Mokculat Immunology,Vol. 27, No. 4, pp. 351-361, 1990 Printedin Great Britain.

0

Oi61-5890/90 $3.00 + 0.00 1990 PergamonPressplc

BINDING SPECIFICITIES OF INULIN-BINDING IMMUNOGLOBULINS FOR SINISTRIN AND OLIGOSACCHARIDES ISOLATED FROM ASPARAGUS ROOTS

Department

BRENDA HALL,*

CONSTANTIN BONA

and

of Microbiology.

Mount

of Medicine,

Sinai School

CAROL FILTER-KOBRIN

New York,

NY 10029, U.S.A.

(First received 18 September 1989; accepted 9 October 1989) Abstract-The major aim of this study was to further investigate the fine specificity of myeloma proteins recognizing epitopes on fructans. Our studies showed that UPC 61, EPC 109, and a hybrid antibody composed of the heavy chain from UPC 61 and the light chain from EPC 109, UPC 61H:EPC 109L. not

only bind to inulin which is a linear fructan of fi(2 + 1) fructofuranosyl linkages. but also bind to sinistrin, a branched molecule consisting of a fi(2 + 1) fructofuranosyl backbone with /I(2 + 6) branch points. The fine binding specificity of these three antibodies for the fi(2 + 1) fructofuranosyl linkages found in inulin-BSA can be further studied by their binding to fructan oligosaccharides isolated from asparagus roots. From a comparative analysis of the amino acid sequences and the apparent affinity constants (a&) of UPC 61, EPC 109, and the hybrid for various fructan oiigosaccharides, it appears that the light chain

of the immunoglobulin molecule makes an important contribution to the binding specificity. Finally we report for the first time that a monclonal antibody specific for B(2 -V 6) fructans can also bind specifically to inulin-BSA with a lower affinity. This antibody derives its V, and V, from the V,X24 and V,lOb gene families, respectively, which are different and V, 11 gene families).

from the gene families utilized by UPC 61 and EPC 109 (V, 5606

oligosaccharide 5a (Fig. 1) (Streefkerk and Glaudemans, 1977). The binding specificity of the hybrid molecule, UPC 61H : EPC 109L, was also previously studied by Streefkerk et al. (1978). Because the affinity of this hybrid for two inulin-related oligosaccharides. a tetrasaccharide and a pentasaccharide, oligosaccharides 4a and 5a, respectively (Fig. l), more closely resembled the affinity exhibited by EPC 109 for these two oligosaccharides rather than that of UPC 61, the authors suggested that the light chains may exert a dominant influence on determining the degree of affinity between the antibody and the polysa~haride antigen (Streefkerk et al., 1978). In the present study, we further define the specificity of UPC 61, EPC 109, and UPC 61H:EPC 109L hybrid for the p(2 3 1) fructofuranosyl linkage by studying their binding affinities to an extensive panel of novel poly- and oligofructans: sinistrin, a polyfructan consisting of a fi(2 -+ 1) fructofuranosyl backbone with @(2 + 6) branch points, and a series of nine isokestose and neokestose oligofructans isolated from the roots of asparagus (Shiomi et al., 1976, 1979; Shiomi, 1981). The data reported in this communication show that sinistrin is capable of inhibiting UPC 61, EPC 109, and the hybrid from binding to inulin-BSA. Tetrasaccharides (4a, 4b and 4e), pentasaccharides @a, Sb, 5c and 5d), and hexasaccharides (6d, and 6d,) have inhibited to varying degrees the binding of these three inulin-binding myeloma proteins to inulin-BSA. The binding specificities of UPC 61 and EPC 109 can lx distinguished by their

INTRODUCTION

Based upon their specificity for fructan linkages, two groups of specific levan-binding myeloma proteins have been defined in the BALB/c mouse. The first group includes the myeloma proteins, UPC 61, EPC 109, and 5606 which are specific for the /3(2 -+ 1) fructofuranosyl linkage. They bind fructans containing this linkage such as inulin and bacterial levan. The second group includes the myeloma proteins ABPC 48 and UPC 10, which are specific for the /?(2 -+ 6) fructofuranosyl linkage and consequently bind only to bacterial levan (Grey er al., 1971; Cisar et al., 1974; Lieberman et al., 1975). Inulin is a linear polymer of entirely p(2 --f I)-linked D-fructofuranosyl residues with sucrose at the nonreducing terminus

(Drew and Haworth, 1928). Bacteria1 levan, on the other hand, is a branched polymer consisting of a /?(2 -+ 6)-linked D-fru~tofuranosy1 backbone with fl(2 -* 1) linkages at the branch points (Feingold and Gehatia, 1957). Immunochemical studies of the binding specificity of UPC 61 and EPC 109 showed that the combining site of these irnmunoglobulins had the highest complementarity for a tetrafructofuranosyl sequence terminated by a (2 -+ 1) linked a-o-glucose moiety, *Author to whom correspondence

should be sent: Depart-

ment of Microbiology; One Gustave 10029, U.S.A.

Mount Sinai School of Medicine, L. Levy Place, Box 1124, New York, NY

351

BRENDA HALL et al.

