THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1987 by The American Society of Biological Chemists, Inc.

Vol. 262, No. 3, Issue of January 25, pp. 1294-1299,1987 Printed in U. S.A.

Concanavalin A Interactions with Asparagine-linked Glycopeptides BIVALENCY OF BISECTED COMPLEX TYPE OLIGOSACCHARIDES*

(Received for publication, May 19, 1986)

Lokesh BhattacharyyaS, Martin HaraldssonP, and C. Fred BrewerSlI From the $Departments of Molecular Pharmacology, and Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461 and the §Department of Chemistry, Arrhenius Laboratory, University of Stockholm, Stockholm, Sweden

In the preceding paper (Bhattacharyya, L., Ceccar- ini, C., Lorenzoni, P., and Brewer, C. F. (1987) J. Biol. Chem. 262, 1288-1293), we have demonstrated that certain high mannose and bisected hybrid type glyco- peptides are bivalent for concanavalin A (ConA) bind- ing. In the present study, we have investigated the interactions of ConA with a series of synthetic nonbi- sected and bisected complex type oligosaccharides and related glycopeptides. The modes of binding of the carbohydrates were studied by nuclear magnetic relax- ation dispersion techniques, and their affinities were determined by hemagglutination inhibition measure- ments. We find that certain bisected complex type oli- gosaccharides are capable of binding and precipitating the lectin. The corresponding nonbisected analogs, however, bind but do not precipitate the protein. The stoichiometries of the precipitin reactions were inves- tigated by quantitative precipitation analyses. The equivalence zones (regions of maximum precipitation) of the precipitin curves indicate that the bisected com- plex type oligosaccharides are bivalent for lectin bind- ing. Data for the nonbisected analogs are consistent with their being univalent. The nuclear magnetic re- laxation dispersion and precipitation data indicate that nonbisected and bisected complex type carbohydrates bind with different mechanisms and conformations. The former class binds by extended site interactions with the protein involving the 2 a-mannose residues on the a(1-6) and a(1-3) arms of the core &mannose residue. The latter class binds by only 1 of these 2 mannose residues, which leaves the other mannose res- idue free to bind to a second ConA molecule. The role of the bisecting GlcNAc residue in affecting the binding properties of complex type carbohydrates to ConA is discussed, and the results are related to the possible structure-function properties of complex type glyco- peptides on the surface of cells.

In the preceding paper (l), we investigated the interactions of high mannose and bisected hybrid type glycopeptides with

* This work was supported by Grant CA-16054 (to C. F. B.) and Core Grant P30 CA-13330 from the National Cancer Institute, De- partment of Health, Education, and Welfare. The NMR facility a t Albert Einstein College of Medicine was supported by Instrumenta- tion Grants l-S10-RR02309 from the National Institutes of Health and DMB-8413723 from the National Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adver- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

ll To whom correspondence and requests for reprints should be addressed.

concanavalin A (ConA).’ The results demonstrated that cer- tain high mannose and bisected hybrid type glycopeptides are divalent with respect to ConA binding and capable of precip- itating the pzotein from solution. In the present study, the interactions of nonbisected and bisected complex type car- bohydrates (Fig. 1) with the lectin are investigated using NMRD techniques, hemagglutination inhibition assays, and quantitative precipitation analyses. The results demonstrate that certain bisected complex type carbohydrates are also capable of precipitating the protein as divalent ligands, but that nonbisected analogs are unable to precipitate the lectin and bind as univalent ligands. The interactions of these two types of carbohydrates with ConA are shown to involve dif- ferent mechanisms of binding. The role of the bisecting GlcNAc in the interactions of complex type carbohydrates with proteins is discussed.

