Molecular basis of recognition by Gal/GalNAc specific legume lectins: influence of Glu 129 on the specificity of peanut agglutinin (PNA) towards C2-substituents of galactose.
The ability to discriminate between galactose and N- acetylgalactosamine, observed in some lectins, is crucial for their biological activity as well as their usefulness as tools in biology and medicine. However, the molecular basis of differential binding of lectins to these two sugars is poorly understood. Peanut agglutinin (PNA) is one of the few galactose-specific legume lectins which does not bind N- acetylgalactosamine at all and is, therefore, ideal for the study of the basis of specificity towards C-2 substituted derivatives of galactopyranosides. Examination of the three-dimensional structure of PNA in complex with lactose revealed the presence of both a longer loop and bulkier residues in the region surrounding the C-2 hydroxyl of the galactopyranoside ring, which can sterically prevent the accommodation of a bulky substituent in this position. One such residue, is a glutamic acid at position 129 which protrudes into the binding site and perhaps directly obstructs any substitution at the C-2 position. Two mutants in bacterially expressed PNA were therefore constructed. These were E129D and E129A, in which Glu129 was replaced by Asp and Ala, respectively. The specificity of the mutants for galactose, galactosamine, and N- acetylgalactosamine was examined through observing the inhibition of hemagglutination and binding of the lectin to immobilized asialofetuin. The results showed that the affinity of E129A and E129D for C-2-substituted derivatives of the galactose varies. The mutant E129D showed significant binding towards N- acetylgalactosamine, suggesting that the residue Glu 129 is crucial in imparting exclusive galactose-specificity upon PNA. This study not only attempts to provide an explanation for the inability of PNA to accommodate C-2-substituted derivatives at its primary subsite, but also seeks to present a basis for engineering lectins with altered specificities.
Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India.
V
V
Sharma
V R
VR
Srinivas
P
P
Adhikari
M
M
Vijayan
A
A
Surolia
D000116:Acetylgalactosamine / Q000378:metabolism*; D000409:Alanine / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D007887:Fabaceae / Q000378:metabolism*; D005690:Galactose / Q000378:metabolism*; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019887:Peanut Agglutinin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D037121:Plant Lectins; D010946:Plants, Medicinal
D017931:DNA Primers; D019887:Peanut Agglutinin; D037121:Plant Lectins; D001224:Aspartic Acid; D018698:Glutamic Acid; D000116:Acetylgalactosamine; D000409:Alanine; D005690:Galactose