Combining site of WBAI is extended and encompasses all the residues of blood group A-reactive trisaccharide [GalNAcα3Galβ4Glc]. Though both of the fucose residues of A-pentasaccharide [GalNAcα(Fucα2)3Galβ(Fucα3)4Glc] do not directly interact with the combining site they thermodynamically favour the interaction of GalNAcα3Galβ4Glc part of the molecule by imposing a sterically favourable orientation of the binding epitope viz. GalNAcα3Galβ4Glc of the saccharide. Binding of sugars is driven by enthalpy and is devoid of heat capacity changes. This together with enthalpy-entropy compensation observed for these processes underscore the importance of water reorganization as being one of the principal determinant of protein-sugar interactions

Puri, Kamal Deep

Surolia, Avadhesha

English

Publications of the IAS Fellows

Pure & Appl. Chem., Vol. 66, No. 3, pp. 497-502, 1994. 
Printed in Great Britain. 
@ 1994 IUPAC 
Thermodynamics of lectin-sugar interaction: 
Binding of sugars to winged bean (Psophocarpus 
tetragonolobus) basic ag g I ut i n i n ( W BAI) 
KAMAL DEEP PURI and AVADHESHA SUROLIA 
MOLECULAR BIOPHYSICS UNIT, INDIAN INSTITUTE OF SCIENCE, 
BANGALORE-560012, INDIA 
Abstract Combining site of WBAI is extended and encompasses all the 
residues of blood group A-reactive trisaccharide [GalNAccrl3GalfMGlc]. 
Though both of the fucose residues of A-pentasaccharide 
[GalNAcol(Fucd2)3Gale(FucoL3)4Glc] do not directly interact with the 
combining site they thermodynamically favour the interaction of 
GalNAca3Galp4Glc part of the molecule by imposing a sterically 
favourable orientation of the binding epitope & GalNAcd3Galb4Glc of 
the saccharide. Binding of sugars is driven by enthalpy and is devoid 
of heat capacity changes. This together with enthalpy-entropy 
compensation observed for these processes underscore the importance of 
water reorganization as being one of the principal determinant of 
protein-sugar interactions. 
Recognition is a key event in many biological phenomena. It is 
also an initiation step in numerous processes, based on cell-cell 
interactions, such as fertilization, embryogenesis, organ formation, 
immune and microbial defences, etc. (1). Carbohydrates perched at the 
surface of the cells provide cells with their individuality and 
recognition patterns that generally play a crucial role in the day to 
day life of an ordinary cell (1,2). Aberrant expression of sugars on 
the surface of the cell is hallmark of diseases such as autoimmune 
disorders and neoplastic transformation (1). The discovery of lectins, 
a class of multivalent proteins that bind sugars selectively much as 
antibodies bind to antigens was a great step in the elucidation of the 
mechanism of cellular recognition in general and the role of sugars 
therein in particular. Because of their sugar selectivity lectins 
display blood group and tumor cell specific agglutination, mitogenicity 
etc. (2). Consequently they are used routinely in clinics and blood 
banks as well as in the isolation and purification of glycoproteins 
( 3 ) .  As all the activities of lectins are manifestation of their sugar 
specificities and as they provide prototype model systems for the 
interactions that occur at cell surfaces as well as protein-sugar 
interactions, the combining sites of lectins have become objects of 
intense scientific scrutiny in our laboratory for the last decade. 
Thermodynamic investigations reported here pertain to the general 
features of protein-carbohydrate recognition and more specific ones of 
Winged bean (Psophocarpus tetrasonolobus) basic agglutinin (WBAI) which 
has been shown by us earlier to interact exquisitely with blood group 
A-reactive sugars (4,5). These studies underscore the important part 
played by sugars not involved directly in binding in favourably 
orienting the interacting regions of sugars and the role of solvent 
(water) re-organization in these interactions. 
