©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mutational Analysis of the Ricinus Lectin B-chains
GALACTOSE-BINDING ABILITY OF THE 2 SUBDOMAIN OF RICINUS COMMUNIS AGGLUTININ B-CHAIN (*)

(Received for publication, February 24, 1995; and in revised form, May 15, 1995)

Nathalie Sphyris J. Michael Lord (§) Richard Wales Lynne M. Roberts

From the Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Ricin B-chain (RTB) is a galactose-specific lectin that folds into two globular domains, each of which binds a single galactoside. The two binding sites are structurally similar and both contain a conserved tripeptide kink and an aromatic residue that comprises a sugar-binding platform. Whereas the critical RTB residues implicated in lectin activity are conserved in domain 1 of Ricinus communis agglutinin (RCA) B-chain, the sugar platform aromatic residue Tyr-248 present in domain 2 of RTB is replaced by His in RCA B-chain.

In this study, key residues in the vicinity of the binding sites of the Ricinus lectin B-chains were altered by site-directed mutagenesis. The recombinant B-chains were produced in Xenopus oocytes in soluble, stable, and core-glycosylated forms. Both sites of RCA B-chain must be simultaneously modified in order to abolish lectin activity, indicating the presence of two independent, functional binding sites/molecule. Activity associated with the domain 2 site of RCA B-chain is abrogated by the conversion of Trp-258 to Ser. Moreover, the domain 2 site appears responsible for a weak binding interaction of recombinant RCA B-chain with GalNAc, not observed with native tetrameric RCA. Finally, the introduction of His at position 248 of RTB severely disrupts but does not abolish GalNAc binding.


INTRODUCTION

The endosperm of castor bean seeds contains two toxic lectins: ricin and the Ricinus communis agglutinin (RCA). (^1)Both proteins are composed of two dissimilar types of subunit, denoted A and B, linked by a single disulfide bond(1) . The corresponding subunits of the Ricinus lectins are highly homologous(2, 3) , although they differ in quaternary structure. Ricin occurs as a dimer of disulfide-linked A- and B-chains, while RCA is a tetramer, composed of two ricin-like heterodimers, held together through noncovalent interactions(4, 5) .

The saccharide-binding affinities and specificities of the Ricinus lectins have been extensively studied. Both proteins bind beta-D-galactopyranoside moieties(6) , although only ricin binds GalNAc(6, 7, 8) . Studies on the binding of fluorescent galactosides to ricin B-chain (RTB) suggested that the two sites were of similar affinities(9) . In contrast, equilibrium dialysis, microcalorimetry, and fluorescence polarization showed RTB to possess two noncooperative lactose-binding sites with different galactoside affinities(10) . Consistent with this latter finding, x-ray data indicated one site to be more highly occupied(11) . More recently, primary structure analysis and x-ray crystallographic studies have shown that RTB folds into two globular domains of similar folding topologies, each binding a single galactoside(12) . It has been proposed (11) that RTB is the product of a series of gene duplications, since it appears that each of the two galactose-binding domains is composed of three copies of an ancestral galactose-binding peptide (termed alpha, beta, and ). Only the 1alpha and 2 subdomains of the present day RTB molecule display lectin activity(13) , and only the latter site accommodates both galactopyranosides and N-acetylgalactosamines(14) . The binding pocket is formed by a sharp bend in the polypeptide backbone corresponding to the tripeptide Asp, Val, and Arg, which accommodates the galactose moiety, and an aromatic amino acid (Trp-37 in the 1alpha/low affinity site and Tyr-248 in the 2/high affinity site), which provides a binding platform for the sugar. In each domain, the hydroxyl groups of the galactose moiety participate in hydrogen bonds with the homologous amides Asn-46 and Asn-255, which are in turn stabilized by hydrogen bonding to Asp-22 and Asp-234(12) , residues that also interact directly with the sugar(13) . Mutagenesis of the key hydrogen bonding residues or the homologous tripeptide in one or other or in both of the RTB binding sites confirmed the presence of two independent galactose-binding sites/RTB molecule(15) .