352

A Series

B Series

C Series

5d 4b

6d,

5b

5C

5a

6dz

Fig. 1. Haworth representation of the nine fructan oligosaccharides. apparent affinity constants (a&) for 4b in particular. The a& values of the hybrid antibody for 4b resemble that of EPC 109 which strengthens the suggestion of Streetlcerk et al. (1978), that the light chains exert a dominant influence on binding specificity. The amino acid sequence determination of UPC 6 1 and EPC 109 (Potter et al., 1977; Vrana et al., 1978) has revealed that these inulin-binding myeloma proteins derive their variable heavy chain (V,) genes from the 5606 V, family and their variable light (V,)

chain genes from the V,ll family. We demonstrate that other V,-V, associations can also create combining sites capable of specifically accommodating inulin. The monoclonal antibody (Mab), 1-5-1, which has its primary binding specificity for /3(2 -+ 6) fructans (Victor-Kobrin et al., 1985) can also bind to inulin. This Mab derives its V, from V,X24 and its V, from VLIOb (Victor-Kobrin ef al., 1990). The binding of l-5-1 to inulin-BSA is specific because it can be inhibited by various oligosaccharides and sinistrin.

Binding specificities of inulin-binding immuno~obul~ns MATERIALS AND METHODS

Immunoglobuiins The inulin-binding BALB/c myeloma proteins, UPC 61 and EPC 109, were purified from ascites as previously described (Lieberman et af., 1975). These proteins are of the IgA class as determined by typing with anti-allotypic antisera (Lieberman et al., 1975). Both UPC 61 and EPC 109 utilize V, and V, genes from the 5606 and V, 11 families, respectively (Kabat et al., 1987). UPC 61, EPC 109 and UPC 61H:EPC 109L (Manjula et al., 1976) were kindly provided by Dr. R. Lieberman (National Institutes of Health, Bethesda, Maryland). The monoclonal antibody, l-5 1 is of the IgM heavy chain class. Its origin and properties have been previously described in detail (Victor-Kobrin et al., 1985, 1990).

Poiysaccharides

353

fructofuranosyl (2 -+ I)-/I-D-fructofuranosyl (2 -+ I)(2 -+ 6) - a - D- glucopyranosyl /? - D - fructofuranosyl (2 -+ 1)./?-D-frUCtOfUranOSyl(2 + 1)-D-fructofuranose (&I,); fi-D-fructofuranosyl (2 4 1)-/?-D-fructofuranosyl (2 + 6)-a-D-glucopyranosyl (2 + 1)-/I-ofructofuranosyl (2 + 1)-p-D-fructofuranosyl (2 -+ l)D-fructofuranose (&I& The structures of these oligosaccharides are shown in Fig. 1. Sinistrin and the three fractions (5, 13 and 21), consisting of a /I(2 -+ 1) fructan backbone with /I(2 -+ 6) fructan side chains, were kindly provided by E. Nitsch (Linz, Austria). Native sinistrin has an average mol. wt of 3300. Preparative gel-permeation chromatography of native sinistrin using three columns of Sephadex G-50 in series was done to obtain fractions 5 (Sin. F5), 13 (Sin. F 13) and 21 (Sin. F21). Sin. F5 has a mol. wt of approximately 9300 and a degree of polymerization (dp) of approximately 56. Sin. F13 has a mol. wt of approximately 3500 and a dp of approximately 2 1. Sin. F2 1 has a mol. wt of approximately 1430 and a dp of approximately 8.5 (Nitsch et al., 1979).