497 
498 K. D. PURl AND A. SUROLIA 
MATERIALS AND METHODS 
Materials : Galactose (Gal), N-acetylgalactosamine (GalNAc), GaltX6Gal 
and lactose were from Sigma, USA. Gala3Ga1, Gala3GaldMe, Gale3GaleMer 
GaldGal, GalcdGalPEt, GalNAcB3GaldMe were products of Carbohydrates 
International, Sweden. Blood group reactive sugars were purchased from 
Biocarb Chemicals, Sweden. GalNAcd3Gal (A-disaccharide) was prepared 
from GalNAcC(Fucd2)3Gal (A-trisaccharide) as described in Mahanta et 
al. (6). N-Dansyl galactosamine (GalNDns) and WBAI were prepared 
according to Khan et al. (4). 
Thermodynamics of ligand binding to WBAI : The association constants 
K ) for various sugars were determined by substitution titration using 
daf!NDns as the indicator ligand as described in Khan et al. (4) and 
Swamy et al. (91.The changes in enthalpies for the binding of sugars 
were evaluated from the temperature dependence of their Ka values using 
van’t Hoff plots. Changes in entropies were then obtained by 
substituting OG and OH in the equation; ffi = AH - T O S  
Molecular modellinq : Molecular models for sugars were obtained using 
the Hard Sphere Exoanomeric Affect algorithm (7). 
Determination of concentrations : Concentrations of WBAI, GalNDns and 
other sugars were detefmined according to Lowry’s method (81, molar 
absorptivity of 4800 cm at 330 nm (4) and weights, respectively. 
RESULTS AND DISCUSSION 
In a previous study we had shown that WBAI recognizes sugars in D- 
galacto configuration and prefers strongly GalalMe and GalNAcaMe over 
their P-linked counterparts (4). Mellibiose (GalPC6Glc) bound better 
than lactose (Galp4Glc). Immunochemical analyses of its interaction 
with blood group substances revealed that its specificity is directed 
towards blood group A and B antigens (5). These investigations 
reporting the binding of a variety of galactose and N- 
acetylgalactosamine containing oligosaccharides not only confirm these 
findings but also provide considerable informations about the ligand 
specificity of WBAI, topography of its combining site and the mechanism 
of its interaction with sugars. 
Data in Table 1 show that thea-linked disaccharides of Gal and 
GalNAc are considerably better ligands than their fi -linked 
counterparts, GaU3Gal&Me, for example, is 95 times more potent as 
compared to Galp3GalpMe. Since the primary specificity of WBAI is 
TABLE 1. Values of association constant Ka agd - A G  for the 
binding of sugars to WBAI at 20 c 
- AG Relative affinity 
kJmol-’ (galactose = 1) 
Ligands l0-~-3 Ka 
(M 1 
Gal 1.05 
L-Fuc 0.18 
GalNAcd3Gal 10.71 
GalNAcp3GaldMe 0.22 
Galor3Gal 4.52 
Ga la3GalaMe 7.54 
Galf33GalPMe 0.08 
L-Fuca2Gal 0.66 
GalollGalPEt 1.93 
Ga loc4Ga 1 1.50 
GalP4Glc 0.04 
Gald6Glc 2.19 
16.94 
12.65 
22.60 
13.13 
20.50 
21.75 
10.67 
15.81 
18.43 
17.82 
8.99 
18.74 
1.00 
0.17 
10.20 
0.21 
4.30 
7.18 
0.076 
0.63 
1.84 
1.43 
0.038 
2.16 
Thermodynamics of lectin-sugar interaction 499 
GalNAca3Gal 
[A-disaccharide (A-Di)] 
GalNAcoSGalp4Glc 
[A-trisaccharide (A-Trill 
GalNAc &( L-Fuca2 1 3Gal 
[Fucosylated A-trisaccharide (A-TriFl] 
GalNAc&( L-Fucd2 1 3Galp4Glc 
[A-tetrasaccharide (A-Tetra)] 
GalNAcol(L-Fucac2)3Gal~(L-Fucd3)4Glc 
[A-pentasaccharide (A-Pentall 
GalNAc~(L-Futx%2)3Gal~3GlcNAc~3Gal~4Glc 
[A-hexasaccharide (A-Hexall 
GalNAca(L-E’uw2)3Gal~~L-Fu~4)3GlcNAc~3Gal~4Glc 
L-Fuca2Gal~(L-Fuc&)4Glc 
[A-heptasaccharide (A-Heptall 
[Difucosyllactose (DiFucLlI 
Fig.1. Structures of blood group A-reactive saccharides. 
