THE JOURNAL. OF BIOLOGKXL CHEMISTRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 30, kssue of October 25, pp. 16429-16434.1990 Printed in U.S. A.

Defined Geometry of Binding between Triantennary Glycopeptide and the Asialoglycoprotein Receptor of Rat Heptocytes”

(Received for publication, May 14, 1990)

Kevin G. Rice, Ora A. WeiszS, Thomas Barthel, Reiko T. Lee, and Yuan C. Lee5 From the Department of Biology and the *Department of Pharmacology and Molecular Sciences, The Johns Hopkins University and School of Medicine, Baltimore, Maryland 21218

Three derivatives of a triantennary glycopeptide, each containing a single uniquely located 6-amino-galactose residue at either position 6’, 6, or 8, were modified at the 6-amino group by attachment of a photolyzable reagent and radiolabeled by iodination of tyrosine. These were allowed to bind to the asialoglycoprotein receptor of isolated rat hepatocytes and photolyzed for affinity labeling.

Gal/i?( 1+4) GlcNAq3(1+2)Mancr( 1+6) 6' 5 4' \

Man@( 1+4)GlcNAcj3(1+4)GlcNAc&R 6 5 4

/ 3 2 1

Galfl(144)GlcNAcj3( 1+2)Mana(1+3)

I GalB( 1+4)GlcNAc@(1+4) 8 7

Each probe specifically labeled either the major (RHLl) or minor (RHL2/3) subunits which comprise the receptor. A photolyzable group attached to galactose residue 6 or 6’ specifically radiolabeled RHLl, whereas a photolyzable group attached to galactose 8 specifically labeled RHL2/3. Photoaffinity labeling of a soluble rat hepatic lectin preparation demonstrated that the minor subunits (RHL2/3) were no longer labeled by the triantennary probe with a photolyzable group at galactose 8.

The inhibitory potency of a variety of complex glycopeptides against radiolabeled ligand binding to both rat hepatocytes and soluble lectin are in agreement with photoaffinity results that galactose 8 of triantennary glycopeptide is of unique importance by binding solely to the minor subunits (RHL2/3) of the asialoglycoprotein receptor on hepatocytes. Conversely, galac- tose residues 6 and 6’ bind specifically to the major subunit (RHLl), indicating a precise binding geometry between the trivalent ligand and lectin.

The asialoglycoprotein receptor was first identified by Ash- well and co-workers (l), who observed that circulating asial- oglycoproteins bind to and are degraded by hepatocytes. Many researchers have concluded that a multi-subunit receptor is responsible for binding galactose residues on de-sialylated glycoproteins (for reviews see Refs. 2-4). Binding studies utilizing glycopeptides and oligosaccharides revealed that a common triantennary (Galp( 1--*4)TRI) structure (5) bound to the lectin with the highest affinity (nanomolar), whereas oligosaccharides lacking a third branch or containing isomeric linkages of galactose bound with much reduced affinity (6-9).

The rat asialoglycoprotein receptor, purified by affinity chromatography and immunoprecipitation, contains three types of subunits (10-12). Drickamer and co-workers (13-15) found that the peptide sequence of the major rat hepatic lectin subunit (RHL1,41.5 kDa) differs from that of both minor subunits (RHL2,49 kDa and RHL3,54 kDa) and demon-

* This work was supported by National Institutes of Health Re- search Grant DK099?6 and P&tdoctoral Fellowship GM13013-01 Bi4 (to K. G. R.). The costs of oublication of this article were defraved in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

!j To whom correspondence should be addressed.

strated that the separation between RHL2 and RHL3 ob- served on SDS-PAGE’ is due to differences in glycosylation of these glycoproteins.

The molecular mass (264 kDa) of detergent-solubilized preparations of the lectin suggests a hexameric state of aggre- gation (16). Studies which have explored the association of subunits have postulated a hetero-oligomeric receptor of RHLl and RHL2/3 on hepatocyte surface (12, 17-19), whereas cross-linking experiments using soluble lectin prep- arations suggested homo-oligomeric receptors of RHLl and RHL2/3 (15). The topography of the receptor on the hepato- cyte cell surface, as determined by lactoperoxidase iodination, revealed that RHL2/3 subunits are prominently exposed, whereas the same treatment showed that all subunits in the soluble lectin are well exposed (21).

Previous photoaffinity labeling experiments from our lab- oratory utilizing a modified triantennary glycopeptide showed that each subunit of the receptor contains at least one galac- tose binding site, and that solubilized lectin produced a dif-

1 The abbreviations used are: SDS-PAGE, sodium dodecyl sulfate- polyacrylamide gel electrophoresis; ANB-NOS, N&azido-2-nitro- benzoyloxy succinamide; HEPES, N-Z-hydroxyethylpiperazine-N’- 2-ethanesulfonic acid; MDE, modified Dulbecco’s Eagle’s medium; Boc, t-butyloxycarbonyl; B-amino-Gal, 6-amino-6-deoxy-D-galactose.