A conjugate of inulin containing only b(2 -+ 1) fructan linkages coupled to bovine serum albumin, inulin-BSA (Inu-BSA), was prepared according to Chien el al. (1979). p(2 -+ 6)-Poly-D-galactose Direct binding assay coupled to BSA, galactan-BSA, was provided by Dr. M. Potter (National Institutes of Health, ~olyvinyl~hioride 96 well plates (Falcon) were Bethesda, Maryland). Bacterial levan, BL, molecular coated with antigen: Inu-BSA 5 or lOpg/ml and BL weight (mol. wt) 2 x lo7 from A. levanicum (ATCC 20 pgg/ml diluted in PBS for 2 hr at 37°C or overnight 1552) was prepared according to Lieberman et al. at 4°C. The plates were then blocked with 1% BSA (1976). Grass levans from Lolium temulentum in PBS for 30min at room temp. The plates were (Darnel) of approximately 10,000 mol. wt, consisting washed three times with PBS before 50~1 of the almost entirely of linear fl(2 --) 6) fructan linkages, antibody diluted in PBS containing 1% BSA and and from stems of cocksfoot (Dactylis glomerata) 0.05% Tween were added to each well of the plate. consisting of a linear a(2 + 6) fructan polymer of After addition of the antibody, the plates were incuapproximately 50,000 mol. wt (Pollock, 1979, 1982) bated at 4°C overnight. The following day the plates were a gift from C. J. Pollock (Welsh Plant-Breeding were washed with PBS, 0.05% Tween. Rat antiStation, U.K.). mouse kappa monoclonal antibody (187.1) (Yelton Nine fructan oligosaccharides consisting of primaret al., 1981) labelled with ‘25I(Greenwood et al., 1963) ily /I(2 -) 1) linked fructans were isolated from the was diluted in PBS containing 1% BSA and 0.05% roots of asparagus (Asparagus oficinalis). Their isolaTween to a final concn of 50,000 cpmj50 ~1. Then tion and characterization were previously described 50 ~1 was added to each well of the plate to measure (Shiomi et al., 1976, 1979; Shiomi 1981). They were the binding of the antibody to the antigen coated kindly provided by Dr. Norio Shiomi (Ebetsu, plates. After a 2-3 hr incubation at room temperaHokkaido, Japan): p-D-fructofuranosyl (2 -+ 1)-/3-D- ture, the plate was washed extensively, and the radiofructofuranosyl (2 + I)-P-D-fructofuranosyl (2 + l)activity was counted in a gamma radiation counter a -D-glucopyranose (4a, nystose); p -D-fructofuranosyl (LKB 1272 Clini Gamma). (2 -+ 1)-/I -D-frUCtOfUranOSy1 (2 -+ 1)-p -D-fructoCompetitive inhibition assay furanosyl (2 --+ l)-/I-D-fructofuranosyl (2 + 1)-a-Dglucopyranose @a); /I-D-fructofuranosyl(2 -+ 1)$-DPolyvinylchloride 96 well plates (Falcon) were fructofuranosyl (2 + 6)-~-D-glucopyranosyl (2 + l)coated with Inu-BSA (10 fig/ml) overnight at 4°C. /? -D-fructofuranose (4b); /I -~-fructofuranosyl The plates were blocked with 1% BSA in PBS fOT (2 + l)-/I-D-frUCtOfUranOSy1 (2 -+ l)-/I-D-frUCtOfUTa- 30 min at room temp. The saccharide inhibitors and nosy1 (2 -+ 6)-a-D-glucopyranosyl (2 + l)-D-fructoantibodies were diluted separately in PBS containing furanose (Sb); /-I-D-fructofuranosyl (2 + 6)-a-D1% BSA and 0.05% Tween to a final concentration glucopyranosyl (2 + I)-/I-D-fructofuranosyl (2 + l)of 2 x . Equal volumes of the inhibitor and antibody D-fructofuranose (4e); /I-D-fructofuranosyl (2 + 6)- were incubated together for 2 hr at room temperaff -D-glucopyranosyl (2 -+ 1)-P-D-fructofuranosyl ture. After the 2 hr incubation, 50 @1of the inhibitor (2 + 1)-~-D-fructofuranosyl(2 -+ 1)-D-fructofuranose and antibody solution was added to each well of the (5e); ~“D-fructofuranosyl (2 -+ l)-/I-D-fructo96 well plate that was pre-coated with 10 gg/mI furanosyl (2 -+ 6)-a -D-glUCOpyranOSy1 (2 + 1)$-DInu-BSA. The plates were then incubated overnight frUctofUranOSy1 (2 + 1)-D-fructofuranose (5d); B-D- at 4”C, washed with PBS containing 0.05% Tween,

BRENDA HALL et al.

354

and ‘251-labelled rat anti-mouse kappa monoclonal antibody (187.1) was added as described above. The apparent affinity constants (aK,) of UPC 61, EPC 109, UPC 61H:EPC 109L and l-5-1 for the various levans and oligosaccharides were determined by the method of Nieto et al. (1984). Briefly, the apparent affinity constant (aK,) is defined as the reciprocal concentration of free inhibitor that is required to inhibit by 50% the antibody from binding to the antigen that has been immobilized. The aK, values determined by this method have been shown to be in close agreement with the intrinsic affinity constant measured by fluorescence quenching of the Farr assay (Nieto et nl., 1984). The average molecular weights of inulin-BSA, bacterial levan, native sinistrin, Sin. F5, Sin. F13, Sin. F21, Lolium temulentum and Dactylis glomerata were used to calculate the concn (mol/l) of the inhibitor that was required to inhibit by 50% the binding of the antibody to the antigen. Column chromatography Monomeric (7s) UPC 61 and UPC 61H:EPC 109L were obtained by passage over a Superose 6 gel filtration column (Pharmacia Inc., Uppsala, Sweden). RESULTS

Direct binding of UPC 61, EPC 61H:EPC 109L to inulin-BSA

109 and UPC

We studied the binding of UPC 61, EPC 109 and the UPC 61H: EPC 109L hybrid to various oligo- and polysaccharides containing /I(2 -+ 6) fructan linkages to further define the specificity of these antibodies. The direct binding of UPC 61, EPC 109, and the hybrid, UPC 61H:EPC 109L, to inulin-BSA is shown in Fig. 2. As can be seen, all three antibodies bind to inulin, a linear fructan polymer with j(2 + 1) linkages.

