Mutation of Asn128 to Asp of Phaseolus vulgaris leucoagglutinin (PHA-L) eliminates carbohydrate-binding and biological activity.
Phytohaemagglutinin (PHA) is the major lectin present in the seeds of the common bean, Phaseolus vulgaris, and PHA-L is the leucocyte-agglutinating form of this lectin. This tetrameric glycoprotein accumulates in the vacuoles of storage parenchyma cells. Based on amino acid sequence comparisons of legume lectins and the three-dimensional structure of lectin-carbohydrate complexes, Asn128 can be identified as a likely candidate for site-directed mutagenesis to create a mutant PHA-L that does not bind carbohydrate. PHA-L N128-->D was obtained and the mutant as well as the wild-type gene expressed in tobacco cells. Lectin (carbohydrate-binding) activity was completely abolished in the mutant protein produced in the tobacco cells. The leucoagglutinating and mitogenic activities characteristic of PHA-L were also eliminated by this mutation, confirming that carbohydrate binding is essential for the biological activities of this protein. The mutant polypeptides formed normal tetramers and these were transported to the vacuoles of the plant cells where they accumulated. This finding indicates that the mutations did not introduce a gross disturbance of the structure of PHA.
Mirkov
T E
TE
Department of Biology, University of California, San Diego, La Jolla 92093-0116.
Chrispeels
M J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000235:genetics / Q000378:metabolism*; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D050260:Carbohydrate Metabolism; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004247:DNA; D007887:Fabaceae / Q000378:metabolism*; D006386:Hemagglutination Tests; D006801:Humans; D007962:Leukocytes / Q000166:cytology / Q000187:drug effects; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010835:Phytohemagglutinins / Q000235:genetics / Q000378:metabolism*; D037121:Plant Lectins; D010946:Plants, Medicinal; D010947:Plants, Toxic; D014026:Nicotiana / Q000235:genetics
D010835:Phytohemagglutinins; D037121:Plant Lectins; C100156:leukoagglutinins, plants; D001224:Aspartic Acid; D001216:Asparagine; D004247:DNA