepharose ['H]CBP30 was incubated with asialofetuin-Sepharose in the pres-

ence or absence of inhibitor, and IDSO was calculated as described under "Experimental Procedures." The inhibitory effect of asialofe- tuin is shown for comparison.

Fragment IDm

Laminin ElXNd P1 E8 Asialofetuin

B M

0.2 0.07 0.1 0.1 3.0

added to cells spread out on laminin or fragment E8 substrate (results not shown). By contrast, a polyclonal antibody raised against BHK cells and recognizing plasma membrane glyco- proteins, including M , = 140,000 integrin subunits (22, 33), inhibited attachment (Fig. 7) and induced extensive cell rounding and detachment (results not shown) on laminin or fragment E8 substrates. Interestingly, exogenously added CBP30 blocked significantly BHK cell attachment to laminin in a dose-dependent manner (Fig. 7). Although CBP3O when added at 10 pg/ml had only a small effect on cell attachment (Fig. 7), the spreading of attached cells was significantly impaired (Fig. 8). Approximately a quarter of the attached cells in presence of 10 pg/ml CBP3O were scored as having a well spread morphology whereas in the control wells more than three-quarters of the attached cells were well spread. In the presence of 45 pg/ml CBP3O most of the attached cells had a rounded or poorly spread morphology (Fig. 8).

DISCUSSION

The inhibition data collected in Table I point out several important aspects of the carbohydrate binding specificity of BHK cell CBP30. The lectin binds efficiently to H type I or type I1 sequences, and binding affinity is much reduced in the type I derivatives Le" and Le". Unfortunately we were unable to compare directly the affinity of CBP30 for the equivalent type I1 chains. However, Le" was a poor inhibitor, and it is likely that LeY would also be ineffective. Blood group epitope A appears to be even more effective in CBP30 binding than

,11 Lectin 1 2 0 1 1

1 0 0 -1 C 0

E8 TJ

- Laminin '5 8 0 - - Fibronect in

c . a,

E3 E 6 0 1 --b E l X N d .- E' I

s i 40 : 2 0 01

-&..., .01 .1 1 1 0 100

Concentration ( pglml )

FIG. 5. Adhesion of BHK cells to various substrata. Wells were coated with human plasma fibronectin. EHS laminin, or laminin fragments P1, ElXNd, E3, and' E8 in phosphate-buffered saline a t the indicated concentration for 1 h, rinsed with phosphate-buffered saline, and blocked with 4% BSA in phosphate-buffered saline for 1 h. BHK cells were released from subconfluent monolayer cultures with trypsin and washed with serum-free medium. Aliquots (approx- imately lo4 cells) were plated and incubated a t 37 "C for 2 h before fixation of adherent cells with 1% glutaraldehyde in phosphate- buffered saline followed by staining with 0.1% crystal violet in H,O. Cells were solubilized in 0.25 ml of 0.2% Triton X-100 and the Asronm measured. The results are expressed as a percentage of the maximum adherence to laminin-coated wells.

FIG. 6. Morphology of BHK cells plated onto EHS laminin (a), E8 fragment (b ) , E3 fragment (c ) , or human plasma fibronectin ( d ) for 2 h at 37 "C before fixation. Magnification, x 100.

H sequences. Although we did not have a B-type oligosaccha- ride for testing, the data suggest that this structure too would bind efficiently, inasmuch as a Gal(cu1-3) terminal substit- uent enhanced the binding of N-acetyllactosamine. The data also show clearly that CBP30 binding is increased in oligo- saccharides in parallel with the content of interactive Gal(@l+4[3])GlcNAc(Glc) units. Straight chain structures containing a given number of disaccharides showed higher affinity for CBP30 than branched structures with the same number of disaccharides. Therefore CBP30 would be expected to bind with highest affinity to glycoconjugates carrying po- lylactosamine glycans. Other members of the @-galactoside binding family of mammalian lectins including lectins of similar molecular size (Mr = 29,000) from rat or human lung have also been shown to bind to glycoproteins containing polylactosamines and to react with fetal cells expressing such glycoproteins at the cell surface (34-36). However, differences are apparent in the carbohydrate binding specificity of BHK CBP30 (Table I) and rat and human lung M , = 29,000 lectins

BHK Cell Lectin 6989

CBPBO 10 uglml

45 pg/ml

ANTI CBPBO 0.1 mg/ml

0.3 mglrnl

ANTI BHK 0.1 mg/rnl

EDTA

0 2 0 4 0 6 0 80 100 120

Adherence ( % of control )

FIG. 7. Effects of CBPBO and antibodies on the adhesion of BHK cells to EHS laminin. Cells were seeded onto plastic dishes coated with 10 pg/ml laminin. As indicated, the cell suspension added to the dishes contained polyclonal antibodies to CBP30 or to BHK cells (100 and 300 pg/ml), CBP30 (10 or 45 pg/ml), or EDTA (0.5 mM). Adherent cells were estimated as described under "Experimental Procedures" after a 2-h incubation at 37 "C.