There are conflicting reports in the literature concerning the stoichiometry of sugar binding to RCA. Houston and Dooley (9) reported the binding of two molecules of 4-methylumbelliferyl galactose or 4-methylumbelliferyl N-acetylgalactosamine to the B-chains of the Ricinus lectins irrespective of the presence of the A-chain. In contrast, earlier studies suggested that there are only two functional carbohydrate-binding sites/RCA tetramer as opposed to the four we might expect from a molecule comprised of two ricin-like heterodimers(16) . Furthermore, RCA does not bind GalNAc(6) , which has been shown to interact exclusively with the 2 subdomain of RTB(14, 17) . The corollary of these observations was that the 2 subdomain of RCA B-chain is devoid of lectin activity. The primary structure of RCA B-chain exhibits the same pattern of subdomain duplication as RTB(13, 18) . However, whereas the critical residues implicated in lectin activity are conserved in domain 1 of RCA B-chain, the sugar platform Tyr-248 is replaced by His in domain 2. Histidine is a smaller residue than tyrosine and thus we would not necessarily expect its presence to introduce steric constraints on the positioning of the sugar within the binding pocket of the 2 subdomain. However, Rutenber and Robertus (13) proposed that the partial charge of the imidazole ring of histidine may disrupt the hydrophobic stacking interactions holding the apolar carbohydrate ring structure in place.

In this paper we describe the production of a series of recombinant wild-type and mutant Ricinus lectin B-chains in Xenopus oocytes. Key binding or structural residues in the potential combining sites have been altered by mutagenesis. The data clearly suggest that both the 1alpha and 2 subdomains of RCA B-chain must be simultaneously modified in order to abolish lectin activity and infer the presence of two independent functional binding sites/molecule. The domain 2 site appears responsible for a weak binding interaction with GalNAc, a sugar-binding activity not observed with native tetrameric RCA.


EXPERIMENTAL PROCEDURES

Chemicals and Reagents

L-[S]Methionine (1000 Ci/mmol) was obtained from Amersham Corp. Endo-beta-N-acetylglucosaminidase H (endo H) was purchased from ICN ImmunoBiologicals, Costa Mesa, California. Immobilized lactose was from Pierce, while protein A-Sepharose, N-acetyl-D-galactosamine, and galactose were obtained from Sigma. Sepharose 6B was from Pharmacia Biotech Inc.

Mutagenesis

All plasmids were constructed by standard cloning and polymerase chain reaction mutagenesis techniques so as to encode a fusion of the ricin N-terminal signal peptide with the appropriate mature B-chain. The vector used, a derivative of pSP64T, contained the SP6 promoter and the 5`- and 3`-untranslated flanking regions of Xenopus beta-globin cDNA reported to provide enhanced stability of the corresponding transcript in injected oocytes (19) . Mature RCA B-chain cDNA was recovered by polymerase chain reaction from clone pUC857 as described previously(3) . The primers used were such that the RCA B-chain sequence was generated on a fragment flanked by a 5`-end HindIII site and a 3`-end SalI site. The appropriately digested fragment was inserted in frame with the ricin presequence coding region within the pSP64T vector. Subsequent mutant pre-B-chain constructs were generated by overlap polymerase chain reaction using mutagenic oligonucleotides, designed such that mismatches were flanked on either side by at least eight bases complementary to the wild-type sequence. The cDNA sequences of all the constructs were verified by dideoxy sequencing.

Synthesis and Expression of Pre-B-chain Transcripts

Pre-B-chain transcripts were synthesized in vitro in the presence of the capping dinucleotide 7-Me(5`)GpppG(5`)OH and SP6 RNA polymerase as described earlier(20) . Purified RNA was dissolved in diethylpyrocarbonate-treated distilled water at a concentration of 1 mg/ml. Microinjection into Xenopus laevis oocytes (30 nl of RNA solution/oocyte) was performed using the Narishige microinjection system IM200. Pulse labeling with L-[S]methionine and oocyte homogenisation were performed as described previously(20) .