SpeciJicity of UPC 61, EPC 109 and UPC 61H:EPC 109L for /I(2 + 1) linked fructan saccharides To investigate the binding specificities of UPC 61, EPC 109 and UPC 61H : EPC 109L hybrid for polysaccharides containing p(2 -+ 1) fructofuranosyl linkages, we carried out a competitive inhibition assay using various oligo- and polyfructans such as sinistrin, Lolium temulentum and Dactylis glomerata grass levans. The results of the UPC 61 inhibition by the polyfructans are shown in Fig. 3A. As can be seen, native sinistrin and sinistrin fraction 5 are more efficient than sinistrin fractions 13 and 21 at inhibiting the binding of UPC 61 to inulin-BSA. Lolium temulentum fructan is a poor inhibitor of this binding, presumably because it contains almost entirely /I(2 + 6) fructofuranosyl linkages. Dactylis glomerata fructan does not inhibit UPC 61 from binding to inulin-BSA at the concentrations used in this assay, demonstrating the specificity of UPC 61 for the fl(2 + 1) fructofuranosyl linkages found in inulin. Similar results were also obtained for EPC 109 and the hybrid, UPC 6 1H : EPC 109L (data not shown); however, Lolium temulentum could not inhibit the hybrid antibody from binding to inulin-BSA (Table 1). Nine oligosaccharides containing primarily /I(2 -+ 1) fructofuranosyl linkages isolated from asparagus roots (Fig. 1) were used to further investigate the binding specificity of UPC 61, EPC 109 and UPC 61H:EPC 109L. The results from using these oligofructans to inhibit the binding of UPC 61 to inulin-BSA are shown in Fig. 3B. Similar results were obtained with EPC 109 and UPC 61H:EPC 109L (data not shown). The nystose oligosaccharides 4a and 5a are linear fructans consisting of 2 and 3 jI(2 -+ 1) fructofuranosyl linkages, respectively, with a (2 + 1) E-p-glucopyranosyl linkage at the nonreducing terminus. Our results agree with the previous studies by Streefkerk and Glaudemans (1977) who have shown that 5a was a better inhibitor than 4a, defining the combining site to be large enough to

UPC 61 EPC 109 U6VE109

.Ol

.l

1

10

100

uglml

Fig. 2. Dose-effect binding of inulin-binding myeloma proteins to inulin-BSA. The counts per minute (cpms) of ‘251-labelled rat anti-mouse kappa bound (ordinate) is plotted vs the antibody concentration @g/ml) bound to inulin-BSA (abscissa).

Binding

specificities

of inulin-binding

immunoglobulins

355

80

Inu-BSA

relative

molecules

of

__t_

Lolium

I

Dactylis

-

Sinistrin

I

Sin F5

Y

Sin F21

I

SinF13

lnhlbltor

4a 80

-

4b

-

4c

7-

5a

-

5b

-5c 5d

-

6dl

-

6d2

_

Sucrose

1

.1 nanomoles

-

of

lnhlbltor

of UPC 61 binding to inulin-BSA by various fructans. The per cent inhibition (ordinate) inhibitor concentration in relative molecules or nanomoles (abscissa). (A) Fructan polysaccharide inhibitors: inulin-BSA, Lolium temulentum, Dactylis glomerata, sinistrin, sinistrin fraction 5, sinistrin fraction 21 and sinistrin fraction 13. The relative molecules of inhibitor were calculated as the mass required for 50% inhibition divided by the average molecular weight of the inhibitor. (B) Fructan oligosaccharides: 4a, 4b. 4c, 5a, 5b, SC, Cd,, 6d,, and sucrose. Fig. 3. Inhibition

is plotted versus the fructan

accommodate a /I(2 + 1) linked tetrafructofuranose for all three antibodies. &I,, 6d,, 5b and 5c inhibited the binding of UPC 6 1, EPC 109 and UPC 61 H : EPC 109L to inulin-BSA fairly well, but 5d was a poor inhibitor. 4c does not inhibit any of the three myeloma proteins from binding to inulin-BSA. While 4b does not inhibit UPC 61, it does inhibit EPC 109 and UPC 61H: EPC 109L from binding to inulinBSA, albeit poorly (data not shown). Sucrose does not inhibit any of these three myeloma proteins from binding to inulin-BSA when up to 5 nmol were used, presumably because it is only a disaccharide. A

summary of the binding specificities of UPC 61, EPC 109 and UPC 61H: EPC 109L for the various oligoand polyfructans is shown in Table 1. The binding specificities are expressed as aK, values which are inversely proportional to the concentration of inhibitor that is required to give 50% inhibition of binding (Nieto et al., 1984) to inulin-BSA. Specificity

of the monoclonal

antibody,

l-5-1

In pilot experiments we have studied the binding of a large panel of b(2 -+ 6) fructan specific monoclonal antibodies to inulin-BSA. Among these antibodies

356

al.

BRENDAHALL~I Table I. Apparent

affinity constants, a& (in I/M), of UPC 61, EPC 109, UPC 61H: EPC 109L and 1-5-l for various fructan inhibitors

Inhibitor

UPC 61

EPC 109

Inulin-BSA Bat. levan 4a

I.8 x IO’

4.5 x IO’ ND 1.6 x IOJ I.4 x I04 0 4.2 x IO* 2.9 x IO’ I.6 x IO’ I.8 x IO’ 2.3 x IO’ 1.4 x I05 I.9 x I06 4.2 x IO” 1.5 x IO’ 8.0 x IO’ I.8 x I04

I.3NxDlos”

