Lectin nature of alpha-galactosidase I from Vicia faba

Abstract

alpha-Galactosidase I (EI) preparations from Vicia faba seeds have been reported to possess glucose/mannose-specific lectin activity. Attempts have been made to obtain more information concerning the structural component responsible for this activity. In particular, the enzyme has been examined to see whether it is contaminated with the 'classical' lectin, favin, which also occurs in V. faba seeds. EI and favin were purified and new methods tried in an endeavour to improve the purification procedures. These included the use of an EI-antibody column for EI and a Sephadex G-100 affinity column for favin. The EI preparation was examined by a number of analytical techniques including SDS-PAGE, isoelectric focusing and chromatofocusing. No evidence of contamination by favin was revealed. Microheterogeneous subunits of EI were detected with both alpha-galactosidase activity and haemagglutinin activity.Immunoprecipitation experiments with antibodies for favin and EI have shown that the two proteins are structurally related. Western blotting experiments with EI using favin antibodies revealed the Mr 40,000 monomeric subunit of the enzyme and a minor component with Mr 20,000 ie. a peptide similar to the beta-subunit of favin, which is known to contain the glucose/mannose-combining sites of this lectin. Western blotting experiments with EI using EI antibodies showed the presence of a Mr 28,000 fragment together with EI monomer. However, the former band was not seen on the Western blot using favin antibodies. When Western blotting experiments were carried out using favin beta-subunit antibodies, as with favin antibodies, the enzyme monomer was stained and the peptide with Mr 20,000 was again detected in the EI lane. However, when this experiment was repeated and an empty well was placed between the wells that contained EI and favin, no band corresponding to Mr 20,000 (or Mr 28,000) was detected in the EI lane. This observation indicated that the Mr 20,000 (or Mr 28,000) component observed in EI in the previous Western blots was the result of excess favin flowing across to the adjoining well containing EI. As EI and favin cross-reacted with antibodies to EI, favin and beta-subunit and as no contaminant appeared to be present it is concluded that the lectin properties displayed by EI is probably due to an amino acid sequence in the enzyme which is homologous to favin.