MATERIALS AND METHODS AND RESULTS’

DISCUSSION

The presence or absence of a bisecting p(1-4) GlcNAc linked to the core @-mannose residue in asparagine-linked complex type carbohydrates appears to influence the intrinsic molecular and biological properties of these molecules in several ways. Brisson and Carver (14, 15) have presented evidence that the bisecting GlcNAc in complex type glycopep- tides affects the rotamer conformation about the a(1-6) link- age in such molecules. Schachter and colleagues (16-19) have shown that at least four different biosynthetically important glycosidases and glycosyltransferases, which are active toward complex type glycopeptides, are inactive toward bisected com- plex type glycopeptides. A galactosyltransferase has also been recently shown to have reduced activity for bisected complex type glycopeptides (20). Bisected and nonbisected complex type carbohydrates also demonstrate selective binding to lec- tins (9, 10,20-23). For example, mixtures of the two types of

The abbreviations used are: ConA, concanavalin A with unspec- ified metal ion content; CMPL, ConA with Mn2+ and Ca2+ at the S1 and S2 sites, respectively, in the locked conformation (2); CZPL, ConA with Zn2+ and Ca2+ at the S1 and S2 sites, respectively, in the locked conformation (2); NMRD, nuclear magnetic relaxation disper- sion, the magnetic field dependence of nuclear magnetic relaxation rates, in the present case, the longitudinal relaxation rate, l/T1, of solvent protons; a-MDM, methyl a-D-mannopyranoside.

Portions of this paper (including “Materials and Methods,” “Re- sults,” and Figs. 2-4) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biolog- ical Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 86M-1662, cite the authors, and include a check or money order for $2.80 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

1294

Precipitation of Concanavalin A by Bisected Complex Type Carbohydrates 1295

glycopeptides have been separated on ConA-Sepharose col- umns, since nonbisected complex type carbohydrates bind well, while the bisected complex type bind weakly, if at all (20, 23). I t is therefore evident that the bisecting GlcNAc residue in these molecules plays a key role in the molecular recognition properties of this class of asparagine-linked car- bohydrates.

In the present study, we have compared the effects of binding of nonbisected and bisected complex type oligosac- charides, and a nonbisected complex type glycopeptide (Fig. 1) to ConA, using NMRD techniques (24, 25). We have also determined their relative affinities for the protein and inves- tigated the ability of certain bisected oligosaccharides to pre- cipitate the lectin. We have observed that bisected complex type oligosaccharide 4 , as well as the related analog 2, is able to bind and precipitate ConA, while the corresponding non- bisected analogs 3 and 1 (as well as 5) bind, but are unable to precipitate the lectin. Analyses of the stoichiometry of the precipitation data indicate that 2 and 4 are bivalent for ConA binding. On the other hand, data suggest that the correspond- ing nonbisected complex type carbohydrates are univalent. The findings in the present study have permitted us to propose two different mechanisms of binding for nonbisected and bisected complex type carbohydrates to ConA, which account for their different binding properties and valencies. Details of these findings are discussed below.

Relative Binding Affinities of Nonbisected and Bisected Complex Type Carbohydrates to Cod-Fig. 1 shows triman- nosy1 oligosaccharide 1, which is the common structural ele- ment present in all complex type glycopeptides and is respon- sible for their binding to ConA (10). Oligosaccharide 5 in Fig. 1 is an analog of nonbisected complex type glycopeptides. Compound 3 is a nonbisected complex type glycopeptide, which is intermediate in structure between 1 and 5. The relative binding affinities of 1,3, and 5 for ConA, as measured by hemagglutination inhibition, are 130, 120, and 20, respec- tively, with respect to a-MDM (Table I).

Fig. 1 also shows the corresponding bisected complex type carbohydrate analogs 2, 4 , and 6. Compounds 2 and 4 have 7- and 10-fold greater relative affinities for the lectin, respec-

M a n MO"

k a " - O " k . 6

/I. 3 /I. 3

G I ~ N A ~ ~ M ~ " - O H

Ma" Man

1 2

G I ~ N A ~ E M ~ ~ G I ~ N A ~ % M ~ ~

k:a"-R P I 4

\ ,6

GlcNAc-

G I ~ N A ~ E Y M ~ ~ L l . 3 L l l f " " - O H GI~NA~EM~~

(Gn Gn) 3 4

G~I-G~NA~LM~~ p14 p l 2

G . I L L G I ~ N A ~ P M ~ ~ Pi 4

k o " - O H Pl,*.6

p1.4 p1.2 Al.3

GlcNAc ~

B1.4 p1.2 / c O " Gal-GIcNAc-Man Gal-GlcNAc-Man

5 6 (Hepto)