4b

0

4c 5s

: 2.3 5.4 3.6 9.3 1.9 8.5 1.4 3.1 9.5 1.4

5b SC 5d 6& 6& Sinistrin Sin. F5 Sin. F13 Sin. F21 Lolium

remulen1um

Dncfylis

glomerara

IO’” I04 IO4 IO’ 105 IO’ 10’ IO’ I04 105 1.0 x I04 x x x x x x x x x 0

0

UPC 6lH: EPC 109L I.4 x IO6 2. INXDIO’” 3.1 x IO’ “, 3.0 105” 1.9 x I04 2.1 x I04 2.5 x IO’ 2.5 x IO4 2.4 x IO4 1.7 x 105 3.4 x 10s 5.7 x 10s 6.8 x IO4 0 0

1-5-l I.1 x IO’ 2.5 x IO9 ND ND ND 1.5 x I04 ND ND ND 1.9 x IO’ 2.5 x IO4 I.2 x 10s 2.6 x IO6 4.2 x IO4 1.9 x IO’ ND ND

“aK, of monomeric immunoglobulin isolated from Superose 6 column. a& was determined as I/inhibitor concentration (mol/l) that gave 50% inhibition of binding to inulin_BSA (Nieto ef al., 1984).

we have found one which binds to both b(2 + 6) and fi(2 + 1) linked fructans. The direct binding of 1-5-l to inulin-BSA, bacterial levan (BL), and arabinogalactan is shown in Fig. 4A. This demonstrates that the binding of l-5-1 to bacterial levan is much stronger than it is to inulin. However, at a higher concentration, this antibody bound specifically to inulin-BSA. The data presented in Fig. 4B shows that the binding to inulin-BSA was specific because 50% inhibition of 1-5-1 binding to inulin-BSA was achieved with 4.5 relative molecules of inulin-BSA, whereas, galactan-BSA did not inhibit the binding to inulin-BSA even at 1 mg/ml (data not shown). Native sinistrin as well as fractions 5. 13 and 21 also inhibit 1-5-1 from binding to inulin-BSA (Fig. 4B). The fi(2 -+ 1) linked fructofuranosyl linked oligosaccharides, 5a, 6d and 6d,, all inhibit 1-5-1 from binding to inulin-BSA (Fig. 4C). A summary of the apparent affinity constants (aK,) of 1-5-1 for the various fructan inhibitors is also shown in Table 1. 1-5-1 is of the IgM heavy chain class and is predominantly in the pentameric form which explains why its aK, values for some of the saccharide inhibitors may be higher than the inulin-binding myeloma proteins. The apparent affinity constant of 1-5-1 for bacterial levan is much higher than it is for inulin or any of the fructan inhibitors. Nevertheless, the binding of 1-5-1 to inulin-BSA can be specifically inhibited with fructans containing fi(2 -+ 1) fructofuranosyl linkages. DlSCUSSlON

In this communication we show that three inulinbinding myeloma proteins, UPC 61, EPC 109 and a hybrid UPC 61H:EPC 109L antibody bind to native sinistrin and three chromatographically isolated molecular weight fractions derived from it. Sinistrin is similar to inulin in that it consists of a linear

backbone of p(2 -+ 1) fructofuranosyl linkages with a terminal nonreducing 2 + 1 linkage to x-D-glucose, but sinistrin also contains p(2 -+ 6) fructofuranosyl branch points. Chromatographic analysis of native sinistrin shows a bell shaped distribution of molecular weight species, ranging from 1430 to 9800, with an average mol. wt of about 3280 (Nitsch et al., 1979). All three inulin-binding myeloma proteins bind very well to native sinistrin and fraction 5. While EPC 109 and the hybrid bind better to inulin-BSA than to any of the sinistrin fractions, UPC 61 shows comparable affinity for inulin-BSA, native sinistrin, and Sin. FS (Table 1). Presumably, the binding to native sinistrin is mediated through a minority of higher molecular weight molecules which is why it can be bound as well as Sin. F5, a high molecular weight fraction. Presumably, the binding of UPC 61, EPC 109 and UPC 61H: EPC 109L to sinistrin could be mediated through the /I(2 + 1) fructofuranosyl linkages found in the backbone of sinistrin and also through the /3(2 -+ 6) fructofuranosyl branch point linkages if they are surface exposed. By studying molecular models of the fi(2 -+ 1) fructofuranosyl linkage and the fl(2 + 6) linkage, Cisar et al. (1974) proposed that an antibody specific for a /?(2 -+ 1) fructofuranosyl linkage could accommodate a p(2 + 6) fructofuranosyl linkage in its combining site. This is because the p(2 -+ 1) fructofuranosyl linkage is much bulkier than the p(2 + 6) fructofuranosyl linkage. Sequencing of EPC 109 and UPC 61 has shown that both antibodies possess a third complementarity determining region (CDR) consisting of only one amino acid (Potter et al., 1977; Vrana et al., 1978). A hypothetical model of the variable region of EPC 109 based upon the amino acid sequence shows that the one amino acid of CDR 3 allows this region to be left wide open providing a wide central valley in the combining site (Potter et al., 1977). This large open combining site could accommodate the bulky p(2 -+ 1) fructofuranosyl linkage. Theoretically,

Binding specificities of inulin-binding immunoglobulins

357

-

ugfml

+

l3L tnu&SA

+

Arabinogal

eb

Sinislrin Sin. FS Sin. F13 Sin. F2l

‘01

.1 relefive

lb

i molecules

of

100

lnhlbltot

c.

