Mutational analysis of pea lectin. Substitution of Asn125 for Asp in the monosaccharide-binding site eliminates mannose/glucose-binding activity

Eijsden, R.R. van; Hoedemaeker, Flip J.; Diaz, C. L.; Lugtenberg, Ben; Pater, B. De Sylvia; Kijne, Jan W.

doi:10.1007/bf00028892

Cited by 41 publications

(40 citation statements)

References 34 publications

(33 reference statements)

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“…However, RI viciae is able to induce infection thread formation and nodulation in a significant percentage of white clover plants with roots transformed with the psl gene, as reported by Díaz et al (1989Díaz et al ( , 1995. This heterologous nodulation does not take place when a psl mutant, which encodes a non-sugar-binding protein, is used for transformation of white clover roots (Van Eijsden et al, 1992. These results strongly suggest that (a) PSL is functionally expressed in transgenic white clover roots, (b) conservation of the sugar-binding activity of PSL is necessary for infection of white clover by RI viciae, and (c) root lectin helps to break a host plant-specific barrier for nodulation.…”

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confidence: 68%

Sugar-Binding Activity of Pea Lectin Expressed in White Clover Hairy Roots

et al. 1995

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Dénarié et al., 1992). Nodulation is regulated by a two-way exchange of plant and bacterial signal molecules (Fisher and Long, 1992;Spaink et al., 1993). Signal exchange begins in the rhizosphere. Rhizobia sense flavonoids secreted by seeds and by the host plant root, which activate the constitutively expressed nodD gene product (reviewed by Schlaman et al., 1992). This step is a determinant of host plant specificity in some Rhizobium-host plant combinations (Horvath et al., 1987; Spaink et al., 1987). Transcription of structural nod genes is under the control of NodD and results in production and secretion of specific lipochitin oligosaccharides (Nod factors), which act as host plant-specific inducers of nodule morphogenesis (reviewed by Dénarié and Cullimore, 1993;Spaink et al., 1993). Most probably, Nod factors constitute the primary determinants of host-plant specificity of nodulation.Root lectins are plant factors involved in nodulation. Lectins are nonenzymatic sugar-binding (g1yco)proteins characterized by sugar-binding specificity. For example, root PSL is Glc/Man specific (Díaz et al., 1990) and white clover root lectin is 2-deoxyglucose specific (Sherwood et al., 1984). In general, the interaction between RI uiciae and white clover does not proceed beyond root hair curling, and these bacteria are not able to penetrate host plant cells by infection thread formation (Yao and Vincent, 1969;Djordjevic et al., 1986;Huang et al., 1993). However, RI viciae is able to induce infection thread formation and nodulation in a significant percentage of white clover plants with roots transformed with the psl gene, as reported by Díaz et al. (1989Díaz et al. ( , 1995. This heterologous nodulation does not take place when a psl mutant, which encodes a non-sugar-binding protein, is used for transformation of white clover roots (Van Eijsden et al., 1992. These results strongly suggest that (a) PSL is functionally expressed in transgenic white clover roots, (b) conservation of the sugar-binding activity of PSL is necessary for infection of white clover by RI viciae, and (c) root lectin helps to break a host plant-specific barrier for nodulation. The relationship between Nod factor recognition by host plants and Rhizobium recognition by host lectin is not yet clear.PSL is an ellipsoidal, nonglycosylated dimeric lectin with two sugar-binding sites, each located near the top of two identical monomers (Einspahr et al., 1988). PSL is encoded by a single gene (Gatehouse et al., 1987;Kaminski et al., 1987) and is synthesized as a single translation product, a prolectin with a molecular mass of 28 kD. Posttranslational modifications result in a protein that dissociates into two p subunits (18 kD each) and two 01 subunits (6 kD each, Higgins et al., 1983)

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confidence: 68%

Sugar-Binding Activity of Pea Lectin Expressed in White Clover Hairy Roots

et al. 1995

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“…START is 10% of total protein, FT is 10% of flow-through fraction, W is last fraction of washing step. analysis, and underlining the importance of the conserved asparagine, both mutations eliminated carbohydrate binding (van Eijsden et al, 1992;Mirkov and Chrispeels, 1993). To test the relationship of ERGIC-53 to leguminous lectins we substituted N156 by alanine.…”

Section: Ergic-53 Mutants Containing An N-terminalmentioning

confidence: 99%

“…These findings provide the first direct experimental evidence for a lectin function of ERGIC-53. To test the hypothesis that ERGIC-53 may be a functional homologue of leguminous lectins , we substituted N156 of ERGIC-53, which is conserved and essential in the carbohydrate binding site of leguminous lectins (Sharon and Lis, 1990;van Eijsden et al, 1992;Mirkov and Chrispeels, 1993). A second mutation that also should affect the binding site was generated to confirm the first mutation.…”

Section: Introductionmentioning

confidence: 99%

ERGIC-53 is a functional mannose-selective and calcium-dependent human homologue of leguminous lectins.