18429

by guest on October 29, 2020

http://ww

w.jbc.org/

Dow

nloaded from

18430 Binding Geometry of the Asialoglycoprotein Receptor

ferent labeling pattern than did lectin on isolated rat hepa- tocytes (20, 21). It was also shown more recently using an equilibrium binding technique that each subunit of the soluble lectin contains two galactose binding sites (22).

In this study we have defined the geometry of binding between the three galactose residues of triantennary glyco- peptide and the three subunits of the asialoglycoprotein recep- tor of rat hepatocytes. This was accomplished by utilizing three well defined triantennary glycopeptide ligands which allow attachment of a single photolyzable group to one of each terminal galactose residue. These triantennary glycopep- tide probes have revealed that the asialoglycoprotein receptor found on hepatocyte cell surfaces is a heteroligomer of RHLl and RHL2/3 subunits which binds triantennary glycopeptide in a single defined geometry.

MATERIALS AND METHODS

Reagents utilized in the preparation of MDE (22) were obtained from Calbiochem. Trypan blue, mineral oil, low molecular weight SDS-PAGE standards were from Sigma. Silicon oil (DC 550) was obtained from Accumetric (Elizabethtown, KY). Carrier-free Na”‘I in 0.1 M sodium hydroxide was from Amersham Corp. Orosomucoid was donated bv Dr. M. Wickenhauser of the American Red Cross National Fractionation Center (Bethesda, MD). N-5-Azido-2-nitro- benzoyloxy-succinimide (ANB-NOS) was purchased from Pierce Chemical Co.

Glycopeptide concentrations were determined by quantitative analysis of glucosamine (24) after hydrolysis with hydrochloric acid. SDS-PAGE was performed by the method of Laemmli (25). Molecular masses were calculated from the linear regression line of migration distance versus molecular mass using low molecular weight markers from Sigma. Thin layer chromatography was performed utilizing Silica Gel 60 Fz5* plates (E. Merck, Darmstadt, Federal Republic of Germany), developed with a mixture of 4:3:2:2 (v/v/v/v) ethyl ace- tate/acetic acid/pyridine/water. Autoradiography was performed at -70 “C in a cassette containing OPTex Tl-3 intensifying screens, utilizing Kodak X-Omat AR film.

Rat hepatocytes were prepared by the collagenase perfusion method based on Seglen (26). Soluble lectin was prepared from frozen rat liver by the method of Hudgin et al. (27) utilizing a immobilized asialo-fetuin-Sepharose column for the affinity purification proce- dure.

A detailed description for the preparation of the asialoglycopeptides structures from the three iv-linked glycosylation sites of bovine fetuin (GPl, GP2, and GP3 from site A: GP4.and GP5 from site B, and GP7 and GP8 from site C) has been nublished (28). The modification of GP2 into three derivatives (GP2-8N, GP2-6N, and GP-G’N, see Table I) containing a single 6-amino-Gal residue at either galactose 8, 6, or 6’ position of triantennary structure was accomplished by oxidation with galactose oxidase followed by reductive amination (29). The preparation and characterization of three triantennary substructures (6.6’TRI. 6,8TRI, and 6’JTRI) of GP5. each missing a single galactose residue,. has been described .(29).

Coupling of ANB-NOS and Iodination of Glycopeptides-The gly- copeptide samples (0.5 nmol of GP2-8N, GP2-6N, and GP2-6’N) were dried and dissolved in 10 pl of 100 nM sodium bicarbonate, pH 8 to which was added 10 ~1 of dimethylformamide containing 50 nmol of ANB-NOS. After reaction for 4 h at room temperature in the dark, the sample was iodinated with 1 mCi of Na’*‘I for 3 min by the Chloramine-T method (30). and chromatographed on a Sephadex G- 15 column (5 x 0.5 cm) eluted with Buffer Ii (100 mM HEPES, 65 mM sodium chloride, 7 mM potassium chloride, 10 mM calcium chloride, pH 7.6).The void volume peak was collected in 2 ml provid- ing a sample of 250 nM in photoaffinity ligand with specific activity of 1 X lo6 cpm/pmol, assuming quantitative recovery.