% B t

”

.c

5a 6dl

s

6d2

nanomoles

of

lnhlbltor

Fig. 4. {A) Dose-effect binding of mono~lonal antibody, t-5-1, to in&in-BSA, bacterial levan, and arabinogalactan. The counts per minute (cpms) of ‘Wabelled rat anti-mouse kappa bound (ordinate) is plotted vs the antibody concentration @g/ml) bound to inulin-BSA or BL (abscissa). (B) Inhibition of the monoclonal antibody, 1-5-1, binding to inulin-BSA by various polysaccharide fructans. The percentage inhibition (ordinate) is plotted vs the fructan inhibitor concentration in relative molecules (abscissa). The relative molecules of inhibitor were calculated as described in Fig. 2. (C) Inhibition of the monoclonal antibody, I-5-1, binding to in&in-BSA by various oligosaccharides. The percentage inhibition (ordinate) is plotted vs the fructan inhibitor concentration in nanomoles (abscissa). The fructan oligosaccharides are as described in Fig. 2.

BRENDAHALL~~

358

either the p(2 -) 1) or the /?(2 -+ 6) fructofuranosyl linkage of sin&ran is capable of entering into the combining site of UPC 61, EPC 109, or UPC 61H:EPC 109L hybrid. Streefkerk et al. (1979) also proposed that the extended confirmation of a /I(2 + 6) fructofuranosyl linkage is capable of entering, at least partially, into the wide combining site based on their studies with Lolium perenne, a grass levan consisting entirely of linear /3(2 -+ 6) fructofuranosyl linkages. They found that the Fab’ fragment of EPC 109 was capable of binding to Lolium perenne; however, the Fab’ fragment of UPC 61 was not (Streefkerk et al., 1979). We have shown that both UPC 61 and EPC 109 are capable of binding weakly to Lolium temulentum, a grass levan consisting almost entirely of b(2 + 6) fructofuranosyl linkages. The differences in binding of UPC 61 and EPC 109 to these two different grass levans could be attributed to the unknown linkages, perhaps fi(2 + 1) fructofuranosyl linkages, present in Lolium temulentum. The affinity of UPC 61 and EPC 109 to the /3(2 + 6) fructofuranosyl linkage is not very strong because neither of these antibodies could bind tightly enough to Dactvlis glomerata, a linear /I(2 + 6) fructan, to allow an affinity measurement. Although it is possible for the fl(2 -+ 6) fructofuranosyl linkage to fit into the combining site of these inulin-binding myeloma proteins, the affinities of these antibodies for sinistrin is most likely mediated through the binding to the fl(2 + 1) fructofuranosyl linkages. We also show that the binding of these three inulin-binding myeloma proteins to inulin-BSA can be inhibited by oligosaccharides containing b(2 -+ 1) fructofuranosyl linkages which were isolated from the roots of asparagus. The fine binding specificity of UPC 61, EPC 109 and UPC 61H:EPC 109L was defined by the extent to which the various fructan oligosaccharides could inhibit the binding of these three antibodies to inulin-BSA. The A series, 4a and 5a, are linear p(2 + 1) linked fructans with a terminal nonreducing 2 + 1 linkage to a-n-glucose (Fig. 1). These two oligosaccharides are the most analogous to the structure of inulin. By

al.

comparatively analyzing the aK, values of 4a and 5a (Table 1) in the case of UPC 61, EPC 109 and UPC 61H:EPC 109L, it is apparent that 5a is the better inhibitor suggesting that the size of the combining site can accommodate four fructose residues linked fi(2 -+ 1) to each other. Gur results obtained for the aK, values of 4a and 5a for monomeric (7s) UPC 61, the hybrid molecule, and for EPC 109 are in agreement with those previously reported by other methods such as fluorescence titration (Streefkerk and Glaudemans, 1977) or fluorescence quenching (Sugii and Kabat, 1980). This strengthens the conclusion that the size of the paratope can accommodate a /?(2 -+ 1) linked tetrafructofuranoside. We also studied the effect of the B series of neokestose inulin-like oligosaccharides (Fig. 1). 4b and 5b (Fig. 1) both contain an r-n-glucose residue that has a /I(2 + 1) linked fructose as well as a fi(2 -+ 6) linked fructose. Streefkerk and Glaudemans (1977) have shown by comparatively analyzing molecular models that the /I-D-fructofuranosyl (2 + 6)-D-glucopyranosyl linkage mimics the /?(2 + 1) fructofuranosyl linkage but not the P-Dfructofuranosyl (2 -+ I)-a-D-glucopyranosyl linkage. 5b is a much better inhibitor than 4b in the case of UPC 61, EPC 109 and UPC 61H:EPC 109L; however, 4b can inhibit the binding of EPC 109 and the hybrid to inulin-BSA at a higher molar concentration with no effect on the binding of UPC 61 to inulin-BSA. Because the fl-D-fructofuranosyl (2 + 6)-D-glucopyranosyl and the /?(2 4 1) fructofuranosyl linkage are indistinguishable according to Streefkerk and Glaudemans (1977) one would predict that 5b should be equivalent to 5a in its ability to inhibit the binding to inulin-BSA; however, this is not the case. We conclude that the additional fl-~fructofuranosyl (2 + I)-a-D-glucopyranosyl linkage in 5b (Fig. 1) may interfere with the binding to this b-II-fructofuranosyl (2 + 6)-D-glucopyranosyl linkage. Therefore, 5b contains the same number of /I(2 + 1) fructofuranosyl linkages as 4a and is probably recognized by the three inulin-binding antibodies in a similar manner as 4a. The explanation for weak binding of