FIG. 1. Structures of nonbisected ( I , 3 (GnGn), 5 (hepta)) and bisected (2 ,4 ,6 ) complex type glycopeptide, oligosaccha- rides, and related analogs. Man, Gal, GlcNAc, and R represent D- mannose, D-galaCtOSe, N-acetyl-D-glucosamine, and core residues, respectively.

tively, than that of a-MDM (Table I). Compound 6 was observed to bind too weakly to detect. The failure of the corresponding glycopeptide of 6 to bind to ConA-Sepharose has been noted (20, 22, 23). Thus, the affinities of the syn- thetic nonbisected complex type oligosaccharides for ConA are generally greater, often by an order of magnitude, than those of the corresponding bisected analogs, in agreement with data for the corresponding glycopeptides (20,23). These results suggest that the higher affinities of the nonbisected complex type carbohydrates, compared to the bisected ana- logs, may be due to different binding mechanisms. In order to investigate this possibility, we have used NMRD techniques to monitor their interactions with ConA.

NMRD Studies of Nonbisected and Bisected Complex Type Oligosaccharides Binding to CMPL-Our previous studies (9, 10) indicated that the two nonreducing terminal mannose residues in 1 simultaneously bind to an extended binding site on each monomer of ConA. Oligosaccharide I induced a partial drop in the NMRD profile of CMPL, compared to the drop induced by a-MDM, which indicated a different confor- mational change in the protein upon binding the trimannosyl moiety. The same change in the NMRD profile of CMPL was also observed for the binding of 5 (hepta) (9, lo), which indicated a similar mechanism of binding. The NMRD results for 3 (Fig. 2) are similar to those for 1 and 5, which indicate that 3 also binds by its trimannosyl group with the same extended site interactions.

Studies with bisected complex type oligosaccharide analog 2 (Fig. 1) showed that it induced a change in the NMRD profile of CMPL similar to that by a-MDM (10). These results suggested that 2 did not bind by extended site interactions like 1 , but rather by a single mannose residue. The reduced affinity of 2, relative to 1 , was consistent with this conclusion. (The slight enhanced affinity of 2 relative to a-MDM is discussed below.) Thus, the NMRD data provided evidence for different mechanisms of binding for 1 and 2.

The NMRD profile of CMPL in the presence of 4 does not resemble those for I , 3, or 5, but rather shows an even larger drop (not shown) in the rate profile compared to that for a- MDM. Details of the effects of 4 on the NMRD profile of CMPL will be presented elsewhere, since the effects are unique and need be discussed at length. However, it is clear that 4 does not bind by the same mechanism as the complex type carbohydrates above. Rather, its affinity is nearly the same as 2, and, like 2, it results in a larger drop in the NMRD profile than that induced by I , 3, or 5.

Precipitation of ConA by Bisected Complex Type Carbohy- drates-Fig. 4 shows that bisected complex type oligosaccha-

TABLE I Inhibitory power of glycopeptides and oligosaccharides for C o d -

mediated hemagglutination of rabbit erythrocytes Minimum

Glycopeptides or oligosaccha-

rides

concentrations required for

complete inhibition of

Relative inhibitory potencQ

hemagglutination

LI-MDM 3.1 mM 1 1 23.8 p M 130' 2 0.17 mM 7b 3 26.0 p M 120 4 0.31 mM 10 5 0.15 mM 20' 6 >10.0 mM

"All data normalized to that of a-MDM. Hieher values indicate Y

greater inhibitory potency. ' Data taken from Brewer and Bhattacharyya (10).

