Sequence studies of peanut agglutinin

Lauwereys, Marc; Foriers, André; Sharor, N.; Strosberg, A. Donny

doi:10.1016/0014-5793(85)80267-2

Cited by 9 publications

(5 citation statements)

References 16 publications

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“…The strategy adopted therefore was to obtain the three acid cleavage fragments and sequence them from their tryptic peptides. The partial sequence [12] gave the overlaps of the acid fragments and these are substantiated by the general similarity of lectins in these areas (see alignment below). Also, the amino acid composition of PNA (Table 1) does not indicate any additional Asp-Pro sites.…”

Section: Sequencing Strategjmentioning

confidence: 73%

“…The partial sequence [12] had two such sites at approximately residues 80 and 130. The strategy adopted therefore was to obtain the three acid cleavage fragments and sequence them from their tryptic peptides.…”

Section: Sequencing Strategjmentioning

confidence: 99%

“…In a preliminary experiment with CNBr, it was discovered that the acid conditions were causing cleavage at Asp-Pro peptide bonds. The partial sequence [12] had two such sites at approximately residues 80 and 130. The strategy adopted therefore was to obtain the three acid cleavage fragments and sequence them from their tryptic peptides.…”

Section: Sequencing Strategjmentioning

confidence: 99%

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The amino acid sequence of peanut agglutinin

Young

Johnston

Watson

1991

European Journal of Biochemistry

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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...

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Section: Sequencing Strategjmentioning

confidence: 73%

Section: Sequencing Strategjmentioning

confidence: 99%

Section: Sequencing Strategjmentioning

confidence: 99%

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The amino acid sequence of peanut agglutinin

Young

Johnston

Watson

1991

European Journal of Biochemistry

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“…Metal-binding residues are conserved in most of these lectins and are essential for the correct folding of the carbohydrate recognition domain. BLASTp searches of the peptides against the non-redundant database indicated a high sequence similarity with the galactose-specific lectin sequences from PNA [43,44], VML [45] , VGL [22] and RPbAI [46] (Fig 3). Sequence alignments of VaL and VML revealed identical residues that make up the carbohydrate-binding domain (Asp87, Gly105, Phe127, and Asn129) [47].…”

Section: Discussionmentioning

confidence: 99%

A galactoside-specific Dalbergieae legume lectin from seeds of Vataireopsis araroba (Aguiar) Ducke

Osterne

Oliveira²,

Schutter³

et al. 2022

Glycoconj J

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The Dalbergieae lectin group encompasses several lectins with significant differences in their carbohydrate specificities and biological properties. The current work reports on the purification and characterization of a GalNAc/Gal-specific lectin from Vataireopsis araroba (Aguiar) Ducke, designated as VaL. The lectin was purified from the seeds in a single step using guar gum affinity chromatography. The lectin migrated as a single band of about 35 kDa on SDS-PAGE and, in native conditions, occurs as a homodimer. The purified lectin is stable at temperatures up to 60 °C and in a pH range from 7 to 8 and requires divalent cations for its activity. Sugar-inhibition assays demonstrate the lectin specificity towards N-acetyl-D-galactosamine, Dgalactose and related sugars. Furthermore, glycan array analyses show that VaL interacts preferentially with glycans containing terminal GalNAc/Galβ1-4GlcNAc. Biological activity assays were performed using three insect cell lines: CF1 midgut cells from the spruce budworm Choristoneura fumiferana, S2 embryo cells from the fruit fly Drosophila melanogaster, and GutAW midgut cells from the corn earworm Helicoverpa zea. In vitro assays indicated a biostatic effect for VaL on CF1 cells, but not on S2 and GutAW cells. The lectin presented a biostatic effect by reducing the cell growth and inducing cell agglutination, suggesting an interaction with glycans on the cell surface. VaL has been characterized as a galactoside-specific lectin of the Dalbergieae tribe, with sequence similarity to lectins from Vatairea and Arachis.

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“…This specificity allows for the use of PNA to detect the T-antigen on erythrocytes [1], and to separate mature and immature thymocytes of mouse [7] and man [8]. A partial sequence of PNA has been reported previously [9] and, while this work was being completed, a seque.nee of 236 amino acids of the leetin has been established by conventional techniques [10]. As expected, it shows extensive homology with the sequences of other legume lectins.…”

Section: Introductionmentioning

confidence: 99%

Cloning, sequence analysis and expression in Escherichia coli of the cDNA encoding a precursor of peanut agglutinin

et al. 1992

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The eDNA coding for pro-peanut agglutinin (PNA) was isolated from a bacterial expression library, It codes for a polypeptide of 273 amino acids composed of a hydrophobie signal peptide of 23 amino acids and a mature protein of 250 amino acids, The sequence of the latter is identi~l to that of native PNA, determined very recently by conventional methods, e~cept that it contains 14 additional amino acids at the C-terminus. Bacterial cells harboring a plasmid with the prePNA--cDNA, produced two PNA cross-reacting proteins: one migrated on SDS-PAGE identically with the native lectin (apparent tool, wt. 31 kDa); the other, at 35 kDa. was a fl-galactosidase pre.PNA fusion protein, The former protein poss~sed an N-terminal sequence identical to that of the mature, native FNA, suggesting that it was processed from the 35 kDa prePNA precursor. Only the 31 kDa protein was exported into the bacterial perilalasmie space, and had the ability to bind to galactose-Sepharos¢, The isolated progcsz,:s~ protein had the same hemagglutinating activity as the native iectin, when assayed with sialidase-treated human erythro~ytcs. Like the native leetin, it did not agglutinate the untreated cells, was not inhibited by N.acetylgalactosamine, and was inhibited by Galfll ~3GalNAc 30-times more strongly than by ~.alactose, In this paper we report the isolation and characterisation of a eDNA encoding prePNA and the e~;pression of the recombinant active lectin in Escherichia coil, demonstrating that the precursor is processed by the bacteria and that only the processed form binds carbohydrate. Our results provide a basis for studies of the Abbre~,iations: PBS, phosphate.buffered saline; PNA, peanut a~gluti-nin; pPNAT, Bluescript plasmid containing prePNA.cDNA from clone C7; rPNA, recombinant PNA.

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Sequence studies of peanut agglutinin

Cited by 9 publications

References 16 publications

The amino acid sequence of peanut agglutinin

The amino acid sequence of peanut agglutinin

A galactoside-specific Dalbergieae legume lectin from seeds of Vataireopsis araroba (Aguiar) Ducke

Cloning, sequence analysis and expression in Escherichia coli of the cDNA encoding a precursor of peanut agglutinin

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