directed towards galactose, it must occupy the primary subsite and as 
L-FucQ2Gal binds reasonably well this subsite must be complementary to 
the subterminal galactose residue. As L-fucose by itself is inactive 
WBAI must be able to bind the reducing end galactose residue in L- 
Fua2Gal. These data also show that fucosylation of galactose residue 
in 051-2 linkage neither impairs the binding of the galactosyl residue 
appreciably nor enhances its affinity. It may be noted that amongst 
disaccharides GalNAcL3Gal is the best ligand followed by Gala3Gal 
which, respectively, are the blood group A and B reactive 
disaccharides. This indicates that the acetamido group at C-2 position 
of qubterminal galactose structure makes a favgyrable contribution to 
the binding affinity which amounts to 2.5 kJmol . Gala4Gal is 3 times 
poorer a ligand as compared to GaU3Gal. This is probably due to the 
close vicinity of the non-reducing end galactose to the reducing one 
which shields a substantial portion of the groups of the latter 
diminishing the affinity of Gald4Gal relative to Gal(Z3Ga1, as shown in 
(61, excepting that in contrast to Jacalin, WBAI binds to the reducing 
Gal. 
TABLE 2. Association constants (at 2OoC) and thermodynamic parameters 
for the binding of WBAI to blood group A antigenic sugars. 
~~~~~~ ~ 
Ligands 10-3XKa Relative - AG-1 - AH -AS1 -1 
affinity kJmol k Jmol -' Jmol K 
(M’ll (GalNAc = 1) 
GalNAc 
A-Di 
A-TriF 
A-Tri 
A-Tetra 
A- Penta 
A-Hexa 
A-Hepta 
DiFucL 
5.26 1.00 
10.71 2.04 
12.41 2.36 
94.14 17.90 
89.50 17.02 
98.50 18.73 
124.24 23.62 
5.36 1.02 
No Binding 
20.87 27.19 
22.60 34.54 
22.96 34.26 
27.90 44.00 
27.78 31.24 
28.01 29.84 
28.57 24.59 
20.92 19.47 
21.54 
40.73 
38.58 
54.95 
11.79 
6.20 
-13.59 
-4.85 
500 K. D. PURl AND A. SUROLIA 
Table 2 lists the association constants and thermodynamic 
parameters of blood group A reactive saccharides together with their 
relative affinities with GalNAc as the reference (Refer Fig. 1 for 
structures). Incorporation of a GalNAc residue inal-3 linkage to Gal 
as in GalNAcd3Ga1, increases the affinity 2.04 times. Addition of a Glc 
residue in pl-4 linkage to Gal of this disaccharide increases the 
binding propensity of the resultant trisaccharide, GalNAcd3GalP4Glc by 
eight fold. A-trisaccharide is thus 17.90 and 8.8 times more potent a 
ligand over GalNAc and A-Di (Fig.11, respectively. However the presence 
of a Fuc in tZ1-2 linkage to Gal residue of A-Di as in 
GalNAcd(Fuca2)3Gal increases the affinity only by 1.16 over the 
corresponding A-Di. Increase in the enthalpies for the binding of A-Tri 
over that observed for A-Di suggests that the lectin has an extended 
combining site complementary to all the constituent monosaccharides of 
A-Tri. 