Direct Bhding Measurement of the Dissociation Constant-Stock solutions of GP2 in water were mixed with 1% lz51-GP2 and were serially diluted into polystyrene test tubes (12 x 75 mm), made up to a total volume with 0.5 ml of MDE. Hepatocytes were added (2.75 X 106/0.5 ml of MDE) and incubated at 2 ‘C! with end-over-end rotation (6 rpm) for 2 h. Aliquots were removed and measured for total radioactivity. Total cell-associated radioactivity was determined by centrifuging 200 ~1 of the cell suspension through silicon/mineral oil mixture (41) at 10,000 x g for 1 min and then measuring “‘1 in the pelleted cells. Nonspecifically associated radioactivity was determined

by pipeting 200 ~1 of the cell suspension into 10 ~1 of 0.5 M EDTA at 2 “C and measuring the remaining cell-associated radioactivity after 30 min. Data were analyzed initially by Scatchard analysis (31), then the best-fix Kd and receptor number were determined with the LI- GAND program utilizing a single site, single receptor model (32).

Inhibition Assays Using Hepatocytes and Soluble Lectin-Relative inhibitory potencies of glycopeptides binding to rat hepatocytes were assessed by comparison of I,, values, which are the ligand concentra- tion which causes 50% reduction in binding of ‘251-asialo-orosomucoid to hepatocytes (9). To measure the 15, of photolyzable glycopeptide derivatives (GPP-6-ANB, GPPS’-ANB, and GPL-&ANB), 10 nmol of glycopeptide (GP2, GP2-6N, GP2-6’N, GP2-8N) in 10 ~1 of sodium bicarbonate was reacted with 50 nmol of ANB-NOS in 10 ~1 of dimethylformamide at room temperature in the dark for 4 h, and 180 gl of MDE was then added. This solution was serially diluted and utilized directly in the inhibition assay. Incubation and handling were performed in subdued light. Inhibition assays for the soluble lectin was performed with ‘251-asialo-orosomucoid as the primary labeled ligand using an ammonium sulfate precipitation procedure (33,34).

Photoaffinity Labeling of Hepatocytes-Photoaffinity labeling ex- periments were performed on isolated rat hepatocytes using an ad- aptation of the procedure previously described (21). Iodinated photo- affinity reagents (GP2-6-ANB, GP2-6’-ANB, and GPZ-8-ANB), 300 p, 75 pmol) were added to an incubation tube (12 x 75 mm) containing 2.7 ml of MDE. Rat hepatocytes (1 ml containing 2 X lo6 cells, routinely 95% viable by trypan blue exclusion) in MDE were added to each. The “affinitv-blank” tube contained in addition 5 nmol of unlabeled triantennary glycopeptide and the “photolysis-blank” tube was kept under subdued light throughout the experiment. The sam- ples were incubated with end-over-end rotation at 2 “C for 2 h after which aliquots (200 ~1) were removed and total radioactivity, total cell-associated radioactivity, and the nonspecifically bound radioac- tivity measured. The samples were flashed five times by three syn- chronized lamps positioned 2 cm from the incubation vial, while photolysis-blank tubes omitted the flashing procedure. Non-EDTA- dissociable radioactivity was measured again after flashing to deter- mine covalently bound radioactivity and to estimate the efficiency of photoaffinity labeling.

After flashing, the cells were pelleted by centrifugation (500 x g for 3 min) and washed twice in cold Buffer I (100 mM HEPES, 65 mM sodium chloride, 7 mM potassium chloride, and 10 mM EDTA, pH 7.6) to dissociate noncovalently bound ligand. The cell pellet was dissolved in 0.5 ml of cell solubilization buffer (10 mM HEPES, 4.8 pM pepstatin, 23 pM leupeptin, 10 mM iodoacetamide, 0.1 mM phenyl- methylsulfonyl fluoride, and 1% Triton X-100, pH 7.6) incubated for 30 min at 4 “C, centrifuged at 10,000 x g for 2 min, and the super- natant stored frozen.

Photoaffinity Labeling of Soluble Pectin-The affinity purified sol- uble lectin (10 pg) was added to a solution containing 25 pmol of photoaffinity reagent in 200 ~1 of buffer (50 mM Tris, 50 mM calcium chloride, 1 M sodium chloride, 0.5% Triton X-100, pH 7.8) in a l-ml polyethylene microcentrifuge vial. Affinity-blank tubes also contained 5 nmol of triantennary glycopeptide as a competitive inhibitor. The samples were incubated at 25 “C for 30 min and then flashed five times as described above. The samnles were diluted with 200 ul of saturated ammonium sulfate, and after 10 min centrifuged at 16,000 x g for 2 min. The supernatant was removed, the pellet washed three times with 200 ~1 of saturated ammonium sulfate, dissolved in 200 ~1 of buffer, and stored frozen.