Lactose Binding Assay

A 1 10-cm disposable polystyrene column was packed with 0.5 ml of lactose immobilized onto 4% beaded agarose. Oocyte homogenates were passed down the column a total of three times. The column was washed with oocyte homogenization buffer (OHB: 20 mM Tris-HCl (pH 7.6), 0.1 M NaCl, 1% (v/v) Triton X-100, and 1 mM phenylmethylsulfonyl fluoride) to remove any unbound proteins, and 1-ml fractions were collected. OHB containing 50 mM galactose or 50 mM GalNAc, was applied to the column to elute any bound proteins in 1-ml fractions. Fractions were immunoprecipitated and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and fluorography.

Immunoprecipitation

Volumes of oocyte homogenates were adjusted to 500 µl with OHB before the addition of 400 µl of ice-cold immunoprecipitation buffer (IPB: 0.1 M Tris-HCl (pH 7.8), 1% (v/v) Triton X-100, 0.5% (w/v) SDS, 5 mM MgCl(2), 0.1 M KCl, 1% (w/v) deoxycholate free acid (pH 8.2), and 1 mM phenylmethylsulfonyl fluoride) and 5 µl of rabbit anti-RTB antibodies. After a 15-min incubation at room temperature, 40 µl of a 1:1 slurry of protein A-Sepharose equilibrated in IPB was added. The samples were mixed gently by rotation either for 1 h at room temperature or overnight at 4 °C. The beads were pelleted by centrifugation and washed 3 times with 500 µl of IPB before boiling in 30 µl of SDS-PAGE loading buffer. The supernatants were analyzed by SDS-PAGE and fluorography.

Other Methods

Ricin and RCA were purified from castor beans as described previously(21) . Published procedures were followed for translation of in vitro generated transcripts in a wheat germ cell-free lysate(22) , SDS-PAGE and fluorography(23) , enzymic deglycosylation using endo H (24) and for the determination of solubility and stability of the recombinant proteins(15) .


RESULTS

Mutagenesis of the Ricinus Lectin B-chains

Mutagenesis of the Ricinus lectin B-chains was performed in order to create a range of mutants in which critical residues in one or other of the putative carbohydrate-binding sites were altered. The mutations introduced are listed in Table 1.



The expression constructs encoding the wild-type (pRTB1) and the first binding site mutant of RTB (pRTB2) have been described previously(15) . In the pRTB2 mutant, critical hydrogen bonding residues Lys-40 and Asn-46 of the 1alpha subdomain have been converted to Met and Gly, respectively, thus rendering the 1alpha/low affinity site devoid of lectin activity. The replacement of Tyr-248 by His created a variant of RTB, termed pRTB3, bearing the characteristic 2 subdomain residues of RCA and the ricin E isoform(25) . In order to determine whether substitution of Tyr-248 by His alone was sufficient to abrogate lectin activity from the high affinity site of RTB, it was necessary to selectively eliminate the contribution of the 1alpha subdomain. The corresponding double binding site mutant (encoded by pRTB4) in which the 1alpha site is inactivated as in pRTB2 and the 2 subdomain possesses His in place of Tyr at position 248, was expected to have greatly diminished lectin activity.

In order to determine whether the 2 subdomain of RCA B-chain is potentially functional, the lectin activity, associated with the 1alpha subdomain, was selectively eliminated through the conversion of the conserved DVT tripeptide of the 1alpha subdomain to the amino acid sequence found at the equivalent position in the primary structure of the 2alpha subdomain, namely the tripeptide QAN. The clones encoding the wild-type (pRCAB1) and the 1alpha subdomain mutant (pRCAB3) of RCA B-chain provided the framework for the conversion of His-248 to Tyr in the 2 subdomain yielding pRCAB2 and pRCAB4, respectively. A second set of RCA B-chain derivatives (pRCAB5-8) contained the above mutations in conjunction with Trp258 Ser. Trp-258 is a residue crucial to the hydrophobic core of the domain 2 globular structure (13) .

