Mutational analysis of the sugar-binding site of pea lectin

Eijsden, R.R. van; Páter, Béla; Kijne, Jan W.

doi:10.1007/bf00731212

Cited by 23 publications

(12 citation statements)

References 33 publications

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“…However, an aspartic acid residue which is well conserved in the legume lectins (Asp 118 in RBL, Asp 111 in PSL and Asp 89 in GS4; see Fig. 2) and is essential for the binding of lectins to carbohydrates (Van Eijsden et al 1994;Zhu et al 1996), is replaced by a glutamic acid residue at position 115 in PnLPK. We previously showed that replacement of Asp 118 by glutamic acid resulted in the loss of the hemagglutination activity of RBL (Nishiguchi et al 1997).…”

Section: Discussionmentioning

confidence: 99%

A receptor-like protein kinase with a lectin-like domain from lombardy poplar: gene expression in response to wounding and characterization of phosphorylation activity

et al. 2002

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Plant receptor-like protein kinases (RLKs) are thought to be involved in various cellular processes mediated via signal transduction pathways. To clarify the initial step in such a signal transduction pathway in woody plants, we cloned a cDNA encoding PnLPK (a P opulus n igra var. italica lectin-like protein kinase) from lombardy poplar. The C-terminal region of the predicted PnLPK protein includes a protein kinase catalytic domain consisting of the 12 subdomains typical of the eukaryotic protein kinase superfamily. Following the signal peptide at the N-terminus, a domain that shows homology to legume lectins retains the putative Mn(2+)- and Ca(2+)-binding amino acids, which are highly conserved among lectin-related proteins. Because a putative hydrophobic transmembrane domain was localized between the lectin-like domain and the protein kinase domain, PnLPK was determined to be a member of the plant RLK subfamily with a lectin-like domain. Transcripts of the PnLPK gene accumulate in roots, mature leaves and calli of lombardy poplar, whereas only trace amounts of the transcripts are detectable in stems, young leaves and apical buds. Wounding of the young leaves increased the amount of PnLPK mRNA, but none of several phytohormones tested had any effect on the transcription of PnLPK. When incubated in the presence of divalent metal cations such as Mn(2+), the C-terminal catalytic domain of PnLPK showed significantly higher autophosphorylation activity than the full-length PnLPK protein. The phosphorylation activity of PnLPK was also detected using beta-casein as substrate. Phosphoamino acid analysis indicated that PnLPK is a serine/threonine kinase.

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Section: Discussionmentioning

confidence: 99%

A receptor-like protein kinase with a lectin-like domain from lombardy poplar: gene expression in response to wounding and characterization of phosphorylation activity

et al. 2002

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“…Mutation of a single hydrogen-bonding or sugar-stacking residue within the lectin site of various legume lectins results in loss of glycan binding. For the lectin from Erythrina corallodendron (ECorL), mutation of the hydrogen-bonding residues Asp 89 or Asn 133 to Ala (44) and mutations in equivalent residues in the Phaseolus vulgaris phytohemagglutinin lectin (45), Pisum sativum lectin (46), and Griffonia simplicifolia lectin II (47) result in a loss of sugar binding. A mannose-specific animal lectin with a role in ER-Golgi trafficking, ERGIC-53, is homologous to the legume lectins, and mutation of the conserved Asn residue also results in the loss of lectin activity (48).…”

Section: Lectin Site Mutants Of Calnexin Retain the Ability To Protecmentioning

confidence: 99%

Lectin-deficient Calnexin Is Capable of Binding Class I Histocompatibility Molecules in Vivo and Preventing Their Degradation

Leach¹,

Williams

2004

Journal of Biological Chemistry

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Calnexin is a membrane-bound lectin of the endoplasmic reticulum (ER) that binds transiently to newly synthesized glycoproteins. By interacting with oligosaccharides of the form Glc 1 Man 9 GlcNAc 2 , calnexin enhances the folding of glycoprotein substrates, retains misfolded variants in the ER, and in some cases participates in their degradation. Calnexin has also been shown to bind polypeptides in vivo that do not possess a glycan of this form and to function in vitro as a molecular chaperone for nonglycosylated proteins. To test the relative importance of the lectin site compared with the polypeptidebinding site, we have generated six calnexin mutants defective in oligosaccharide binding using site-directed mutagenesis. Expressed as glutathione S-transferase fusions, these mutants were still capable of binding ERp57, a thiol oxidoreductase, and preventing the aggregation of a nonglycosylated substrate, citrate synthase. They were, however, unable to bind Glc 1 Man 9 GlcNAc 2 oligosaccharide and were compromised in preventing the aggregation of the monoglucosylated substrate jack bean ␣-mannosidase. Two of these mutants were then engineered into full-length calnexin for heterologous expression in Drosophila cells along with the murine class I histocompatibility molecules K b and D b as model glycoproteins. In this system, lectin sitedefective calnexin was able to replace wild type calnexin in forming a complex with K b and D b heavy chains and preventing their degradation. Thus, at least for class I molecules, the lectin site of calnexin is dispensable for some of its chaperone functions. Calnexin (CNX)1 is a type I transmembrane protein of the ER that binds transiently to newly synthesized glycoproteins. Its ER luminal portion is comprised of a globular lectin domain that binds Ca 2ϩ and recognizes the monoglucosylated Glc 1 Man 9 GlcNAc 2 oligosaccharide (1, 2) and an arm-like extension that binds the thiol oxidoreductase ERp57 (3, 4). Calnexin has high sequence similarity to calreticulin (CRT), a soluble ER lectin of the same specificity (5), and considerable functional overlap (6 -8). It is also similar to a testis-specific membrane protein termed calmegin (9). CNX and CRT function as chaperones by enhancing the in vivo folding of their substrates as has been demonstrated in a number of model systems including the assembly of major histocompatibility complex class I molecules (8, 10), the nicotinic acetylcholine receptor (11), and influenza hemagglutinin (12). They also act as components of the quality control machinery, retaining misfolded or incompletely folded substrates within the ER (13, 14). Recently it has been shown that CNX has a role in ER-associated degradation by binding and delivering substrates to the ER degradationenhancing mannosidase-like protein, a mannose-specific lectin that targets glycoproteins for retrotranslocation to the cytosol where they are degraded by the proteosome (15, 16). The signal for degradation is the conversion of the oligosaccharide from Man 9 GlcNAc 2 to a Man 8 GlcN...