1296 Precipitation of Concanavalin A by Bisected Complex Type Carbohydrates

rides, 2 and 4, are capable of precipitating the lectin, under appropriate conditions. The equivalence zones in the precip- itin curves of 4 (Fig. 4a) show a nearly 1:2 stoichiometry of oligosaccharide to ConA monomer (Table 11). Since each monomer of ConA has one sugar binding site (cf. Ref. 26), the results indicate that 4 is bivalent for ConA. The equivalence zone of the precipitin curve of 2 (Fig. 46) shows a stoichiom- etry of 1:1.4 of oligosaccharide to ConA monomer (Table I), which is lower than that observed for 4 . This appears to be due to the weaker binding of 2 (Table I). Nevertheless, the results are similar to 4, and we conclude that 2 is also bivalent for ConA.

In contrast, 1,3, and 5 do not precipitate ConA. However, in order to determine if soluble aggregates were formed, a further check was made by carrying out NMRD measure- ments of the oligosaccharides bound to CZPL (Ca2+-Zn2+- ConA). Lindstrom and Koenig (27) have shown that the NMRD profiles of diamagnetic proteins are sensitive to pro- tein aggregation in solution. Fig. 3 shows the profile of CZPL which is a tetramer at pH 7.2 (12). The profile is relatively flat below 1 MHz, indicating lack of protein aggregation. Addition of various amounts of a-MDM did not alter the profile. Similar results were found for I , 3, and 5. However, Fig. 4 shows the profile of CZPL in the presence of near stoichiometric amounts of 4, which rises steeply below 1 MHz. The shape is diagnostic of the aggregation of macromolecules in solution (27). Similar results were obtained for 2, as well as for high mannose and bisected hybrid type glycopeptides which precipitate the protein (previous paper (1)). These results confirm the lack of aggregation of the protein by 1,3, and 5 , which is consistent with their binding as univalent ligands.

Mechanisms of Binding of Nonbisected and Bisected Com- plex Type Carbohydrates-The present results indicate that the nonbisected and bisected complex type carbohydrates in Fig. 1 bind to ConA via different mechanisms. Insight into these differences comes from examining the data for 1 and 2 (10). The NMRD profile of CMPL in the presence of 1 resembles those in the presence of high mannose, bisected hybrid, and nonbisected complex type glycopeptides and oli- gosaccharides (10). Oligosaccharide 1 undergoes extended site interaction with the protein which accounts for its high affin- ity (9, 10). These results are consistent with its univalence, since both of its nonreducing mannose residues are simulta- neously bound to the same ConA monomer. Bisected analog 2, on the other hand, gives rise to an NMRD profile which is indistinguishable from that for a-MDM (lo), suggesting that 2 binds to a monomer of ConA by only 1 of its nonreducing mannose residues. This leaves the other nonreducing man- nose residue of 2 free to bind to a second ConA molecule, which, under the appropriate conditions, results in cross- linking and precipitation of the protein (Fig. 46).

Analysis of Corey-Pauling-Koltun space-filling models of 1 and 2 (Fig. 5) indicate that the orientations of their 41-6)

TABLE I1 Stoichiometry ofprecipitin reaction between ConA and bisected

complex oligosaccharides

Concentration of oligosaccha- Protein concentration

Oligosaccharides ride at equiva-

Ratio of

concentration of glycopeptide lence point to protein

monomer

UH 1.2 DH 5.6 DH 1.2 pH 5.6 pH 1.2 pH 5.6

mM mM 2 0.14 0.20 1:1.4 4 0.12 0.13 0.22 0.24 1:Z.O 1:1.8

arms when bound to the protein must be different in order to be consistent with the NMRD and precipitation data. Evi- dence from proton NMR studies and minimum energy cal- culations suggests that the a(1-6) arm of the methyl a- glycoside of 1 exists predominantly in two rotamer confor- mations with values of w = -60" and 180" (14, 15). Since our data are consistent with both nonreducing terminal mannose residues of 1 binding to an extended binding site of ConA (9, lo), this requires that the a(1-6) arm possess a value of w = 180" for binding, as shown in Fig. 5A. In this conformation, both the a(1-3) and a(1-6) mannose residues of 1 have their 3-, 4-, and 6-hydroxyl groups facing the same direction, which is required for binding to a common protein surface. This, therefore, appears to be the conformation of 1 bound to the protein. The high energy barriers to rotation about the a(1- 3) arm preclude alterations in the conformation of this portion of the molecule (14, 15).

