Amino acid sequence of the winged bean (Psophocarpus tetragonolobus) basic lectin. Adenine binding and identification of the active-site tryptophan residue.

K D Puri,A Surolia

doi:10.1016/s0021-9258(18)47369-3

K D Puri, A Surolia

Open Access

https://doi.org/10.1016/s0021-9258(18)47369-3

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Abstract

The complete amino acid sequence of winged bean basic agglutinin (WBA I) was obtained by a combination of manual and gas-phase sequencing methods. Peptide fragments for sequence analyses were obtained by enzymatic cleavages using trypsin and Staphylococcus aureus V8 endoproteinase and by chemical cleavages using iodosobenzoic acid, hydroxylamine, and formic acid. COOH-terminal sequence analysis of WBA I and other peptides was performed using carboxypeptidase Y. The primary structure of WBA I was homologous to those of other legume lectins and more so to Erythrina corallodendron. Interestingly, the sequence shows remarkable identities in the regions involved in the association of the two monomers of E. corallodendron lectin. Other conserved regions are the double metal-binding site and residues contributing to the formation of the hydrophobic cavity and the carbohydrate-binding site. Chemical modification studies both in the presence and absence of N-acetylgalactosamine together with sequence analyses of tryptophan-containing tryptic peptides demonstrate that tryptophan 133 is involved in the binding of carbohydrate ligands by the lectin. The location of tryptophan 133 at the active center of WBA I for the first time subserves to explain a role for one of the most conserved residues in legume lectins.

Highlights

Kamal Deep PuriS and Avadhesha SuroliaO From the Molecular Biophysics Unit, Indian Instituteof Science, Bangalore 560 012, India
The location of tryp- In addition to the identification of an adenine-bindingsite tophan 133 at the active center of WBAI for the first time formed at its monomer-monomer interface, we report on subsewes to explaina role for oonfethe most conserved the indispensability of tryptophan 133 in the sugar binding residues in legume lectins
Isoelectric focusing ofWBA I gave three bands in the ratio 13.3:32.0:54.7,with theisolectins corresponding to PI values of 9.94, 10.01, and 10.11 designated as B1, B2 and B3, respectively (Fig. 2)

Summary

EXPERIMENTAL PROCEDURES

Multivalent cell-agglutinating proteins by virtue of their exquisite sugar specificities, have been found useful in widespread applications for monitoring the expression of cellsurface carbohydratesas well as for the purification and char-. S. aureus V8 Endoproteinase Digestion-S. aureus V8 endoprotein- concentration of NBS, 1.83 tryptophan residues/molecule/subunitare ase was added to denatured WBA I in 0.1% SDS and 50 mM sodium oxidized without the oxidation of tyrosine or other amino acids. The peptides were purified on a C,-C,, digest was made to 0.1% trifluoroaceticacid and 5 M GdmCl and purified Pep-RPC column,and elution was monitored both at 254 and 280 nm. To WBA I (2-5 nmol) or peptides (1-2 WBA I-Association constants for the binding of GalNDns to unmodinmol) in 49 pl of 50 m sodium acetate buffer (pH 5.5), carboxypepti- fied and modified WBA I samples were determined as described by dase Y was added to an enzyme/peptideratio of 1:250,and theresulting Khan et al (7). The peptides generated were desalted on a Sephadex G-10 column (1.0 x 30.0 cm) and purified on a C,-C,, Pep-RPC column

RESULTS

V 1 KY DASS

DISCUSSION