(Received for publication, February 24, 1995; and in revised form, May 15, 1995)
From the
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.
The endosperm of castor bean seeds contains two toxic lectins:
ricin and the Ricinus communis agglutinin (RCA). ()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
-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
,
, and
). Only the 1
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 1
/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 1 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.
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 1 subdomain have been converted
to Met and Gly, respectively, thus rendering the 1
/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
1
subdomain. The corresponding double binding site mutant (encoded
by pRTB4) in which the 1
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 1
subdomain,
was selectively eliminated through the conversion of the conserved DVT
tripeptide of the 1
subdomain to the amino acid sequence found at
the equivalent position in the primary structure of the 2
subdomain, namely the tripeptide QAN. The clones encoding the wild-type
(pRCAB1) and the 1
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) .
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.
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.
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 1 subdomain of RCA B-chain with its 2
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 1
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 1
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 1
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
1 subdomain mutation.
pRCAB8 is inactive as expected for a
mutant bearing the combined mutations of the QAN tripeptide in the
1 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 1
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 1
subdomain.
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) -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 1 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 1
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.
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-
-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 1 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 1
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 -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
1
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 1
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 1
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 1 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.