MUTATION OF ASN(128) TO, ASP OF PHASEOLUS-VULGARIS-LEUKOAGGLUTININ (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-agglut inating form of this lectin. This tetrameric glycoprotein accumulates in the vacuoles of storage parenchyma cells. Based on amino acid seque nce comparisons of legume lectins and the three-dimensional structure of lectin-carbohydrate complexes, Asn(128) can be identified as a like ly candidate for site-directed mutagenesis to create a mutant PHA-L th at does not bind carbohydrate. PHA-L N-128-->D obtained and the mutant as well as the wild-type gene expressed in tobacco cells. Lectin (car bohydrate-binding) activity was completely abolished in the mutant pro tein produced in the tobacco cells. The leucoagglutinating and mitogen ic activities characteristic of PHA-L were also eliminated by this mut ation, confirming that carbohydrate binding is essential for the biolo gical activities of this protein. The mutant polypeptides formed norma l tetramers and these were transported to the vacuoles of the plant ce lls where they accumulated. This finding indicates that the mutations did not introduce a gross disturbance of the structure of PHA.