Binding specificity of R-10G and TRA-1-60/81, and substrate specificity of keratanase II studied with chemically synthesized oligosaccharides

Binding specificity of R-10G and TRA-1-60/81, and substrate specificity of keratanase II studied... Recently, we established a mouse monoclonal antibody specific to hiPS/ hES cells, R-10G, which recognizes a type of keratan sulfate. Keratan sulfates (KS) comprise a family of glycosaminoglycans consisting of the repeating unit of [Gal-GlcNAc(6S)]. However, there is a diversity in the degree of sulfation at Gal and GlcNAc residues, and also in the mode of linkage, Galβ1 − 3GlcNAc (type 1) or Galβ1 − 4GlcNAc (type 2). To gain more insight into the binding specificity of R-10G, we carried out an ELISA test on avidin-coated plates using polyethylene glycol (PEG)3-biotinylated derivatives of a series of N-acetyllactosamine tetrasaccharides (keratan sulfates (KSs)). The results suggested that the minimum epitope structure is Galβ1 − 4GlcNAc(6S)β1 − 3Galβ1 − 4GlcNAc(6S)β1 (type 2- type 2 keratan sulfate). Removal of sulfate from GlcNAc(6S) or addition of sulfate to Gal abolished the binding activity almost completely. We also examined the binding specificity of TRA-1-60/81 in the same assay system. The minimum epitope structure was shown to be Galβ1 − 3GlcNAcβ1 − 3Galβ1 − 4GlcNAcβ1 in agreement with the previous study involving glycan arrays (Natunen et al., Glycobiology, 21, 1125–1130 (2011)). Interestingly, however, TRA-1-60/81 was shown to bind to Galβ1 − 3GlcNAc(6S)β1 − 3Galβ1 − 4GlcNAc(6S)β1 (type 1- type 2 keratan sulfate) dose-dependently, being more than one-third the binding activity toward Galβ1 − 3GlcNAcβ1 − 3Galβ1 − 4GlcNAcβ1 than in the case of TRA-1-60. In addition, a substrate specificity study on keratanase II revealed that keratanase II degraded not only “type 2-type 2 keratan sulfate” but also “type 1-type 2 keratan sulfate”, significantly. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Glycoconjugate Journal Springer Journals

Binding specificity of R-10G and TRA-1-60/81, and substrate specificity of keratanase II studied with chemically synthesized oligosaccharides

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Publisher
Springer Journals
Copyright
Copyright © 2017 by Springer Science+Business Media New York
Subject
Life Sciences; Biochemistry, general; Pathology
ISSN
0282-0080
eISSN
1573-4986
D.O.I.
10.1007/s10719-017-9765-8
Publisher site
See Article on Publisher Site

Abstract

Recently, we established a mouse monoclonal antibody specific to hiPS/ hES cells, R-10G, which recognizes a type of keratan sulfate. Keratan sulfates (KS) comprise a family of glycosaminoglycans consisting of the repeating unit of [Gal-GlcNAc(6S)]. However, there is a diversity in the degree of sulfation at Gal and GlcNAc residues, and also in the mode of linkage, Galβ1 − 3GlcNAc (type 1) or Galβ1 − 4GlcNAc (type 2). To gain more insight into the binding specificity of R-10G, we carried out an ELISA test on avidin-coated plates using polyethylene glycol (PEG)3-biotinylated derivatives of a series of N-acetyllactosamine tetrasaccharides (keratan sulfates (KSs)). The results suggested that the minimum epitope structure is Galβ1 − 4GlcNAc(6S)β1 − 3Galβ1 − 4GlcNAc(6S)β1 (type 2- type 2 keratan sulfate). Removal of sulfate from GlcNAc(6S) or addition of sulfate to Gal abolished the binding activity almost completely. We also examined the binding specificity of TRA-1-60/81 in the same assay system. The minimum epitope structure was shown to be Galβ1 − 3GlcNAcβ1 − 3Galβ1 − 4GlcNAcβ1 in agreement with the previous study involving glycan arrays (Natunen et al., Glycobiology, 21, 1125–1130 (2011)). Interestingly, however, TRA-1-60/81 was shown to bind to Galβ1 − 3GlcNAc(6S)β1 − 3Galβ1 − 4GlcNAc(6S)β1 (type 1- type 2 keratan sulfate) dose-dependently, being more than one-third the binding activity toward Galβ1 − 3GlcNAcβ1 − 3Galβ1 − 4GlcNAcβ1 than in the case of TRA-1-60. In addition, a substrate specificity study on keratanase II revealed that keratanase II degraded not only “type 2-type 2 keratan sulfate” but also “type 1-type 2 keratan sulfate”, significantly.

Journal

Glycoconjugate JournalSpringer Journals

Published: Mar 14, 2017

References

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