Caenorhabditis elegans N-glycans containing a Gal-Fuc disaccharide unit linked to the innermost GlcNAc residue are recognized by C. elegans galectin LEC-6

Takeuchi, Tomoharu; Hayama, Ko; Hirabayashi, Jun; Kasai, Ken‐ichi

doi:10.1093/glycob/cwn077

Cited by 45 publications

(42 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…[16][17][18][19][20][21] Recently, we found that another C. elegans galectin LEC-6 strongly interacts with N-glycans containing a Gal-Fuc disaccharide unit attached at position 6 of the innermost GlcNAc residue. 22) We also found that LEC-6 interacts with Galb1→4Fuc more strongly than does Galb1→ 3Fuc 23) by using chemically synthesized Galb1→4Fuc and Galb1→3Fuc. 24) Furthermore, the tandem repeat-type nematode galectin LEC-1 also shows preferential binding to Galb1→4Fuc.…”

Section: )mentioning

confidence: 51%

Cross-Link Formation between Mutant Galectins of Caenorhabditis elegans with a Substituted Cysteine Residue and Asialofetuin via a Photoactivatable Bifunctional Reagent

Tamura

Takeuchi

Nonaka

et al. 2011

Biological & Pharmaceutical Bulletin

View full text Add to dashboard Cite

Galectins are a group of animal lectins characterized by their specificity for b-galactosides and an evolutionarily conserved sequence motif in the carbohydrate-binding site. Galectins are involved in a wide variety of biological phenomena, including development, cell differentiation, tumor metastasis, apoptosis, RNA splicing, and regulation of immune functioning. [1][2][3][4][5][6] Galectins can be classified into three types on the basis of their molecular architecture: proto, chimera, and tandem-repeat types. LEC-1, the 32-kDa galectin of the nematode, Caenorhabditis elegans (C. elegans), was the first example of a tandem repeat-type galectin composed of two homologous regions. 7,8) A variety of pyridylaminated oligosaccharides were used as analytes in frontal affinity chromatography, 9) and the result showed that the two lectin domains of LEC-1 have different sugar-binding profiles.10) This finding suggests that the endogenous ligand glycoconjugates for the two domains of LEC-1 differ. However, it is difficult to confirm this finding, especially when the binding ability of each domain is not adequately strong. We developed a new approach to overcome this problem. We prepared LEC-1 mutants in which a cysteine residue was introduced near the binding site of the Nterminal half domain; after incubation with a model glycoprotein ligand, asialofetuin, cross-linked products were formed using a photoactivatable bifunctional reagent, benzophenone-4-maleimide (BPM). Among the five LEC-1 mutants produced, one of them (LEC-1 Q38C) produced the cross-linked product.11) This method was also applied to human galectin-1 (Gal-1). At first, all cysteine residues were substituted with Ser residues, and Lys 28 of the resultant cysteine-less Gal-1 was substituted with a cysteine residue. The position of Lys 28 corresponds to Gln 38 of LEC-1. Crosslinked products between hGal-1 K28C and asialofetuin via BPM were observed. 12) In the present study, we produced several LEC-1 mutants, each with a cysteine substitution in the C-terminal lectin domain (Ch) to detect interactions with asialofetuin. Two mutants, K177C and S268C, produced cross-linked products. This procedure is very promising for capturing and identifying endogenous ligands for each lectin domain of the tandem repeat-type galectin, LEC-1. MATERIALS AND METHODS ChemicalsRabbit anti-bovine fetuin polyclonal antibody was purchased from Chemicon International (Temecula, CA, U.S.A.). BPM and bovine fetuin were purchased from Sigma (St. Louis, MO, U.S.A.). Sialidase and b-galactosidase were purchased from Seikagaku Co. (Tokyo, Japan).Construction of Mutant Lectin Genes A cysteine residue was introduced using site-directed mutagenesis of LEC-1 cDNA (LEC-1 contains no cysteine residues). 8)Residues subjected to substitution were selected on the basis of the X-ray crystal structure of LEC-1 in complex with galactose.13) To facilitate cross-link formation, hydrophilic amino acid residues with side chains extending from the surface of the protein toward the bound galactose but not...