Expression

Consistent with previous studies(15, 20) , recombinant B-chain expression constructs were designed to encode subunit precursors consisting of a fusion of the mature B-chain coding region with the preproricin leader sequence, required for co-translational targeting to the lumen of the endoplasmic reticulum. Encoded in this context, oocyte-expressed RTB was soluble, core-glycosylated, biologically active, and capable of reassociating with recombinant ricin A-chain.

All of the transcripts generated in this study yielded a single product of the expected size when translated in a wheat germ cell-free translation system (data not shown). When expressed in oocytes, we obtained no evidence for oligomerization of the recombinant B-chains; a single band corresponding in size to monomer was exclusively seen on nondenaturing gels (data not shown).

The oocyte-expressed B-chains were immunoprecipitated with rabbit anti-RTB sera, which cross-react with both types of Ricinus lectin B-chains, and subsequently treated with endo H (Fig. 1). The corresponding increase in gel mobility of the endo H-treated samples in comparison with untreated counterparts signifies the core-glycosylated status of the recombinant B-chains. The apparent molecular weights of the agglutinin derivatives (e.g.Fig. 1, lanes9 and 10, denoted -) are slightly greater than their ricin counterparts (e.g.Fig. 1, lanes7 and 8, denoted -). The position of the glycosylation sites within the primary structures of RCA and ricin are conserved, although the amino acid sequence of the agglutinin B-chain encodes three potential glycosylation sites, one extra to its ricin counterpart(3) . It is the presence of this additional N-linked glycan substituent on the polypeptide chain of the RCA B-chain derivatives that accounts for the apparent size difference between the two recombinant Ricinus lectin B-chains(26) . The faint higher molecular weight band, apparent in some of the immunoprecipitated samples of the recombinant B-chains, possibly represents a glycoform as the band is not detected in endo H-treated samples (Fig. 1, lanes denoted +).


Figure 1: Treatment of recombinant B-chains with endo H. In vitro generated transcripts were injected into Xenopus oocytes, which were subsequently incubated overnight in the presence of [S]methionine. Duplicate homogenate samples were immunoprecipitated using rabbit anti-RTB antisera. Lanes labeled + and - represent endo H-treated samples and untreated controls, respectively. Samples were analyzed by reducing SDS-PAGE and fluorography. Lanes 1-10, samples from homogenates of oocytes expressing the Ricinus lectin B-chain transcripts: pRCAB1 (1); pRCAB2 (2); pRCAB3 (3); pRCAB4 (4); pRCAB7 (5); pRCAB8 (6); pRTB4 (7); pRTB3 (8); pRCAB5 (9); pRCAB6 (10); lane 11, wheat germ translation product of pRCAB5.



The solubility of the wild-type and mutant B-chains was assessed by the criterion of centrifugation at 100,000 g (data not shown). The recombinant B-chains were recovered in the supernatant fraction from freshly prepared oocyte homogenates, indicating that they were produced in a soluble form. The majority of the B-chain molecules retained solubility in samples analyzed 7 days after homogenization.

Lectin Activity of the Recombinant Ricinus Lectin B-chains

Elution of RTB Variants with Galactose

In order to qualitatively assay lectin activity, the oocyte-expressed B-chains were applied to a column of immobilized lactose. Fig. 2shows the results from the binding of the recombinant wild-type RTB and its mutant derivatives to the immobilized lactose matrix and their elution with galactose. pRTB3 retains sugar-binding ability as expected for a mutant bearing an undisrupted 1alpha subdomain. It is known that simultaneous conversion of the key hydrogen bonding residues Lys-40 and Asn-46 to Met and Gly, respectively, abrogates lectin activity associated with the 1alpha subdomain(15) . Thus, pRTB2 retains lectin activity by virtue of the unaltered 2 subdomain ( (15) and data not shown). The double binding site mutant pRTB4 appears to have been retarded slightly by the column, although there is no evidence of galactose-specific elution.