RESULTS

Direct binding of Y-labeled triantennary glycopeptide (GP2, Table I) to rat hepatocytes revealed 1.5 x lo6 binding sites/cell with a dissociation constant of 4.0 nM (Fig. 1). This is comparable to the value (2 nM) reported for the binding of an oligosaccharide with the same sugar branching structure as GP2 to rabbit hepatocytes (5). The inhibitory potency of a variety of triantennary and biantennary glycopeptides (Table I) to isolated rat heptocytes and purified soluble lectin was evaluated by inhibition assays. The inhibition assay on iso- lated hepatocytes showed that the IS0 (40 nmol) under the present conditions differed from the Kd (4 nmol) for GP2 by IO-fold, and that each Gal@(l+QTRI tested (GP2, GP5, and GP8 from Table I) had similar inhibitory potencies (Table II). The ISo of Gal/I(1+3)TRI structures (GPl, GP4, and GP7

by guest on October 29, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Binding Geometry of the Asialoglycoprotein Receptor

TABLE I The structure of GalP(l4) TRI and Gal@ (14) TRZ, the latter of which contains a variant linkage at position 8

The peptide portion R = t-Boc-Tyr-Asn-Asp-Ser-Arg for GPl, GP2, GP3, GPZ-6-ANB, GP2-6’-ANB, and GP2- S-ANB (29). Ala-Asn for GP4, GP5, 6,6’TRI, 6,8TRI, and 8,6TRI (29) and R = t-Boc-Tyr-Asn-Gly-Ser for GP7 and GP8 (28).

18431

CGlcNAc@( l+Z)Marm( l-6) 6’ 5’ 4’ \

Manp( 1+4)GlcNAcp( 1+4)GlcNAc+R 3 2 1

6 5 4 / G-GlcNAcP( l+Z)Mancu( l-3)

/ G-GlcNAcP( l-4) 8 7

Status of galactose residue Derivatives

G6’ G6 GS

Gal@(1+4)TRI (GP2, GP5, GP8) Galfl( 14) Galfl( 1+4) Galp( 14) Gal/3(1+3)TRI (GPl, GP4, GP7) Galfl( 14) Galfl(1+4) Gal@( 143) Biantennary” (GP3) Galfi( 14) Gal@(1+4) NP* TRI substructures

6,6’ TRI Galfl( l-+4) Gal@(1+4) NP 6,8 TRI 6’,8 TRI Gal;:+4)

Gal@(1+4) Galfl( l--*4) NP Galfl( 14)

Photoaffinity probes’ GP2-6N Gal/3( l--*4) 6NH2-Gal Gal@( 14) GP2-6’N 6NHz-Gal GalP(1+4) Gal@(1+4) GPZ-8N Gal@( 14) Gal@(1+4) 6NH2-Gal

0 Biantennarv also lacks GlcNAc residue 7. b Indicated residue is not present. ’ Photoaffinity probes were activated by addition of ANB-NOS to the 6NH2-Gal on the branch indicated.

7-

II II II ,I ,I II 0 2b 40 60 80 100 120 140 160

Dose (nbiolar) FIG. 1. Direct binding of GP2 to rat hepatocytes. A, the

bound versus dose curve determined as described under “Materials and Methods,” and inset B is the Scatchard transformation of the data with the optimized tit line as determined using the LIGAND program. The analysis indicates a high affinity binding site with Kd = 4 nM and 1.5 X lo6 sites/cell.

from Table I) was lo-20-fold higher than that of Gal@(l+ 4)TRI. Biantennary glycopeptide (GP3) was even less inhib- itory than Gal/3( 1+3)TRI and exhibited approximately a 200- fold higher Ih0 than Galfi( 1-+4)TRI.

Not surprisingly, 6,6’TRI structure showed inhibitory po- tency coincidental with biantennary GP3, whereas 6,8TRI or 6’,8TRI were equal in potency and displayed a moderate (2.5- fold) increased in inhibitory effect over GP3 or 6,6’TRI (Table

TABLE II Concentration at 50% inhibition (I& ‘251-asialo-orosomucoid binding

‘251-Asialo-orosomucoid concentration was 125 PM. Cell density of 2 x 106/ml was used.

Inhibit& Soluble lectin Hepatocytes

PM Biantennary

GP3 6,6’TRI 6,8TRI 8,6’TRI

Gal/3(1+3)TRI GPl GP4 GP7

Galb( 1+4)TRI GP2 GP5 GP8 GP2-6’-ANB GPP-6-ANB GP2-8-ANB

NDb 5 ND 5 ND 2 ND 2

26 0.9 9.6 0.2

19 0.6

1 0.040 2 0.035 2.4 0.025

ND 0.045 ND 0.200 ND 0.050

’ See Table I for structural nomenclature. * Not determined due to limited sample.

II). The IbO for Gala(l+4)TRI and Gal@( l+B)TRI glycopep- tides on solubilized lectin was 25-loo-fold higher than the Is0 obtained using hepatocytes.