show abstract

Section: )mentioning

confidence: 51%

Cross-Link Formation between Mutant Galectins of Caenorhabditis elegans with a Substituted Cysteine Residue and Asialofetuin via a Photoactivatable Bifunctional Reagent

Tamura

Takeuchi

Nonaka

et al. 2011

Biological & Pharmaceutical Bulletin

View full text Add to dashboard Cite

show abstract

Section: -15)mentioning

confidence: 99%

“…Two pyridylaminated-LEC-6-binding glycans, D3 and E3, 16) were subjected to FAC analysis by using LEC-1-, LEC-1Nh-, and LEC-1Ch-immobilized columns (results are shown in Fig. 2).…”

Section: Of Ref 18)mentioning

confidence: 99%

“…We found that LEC-6 interacts strongly with N-glycans containing a Gal-Fuc disaccharide unit attached to position 6 of the innermost GlcNAc residue. 16) In order to identify the linkage between the galactose and fucose residues, and examine the structure of the endogenous glycoepitope recognized by LEC-6, we compared the binding strengths of the chemically synthesized Galb1-4Fuc and Galb1-3Fuc, 17) and found that LEC-6 interacts more strongly with Galb1-4Fuc than with Galb1-3Fuc.18) LEC-1, another major galectin from C. elegans, also showed preferential binding to 18) These results suggest that Galb1-4Fuc is the endogenous unit recognized by the C. elegans galectins.LEC-1 is a tandem-repeat type galectin, wherein two homologous carbohydrate recognition domains exist within a single polypeptide chain. 7,8) We previously reported that although the two domains of LEC-1 showed affinity for sugars containing Galb1-4GlcNAc units, detailed analysis using fluorescence-labeled oligosaccharides (pyridylaminated (PA) sugars) in frontal affinity chromatography (FAC) 19) revealed these domains had different sugar binding properties.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Sugar-Binding Properties of the Two Lectin Domains of LEC-1 with Respect to the Gal.BETA.1-4Fuc Disaccharide Unit Present in Protostomia Glycoconjugates

Takeuchi

Sugiura

Nishiyama

et al. 2011

Biological & Pharmaceutical Bulletin

View full text Add to dashboard Cite

Galectins are a group of animal lectins characterized by their specificity for b-galactosides and by an evolutionarily conserved sequence motif in their carbohydrate-binding site. Galectins are involved in a wide variety of biological phenomena, including development, cell differentiation, tumor metastasis, apoptosis, RNA splicing, and regulation of immune function.1-6) A Galb1-4GlcNAc (N-acetyllactosamine) disaccharide unit is thought to be the endogenous glycoepitope recognized by vertebrate galectins. This recognition has also been observed in galectins from invertebrate species such as Caenorhabditis elegans (C. elegans), including the galectins LEC-1 and LEC-6, which we have isolated previously. [7][8][9] However, the existence of a glycan containing Nacetyllactosamine units has not yet been confirmed in C. elegans. 10-15)Recently, we isolated N-type glycoproteins captured by the C. elegans galectin LEC-6 and analyzed their sugar structure by matrix-assisted laser desorption-time-of-flight mass spectrometry (MALDI-TOF MS/MS) in conjunction with glycosidase digestion. We found that LEC-6 interacts strongly with N-glycans containing a Gal-Fuc disaccharide unit attached to position 6 of the innermost GlcNAc residue. 16) In order to identify the linkage between the galactose and fucose residues, and examine the structure of the endogenous glycoepitope recognized by LEC-6, we compared the binding strengths of the chemically synthesized Galb1-4Fuc and Galb1-3Fuc, 17) and found that LEC-6 interacts more strongly with Galb1-4Fuc than with Galb1-3Fuc.18) LEC-1, another major galectin from C. elegans, also showed preferential binding to 18) These results suggest that Galb1-4Fuc is the endogenous unit recognized by the C. elegans galectins.LEC-1 is a tandem-repeat type galectin, wherein two homologous carbohydrate recognition domains exist within a single polypeptide chain. 7,8) We previously reported that although the two domains of LEC-1 showed affinity for sugars containing Galb1-4GlcNAc units, detailed analysis using fluorescence-labeled oligosaccharides (pyridylaminated (PA) sugars) in frontal affinity chromatography (FAC) 19) revealed these domains had different sugar binding properties. 20)However, the oligosaccharides used in the previous study 20) differed in that they were mainly of vertebrate origin and commercially available in pyridylaminated forms.For this report, we used recombinant proteins of the whole molecule (LEC-1), the N-terminal lectin domain (LEC-1Nh), and the C-terminal lectin domain (LEC-1Ch), and compared their binding affinities for the Galb1-4Fuc and Galb1-3Fuc disaccharide units. Although the two lectin domains showed preferential binding to Galb1-4Fuc compared to Galb1-3Fuc and N-acetyllactosamine-containing structure LNnT (Galb1-4GlcNAcb1-3Galb1-4Glc), the binding profiles of the two domains were different. While LEC-1Nh demonstrated a very high affinity to Galb1-4Fuc with a very low affinity for Galb1-3Fuc and LNnT, LEC-1Ch displayed a moderate affinity for both Galb1-3Fuc and LNnT....