GalNAc ? 
’ Glc 
Gal& d 3 G a l  P4Glc 
GulNAc ~ ( L - F u c d 2 ) 3 G a l  p(L-Fucm3)4Glc 
Fig.2. Pluto plots of the preferred orientation of sugar residues in 
non-fucosylated A-Tri [GalNAcQ;3Gal@4Glcl and the difucosylated A-Penta 
[GalNAcd(L-E’ucd)3Gal~(L-Fuc&)4Glc]. The carbon atoms of the terminal 
GalNAc are shown by filled circles (01, subterminal Gal and Glc by half 
filled circles ( 0 )  and those of fucoses by circles with a bar (el .  The 
combining site of WBAI is complementary to the linear non-fucosylated 
trisaccharide. Terminal GalNAc is indispensable and it occupies the 
deep groove of the combining site. However, subterminal Gal and Glc 
resides are in the shallow region of the binding site. Both fucoses of 
the A-Penta project away from the binding site. Note that upon 
fucosylation the structure of the trisaccharide shown left becomes more 
favourable for the interaction to take place as shown in the right side 
of the figure. 0-0, oxygen atoms; 0-0, nitrogen atoms. Hydrogen atoms 
have been omitted. 
Non-fucosylated A-Tri EGalNAca3Gal~4Glc1, monofucosylated A-Tetra 
[GalNAca(Fuca2)3Gal~4Glcl, and difucosylated A-Penta CGalNAcd(Fucd2) 
3Gal~(Fucd3)4Glc] have very similar free-energy values, which would 
mean that both of the fucose residues of this pentasaccharides do not 
make any direct contribution to the binding energy of the ligands to 
WBAI. This is also evident from the fact that addition of fucose 
residues does not change enthalpy. Since difucosyllactose, which lacks 
the terminal GalNAc of the A-Penta is inactive, the terminal al-3 
linked GalNAc of blood group A reactive pentasaccharide must make a 
major contribution to the binding energy. Hence the terminal ul-3 
linked GalNAc must be buried in a deep cleft in the combining site of 
the lectin. The subterminal Gal and Glc residues of A-Tri 
Thermodynamics of lectin-sugar inferaction 501 
(GalNAca3Gal@4Glc) make marginal contribution to the binding energies. 
Interestingly enough, despite the non-interaction of the fucosyl 
residues of A-Penta with the lectin they entropically favour the 
binding of oligosaccharide by suitably orienting the binding epitope 
(GalNAca!3Gal~4Glc) of the saccharide. 
A-Hexa [GalNAcd(Fucd2)3Gal~3GlcNAc~3Gal~4Glc], which has p1-3 
linked GlcNAc~3Gal~4Glc to the Gal is about 1.4 times better ligand 
than A-Tetra, which would imply that the replacement of p4Glc of A- 
Penta with p3GlcNAc favours the binding of the A-Hexa. Alternatively 
additional residues make some favourable contacts with the protein. 
Addition of a fucose residue in 41-4 linkage at B3GlcNAcP of A-Hexa 
giving A-Hepta [ G a l N A c d ( L - F u c ~ 2 ) 3 G a l ~ ~ L - F u c d 4 ) 3 G l c N A c  3Galp4Glcl 
reduces the affinity of the latter by 23.18 times. Thus the presence of 
a fucose residue in oc1-4 linkage highly destabilizes the protein-ligand 
complex, which may be due to steric reasons. Due to this steric 
hinderance A-Hepta presumably interacts with its terminal GalNAc alone 
which is also attested to by its affinity which is close to that of 
GalNAc itself. Fucose intX1-4 linkage in A-Hepta may also impart an 
unfavourable orientation to the binding epitope in contrast to the a1-2 
and al-3 linked fucose residues of A-pentasaccharide allowing the 
interaction of terminal GalNAc alone. 