Radiolabeled GP2 and GP2-6N (GP2 whose galactose 6 has been converted to a 6-amino-Gal) were reacted with ANB- NOS, chromatographed on TLC, and visualized by autoradi- ography (Fig. 2). GP2-6N migrated slower (RF = 0.45) than GP2 (RF = 0.5), ANB-NOS converted GP2-6N completely to a faster moving GP2-6-ANB (RF = 0.7), whereas migration of GP2 was unaffected by reaction with ANB-NOS. Inhibition assays using the photoaffinity probes, GPS-6-ANB, GP2-6’- ANB, and GP2-S-ANB, were performed on hepatocytes di-

by guest on October 29, 2020

http://ww

w.jbc.org/

Dow

nloaded from

18432 Binding Geometry of the Asialoglycoprotein Receptor

c Front

A B C D

FIG. 2. Analysis of photoaffinity coupling product by TLC with autoradiograuhv detection. Iodinated GP2-6N after (lane A) and before (la& Z3j coupling wigh ANB-NOS, and GP2 after (lane C) and before (lane n) coupling with ANB-NOS.

TABLE III

Quantitative analysis of photoaffinity labeling rat hepatocytes

SalTlple Nonspe- Covalently SIgnal/ Dose Bound” cifically bound” Effimncy

bound” noise

TlM %

GP2-A-ANB 18.8 2.4 0.2 0.5 2.5 11 GP2-6’-ANB 18.8 12.5 0.3 0.6 2.2 2.8 GP2-8-ANB 18.8 11.0 0.3 0.7 2.4 3.6

” Expressed as percent of dose based on counts/min of lp51 meas- ured.

rectly after coupling ANB-NOS as described under “Materials and Methods.” The Is0 value for GPB-6-ANB and GP2-8-ANB was comparable to that of the parent compound (GP2) sub- jected to identical reaction conditions, whereas the Iso of GP2- 6-ANB was 4-fold higher (Table II).

Photoaffinity labeling was performed on intact hepatocytes after incubating with the iodinated glycopeptide probes at 2 “C for 2 h. Approximately 12% of GPB-6’-ANB and GP2-8- ANB, and 2.4% of the GP2-6-ANB isomer were specifically bound to the receptor before photolysis (Table III). Flashing resulted in the specific covalent attachment of radiolabeled ligand except when excess competitive inhibitor was present. This increase in radiolabel nondissociable with EDTA was used to estimate the efficiency of photoaffinity labeling (Table III). The efficiency of labeling was not influenced by substi- tuting MDE as buffer (which produced lower background relative to the use of Buffer II) or by flashing in polyethylene incubation tubes (instead of quartz tubes containing a reflec- tion rod (21)). Interestingly, the labeling efficiency of GP2-6- ANB was consistently higher (11%) than that of GP2-6’- ANB and GP2-B-ANB (3%) (Table III). Typically, a signal (experimental) to noise (blank) ratio of 2.5:1 was observed. The background was predominantly noncovalently adsorbed glycopeptide which was removed after SDS-PAGE as de- scribed below.

The overall efficiency of photoaffinity labeling of the solu- bilized lectin was equivalent for each of the glycopeptide probes as determined by the radiolabel incorporated in the photoaffinity experiments uersus affinity-blank controls. The faction of glycopeptide bound to the receptor before flashing could not be evaluated due to dissociation of the ligand during ammonium sulfate precipitation and filtration. The nonspe- cifically associated ligand could not be completely removed by extraction with saturated ammonium sulfate, resulting in photoaffinity labeled samples with signal to noise ratio of 2:l. However, this nonspecific radiolabel was removed after per- forming SDS-PAGE as described below.

The products of photoaffinity labeling were analyzed by

SDS-PAGE on an 8% gel with iodinated molecular weight markers included in the two outermost lanes. Typically, 10 ~1 (10,000 cpm) of solubilized cell supernatant containing photo- affinity labeling product or control (affinity-blank and photo- blank) were boiled in an equal volume of 2 x sample buffer, and electrophoresed, and the gel was treated for 24 h in 20% trichloroacetic acid. This procedure removed iodinated photo- affinity probe that migrated near the labeled receptor bands. The gel was washed for 15 min in 10% glycerol and dried on Whatman 3M paper and then autoradiographed for 24-48 h.

A single radiolabeled band located with an apparent molec- ular mass of 48 kDa was detected when GPB-6-ANB or GP2- 6’-ANB were used as photoaffinity probes, whereas a band at 52 kDa and a broader band at 58 kDa were observed when GP2-S-ANB was used (Fig. 3, lanes A, B, and C). Scanning of the autoradiography film illustrated that no 52- or 58-kDa bands were labeled by GPB-6-ANB or GPS-6’-ANB, whereas only a slight amount (~5%) of the 48-kDa band was labeled by GP2-8-ANB (Fig. 3). Affinity-blank (Fig. 3, lanes A’, B’, and C’) and photo-blank experiments showed no radiolabeled bands.