HEAVY CHAIN

FR2 ---

LIGHT CHAIN

FR3 ----~--~~---~

FR4 --_

CDRl ----

CDR2 ---_

110

30

56

FR3 ---

48

79

UPC 61

VAL

TYR

TYR

PRO

SER

ASP

SER

EPC 109

ILE

PHE

HIS

THR

ASN

ALA

ARG

Fig. 5. The amino acid differences the framework

90

65

between the V, and V, chains of UPC 61 and EPC 109. FR indicates regions. CDR indicates the complementarity determining regions. The amino acid positions are defined by numbers (Kabat et al., 1987).

Binding specificities of inulin-binding immunoglobulins

4b to EPC 109 and the the hybrid may be that 4b is recognized as a disaccharide because the /?-D-fructofuranosyl (2 + I)-a-D-glucopyranosyl linkage may interfere with the fl(2 + 6) linkage between fructose and glucose from binding to the antibody. Alternatively 4b could be recognized as a trisaccharide, but the /?(2 + 1) linkage between the glucose and fructose may interfere with the binding as we proposed for 5b. Therefore, because the size of the combining site is that of a fl(2 + I) linked tetrafructofuranoside, the binding is not as strong to 4b. Study of the C series of neokestose fructan oligosaccharides 4c and 5c (Fig. 1) which contain both a fructose p(2 -+ 6) and a fructose b(2 + 1) linked to a-D-glucose showed that only 5e was capable of inhibiting the binding to inulin-BSA. 4e would be expected to inhibit as if it were a disaccharide consisting of one /?(2 + 1) fructofuranosyl linkage or one b-p-fructofuranosyl (2 + 6)-D-glucopyranosyl linkage, but no inhibition was observed at the concentrations tested (up to 3 nmol). In contrast to the situation previously described for the B series of oligosaccharides, the fi(2 + 6) linkage between fructose and glucose does not interfere with the ability of the C series oligosaccharides to inhibit the binding of these antibodies to inulin-BSA. In fact, this linkage should be recognized as a disaccharide. 5c contains two /3(2 + 1) fructofuranosyl linkages as does 4a and is probably recognized by these antibodies as if it was 4a (Fig. 1). Our final study of the D series of neokestose fructan oligosaccharides represented by 5d, 6d, and 6dZ, also contains both a /I(2 + 6) and a /3(2 -+ 1) fructose linkage to cc-n-glucose (Fig. 1). 5d is a poor inhibitor of UPC 61, EPC 109 and UPC 61H:EPC 109L from binding to inulin-BSA (Table 1). The aK, values of 5d for the three inulin-binding antibodies are much lower than for any of the other oligosaccharides. It is probable that 5d is recognized as an exposed disaccharide or as a trisaccharide with interference from the remaining /?(2 + 1) linked difructofuranoside. Perhaps the second potential of inhibiting as a disaccharide is what gives 5d a higher affinity than 4b in the case of UPC 61, the same affinity in the case of the hybrid, and a IO-fold lower affinity in the case of EPC 109. Thus 5d can distinguish the binding specificities of the three antibodies. For each antibody, the affinity constants for 6dl are comparable to 5b within a factor of two, and the affinity constants for 6d, are comparable to 5c (Table 1). Therefore, the additional p(2 -+ 1) linked fructose residue does not significantly enhance the ability of the antibodies to bind much better to either 6d, or 6d2 as compared with 5b and SC, respectively. We conclude that the antibodies recognize 6d, and &I2 in a similar manner as they recognize 5b and SC, respectively. These naturally occurring oligosaccharides from the B, C and D series of p(2 + 1) linked fructofuranosides (Fig. 1) give further insight as to how