FIG. 8. Spreading patterns of BHK cells on EHS laminin in the presence of CBPSO. a, control cells without CBP30; b, cells plus 10 pg/ml CBP3O; c, cells plus 45 pg/ml CBP30.

(34, 35). In particular, substitution of reactive oligosaccha- rides a t C6 of the terminal galactose with sialic acid com- pletely abolished binding to rat and human M , = 29,000 lectins whereas it had no significant effect on CBP30 binding. In other respects, however, where a direct comparison can be made, the affinities of CBP30, rat, and human M , = 29,000 lectins display a broadly similar relative affinity for several of the oligosaccharides tested (Table I, compounds 1, 2, 3, 4, 7, 9, 14, 26, and 27) although quantitative differences can be seen. For example, the tetraantennary complex-type glycans of a1 acid glycoproteins were 5-7-fold more active in inhibiting CBP30 than the other lectins, and the relative affinities of CBP30 for lactose and N-acetyllactosamine were more similar than for the rat or human lung lectins. Therefore, our present data together with other work showing differences in the binding specificities of the rat and human lung M , = 29,000 lectins (34, 35) suggest that the BHK cell CBP30 is another member of the expanding family of p-galactoside-binding proteins of mammalian cells. Interestingly, unlike some other members of this lectin family (37-39) the BHK cell CBP3O does not bind human IgE with or without sialic acid as determined by Western blotting.*

The endogenous ligands recognized by CBP30 in hamster kidney are under investigation. The binding specificity data would suggest that the ligands contain AB H type I or I1 structures, polylactosamine sequences, branched complex- type asparagine-linked glycans, or a combination of such structures. The expression of blood group epitopes in hamster kidney is not known. However, in human kidney AB H and related antigens are present in distal convoluted tubules, a

* S. Sato, unpublished results.

cellular region that also expresses the Tamm-Horsfall glyco- protein (40). The glycoprotein is synthesized by BHK cells, suggesting a tubular origin (16). In embryonic and newborn hamster kidney CBP30 is expressed a t these sites and is lost soon after birth, making it of interest to examine the expres- sion and cellular location of AB H type 1/11 structures in the developing hamster kidney for comparison with that of CBP30 and to identify putative ligands capable of interacting with the lectins. The finding that Leh oligosaccharides have greatly reduced affinity for CBP30 may also have significance because any interactions involving CBP3O in the renal tubules could be modulated by enzymatic conversion of the reactive H type I sequence into an Leh structure.

CBP3O is also expressed at the apical domain of intestinal epithelium although in that tissue expression continues at maximal levels into the adult animal (16). Recently the glycan structures of aminopeptidase N, a resident glycoprotein of the apical membrane of intestinal brush borders, were determined (41). This glycoprotein contains large amounts of H type 1/11 structures on branched complex-type glycans, raising the possibility that such components may be endogenous ligands for CBP3O. Resident glycoproteins of renal brush borders of adult rat, y-glutamyl transpeptidase, amino peptidase N, and dipeptidylpeptidase IV, are lacking, however, in H-type struc- tures, and carry nonreducing terminal GlcNAc residues, prob- ably without affinity for CBP3O. It would be interesting to know if the glycosylation patterns of these enzymes change during kidney development, in line with CBP30 expression, and if other kidney brush border enzymes contain ABH- related epitopes that could be effective in CBP30 interactions. The role of such interactions remains to be determined but may involve the vectorial secretion of glycoproteins to the apical domain of polarized epithelia in conjunction with other targeting signals such as phosphatidylinositol glycan tails (42).