Figure 2: Lectin activity of oocyte-expressed ricin B-chains. Homogenates from oocytes expressing the appropriate ricin B-chain were applied to columns of immobilized lactose. The columns were washed first with OHB and subsequently with OHB containing 50 mM galactose, added as indicated by the arrow. RTB was recovered from the collected fractions by immunoprecipitation. Samples were analyzed by reducing SDS-PAGE and fluorography. Lane 1, column flow-through; lanes 2-4, successive wash fractions; lanes 5 and 6, galactose eluate.



Elution of RCA B-chain Derivatives with Galactose

Wild-type recombinant RCA B-chain (pRCAB1) and pRCAB2 bind efficiently to the immobilized lactose matrix and are eluted with galactose (Fig. 3A).


Figure 3: Lectin activity of oocyte-expressed RCA B-chains. Homogenates from oocytes expressing RCA B-chain derivatives (A) or RCA B-chain variants with the Trp-258 Ser mutation (B) were applied to columns of immobilized lactose. Bound material was eluted with OHB containing 50 mM galactose, added as indicated by the arrow. RCA B-chain was recovered from the collected fractions by immunoprecipitation. Samples were analyzed by reducing SDS-PAGE and fluorography. A, lane 1, column flow-through; lanes 2-5, successive wash fractions; lanes 6 and 7, galactose eluate. B, lane 1, column flow-through; lanes 2-3, successive wash fractions; lanes 4 and 5, galactose eluate.



The replacement of the DVT tripeptide in the 1alpha subdomain of RCA B-chain with its 2alpha subdomain equivalent, i.e. the sequence QAN, was expected to abolish activity associated with this site by analogy with a similar mutation of RTB(15) . The simultaneous introduction of the QAN tripeptide in the 1alpha subdomain and the conversion of Trp-258 to Ser, in the region of the 2 subdomain, resulted in a protein (pRCAB6) devoid of galactose-binding ability (Fig. 3B). However, pRCAB3, the 1alpha subdomain mutant of RCA B-chain, exhibits weak binding activity, and a proportion of the molecules is displaced from the matrix by galactose (Fig. 3A). Taken together, these results suggest the presence of two independent binding sites corresponding to the 1alpha and 2 subdomains of RCA B-chain.

The amount of pRCAB3 in the column flow-through and the wash fractions is disproportionately high in comparison with the bound product. The reason for this remains unclear as there is no evidence to suggest that the introduction of the QAN tripeptide in place of the DVT sequence of RCA B-chain would promote instability, particularly as the same pattern is not obtained with pRCAB4, which possesses the same 1alpha subdomain mutation.

pRCAB8 is inactive as expected for a mutant bearing the combined mutations of the QAN tripeptide in the 1alpha subdomain and the Trp-258 Ser conversion in the 2 subdomain. Furthermore, introduction of tyrosine in the domain 2 site does not rescue activity, which suggests that the effect of the Trp-258 Ser mutation is exerted indirectly, possibly through a localized conformational change. pRCAB5 and pRCAB7 retain activity that is attributed solely to the 1alpha subdomain, confirming yet again that the two binding sites of RCA B-chain are independent and that the mutations introduced in the vicinity of the 2 subdomain are without notable effect on the activity of the 1alpha subdomain.

Specificity toward GalNAc

The widely adopted method of preparation of the Ricinus lectins involves application of the castor bean endosperm extract onto a Sepharose 6B column and sequential elution of ricin with GalNAc and subsequently RCA with galactose(21) . Fig. 4A shows the elution profile from this purification. Fractions from the Sepharose 6B column were analyzed by denaturing reducing/nonreducing SDS-PAGE and visualized by staining with Coomassie Brilliant Blue (Fig. 4B). Denaturing SDS-PAGE is unsuitable for the separation of the dimeric and tetrameric forms of the Ricinus lectins, as the RCA tetramer dissociates when boiled in SDS. Heating RCA above 70 °C for 10 min, in the absence of reducing agents but in the presence of SDS, causes the 120 kDa band to disappear (5) with concomitant generation of forms of intermediate apparent molecular weights of 60,000 and, to a lesser extent, 90,000 and 30,000. However, a small proportion of the purified RCA (Fig. 4B), boiled only for 3 min in the presence of SDS, appears resistant to dissociation and retains its apparent molecular mass of 120 kDa in agreement with earlier findings(27) . The electrophoretic analysis of the purified fractions clearly demonstrates the exclusion of the RCA species from the GalNAc eluate and the exclusive displacement of native RCA from the matrix with galactose.