359

residues near the sucrose moiety of the parent fructan molecule are recognized. The p(2 + 6) linkage between fructose and glucose in the B series of oligosaccharides is not avidly recognized as a p(2 + 1) fructofuranosyl linkage presumably due to interference of the b(2 + 1) linkage between fructose and the same glucose residue. However, The C series of oligosaccharides (Fig. 1) can be avidly recognized when there are at least two p(2 -+ 1) fructofuranosyl linkages extending from the same parent sucrose moiety (5e). From examining the affinity constants to the D series of oligosaccharides, we conclude that avid recognition can occur on either side of the parent sucrose moiety as long as there are at least two additional /?(2 --* 1) fructofuranosyl linkages on any one side (6d, and &I* vs 5d). The fine binding specificity of UPC 61, EPC 109, and the hybrid, UPC 61 H : EPC 109L for the b(2 -+ 1) fructofuranosyl linkage can be related to the amino acid sequence differences between UPC 61 and EPC 109. Figure 5 shows the amino acid differences between the variable heavy chains (V,) and the variable light chains (V,) of UPC 61 and EPC 109. The VH amino acid sequences of both inulin-binding myeloma proteins are identical in their CDRs and contain two major changes within the framework regions (FR). One of these changes is in FR 3 where a tyrosine exists at amino acid position 90 in UPC 61, and a histidine occurs at the same position in EPC 109. The other change is in FR 4 where a proline exists at amino acid position 110 in UPC 61, and a threonine occurs at the same position in EPC 109. On the other hand, the VL amino acid sequences of UPC 61 and EPC 109 contain different amino acids at position 30 in CDR 1 and at position 56 in CDR 2. FR 1 and FR 2 are identical and one conservative change exists in FR 3. Streefkerk et al. (1978) suggested from their binding studies of the hybrid, UPC 61H: EPC 109L, to 4a and Sa that the EPC 109 light chain may predominantly determine the binding specificity of the hybrid. The aK, values of the hybrid for 4b and 5d resemble the aK, values of EPC 109 for both of these oligosaccharides supporting the suggestion of Streefkerk et al. (1978). However, the aK, values of the hybrid and EPC 109 for 4b and 5d are not identical which leads us to propose that the heavy chain also makes a contribution in determining the binding specificity. This is supported by the fact that the hybrid antibody could not bind to Lolium temulentum while the parental antibodies were able to bind (Table 1). The framework sequences of the EPC 109 and UPC 61 heavy chains may directly influence how the CDRs of the respective heavy chain and light chains fit together to form the combining site specific for the fl(2 + 1) fructofuranosyl linkage. Only by studying the binding of a fructan consisting of almost entirely fl(2 46) fructofuranosyl linkages, Lolium temulenturn, were we able to observe the functional differences between the EPC 109 and UPC 61 heavy chains

BRENDA HALL et al.

360

when combined with the EPC 109 light chain as in the hybrid. The monoclonal antibody, 1-5-1, derives its Vu and V, genes from the V,X24 and V,lOb genes, respectively. This is different from UPC 61 and EPC 109 which utilize the Vu genes from the 5606 V, and the V, genes from the V, 11 families, respectively. The antigen binding specificity of 1-5-1 is predominantly for /3(2 --* 6) fructans with an aK, value (I/M) of 2.5 x lo9 for bacterial levan. However, we were able to show that this antibody can also bind to inulinBSA (Fig. 4A). The binding to inulin-BSA is specific and can be inhibited with the fructan oligosaccharides, 5a, 6d, and &I,, but not with galactan-BSA even at high concentrations of 1 mg/ml. Sinistrin can also inhibit the binding of 1-5-1 to inulin-BSA. This is the first report that different V, and V, gene families could form a combining site capable of binding to insulin-BSA. Generally, the combining site of antibodies specific for polysaccharides is encoded by V genes derived from a single V gene family and restricted V,-V, pairing: for example, galactan antibodies utilize V,X24V,4, (Rudikoff et al., 1983); a(1 -+ 3) dextran antibodies utilize V,J558-V,, (Schilling et al., 1980); and 3-fucosyllactosamine antibodies utilize V, X24-v, 24B (Kimura et al., 1988). Acknowledgement-We would like to thank MaryAnn DiLiberti for her excellent technical assistance throughout this project.

REFERENCES Chien C. C., Lieberman R. and Inman J. H. (1979) Preparation of functionalized derivatives of inulin: conjugation or erythrocytes for hemagglutination and plaque-forming assays. J. Immunol. Meth. 26, 3946. Cisar J., Kabat E. A., Liao J. and Potter M. (1974) Immunochemical studies on mouse myeloma proteins reactive with dextrans or with fructosans and on human antilevans. J. exp. Med. 139, 159-179. Drew H. D. K. and Haworth W. N. (1928) Polysaccharides. III. The molecular complexity of inulin. J. them. Sot. 2690. Feingold D. S. and Gehatia M. (1957) The structure and properties of levan, a polymer of D-fructose produced by cultures and cell-free extracts of Aerobacter leoanicum. J. Polymer Sci. 23, 783. Greenwood F. C., Hunter W. M. and Glover T. S. (1963) The preparation of i3’I-labelled human growth hormone of high specific radioactivity. Biochem. J. 89, 114. Grey HI M.1 Hirst J. W. and Cohen M. (1971) A new mouse immunoalobulin: IaG3. J. exn. Med. 133. 289-304. Kabat E. A., Wu T. T., Reid-Miller M., Perry H. M. and Gottesman K. S. (1987) Sequences of Immunological Interest. U.S. Department of Health and Human Services Bethesda, MD. Kimura H., Cook R., Meek K., Umeda M., Ball E., Capra J. D. and Marcus D. M. (1988) Sequences of the V, and V, regions of murine monoclonal antibodies against 3-fucosyllactosamine. J. Immunol. 140, 1212-1217. Lieberman R., Potter M., Humphrey W. Jr. and Chien C. C. (1976) ldiotypes of inulin-binding antibodies and myeloma proteins controlled by genes linked to the allotype locus of the mouse. J. Immunol. 117, 2105-2111.

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Binding specificities of inulin-binding immunoglobulins Vrana M., Rudikoff S. and Potter M. (1978) Sequence variation among heavy chains from inulin binding myeloma proteins. Proc. nom. Acad. Sci. U.S.A. 75, 1957-1961.

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Yelton D., Desmaymard C. and Scharff M. D. (1981) Use of monoclonal anti-mouse immunoglobulin to detect mouse antibodies. Hybridoma 1, 5.