The identity of the radiolabeled bands were assigned by comparison with three Coomassie-stained bands from purified soluble receptor which showed apparent molecular masses of 44 kDa (RHLl), 49 kDa (RHL2), and 54 kDa (RHL3) on an 8% gel. Taking into account the increase in molecular mass of covalently bound glycopeptide (-iUr = 2500) to the lectin subunits, the radiolabeled bands at 48, 52, and 58 kDa were considered to have originated from RHLl, RHLB, and RHL3, respectively.

The GP2-8-ANB photoaffinity probe did not specifically label RHL2/3 in the soluble lectin preparation. All of the isomers produced a single radiolabeled band at 48 kDa, the predicted mass for labeled RHLl (Fig. 4, lanes A, B, and C). The GP2-8-ANB probe also produced an unidentified band of high molecular mass (66 kDa) but clearly bound predomi- nantly to RHLl. The affinity-blank experiments (Fig. 4, lanes A’, B’, and C’) showed only trace amounts of nonspecific labeling.

DISCUSSION

The asialoglycoprotein receptor binds with high affinity both to naturally occurring complex oligosaccharides found in desialylated glycoproteins and to synthetic clustered gly- cosides which mimic the natural ligands (2, 34). Asialo-oli- gosaccharides or glycopeptides show comparable affinity to the receptor as the glycoproteins from which they were derived (5-9). Thus, glycopeptides of well defined structures, being amenable to purification and structural modification, are use- ful as probes to study the glyco-specificity of the lectin.

Of the various oligosaccharides and glycopeptides tested, a common triantennary structure has been noted to bind to hepatocytes with highest affinity per galactose residue (5). We chose this GalP(l+)TRI complex glycopeptide ligand to perform more in-depth analysis of its binding specificity to the lectin.

Binding of GP2 to rat hepatocytes was of high affinity (& = 4 nM) and comparison of the Iso of three related Galp(l+ 4)TRI glycopeptides (differing only in the peptide sequence) demonstrated that the peptide portion had negligible influ- ence on the inhibition potency. The Is0 for highly purified Gal@( l+S)TRI structures was lo-20-fold higher than Gal6( 1+4)TRI structures which is somewhat lower than pre- viously determined Lo values using glycopeptide mixtures (9). Binding of biantennary glycopeptide which lacks galactose 8 resulted in a further lo-fold reduction in binding potency

by guest on October 29, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Binding Geometry of the Asialoglycoprotein Receptor

FIG. 3. SDS-PAGE and autoradi- ography analysis of photoaffinity labeled rat hepatocytes. Left, lanes A, B, and Care GP2-6-ANB, GPZ-6’-ANB, and GPZ-&ANB, respectively, whereas lanes A’, B’, and C’ are the affinity- blank control experiments for each gly- copeptide reagent. The migration posi- tion of iodinated standard proteins are indicated on the left axis. Right, scanning densitometry of the x-ray film at 580 nm provided trace A, B, and C for the exper- imental lanes. Affinity-blank lanes A’, B’, and C’ each indicated a blank trace.

A A’ B B’ C C’ A I

RHLl n

W (kDi1)

66 45 36 29 Molecular Weight (kDa)

A A’ B B’ C C’

RIW (kDa)

A RtlLl

/b%+lL?

RHLl

L

RHLl

A

6b

Molecular 29

Weigh?(kDo)

compared to Gal/3(1+3)TRI. These data suggest that al- though galactose 8 does not bind optimally to the lectin in Galp( 1+3)TRI it still participates in binding to some extent, as previously noted (9).

binding of galactose 8 from that for galactose 6 or 6’.

Comparison of the ISo of GalB(1+4)TRI and Gal@(l+ 3)TRI glycopeptides in an inhibition assay utilizing soluble lectin indicated that each of the isomers exhibited a 25-100- fold reduction in inhibitory potency. Interestingly, the relative difference in inhibitory potency observed between Gal6(1+ 3)TRI and Gal/l(l+t)TRI was maintained. These two char- acteristics of the soluble lectin preparations have been previ- ously noted for different ligands binding to the soluble rat lectin (8) and for synthetic neoglycopeptides binding to solu- ble rabbit lectin (34).

Binding studies using substructures of Gal@( 1+4)TRI each missing a single galactose residue showed that 6,6’TRI gave an IS0 which was 2.5 times higher than that of 6,8TRI or 8,6’TRI. Although this difference is small, it distinguishes the

The inhibition assay performed using the photoaffinity probes established that only one isomer (GP2-6-ANB) showed a 4-fold higher IS0 value compared to GP2. Because this loss of inhibitory potency is minor, it is most likely that the photolyzable group is not interfering with the natural binding configuration of triantennary glycopeptides to the hepatic lectin. However, the slightly higher Iso of GPZ-6-ANB distin- guishes the galactose 6 binding site from the galactose 6’ although both GPZ-8ANB and GPPB’-ANB photoaffinity labeled subunit RHLl. Also, photoaffinity labeling with GP2- 6-ANB was of higher efficiency relative to either GP2-6’- ANB or GPB-8-ANB. This could be explained if the structural feature which weakens binding of GPPS-ANB (such as a slight steric hindrance) also more efficiently captures the photo-activated probe. These distinctions might be due to the ability of GPPS-ANB and GPS-6’-ANB to photoaffinity label two different galactose binding sites on RHLl, as previously proposed (22).

