Destabilization of pea lectin by substitution of a single amino acid in a surface loop.
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.
RUL-TNO Centre for Phytotechnology, Leiden University, Netherlands.
F J
FJ
Hoedemaeker
R R
RR
van Eijsden
C L
CL
Díaz
B S
BS
de Pater
J W
JW
Kijne
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D002152:Calorimetry, Differential Scanning; D003001:Cloning, Molecular; D004926:Escherichia coli; D007887:Fabaceae / Q000737:chemistry*; D006386:Hemagglutination Tests; D015151:Immunoblotting; D037102:Lectins / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D037121:Plant Lectins; D010946:Plants, Medicinal; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D000596:Amino Acids; D037102:Lectins; D037121:Plant Lectins