The conformation of 1 must also be the same as that of the outer trimannosyl moiety of high mannose and bisected hy- brid type glycopeptides, which binds by the same extended site interactions and is the primary ConA binding determi- nant in these molecules (as shown in Fig. 8 of the preceding

Our data for 2 are consistent with only 1 of its mannose residues binding to one ConA molecule. Rotation of the a (1- 6) arm of 2 to a value of w = -60" results in the 2 nonreducing terminal mannose residues facing away from each other, as shown in Fig. 5B. This allows either the a(1-3) or a(1-6) mannose residue to bind to one protein molecule, while the other nonreducing mannose residue of 2 can bind to a second ConA molecule from the opposite side. I t appears, then, that the perferred binding conformation of the a( 1-6) arm of 2 is with w = -60" (Fig. 5B). Thus, the binding conformations of 1 and 2 appear to be different, which explains their different binding properties. These conclusions also apply to the other nonbisected and bisected complcx type oligosaccharides in Fig. 1, in which their binding determinants are the same as those of 1 and 2, respectively. This indicates that the regions about the binding site of ConA must accommodate the differ- ent relative orientations of the a( 1-6) arms in these molecules (this hints at the reason for the lack of binding of 6, which will be discussed at length elsewhere).

It is important to point out that we do not know the reason for the different binding mechanisms of 1 and 2. We have previously noted that it is possible that the rotamer popula- tion of the a(1-6) arm of 2 is affected by the presence of the bisecting GlcNAc, or that unfavorable steric interactions oc- cur between the GlcNAc residue of 2 and the protein binding site when the rotamer angle about the a(1-6) arm is w = 180" (10).

The relative affinities of the a(1-3) and 4 - 6 ) mannose residues of 2 can be estimated as a first approximation to be comparable to that for a-MDM (28). The slight enhanced affinity of 2, a factor of 7 (Table I) relative to the monosac- charide, is comparable to that of a(1-2) mannobiose (29) and appears to be due to the enhanced probability of binding of a molecule possessing 2 mannose residues with free 3-, 4-, and 6-hydroxyl groups, and not to extended site binding interac- tions (25, 30). The same mechanism of binding also explains the enhanced affinity of 4 (Table I).

Additional Comments-The results in the present study suggest that the nonbisected and bisected complex type oli- gosaccharides in Fig. 1 bind to ConA by two distinct mecha- nisms, which strongly suggests that the rotational angle, w , about their a( 1-6) arms, be 180" and -60", respectively, when bound to the protein. It has been concluded (14, 15) from

paper).

Precipitation of Concanavalin A by Bisected Complex Type Carbohydrates 1297

FIG. 5. Corey-Pauling-Koltun space-filling models of I and 2 (Fig. 1). A, 1 at the rotation angle of a(1- 6) set to w = 180"; B, 2 at w = -60" (14, 15). The angle, w, is the dihedral angle formed by the H-5, C-5, C-6, and 0-6 atoms of the core &mannose residue. The numbers 2,3,4, and 6 indicate 2-, 3-, 4-, and 6-hydroxyl groups of mannose residues. Man3 and Man6 stand for mannose residues on a(1-3) and a(1-6) arms of core p-mannose (Mano). GkNAc represents the bisecting N-aCetyl-D-glUCOSamine residue linked p(1-4) to core p-mannose.

proton NMR experiments and minimum energy calculations that the rotational angle of the a( 1-6) arm of bisected complex type glycopeptides in solution is w = 180". If these results are correct (the signal to noise ratio of the NMR difference spectra were very low), and the data for the glycopeptide hold for the corresponding oligosaccharides, our present findings indicate that the conformation of bound bisected complex type carbohydrates (w = -60" for the a(1-6) arms) differs from that of the predominant solution conformation. Thus, the conformations of these types of carbohydrates in solution may not be the same as their bound conformations.