0 A - D i  0 A - P e n t a  
n 4 5 t  0 A - T r i  A - H e x a  
A A - T r i  F X A - H e p t a  
A A - T e t r a  
A 
X 
- -  
-5 0 5 10 15 20 
-T AS" ( k J  mole') 
Fig.3. A plot of - AHo versus -T ASo at 2OoC for blood 
group A reactive sugars. The straight line is the 
least squares fit of the data to a straight line. 
An examination of the thermodynamic parameters for blood group A- 
reactive sugars also reveals an enthalpy-entropy compensation for the 
binding process, indicating that these interactions which are primarily 
enthalpically ariven are accompanied by reorganization of water around 
both the lectin and carbohydrate. Such enthalpy-entropy compensation 
have also been observed by us in many other lectin-sugar interactions 
(11). Precedences for water being the predominant source determining 
the thermodynamics of protein-ligand interactions are also speculated 
in other systems (12-14). 
Failure to observe appreciable conformational rearrangement(s1 for 
either protein or sugar as well as the complex (4,9,ll) together with 
the linearity of van't Hoff plots for each of the sugar examined 
indicates that these interactions are accompanied by little heat 
capacity changes which would give credence to our suggestion that 
reorganization of water is the major driving force for the lectin- 
carbohydrate recognition. 
502 K. D. PURl AND A. SUROLIA 
Acknowledgements 
Science 
and Technology, Government of India (to A.S.). K.D.P. is a Senior 
Research Fellow of the Council of Scientific and Industrial Research, 
India. 
This work was supported by a grant from the Department of 
REFERENCES 
1. N. Sharon and H. Lis, Science 246, 227-234 (1989). 
2. N. Sharon and H. Lis, Lectins Chapman and Hall, New York (1989). 
3. I.E. Liener, N. Sharon-J. Goldstein, In Lectins : Properties, 
lications & Bioloqy and Maicine, Aczdemic Press, 
4. M.I. Khan, M.V.K. Sastry and A. Surolia, J. Biol. Chem. 261, 3013- 
5. T. Matsuda, E.A. Kabat and A. Surolia, & Immunol. 26, 189-195 
6. S.K. Mahanta, M.V.K. Sastry and A. Surolia, Biochem. J. 265, 831- 
7. S. Acharya, S.R. Patanjali, S.U. Sajjan, B. Gopalakrishnan and A. 
8. O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, J. Biol. 
9. M.J. Swamy, M.V.K. Sastry, M.I. Khan, and A. Surolia, Biochem, J. 
10. F.P. Schwarz, K.D. Puri and A. Surolia, J. Biol. Chem. 266, 24344- 
11. K.D. Puri, M.I. Khan, D. Gupta and A. Surolia J. Biol. Chem. 
12. U. Spohr, E. Paszkiewicz-Hnatiw, N. Morishima and R.U. Lemieux, 
13. J. Becvar and G. Palmer, J. Biol. Chem. 257, 5607-5617 (1992). 
14. H.-J. Hinz, Annu. Rev. Biophys. Bioeng. 12, 285 (1983). 
Functions and A 
New York (m6- 
3019 (1986). 
(1989). 
840 (1990). 
Surolia, J. Biol. Chem. 265, 11586-11594 (1990). 
Chem. 193, 265-275 (1951). 
234, 515-522 (1986). 
24350 (1991). 
-- 
-
' (under revision). 
Can. J. Chem. 70, 254-271 (1992). ---- 


Thermodynamics of lectin-sugar interaction: binding of sugars to winged bean (Psophocarpus tetragonolobus) basic aggIutinin (WBAI)

http://repository.ias.ac.in/56444/1/8-pub.pdf

Thermodynamics of lectin-sugar interaction: binding of sugars to winged bean (Psophocarpus tetragonolobus) basic aggIutinin (WBAI)

Abstract

Similar works

Full text

Available Versions

Publications of the IAS Fellows