The amino acid sequence of peanut agglutinin

Abstract

The amino acid sequence of peanut (Arachis hypogaea) agglutinin was determined from three major fragments obtained by mild acid cleavage at Asp-Pro peptide bonds. The sequence of 236 amino acids has residues identical to those that form the metal-binding site and the hydrophobic pocket in concanavalin A and other lectins, although the overall similarity is only 42%. In the segments of peanut agglutinin that correspond to the four loops that form the carbohydrate-binding site in concanavalin A and favin, several central residues are homologous, while others show changes to smaller side chains, such as Tyr --f Gly. The carbohydrate-binding site of peanut agglutinin may therefore have a similar peptide-backbone architecture, but form a considerably more open cleft.Peanut agglutinin (PNA) is a legume seed lectin that is specific for D-galactose residues at non-reducing terminal positions of glycoconjugates [l]. This specificity has made it useful for histochemical and cell-surface studies. The lectin recognises both a-and P-galactosides, and the disaccharide of highest known affinity is the T-antigen structure Gal(b1-3)GalNAc [2] (GalNAc, N-acetyl-D-galactosamine). Detailed studies of the interactions of PNA with monosaccharides and disaccharides have been made by UV difference [3,4], fluorescence [5, 61 and NMR [7, 81 spectroscopy, and a model of the binding process has been proposed [8]. PNA is therefore one of the best characterized lectins in its functional aspects, but far less is known about its structure. It is a tetramer of subunits of molecular mass 27 kDa [9], each with one bindingsite [3], and is not a glycoprotein [I]. PNA has been successfully crystallized for X-ray studies in two laboratories [lo, 1 I].A partial amino acid sequence was reported by Lauwereys et al. [I21 who encountered difficulties with multiple genetic forms and poorly soluble peptides. The former are to be expected in view of the multiple bands seen in isoelectric focussing and chromatofocussing experiments [13,14]. We have reinvestigated the amino acid sequence of PNA and report a complete sequence together with data on the genetic variation at some positions. The sequences of the peptide loops in the putative carbohydrate-binding-site region are discussed in relation to the X-ray results for the D-mannose-specific lectins concanavalin A Abbreviations. PNA, peanut agglutinin; GalNAc, N-acetyl-Dgalactosamine. MATERIALS AND METHODS Purqication of the proteinPeanut agglutinin was purified from buffer extracts of delipidated peanut meal by affinity chromatography on a lactose-substituted agarose matrix [3]. The initial PNA sample was material used for spectroscopic studies [3,4,7, 81; subsequent samples were prepared identically from the same batch of peanuts. Chemical cleavageThe protein was cleaved at Asp-Pro peptide bonds by limited acid hydrolysis [19]. PNA (22 mg) was dissolved in 2.28 ml 88% formic acid, 1.72 ml water was added and the solution was incubated at 37°C for 96 h. The digest was diluted and freeze-dried. The fragments...

Molten Globule-like State of Peanut Lectin Monomer Retains Its Carbohydrate Specificity

“…would promote deoligomerization of PNA (30). The Molten Globule-like Intermediate Retains Its Carbohydrate Binding Activity-Finally, the FPLC-purified monomeric intermediate in 2 M GdnHCl, which was confirmed to retain its molten globule-like characteristics at the concentrations used for titrations, bound to the sugar ligands almost akin to its native counterpart (Fig.…”

Simian Immunodeficiency Virus from the Sooty Mangabey and Rhesus Macaque Is Modified with O-Linked Carbohydrate

“…Jacalin also binds the Tn antigen and monosialylated core 1 carbohydrate (48,53). PNA recognizes only nonsialylated, not sialylated, core 1 mucin-type O-linked carbohydrate (35,47,54). In this report, we identify an O-linked carbohydrate attachment region in gp120 whose mutation imparts high-level resistance to the inhibitory effects of jacalin to cloned SIVmac239.…”

“…Jacalin binds the Tn antigen, core 1, and monosialylated core 1 mucin-type carbohydrate (26,42,48,53). PNA binds to nonsialylated core 1 carbohydrate (31,35,47,54). There are eight core mucin-type O-linked carbohydrate structures, of which core 1 and core 2 are the most common (25,49) (Fig.…”

Crystal structure of peanut lectin, a protein with an unusual quaternary structure.

“…Each subunit in the protein is 236 amino acid residues long and is homologous to the subunits in other legume lectins (12). As in the case of Con A (13,14) and other well-known tetrameric proteins such as hemoglobin (15,16), the molecule dissociates at low pH into dimers which bind sugar with the same stoichiometry as the tetramer (one binding site per protomer) but with an association constant one order of magnitude lower than that for the tetramer (17,18).…”