It has been observed that the presence of the bisecting GlcNAc residue in complex type glycopeptides affects the activities of certain glycosylases on these molecules (16-20). An explanation for these effects has been given in terms of the bisecting GlcNAc sterically shielding the a(1-3) arms of the carbohydrate moieties from binding to the enzymes. An alternative explanation, which follows from the present study, is that the binding orientation of the a(1-6) arm in such molecules is affected by the presence of a bisecting GlcNAc residue. Such changes in the orientation of the a(1-6) arm upon binding may be allowed by certain glycosylases, but not by others, which could account for their binding selectivity. A somewhat similar idea has been advanced by Montreuil and colleagues (31, 32).

CONCLUSIONS

The present study demonstrates that nonbisected and bi- sected complex oligosaccharides bind to ConA with different mechanisms, conformations, and valencies. Certain bisected complex type carbohydrates are bivalent for the protein, while their nonbisected analogs are univalent. As noted in the preceding paper (l), the presence of the bisecting GlcNAc residue in hybrid type glycopeptides has little effect on the interactions of these molecules with ConA. However, the bisecting GlcNAc in the complex type carbohydrates has a profound influence on their mode of binding to the lectin, which may relate to the interaction of these molecules with other proteins and enzymes.

Nonbisected and bisected complex type carbohydrates ap- pear to be receptors on the surface of normal and transformed cells (33,34). Their affinity and valency for ConA may reflect some of their possible functions as recognition determinants

involved in cell-cell interactions and signal transduction mechanisms.

Acknowledgments-We wish to thank Dr. Jorgen Lonngren of Pharmacia Fine Chemicals, Uppsala, Sweden, for generous gifts of the synthetic oligosaccharides 1, 2, 4, 5, and 6, and Drs. Saroja Narashimhan and Harry Schachter of the University of Toronto, Canada, for their kind gift of the glycopeptide GnGn. We also wish to thank Drs. Seymour Koenig and Rodney Brown, 111, of the IBM Thomas J. Watson Research Center, Yorktown Heights, NY, for helpful discussions and the use of their NMRD facilities.

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15. Brisson, J.-R., and Carver, J. P. (1983) Biochemistry 22, 3680- 3686

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1298 Precipitation of Concanavalin A by Bisected Complex Type Carbohydrates

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2555-2562

CONCANIVALIN A INTERACTIONS WITH RSPA?AGINE-LINXPD GLYCOPEPTIDES. BIVALENCY

SUPPLEMENTARY MATERIAL TO

OF BISECTED COMPLEX TYPE GLYCOPEPTIDES

by

Lokesh Bhattacharyya. n a r t l n Haraldsson,and c. Fred Brewer

MATERIALS AND METHODS

prepared and characterized as previou~ly described (2). Protein COnCentrd- Materials. Con A was purchased from Miles-Veda. CMPL and CZPL were

&1%a1Cm=12.4 at 28Q rn ( 3 ) . and expressed ln terms of the rnanorner (Mr26,000 tions were determined spectr~photonetri~ally at pH 5.6 using an absorbance

glfts from ~ r . 3orqen Lonngren. Glycopeptide 2 Iflqure 11 was klndly [ I l l . The Synthetic oligosaccharides 1, 2, 1, ?,and 6 (Figure 11 were

provided by Dr. Harry Sehacter. Concentrations of these compounds Were

measured by the phenol-sulfurlc acid method ( 5 ) using F-mannase as standard. Purity of these compounds were checked by the high resolution 1H NNR. o-MDH

was obtained from Pfanstiehl Laboratoiles.

oljgosaccharide in 200 uL Of buffer were added to 200 YL Of Con ii solution Cuantifarive Precipitation Assays. Increasing dmoUntI Of glycopeptide or

allowed to stand at 21°C for 2 4 h, then centrifuged and the COnCentrdtiOnS Of (about 10 rnglml l and mlxed >mediately ~n a vortex m l x e r . The SolYtlDnI were

Con A measured I" the Supernatants to obtain the percent Con A preclpltated. The buffers at pH 1 . 2 and 5 . 6 were 0.1 M Tris-HC1 and 0.1 M pOt1551Um acetate, respectively, both contdinlnq 0.3 H KC1, 1 mM MnC12. and 1 mM CaC12. Control experlrnents were performed with *"DM added to the SOlutionS.