Recently, considerable attention has been given to the potential roles of p-galactoside-binding lectins in cellular in- teractions with laminin. Calf heart lectin (MI = 14,000) and the macrophage surface antigen Mac 2 (Mr = 35,000) have been shown to bind to murine EHS tumor laminin (7-9). In the former case, it was shown that binding involves mainly polylactosamine glycans (36) and the lectin is associated with a laminin matrix in adult mouse muscle (43), strongly imply- ing a functional relationship in that system. Our data suggest that BHK cell CBP3O binds to EHS laminin at glycosylation sites present in different proteolytic fragments spanning the whole laminin molecule. Binding appears to involve both endo-/3-galactosidase-sensitive polylactosamine glycans as well as resistant complex-type glycans, presumably of tri- or tetraantennary type. Sequencing data have shown that these glycans are widely distributed on all proteolytic fragments of laminin so far examined (27). However, the EHS laminin structure is not ubiquitous, and several laminin-like molecules that differ in subunit composition have been identified, indi- cating the existence of a family of diverse but related proteins (10,25). In kidney the B subunits of laminin are constitutively expressed whereas expression of the A subunit is develop- mentally regulated and correlates with the establishment of epithelial polarization (13, 14). The glycosylation status of these different laminin isoforms is unknown as is their poten- tial for functional interactions with CBP30. Furthermore, because laminin is a component of basement membranes (10, 25) it would be insulated in the tissue from apical domains containing CBP3O (16). However, it cannot be excluded that laminin-CBP30 interactions do occur transiently during de- velopment or tissue remodeling under conditions in which

6990 BHK Cell Lectin

epithelial polarization breaks down. Recently laminin B chains have been detected at apical intracellular domains in embryonic mouse kidney (44).

Because BHK cells produce laminin: albeit of an as yet undetermined subunit structure and glycosylation status, we have made efforts to identify any functional role of CBP30 in interactions of BHK cells with EHS laminin, for which CBP30 clearly has high affinity. The data indicate that CBP30 does not function directly as a laminin receptor to induce cell adhesion to substratum. BHK cell attachment to laminin was not inhibited by CBP30-specific antibodies and was dependent on calcium ions whereas CBP3O binding activ- ity is Ca2+-independent (16). Furthermore, although CBP3O was shown to interact with glycans present on all laminin fragments tested, only fragment E8 and to a lesser extent fragment E3 supported BHK cell adhesion whereas fragments ElXNd and P1 were inactive. These data could not be a result of major differences in the coating efficiencies of the various fragments inasmuch as other work has shown that certain cell types attach efficiently to ElXNd and P1 fragments under conditions similar to those used in our experiments (45). A likely candidate for the receptor mediating BHK cell inter- actions with fragment E8 is the a& integrin (46, 47), but other integrins interact with this site, and further work is needed to establish which is utilized in BHK cell adhesion to laminin. The sensitivity to exogenously added CBP30 of BHK cell interactions, particular spreading, with a laminin substra- tum is consistent with other studies (48,49) showing inhibi- tion of laminin-mediated adhesion of various cell lines by wheat germ agglutinin and Griffonia simplicifoliu agglutinin I lectins with affinities similar to CBP3O for either polylacto- samine or terminally a-galactosylated complex-type glycans, respectively. However, the CBP30 concentrations required to inhibit cell attachment to laminin significantly were very high although at lower concentrations cell morphology on laminin was clearly affected. Further work is needed to establish any functional significance of these findings. It is interesting that fragment E3 supports BHK cell adhesion. Several other cell types also recognize this fragment, but the receptor has not been characterized as yet. Candidate molecules may include integrins, or alternatively, cell surface heparan sulfate proteo- glycans present in BHK cells (50).

Finally, our data extend the number of identified matrix glycoproteins with affinity for mammalian p-galactoside- binding lectins to include fibronectin. The fibronectins most readily available originate from plasma and display a simple glycosylation pattern based on a biantennary complex-type structure. Our finding that human amniotic fluid fibronectin, synthesized by fetal epithelial cells of amniotic membranes, binds CBPBO presumably reflects the presence of more highly branched glycans and polylactosamine glycans, as shown by L. esculentum lectin and D. stramonium lectin binding (Table 11). Human placental fibronectin, also a product of fetal cella, contains larger amounts of polylactosamine chains (32) and probably would show high affinity for CBP30. Unfortunately we were unable to test this possibility for lack of material. Recently an M, = 14,000 p-galactoside-binding lectin was identified in human placenta and shown to be released from the cytomatric only with lactose, indicating interactions in situ with some structural component (51). Our data suggest that placental fibronectin would be a candidate ligand for such lectins in placenta.

G. Mills and R. C. Hughes, unpublished results.

Acknowfedgments-We thank Rupert Timpl for gifts of EHS lam- inin and fragments, John Aplin for collecting human amniotic fluid, Sulemana Bawumia for large scale cell culture, and Marilyn Brennan for preparing the manuscript.

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122; 9381944