show abstract

“…To search for it, Takeuchi et al isolated N-type glycoproteins bound by C. elegans galectin LEC-6. 15) Matrix assisted laser desorption/ionization-time of flight (MALDI-TOF)/TOF analysis in conjunction with glycosidase digestion elucidated that the glycoproteins contain a Gal-Fuc disaccharide unit attached to the innermost GlcNAc (N-acetylglucosamine) residue. Among several Gal-Fuc units, in which D-galactose and L-fucose are differentially linked at some positions, we assumed that Galb1-4Fuc should be the endogenous glyco-epitope because its presence had been confirmed in C. elegans N-glycans.…”

mentioning

confidence: 99%

Synthesis of Fluorescence-Labeled Gal.BETA.1-3Fuc and Gal.BETA.1-4Fuc as Probes for the Endogenous Glyco-Epitope Recognized by Galectins in Caenorhabditis elegans

Nishiyama

Yamada

Takahashi

et al. 2010

CHEMICAL & PHARMACEUTICAL BULLETIN

View full text Add to dashboard Cite

To search for the endogenous glyco-epitope in Caenorhabditis elegans, we synthesized labeled Galb b1-3Fuc and Galb b1-4Fuc and examined their binding affinity for C. elegans galectin LEC-6 using frontal affinity chromatography analysis. We developed a new strategy for synthesizing the labeled saccharides, in which the labeling unit, the 2-aminopyridine moiety, is coupled with a spacer unit derived from D-mannitol. Our results indicate that Galb b1-4Fuc is the endogenous glyco-epitope present in C. elegans N-glycans.

show abstract

Caenorhabditis elegans N-glycans containing a Gal-Fuc disaccharide unit linked to the innermost GlcNAc residue are recognized by C. elegans galectin LEC-6

Cited by 45 publications

References 35 publications

Cross-Link Formation between Mutant Galectins of Caenorhabditis elegans with a Substituted Cysteine Residue and Asialofetuin via a Photoactivatable Bifunctional Reagent

Cross-Link Formation between Mutant Galectins of Caenorhabditis elegans with a Substituted Cysteine Residue and Asialofetuin via a Photoactivatable Bifunctional Reagent

Sugar-Binding Properties of the Two Lectin Domains of LEC-1 with Respect to the Gal.BETA.1-4Fuc Disaccharide Unit Present in Protostomia Glycoconjugates

Synthesis of Fluorescence-Labeled Gal.BETA.1-3Fuc and Gal.BETA.1-4Fuc as Probes for the Endogenous Glyco-Epitope Recognized by Galectins in Caenorhabditis elegans

Contact Info

Product

Resources

About