Figure 4: Sequential elution of the native Ricinus lectins with GalNAc and galactose. The soluble fraction (10 ml) of the crude castor bean endosperm extract was passed down a Sepharose 6B column and 40 ml of Nonidet P-40 buffer was applied until all unbound proteins were removed. Ricin was eluted with 70 ml of 50 mM GalNAc in Nonidet P-40 buffer. The column was washed with 40 ml of Nonidet P-40 buffer before application of 50 mM galactose and elution of RCA. A, elution profile of the Ricinus lectins from Sepharose 6B. The numbers corresponding to the wash fractions are indicated by the prefix W. The point of application of GalNAc was at fraction 1 and application of galactose (Gal) was at the point corresponding to fraction 78. B, selected fractions from the two peaks of the elution profile were analyzed by denaturing/nonreducing SDS-PAGE and visualized by staining with Coomassie Brilliant Blue. LaneR, 2 µg of ricin; lanes1 and 2, respectively, represent samples of fractions 25 (ricin) and 88 (RCA) reduced with 1% (v/v) beta-mercaptoethanol. The fraction numbers and the relative migration of the Ricinus lectins through the gel are indicated.



Sequential elution of the oocyte-expressed B-chains from immobilized lactose first with GalNAc and second with galactose was performed (Fig. 5). Consistently, a proportion of the RCA B-chains was eluted in the presence of GalNAc in a manner indicative of a weak affinity interaction, whilst the majority of the molecules remained on the column and was subsequently eluted with galactose. In contrast, recombinant wild-type RTB (pRTB1) is eluted with GalNAc, and the majority of the displaced molecules appears in the first GalNAc fraction. The pRTB2 mutant, which is inactive with respect to the 1alpha subdomain, was completely displaced from the matrix with GalNAc, confirming that this sugar interacts with the 2 subdomain of RTB. The ricin mutant pRTB3 is only weakly displaced by GalNAc. In contrast, its ability to bind galactose is unaffected as this interaction is facilitated by means of the unaltered 1alpha subdomain. This is consistent with the notion that the presence of histidine at position 248 may hinder the interaction of GalNAc with the 2 subdomain.


Figure 5: Sequential elution of the recombinant Ricinus lectin B-chains with GalNAc and galactose. Homogenates obtained from oocytes expressing recombinant B-chain were applied to columns of immobilized lactose. The columns were washed with OHB before the addition of 3-5 ml of OHB containing 50 mM GalNAc. The columns were subsequently washed with 5 ml of OHB; these intermediate washes were discarded. The remaining protein was eluted with 2 ml of OHB containing 50 mM galactose. Recombinant B-chain was immunoprecipitated with rabbit anti-RTB antibodies and analyzed by reducing SDS-PAGE and fluorography. Lane 1, column flow-through; lanes 2-4 (as appropriate), sequential wash fractions; lanes GalNAc 1-5 (as appropriate), successive fractions eluted with GalNAc; lanes Gal 1-2, fractions eluted with galactose.



As the Trp-258 Ser conversion appears to inactivate the 2 subdomain of RCA B-chain and GalNAc is presumed to interact albeit weakly with this site, it was anticipated that pRCAB5 would not show the same binding pattern as the remaining RCA B-chain derivatives. This was indeed the case confirming, that the interaction of the recombinant RCA B-chains with GalNAc is mediated specifically by the 2 subdomain and demonstrating that the presence of His in this site is not incompatible with an interaction, even weak, with GalNAc.

NMR analysis of the GalNAc sample used in the elution of the Ricinus lectins from the immobilized lactose matrix showed conclusively the absence of galactose or galactose-containing contaminants (data not shown), thus confirming that the effect observed with recombinant RCA B-chains was due to a genuine interaction with GalNAc.


