As part of a strategy to determine the precise role of pea (Pisum sativum) lectin, Psl, in nodulation of pea by Rhizobium leguminosarum, mutations were introduced into the genetic determinant for pea lectin by site-directed mutagenesis using PCR. Introduction of a specific mutation, N125D, into a central area of the sugar-binding site resulted in complete loss of binding of Psl to dextran as well as of mannose/glucose-sensitive haemagglutination activity. As a control, substitution of an adjacent residue, A126V, did not have any detectable influence on sugar-binding activity. Both mutants appeared to represent normal Psl dimers with a molecular mass of about 55 kDa, in which binding of Ca2+ and Mn2+ ions was not affected. These results demonstrate that the NHD2 group of Asn125 is essential in sugar binding by Psl. To our knowledge, Psl N125D is the first mutant legume lectin which is unable to bind sugar residues. This mutant could be useful in the identification of the potential role of the lectin in the recognition of homologous symbionts.

Legume lectin stimulates infection of roots in the symbiosis between leguminous plants and bacteria of the genus Rhizobium. Introduction of the Pisum sativum lectin gene (psl) into white clover hairy roots enables heterologous infection and nodulation by the pea symbiont R. leguminosarum biovar viciae (R.l. viciae). Legume lectins contain a specific sugar-binding site. Here, we show that inoculation of white clover hairy roots co-transformed with a psl mutant encoding a non-sugar-binding lectin (PSL N125D) with R.l. viciae yielded only background pseudo-nodule formation, in contrast to the situation after transformation with wild type psl or with a psl mutant encoding sugar-binding PSL (PSL A126V). For every construct tested, nodulation by the homologous symbiont R.l. trifolii was normal. These results strongly suggest that (1) sugar-binding activity of PSL is necessary for infection of white clover hairy roots by R.l. viciae, and (2) the rhizobial ligand of host lectin is a sugar residue rather than a lipid.

Comparison of x-ray crystal structures of several legume lectins, co-crystallized with sugar molecules, showed a strong conservation of amino acid residues directly involved in ligand binding. For pea (Pisum sativum) lectin (PSL), these conserved amino acids can be classified into three groups: (I) D81 and N125, present in all legume lectins studied so far; (II) G99 and G216, conserved in almost all legume lectins; and (III) A217 and E218, which are only found in Vicieae lectins and are possibly determinants of sugar-binding specificity. Each of these amino acids in PSL was changed by site-directed mutagenesis, resulting in PSL molecules with single substitutions: for group I D81A, D81N, N125A; for group II G99R, G216L; and for group III A217L, E218Q, respectively. PSL double mutant Y124R; A126S was included as a control. The modified PSL molecules appeared not to be affected in their ability to form dimeric proteins, whereas the sugar-binding activity of each of the PSL mutants, with the exception of the control mutant (as shown by haemagglutination assays), was completely eliminated. These results confirm the model of the sugar-binding site of Vicieae lectins as deduced from X-ray analysis.

Legume lectins are considered to be antinutritional factors (ANF) in the animal feeding industry. Inactivation of ANF is an important element in processing of food. In our study on the stability of Pisum sativum L. lectin (PSL), a conserved hydrophobic amino acid (Val103) in a surface loop was replaced with alanine. The mutant lectin, PSL V103A, showed a decrease in unfolding temperature (Tm) by some 10 degrees C in comparison with wild-type (wt) PSL, and the denaturation energy (delta H) is only about 55% of that of wt PSL. Replacement of an adjacent amino acid (Phe104) with alanine did not result in a significant difference in stability in comparison with wt PSL. Both mutations did not change the sugar-binding properties of the lectin, as compared with wt PSL and with PSL from pea seeds, at ambient temperatures. The double mutant, PSL V103A/F104A, was produced in Escherichia coli, but could not be isolated in an active (i.e. sugar-binding) form. Interestingly, the mutation in PSL V103A reversibly affected sugar-binding at 37 degrees C, as judged from haemagglutination assays. These results open the possibility of production of lectins that are active in planta at ambient temperatures, but are inactive and possibly non-toxic at 37 degrees C in the intestines of mammals.