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Roche

Monsigny

et al. 1996

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Based on sequence homologies with leguminous lectins, the intermediate compartment marker proposed to be a member of a putative new class of animal lectins associated with the secretory pathway. Independently, a promyelocytic protein, MR60, was purified by mannose-column chromatography, and a cDNA was isolated that matched MR60 peptide sequences. This cDNA was identical to that of and homologies with the animal lectin family of the galectins were noticed. Not all peptide sequences of MR60, however, were found in ERGIC-53, raising the possibility that another protein associated with ERGIC-53 may possess the lectin activity. Here, we provide the first direct evidence for a lectin function of ERGIC-53. Overexpressed ERGIC-53 binds to a mannose column in a calcium-dependent manner and also co-stains with mannosylated neoglycoprotein in a morphological binding assay. By using a sequential elution protocol we show that ERGIC-53 has selectivity for mannose and low affinity for glucose and GlcNAc, but no affinity for galactose. To experimentally address the putative homology of ERGIC-53 to leguminous lectins, a highly conserved protein family with an invariant asparagine essential for carbohydrate binding, we substituted the corresponding asparagine in ERGIC-53. This mutation, as well as a mutation affecting a second site in the putative carbohydrate recognition domain, abolished mannosecolumn binding and co-staining with mannosylated neoglycoprotein. These findings establish ERGIC-53 as a lectin and provide functional evidence for its relationship to leguminous lectins. Based on its monosaccharide specificity, domain organization, and recycling properties, we propose ERGIC-53 to function as a sorting receptor for glycoproteins in the early secretory pathway.

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“…This residue is the most conserved amino acid present in legume lectins [3]. Also, mutation of Asn to Asp in both pea lectin and PHA-L eliminated carbohydrate binding and leucoagglutinating and mitogenic activities [4,20]. Tyr TM is not highly conserved in the primary sequence of legume lectins.…”

Section: Urea-treated Gs-iimentioning

confidence: 99%

Identification of N‐acetylglucosamine binding residues in Griffonia simplicifolia lectin II

et al. 1996

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Primary structure and crystallographic data of several legume lectins were used to predict the involvement in carbohydrate binding of six amino acid residues (Asp s8, Glu 1°s, Tyr 134, Asn 136, Leu 226 and Gin 227) in Griffonia simpficifofia lectin H (GS-II). The functional involvement of these residues was evaluated by assessing GIcNAc binding of modified forms of GS-II in which these residues were eliminated in truncated peptides or systematically substituted with other amino acids by site-specific mutations. Mutations at Asp s8, Tyr 134 or Asn 1~6 eliminated GIcNAc binding activity by GS-II, while those at Glu l°s, Leu 226 or Gin 227 did not alter the activity. The former three amino acids were functionally essential for carbohydrate binding by GS-II presumably through hydrogen bonding to and hydrophobic interactions with GIcNAc. Although an Asp or Gly substitution for Tyr 134 eliminated GIcNAc affinity, substitution with Phe did not appreciably affect binding. Despite the fact that mutations to Leu 226 and Gln 227 did not alter carbohydrate binding, a truncated form of GS-II lacking these residues no longer exhibited carbohydrate binding affinity.

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Mutational analysis of pea lectin. Substitution of Asn125 for Asp in the monosaccharide-binding site eliminates mannose/glucose-binding activity

Cited by 41 publications

References 34 publications

Sugar-Binding Activity of Pea Lectin Expressed in White Clover Hairy Roots

Sugar-Binding Activity of Pea Lectin Expressed in White Clover Hairy Roots

ERGIC-53 is a functional mannose-selective and calcium-dependent human homologue of leguminous lectins.

Identification of N‐acetylglucosamine binding residues in Griffonia simplicifolia lectin II

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