DISCUSSION

The bivalency of RCA with respect to galactopyranosides (16) and its inability to bind GalNAc(6) , known to interact with the 2 subdomain of ricin B-chain(13, 17) , suggest the loss of binding ability from the 2 subdomain of each RCA B-subunit. Consistently with this, the binding of 1.94 mol of 4-methylumbelliferyl-beta-D-galactopyranoside/mol of RCA tetramer has been reported(28) . In contrast, another study showed that the agglutinin was tetravalent with respect to 4-methylumbelliferyl galactose and 4-methylumbelliferyl GalNAc, although the authors did suggest that one site/B-chain moiety of the Ricinus lectins may be unstable(9) . A stoichiometry of four sugar residues/RCA tetramer clearly requires two functional binding sites/B-subunit.

In the present study we have used site-directed mutagenesis to alter key residues in the sugar-binding sites of the Ricinus lectin B-chains in an attempt to resolve the controversy of sugar binding to RCA B-chain. Our data indicate that both the 1alpha and 2 subdomains of RCA B-chain must be simultaneously modified in order to abolish lectin activity and demonstrate the presence of two independent, functional binding sites/molecule of RCA B-chain. Interestingly, the conversion of His-248 to Tyr in RCA B-chain confers properties similar to those of wild-type RTB, while the conversion of Tyr-248 to His in RTB gives it properties similar to those of RCA B-chain.

Significantly, activity associated with the 2 subdomain of RCA B-chain is abolished by the conversion of Trp-258 to Ser. This is suggested by comparison of pRCAB6, which is devoid of lectin activity, and pRCAB3, in which both Trp-258 and lectin activity are intact. The x-ray data for ricin B-chain indicate that Trp-258 is not in the immediate vicinity of the combining site but is in fact located behind the binding pocket, lying deeply buried in the molecular interior at the center of a hydrophobic region. Examination of the subdomain alignment of RTB (13) reveals that the sequence Gln-X-Trp, where X is any residue, is conserved in five of the RTB subdomains, and the Trp itself is invariant. This conclusion also applies to a sequence alignment of the subdomains of RCA B-chain based on amino acid identity (not shown). Trp-258 is one of these six highly conserved residues believed to operate at two levels within the tertiary structure of the B-chain. First, within each subdomain these Trp residues stabilize the carboxyl-terminal loop by interacting with conserved downstream Ile residues. Second, these Trp-Ile van der Waals' interactions establish contacts between the subdomains of the same domain, arranging them around a pseudo 3-fold axis to create a compact hydrophobic core. Serine is a less bulky residue than tryptophan and is also considerably more polar by virtue of its ability to participate in hydrogen bonds. Thus, its presence within the highly conserved hydrophobic core is likely to incur destabilization, which is translated at the protein level as abolition of lectin activity associated with the 2 subdomain. In the absence of any crystallographic data for this series of mutants, or indeed native RCA B-chain itself, nothing further can be said as to the nature of the presumably destabilizing effect of this mutation on the conformation of the 2 subdomain. It is clear, however, that the effect of the Trp-258 Ser mutation is confined to the 2 subdomain as pRCAB5 binds efficiently to the immobilized lactose matrix and is eluted with galactose, presumably due to binding via the unaltered 1alpha subdomain (Fig. 3B).

The B-chain of ricin E (a natural isoform of ricin D) is composed of the N-terminal half of ricin D and the C-terminal half of RCA(29) . Equilibrium dialysis and spectroscopy revealed the presence of two independent galactose-binding sites in ricin E with differing affinities. These were accordingly designated as a high and a low affinity site in correspondence with the sites of ricin D. The low affinity sites of the two ricin isoforms, both of which possess a crucial tryptophan residue, appear to be of equivalent binding strength (25) . The proposed high affinity site of ricin E, which lies in the RCA-like part of the molecule, binds sugars with only half the affinity of its ricin D counterpart(25) . Thus by extrapolation it could be predicted that the affinity of the 2 subdomain of RCA B-chain is less than that of the equivalent site in ricin D. Yet, the 2 subdomain of ricin E exhibits dual specificity with respect to galactose and GalNAc despite the fact that the role of the sugar-binding platform is performed by the partially charged imidazole ring of histidine. Furthermore, the ability of histidine to act as a sugar platform, by participation in a hydrophobic stacking interaction with the galactopyranose moiety of the ligand is undisputed, as evidenced from its role in precisely this capacity in wheat germ agglutinin(30) . Thus the presence of histidine in place of tyrosine at position 248 of the Ricinus lectin B-chains does not preclude lectin activity. However, the evidence suggests that the charge of the histidine side chain may drastically reduce the binding ability exhibited by the 2 subdomain.

