Bifunctional Labeling Reagent for Oligosaccharides To Incorporate Both Chromophore and Biotin Groups

Shinohara, Yasuro; Sota, Hiroyuki; Gotoh, Masao; Hasebe, Masaharu; Tosu, Mariko; Nakao, Junko; Hasegawa, Yukio; Shiga, Masanobu

doi:10.1021/ac960004f

Cited by 58 publications

(43 citation statements)

References 24 publications

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“…Quantitation of the lectin was carried out with ultraviolet absorbance at 280 nm monitored by a SMART ® (Amersham Biosciences) UV monitor. 4-(Biotinamido) phenylacetylhydrazide (BPH) was synthesized as previously reported (18).…”

Section: Methodsmentioning

confidence: 99%

The High Specificities of Phaseolus vulgaris Erythro- and Leukoagglutinating Lectins for Bisecting GlcNAc or β1–6-Linked Branch Structures, Respectively, Are Attributable to Loop B

Kaneda¹,

Whittier²,

Yamanaka³

et al. 2002

Journal of Biological Chemistry

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Despite very similar tertiary structures based upon a common framework, legume lectins exhibit an amazing variety of sugar binding specificities. While most of these lectins recognize rather discrete sugar linkages, Phaseolus vulgaris erythroagglutinating and leukoagglutinating lectins (E 4 -and L 4 -PHA) are unique in recognizing larger structures. E 4 -and L 4 -PHA are known to recognize complex type N-glycans containing bisecting GlcNAc or a ␤1,6-linked branch, respectively. However, the detailed mechanisms of molecular recognition are poorly understood. In order to dissect the contributions of different portions of each lectin, we carried out region-swapping mutagenesis between E 4 -and L 4 -PHA. We prepared six chimeric lectins by exchanging different combinations of loop B and the central portion of loop C, two of four loops thought to be important for the recognition of monosaccharides (Sharma, V., and Surolia, A. (1997) J. Mol. Biol. 267, 433-445). The chimeric lectins' sugar binding activities were evaluated quantitatively by surface plasmon resonance. These comparisons indicate that the high specificities of E 4 -and L 4 -PHA toward bisecting GlcNAc and ␤1,6-linked branch structures are almost solely attributable to loop B. The contribution of the central portion of loop C to the recognition of those structural motifs was found to be negligible. Instead, it modulates affinity toward LacNAc residues present at the nonreducing terminus. Moreover, some of the chimeric lectins prepared in this study showed even higher specificities/affinities than native E 4 -and L 4 -PHA toward complex sugar chains containing either a bisecting GlcNAc residue or a ␤1,6-linked branch.The legume lectins are a family of sugar-binding proteins found mainly in the seeds of plants belonging to the Leguminosae family (1-3). Lectins from leguminous plants constitute a large family of homologous proteins displaying remarkable divergence in their carbohydrate specificity. Elucidation of the mechanism by which these lectins can possess such a broad range of binding specificities while maintaining a strikingly similar threedimensional monomer structure will be key to understanding the essence of carbohydrate-protein interactions.At present, the crystal structures of ϳ20 legume lectins have been solved.1 These various lectin monomers share a common so-called "jellyroll" structure composed primary of a six-and a seven-stranded antiparallel ␤-sheet. Each monomer binds a manganese and a calcium ion that are both essential for carbohydrate binding. Amino acid sequence comparisons, x-ray crystallographic analysis, and mutagenesis studies revealed that the differences in carbohydrate specificity appear to be due primarily to differences in amino acid residues residing in loops adjacent to the carbohydrate binding site.The primary carbohydrate-binding site of legume lectins is a shallow depression on loops associated with the concave face of the seven-stranded curved ␤-sheet (5). It is constructed mainly by residues from four sequentially se...

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Section: Methodsmentioning

confidence: 99%

The High Specificities of Phaseolus vulgaris Erythro- and Leukoagglutinating Lectins for Bisecting GlcNAc or β1–6-Linked Branch Structures, Respectively, Are Attributable to Loop B

Kaneda¹,

Whittier²,

Yamanaka³

et al. 2002

Journal of Biological Chemistry

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“…As a result of continuous developments, the traditional protocol of overnight PNGase F digestion at 37°C has been successfully accelerated by microwave irradiation [16], pressure cycling technology [17], or with the utilization of immobilized PNGase F microreactors [18,19]. The released carbohydrates can be tagged at their reductive ends in various ways, such as by Michael-type addition [20] or hydrazine labeling [21], but the most commonly used method is reductive amination in a twostep-reaction. During the first step a Schiff-base is formed in the presence of an acid catalyst, followed by reduction to form a stable conjugate in the second step.…”

Section: Introductionmentioning

confidence: 99%

Evaporative fluorophore labeling of carbohydrates via reductive amination

Reider

Szigeti

Guttman

2018

Talanta

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As analytical glycomics became to prominence, newer and more efficient sample preparation methods are being developed. Albeit, numerous reductive amination based carbohydrate labeling protocols have been reported in the literature, the preferred way to conduct the reaction is in closed vials. Here we report on a novel evaporative labeling protocol with the great advantage of continuously concentrating the reagents during the tagging reaction, therefore accommodating to reach the optimal reagent concentrations for a wide range of glycan structures in a complex mixture. The optimized conditions of the evaporative labeling process minimized sialylation loss, otherwise representing a major issue in reductive amination based carbohydrate tagging. In addition, complete and uniform dispersion of dry samples was obtained by supplementing the low volume labeling mixtures (several microliters) with the addition of extra solvent (e.g., THF).Evaporative labeling is an automation-friendly glycan labeling method, suitable for standard open 96 well plate format operation. Graphical abstract 2/13

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“…77,78 Labeling with biotin-hydrazide enables the immobilization of glycans to streptavidin-coated carriers. 79 …”

Section: Specific Entrapment Of Glycansmentioning

confidence: 99%

Recent Developments in Liquid Chromatography and Capillary Electrophoresis for the Analysis of Glycoprotein Glycans

Suzuki

2013

ANAL. SCI.

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Bifunctional Labeling Reagent for Oligosaccharides To Incorporate Both Chromophore and Biotin Groups

Cited by 58 publications

References 24 publications

The High Specificities of Phaseolus vulgaris Erythro- and Leukoagglutinating Lectins for Bisecting GlcNAc or β1–6-Linked Branch Structures, Respectively, Are Attributable to Loop B

The High Specificities of Phaseolus vulgaris Erythro- and Leukoagglutinating Lectins for Bisecting GlcNAc or β1–6-Linked Branch Structures, Respectively, Are Attributable to Loop B

Evaporative fluorophore labeling of carbohydrates via reductive amination

Recent Developments in Liquid Chromatography and Capillary Electrophoresis for the Analysis of Glycoprotein Glycans

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