FIG. 4. SDS-PAGE and autoradi- ography analysis of photoaffinity labeled solubilized rat asialoglyco- protein receptor. Photoaffinity re- agents and molecular weight markers are the same as described in Fig. 3. Scanning densitometry (right) of experimental lanes A, B, and C indicated that each glycopeptide isomer photoaffinity la- beled RHLl and not the minor subunits RHL2/3. Affinity-blank lanes A’, B’, and C’ indicated only trace background labeling of protein >66 kDa.

by guest on October 29, 2020

http://ww

w.jbc.org/

Dow

nloaded from

18434 Binding Geometry of the Asialoglycoprotein Receptor

The results of photoaffinity labeling hepatocytes demon- strate that a highly ordered mode of binding occurs between all three subunits of the lectin and the three branches of the triantennary. If the mode of binding were completely random, we would anticipate each glycopeptide probe to photoaffinity label each subunit to a similar extent. The high specificity of labeling detected between galactose 8 and RHL2/3 requires that galactose 6 and 6’ binding sites must also be oriented in a precise geometry. It is apparent that some type of structural variation between RHLl and RHL2/3 is responsible for or- ganizing the subunits into a geometry well suited for binding triantennary structures. Since GPS-8ANB photoaffinity la- bels RHL2 and RHL3 equally, the difference in carbohydrate structures which distinguish these two subunits (15) are ap- parently not involved in defining their organization with RHLl on the hepatocyte cell surface. Previous results from our laboratory utilizing a mixture of photoaffinity probes prepared from fetuin glycopeptides preferentially labeled RHL2 on rat hepatocytes (21). Perhaps sample heterogeneity with respect to oligosaccharide structure and the number and position of modified galactose residues or differences in ex- perimental protocol have led to this difference in results.

Several studies have examined the oligomeric state of sub- units of the asialoglycoprotein receptor on rat hepatocytes and in solubilized form (12, 15). In the present study, GP2-8- ANB photoaffinity labeled RHLl in the soluble lectin, sug- gesting that the increased Is0 determined in the soluble lectin inhibition assay for each glycopeptide may be a direct result of the loss of binding of galactose 8 to RHL2/3 and its new association with RHLl. It appears from these data that the lectin organization in the solubilized preparation considerably deviates from that of the lectin found on hepatocyte cell surface.

The hypothesis that the asialoglycoprotein receptor exists as a hetero-oligomer of the RHLl and RHL2/3 subunits on hepatocytes is supported by our photoaffinity labeling data and by several other earlier reports (12,17,19). Although the triantennary glycopeptide probes demonstrate a highly precise mode of binding to the hepatic lectin, it is possible that other hetero- or homo-oligomeric arrangements of receptor subunits co-exist which bind different asialo-complex oligosaccharides. This hypothesis appears unlikely since structurally variant triantennary oligosaccharides or glycopeptides bind with much reduced affinity, suggestive that a galactose residue on one or more branches is not interacting optimally with the organized lectin binding sites. Furthermore, photoaffinity labeling performed at receptor-saturating doses with these triantennary glycopeptide probes demonstrated the same high specificity of labeling as experiments performed at lower doses. These results suggest that most or all of the asialogly- coprotein receptors on the rat hepatocyte cell surface are capable of being organized into a defined geometry to accept Gal6(1+4)TRI oligosaccharides in a highly specific fashion.

The rat asialoglycoprotein receptor has an unusual subunit stoichiometry with two forms of the minor subunit as com- pared to both the rabbit and human receptor which possess a single type of minor subunit (15, 20). It will be of interest to probe the asialoglycoprotein receptor from these sources to

see if these lectins are organized similarly. Acknowledgment-We gratefully acknowledge Dr. Ronald Schnaar

for his laboratory’s support in the preparation of rat hepatocytes.