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“…Furthermore, in all complexes of the mannose/glucose-specific lectins with their ligands, the above aspartic acid and the asparagine form hydrogen bonds not with the 3-OH and 4-OH of the ligand, as found for the galactose-specific legume lectins, but with the 4-OH and 6-OH groups instead. In pea lectin, one of these mannose/glucose-specific lectins, the above hydrogen bonds were shown to be essential for ligand binding, since substitution of Asp81 (which corresponds to Asp89 in E. corallodendron lectin) by alanine or asparagine, or of Asn125 (corresponding to Am133 in the lectin) by alanine, resulted in loss of activity, without loss of metal binding (van Eijsden et al, 1994). Furthermore, in PHA-L, the leucoagglutinin of Phuseolus vulgaris, a lectin with specificity for N-acetyllactosamine (complex) type oligosaccharides, changing of the combining site Asnl25 for aspartic acid eliminated activity without affecting binding of calcium (van Eijsden et al, 1992).…”

Section: Physicochemical Properties the 19 Refolded Mutants Generallymentioning

confidence: 99%

Mutational Studies of the Amino Acid Residues in the Combining Site of Erythrina corallodendron Lectin

Adar

Sharon

1996

European Journal of Biochemistry

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High-resolution X-ray crystallography of the complex of the Gal/GalNAc-specific Erythrinu corullodendron lectin with lactose identified the amino acid side chains that form contacts with the galactose moiety of the disaccharide. The contribution of these amino acids to the binding of different monosaccharides and oligosaccharides by the lectin was examined by site-directed mutagenesis.Replacement of Phel31, on which the galactose is stacked, by tyrosine, gave a mutant with the same hemagglutinating activity and carbohydrate specificity as the parent lectin, but replacement by alanine or valine resulted in loss of activity. Mutations of Ala88, Asp89, and Asn133 produced mutants that were also inactive whereas those of the other combining site residues, Tyr106, Ala218, and Gln219, were biologically active. None of the active mutants interacted with mannose or glucose. Thus, conti-ary to an earlier assumption, Ala218 is not responsible for the inability of E. corullodendron lectin to bind these sugars. Our findings also demonstrate that Gln219 is not involved in galactose binding in solution, even though this is implicated by the crystal data. Instead, our data suggest that Gln219 assists in the ligation of N-acetyllactosamine to the lectin, by interacting with the acetamide group of the disaccharide.Coinparison with other legume lectins specific for niannoselglucose, galactose, N-acetylgaiactosamine, L-fucose or N-acetylglucosamine, shows that only three of the combining site residues of E. corallodendron lectin occupy invariant positions both in their primary and tertiary structures. These residues are an aspartic acid and an asparagine corresponding to positions 89 and 133, respectively, in E. corallodendron lectin, and an aromatic residue, either phenylalanine (as Phel31 in this lectin), tyrosine or tryptophan. We therefore postulate that these three residues are essential for ligand binding by all such lectins, irrespective of their specificity.Keywords: legume lectins ; combining site ; mutagenesis ; contact residues.Legume lectins are members of a large family of structurally similar proteins with distinct carbohydrate specificities. Over 80 of these lectins have been isolated and characterized to varying extents. Almost all are comprised of two or four identical or nearly identical subunits, of 25-30 kDa each, held together by non-covalent forces (Jordan and Goldstein, 1994;Konami et al., 1995;Sharon and Lis, 1990). Each subunit contains tightly bound metal ions (one Ca" and one Mn") and possesses a single combining site to which different carbohydrates are bound with high specificity, although with low affinity, usually in the millimolar range. Many, but not all, are glycoproteins, with one or two N-linked glycans per subunit. In the primary structures of some 40 of the legume lectins that have been determined, 40% of the amino acid residues are invariant, or conservatively substituted. Moreover, the tertiary structures of nine of these lectins that have been solved by X-ray crystallography are superimposa...

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Mutational analysis of the sugar-binding site of pea lectin

Cited by 23 publications

References 33 publications

A receptor-like protein kinase with a lectin-like domain from lombardy poplar: gene expression in response to wounding and characterization of phosphorylation activity

A receptor-like protein kinase with a lectin-like domain from lombardy poplar: gene expression in response to wounding and characterization of phosphorylation activity

Lectin-deficient Calnexin Is Capable of Binding Class I Histocompatibility Molecules in Vivo and Preventing Their Degradation

Mutational Studies of the Amino Acid Residues in the Combining Site of Erythrina corallodendron Lectin

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