Inhlbltlon AS-. These were done a t 21'C by 2-fold serial dllurlon technique in 10 mM sodium phosphate buffer. pH 7 . 2 , contdlnlng 0.15 n NaCl, u51ng 3% 5USpenSiOnS Of rabbit erythrocytes ( 6 ) .

~~

solvent water proron relaxation rates ~ilTl,. L . P . , ""clear magnetic relax-

NMRO Measurements_. Measurements of the magnetic fleld dependence of the

atLon dispersion I N M R D l , corresponding to the proton Lamor frequencies from

previously descrlbed 1 7 , 8 ) . Reproducibility of t h e data "11 generally better 0.01 to 40 NHz, were made at 25'C using an unporved fleld cycling device

than t 1%. Sample Solutions ,100 u L 1 contained known concentrations of CMPL and CZPL together With appropriate a m o ~ n t s Of carbohydrates ~n pX 5 . 6 10.1 H

potasslum a c e t a t e , 0.9 M KC11 or 7.2 buffer 10.1 M Trls-HC1, 0.3 M KC11.

RESULTS

NMRD Experiments. Figure 2 shows the proton NMRD profile of CNPL In the presence of nonbisected complex type glycopeptide 2 in pH 5.6 buffer. The prof3185 of CWPL in the absence and presence of o-MDM are also included for comparison. The solvent water proton r e l a x a t m n rates at all values of mag-

compared to the decrease observed ln the presence Of a-MDH. netx fleld (expressed as proton Lamor frequency) undergo a partial drop

jence of n - m M and blsected aomplex type ol>gosaccharide 1 Ln pH 1 . 2 buffer Increaslng amounts of " " D M did not perturb the profile SignLficantly.

Fiqure 3 shows the proton NMRD proflle of CZPL In the absence and pre-

smilar re+ults were Obtalne0 with 1, 2, and 5 . However, the proflle ln the presence of an approxrmately sto1chiometrx amount Of j 1 1 Observed to have progressively higher rates trm 1 to 0.01 MHz, Which is a region that 15 relatively flat for CZPL alone [figure 3 ) . A similar result was Obtained in the presence of oligoeaccharlde 1. Both protein solutions of 2 and 4 dis- played vlsible cloudiness.

glutlnatron of rabbit red blood cells by Con R vslnq the glycopeptide and oi~gosaccharldes in Figure 1. The results for 1, 2, and 5 , which have been previously reported 13.10). are listed tor canparisan. Ollgonaccharrde 4

lnhlbitlon Assays. Table I shows the results of the inhibition of hemaq-

27. Lindstrom, T. R., and Koenig, S. H. (1974) J. Magnetic Res. 15, 344-353

28. Van Landschoot, A., Loontiens, F. G., and de Bruyne, C. K. (1980) Eur. J. Biochem. 103,307-312

29. So, L. L., and Goldstein, I. J. (1968) J. Biol. Chem. 243, 2003- 2007

30. Van Landschoot, A., Loontiens, F. G., Clegg, R. M., and Jovin, T. M. (1980) Eur. J. Biochem. 103 , 313-321

31. Montreuil, J., Fournet, B., Spik, G., and Strecker, G. (1978) C. R. Hebd. Seances Acad. Sci. 287,837-840

32. Montreuil, J. (1980) in Advances in Carbohydrate Chemistry and Biochemistry (Tipson, R. S., and Horton, D., eds) Vol. 37, pp. 158-223, Academic Press, New York