REFERENCES

1. Pricer, W. E., and Ashwell, G. (1971) J. Biol. Chem. 246, 4825- 4833

2. Ashwell, G., and Harford, J. (1982) Annu. Reu. Biochem. 51, 531-554

3. Breitfeld, P. P., Simmons, C. F., Strous, G. J. A. M., Geeuze, H. J., and Schwartz, A. L. (1985) Znt. Reu. Cytol. 97,47-95

4. Lee, Y. C. (1989) Ciba Found. Symp. 145,80-95 5. Lee. Y. C.. Townsend. R. R.. Hardv. M. R.. Lonneren. J.. Arnarn.

J.; Haraldsson, M.,‘and Lonn, H. (1983) J. Byol. khem. 25& 199-202

6. Regoeczi, E., Taylor, P., Debanne, M. T., Marz, F., and Halton, M. W. C. (1979) Biochem. J. 184,399-407

7. Baenziger, J. U., and Fiete, D. (1980) Cell 22, 611-620 8. Baenziger, J. U., and Maynard, Y. (1980) J. Biol. Chem. 255,

4607-4613 9. Townsend. R. R.. Hardv. M. R., Worm. T. C.. and Lee, Y. C.

(1986) B>ochem&y 25; 5716-5725 -’ 10. Tanabe. T.. Pricer. W. E.. Jr.. and Ashwell. G. (1979) J. Biol.

Chem. 2g4, 10381043 ’ ’ 11. Schwartz, A. L., Rothstein, A. M., Rup, D., and Lodish H. F.

(1981) Proc. Natl. Acad. Sci. U. S. A. 78. 3348-3352 12. Sawyer,‘J. T., Sanford, J. P., and Doyle, D: (1988) J. Biol. Chem.

263,10534-10538 13. Drickamer, K., Mamon, J. F., Binns, G., and Leung, J. 0. (1984)

J. Biol. Chem. 259, 770-778 14. Holland, E. C.. Leung, J. 0.. and Drickamer. K. (1984) Proc. Natl.

Acad. &i. Si, 7338-7342 15. Halbere. D. F.. Waeer. R. E.. Farell. D. C.. Hildreth. J.. IV.

“ , I YI

Quesenberry, M., Loeb, J. A., Holland, E. C., and Drickamer, K. (1987) J. Biol. Chem. 262,9828-9838

16. Anderson, T. T., Feytag, J. W., and Hill, R. L. (1982) J. Biol. Chem. 257,8036-8041

17. Herzig, M. C. S., and Weigel, P. H. (1989) Biochemistry 28,600- 610

18. McPhaul, M., and Berg, P. (1986) Proc. Natl. Acad. Sci. U. S. A. 83.8863-8867

19. Braiterman, L. T., Chance, S. C., Porter, W. R., Lee, Y. C., Townsend, R. R., and Hubbard, A. L. (1989) J. Biol. Chem. 264,1682-1688

20. Lee, R. T., and Lee, Y. C. (1986) Biochemistry 25, 6835-6841 21. Lee, R. T., and Lee, Y. C. (1987) Biochemistry 26,6320-6329 22. Lee, R. T., and Lee, Y. C. (1988) Biochem. Biophys. Res. Commun.

155.1444-1451 23. Schnaar, R. L., Weigel, P H., Kuhlenschmidt, M. S., Lee, Y. C.,

and Roseman. S. (1978) J. Biol. Chem. 253, 7940-7951 24. Hardy, M. R., Townsend, R. R., and Lee, %. C. (1988) Anal.

Biochem. 157, 179-185 25. Laemmli, U. K. (1970) Nature 227,680-685 26. Seglen, P. 0. (1976) Methods Cell Biol. 13, 29-83 27. Hudgin, R. L., Pricer, W. E., Jr., Ashwell, G., Stockert, R. J., and

Morell, A. G. (1974) J. Biol. Chem. 249, 5536-5543 28. Rice, K. G., Rao, N. B. N., and Lee, Y. C. (1990) Anal. Biochem.

184,249-258 29. Rice, K. G., and Lee, Y. C. (1990) J. Biol. Chem. 265, 18423-

18428 30. Greenwood, F. C., Hunter, W. M., and Glover, J. S. (1963)

Biochem. J. 89. 114-123 31. Scatchard, G. (1949) Ann. N. Y. Acad. Sci. 51,660-672 32. Munson. P. J.. and Rodbard, D. (1980) Anal. Biochem. 107,220-

239 ’ 33. Connolly, D. T., Townsend, R. R., Kawaguchi, K., Bell, W. R.,

and Lee, Y. C. (1982) J. Biol. Chem. 257,939-945 34. Lee, R. T., Lin, P., and Lee, Y. C. (1984) Biochemistry 23,4255-

4261

by guest on October 29, 2020

http://ww

w.jbc.org/

Dow

nloaded from

K G Rice, O A Weisz, T Barthel, R T Lee and Y C Leeasialoglycoprotein receptor of rat heptocytes.

Defined geometry of binding between triantennary glycopeptide and the

1990, 265:18429-18434.J. Biol. Chem. 

  http://www.jbc.org/content/265/30/18429Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/265/30/18429.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on October 29, 2020

http://ww

w.jbc.org/

Dow

nloaded from