Abstract
Leguminous lectins have a conserved carbohydrate recognition site comprising four loops (A–D). Here, we randomly mutated the sequence and length of loops C and D of peanut agglutinin (PNA) and expressed the proteins on the surface of mouse green fluorescent protein (GFP)-reporter cells. Flow cytometry, limiting dilution, and cDNA cloning were used to screen for several mutated PNAs with distinct properties. The mutated PNA clones obtained using NeuAcα2-6(Galβ1-3)GalNAc as a ligand showed preference for NeuAcα2-6(Galβ1-3)GalNAc rather than non-sialylated Galβ1-3GlcNAc, whereas wild-type PNA binds to Galβ1-3GlcNAc but not sialylated Galβ1-3GalNAc. Sequence analyses revealed that for all of the glycan-reactive mutated PNA clones, (i) loop C was eight amino acids in length, (ii) loop D was identical to that of wild-type PNA, (iii) residue 127 was asparagine, (iv) residue 125 was tryptophan, and (v) residue 130 was hydrophobic tyrosine, phenylalanine, or histidine. The sugar-binding ability of wild-type PNA was increased nine-fold when Tyr125 was mutated to tryptophan, and that of mutated clone C was increased more than 30-fold after His130 was changed to tyrosine. These results provide an insight into the relationship between the amino acid sequences of the carbohydrate recognition site and sugar-binding abilities of leguminous lectins.
Highlights
Numerous types of glycans are displayed on the surface of mammalian cells and glycans play various important roles in the biological functions of the cell
The carbohydrate recognition sites of various leguminous lectins, which consist of four peptide loops named A,B,C and D [14], share structural similarities
Because Gal1-3GalNAc is a well-known ligand of peanut agglutinin (PNA) [18,19], these results indicate that exogenous PNA displayed on the surface of 2B4 cells was capable of transducing a signal into the cells via cross-linking induced by an immobilized sugar ligand
Summary
Numerous types of glycans are displayed on the surface of mammalian cells and glycans play various important roles in the biological functions of the cell. Especially on the cell surface, has been performed via either mass spectrometric analyses of released oligosaccharides [1] or lectin microarray analyses of the cell itself [2,3,4,5]. Both methods are highly sensitive and reproducible, mass spectrometric analyses are associated with difficulties in quantitative determinations and specifying anomer structures, whereas lectin microarrays are unable to accommodate some glycan structures. We identified the sugar-binding peptides of several leguminous lectins by affinity chromatography analyses of trypsin or lysylendopeptidase digests on haptenic sugar-immobilized columns [7,8,9]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