33. Stanley, P. (1980) in The Biochemistry of Glycoproteins and Proteoglycans (Lennarz, W. J., ed) pp. 161-189, Plenum Pub- lishing COT., New York

34. Kobata, A. (1984) in Biology of Carbohydrates (Ginsburg, V., and Robbins, P. W., eds) Vol. 2, pp. 87-161, John Wiley & Sons, New York

MAGNETIC FIELD (T)

PROTON LARMOR FREQUENCY (MHz)

with 10 mM o"OM ( A I at 25'C in pH 5 . 6 buffer. The COncentration Of CMPL was

w. NlRD profiles of C1QL ('31, C W L with 3 . 3 m 1 !cnGnI !.I a n i CI'PL

0.40 mu. The Concentrationsof Carbohydrates used were sufficient to sat~rate the bindinq Eites af con A .

MAGNETIC FIELD (T)

, 1 1 1 1 r , , I I I I , [ I I , , I 0.001 0.01 0.1 1

.- A

I M v

Con A

W 2.0 -

I- 4 - @L

pH 7.2 - 25 'C - 0

z 1.5 0 0

- 0 54

5 - V No saccharid.

0 a-UDY

?j 1.0 - 0

W @L

$ 0.5 -

I- o - @L

e A 8

8 ' 6

a 0 ' ' ' I ' ' ' ' I v s m

0.01 0.1 10 100

PROTON LARMOR FREQUENCY (MHz) m. N R l O profiles of CZPL I P I , CZPL with 1 mI44101 and CZPL with 10 INI =-MOM ( 0 1 at 25'C in pH 7 . 2 buffer. The ~Oncentratlon of ClPL was 0 . 4 0 mM.

Precipitation of Concanavalin A by Bisected Complex Type Carbohydrates

lnhlbits 10-fold better than "-@DM. The relative lnhlbltory actlvity of 2 is

glycopeptide analog Of 5 Was previously observed by Debray and MOntreuil 1111. 120-fold greater than a-HDM. A slmilar difference in binding between 2 and a

Oligosaccharide 6 was observed to bind weakly. if at all, with an affinity at least three tlmes weaker than m-MDM.

Quantltative Precipltation Analyses. Figure 4 shws the quantitative precrpltln curves for Con A and ologosaccharide A at pH 5 . 6 and 7.2 (Figure 4al. and ollqosaccharide 2 at pH 7.2 (Figure 4b). As was found for high mannose and bisected hybrid type glycopeptides, the data in ~iqvre la Show a

greater percentage Of precipitated Con A at p~ 7.2 than at pH 5.6 due to

the proteln dimer at the lower pH 1 1 2 ) . The shapes of the curves in FlqUre 4 f a m a t L o n of protein tetramer at the higher pH, compared to the presence of

are different from those obtained wlth the hlqh mannose and hybrid type glycopeptides l ~ q u r e s 6 and 7, prevmus paper 11)). The curves are broader. and the equivalence zone i n each curve appears to be shifted toward the carbohydrate-excess reqlon. These features are indicative Of w e a k e r inter- actLon$ between the ollqosaccharides and Con A 113). compared to the inter-

protein. The preclpitates formed with ? and ! and the protein are induced by actions of high mannose and blseoted hybrid type glycopeptides with the

concentrarian of oligosaccharide at the equivalence zone lmaximum precipita- speclflc blndlng since the presence of o"DM inhibits their formation. The

tlon) of each precipitin Curve and the concentration of proteln monomer are listed In Table 11.

1, 2, and 5 failed to precipitate the proteln under slmilar condltionl. 20 -J

0.24 0. 40 0.56 0.72 Conc. of OligoxKcharlde (mM)

1299

Figure. Precipitin curves for precipitation of Con I\ by bisected cmplex

21OC. Concentrations of protein are given in Table 11. type oligosaccharides 4 la ) and 2 lb) in pH 7.2 I o ) and 5 . 6 1.1 buffers at