Studies on the binding of derivatives of methyl beta-lactoside to RCA suggest the existence of a steric hindrance with regard to accommodating the methyl group at the C-2` position of the galactose moiety, not observed with ricin. It was predicted that the binding of an N-acetyl group at this position would encounter an even greater steric hindrance(31) . Thus the different specificities of ricin and RCA with respect to GalNAc binding seem to result from a different topology of the binding sites at this position. It should be emphasized that these proposals (31) do not exclude the possibility of an interaction of GalNAc with RCA but merely predict it to be of weak affinity. Within RTB itself, the binding of GalNAc is restricted to the 2 subdomain as a consequence of the less accommodating topological features of the 1alpha subdomain(13, 17) . Rutenber and Robertus (13) commented that the bound galactose in the 2 subdomain is rotated by approximately 15° relative to the orientation it acquires in the 1alpha site. The authors proposed that in the 2 site the N-acetyl substituent on C-2 of the galactose moiety would extend freely into the solvent, while in the 1alpha site, the extra group would clash irreconcilably with the side chain of Asp-44.

It seems likely that, as with ricin E(25, 32) , the prevalence of histidine in the 2 subdomain of RTB severely disrupts but does not abolish binding, thus reducing the observed affinity to the extent that only a retardation during the column washes is seen with the double binding site mutant pRTB4. Altogether these studies suggest that despite its partial charge, histidine is able to fulfill the role of sugar platform albeit with reduced efficiency compared with tyrosine. This conclusion is corroborated by the role of histidine as a sugar platform in the carbohydrate-binding mechanisms of ricin E (25) and wheat germ agglutinin(30) .

Furthermore, it would appear that two of the four potential combining sites are incapable of a productive interaction in the context of the RCA tetramer. Two mechanisms of inactivation are envisaged. First, the sites of RCA B-chain may be buried at the interior of the tetrameric structure of RCA in a manner preventing their accessibility to carbohydrates. Conversely, the topology of the combining sites may be altered as a result of conformational changes incurred by tetramer formation. Which of the two binding sites is rendered nonfunctional in the context of the tetramer is an issue awaiting investigation. However, as the pattern of weak GalNAc binding obtained with recombinant RCA B-chains is not observed with intact tetramers, inactivation of the 2 subdomain is predicted. Although the results suggesting a stoichiometry of four sugar residues/molecule of RCA tetramer (9) apparently contradict this theory, the implication from that data that one site per B-chain moiety may be unstable is consistent.

Reconstitution of tetramers containing mutant B-chains would provide a means to test the hypothesis put forward here. Thus the 1alpha subdomain mutant of RCA B-chain (pRCAB3) should yield a tetramer devoid of lectin activity if the proposal that the 2 subdomain is inactive in the tetramer holds true. Furthermore, taking into account the inability of the tetrameric agglutinin to bind GalNAc(21) , it would be of interest to investigate the specificity of the recombinant RCA B-chains within the context of a reconstituted tetramer.


FOOTNOTES

*
This work was supported by the United Kingdom Science and Engineering Research Council Grant GR/G00877 and a research studentship (to N. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 44-203523598; Fax: 44-203523701.

(^1)
The abbreviations used are: RCA, R. communis agglutinin; RTB, ricin toxin B-chain; endo H, endo-beta-N-acetylglucosaminidase H; PAGE, polyacrylamide gel electrophoresis.


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