Structural determination of novel sulfated octasaccharides isolated from chondroitin sulfate of shark cartilage and their application for characterizing monoclonal antibody epitopes

Sarama S. Deepa, Shuhei Yamada, Shigeyuki Fukui, Kazuyuki Sugahara

Research output: Contribution to journalArticle

41 Citations (Scopus)

Abstract

Twelve octasaccharide fractions were obtained from chondroitin sulfate C derived from shark cartilage after hyaluronidase digestion. Their sugar and sulfate composition was assigned by matrix-assisted laser desorption ionization time of flight mass spectrometry. The sequences were determined at low picomole amounts by a combination of enzymatic digestions with high-performance liquid chromatography, and were composed of disaccharide building units including O [GlcUAβ1-3GalNAc], C [GlcUAβ1-3GalNAc(6S)], A [GlcUAβ1-3GalNAc(4S)], and/or D [GlcUA(2S)β1-3GalNAc(6S)], where 2S, 4S, and 6S represent 2-O-, 4-O-, and 6-O-sulfate, respectively. As many as 24 different sequences including minor ones were revealed, exhibiting a high degree of structural diversity reflecting the enormous heterogeneity of the parent polysaccharides. Nineteen of them were novel, with the other four reported previously as unsaturated counterparts obtained after digestion with chondroitinase. Microarrays of these structurally defined octasaccharide fractions were prepared using low picomole amounts of their lipid-derivatives to investigate the binding specificity of four commercial anti-chondroitin sulfate antibodies CS-56, MO-225, 2H6, and LY111. The results revealed that multiple unique sequences were recognized by each antibody, which implies that the common conformation shared by the multiple primary sequences in the intact chondroitin sulfate chains is important as an epitope for each monoclonal antibody. Comparison of the specificity of the tested antibodies indicates that CS-56 and MO-225 specifically recognize octasaccharides containing an A-D tetrasaccharide sequence, whereas 2H6 and LY111 require a hexasaccharide as a minimum size for their binding, and prefer sequences with A- and C-units such as C-C-A-C (2H6) or C-C-A-O, C-C-A-A, and C-C-A-C (LY111) for strong binding but require no D-unit.

Original languageEnglish (US)
Pages (from-to)631-645
Number of pages15
JournalGlycobiology
Volume17
Issue number6
DOIs
StatePublished - Jun 2007
Externally publishedYes

Fingerprint

Sharks
Chondroitin Sulfates
Cartilage
Epitopes
Digestion
Monoclonal Antibodies
Sulfates
Antibodies
Chondroitinases and Chondroitin Lyases
Hyaluronoglucosaminidase
Antibody Specificity
Disaccharides
High performance liquid chromatography
Microarrays
Sugars
Ionization
Mass spectrometry
Polysaccharides
Conformations
Desorption

Keywords

  • Antibody epitope
  • Chondroitin sulfate
  • Octasaccharides
  • Sugar sequencing
  • Sulfation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Structural determination of novel sulfated octasaccharides isolated from chondroitin sulfate of shark cartilage and their application for characterizing monoclonal antibody epitopes. / Deepa, Sarama S.; Yamada, Shuhei; Fukui, Shigeyuki; Sugahara, Kazuyuki.

In: Glycobiology, Vol. 17, No. 6, 06.2007, p. 631-645.

Research output: Contribution to journalArticle

@article{ed80ab9a94ea4b69bd9d9230459d882a,
title = "Structural determination of novel sulfated octasaccharides isolated from chondroitin sulfate of shark cartilage and their application for characterizing monoclonal antibody epitopes",
abstract = "Twelve octasaccharide fractions were obtained from chondroitin sulfate C derived from shark cartilage after hyaluronidase digestion. Their sugar and sulfate composition was assigned by matrix-assisted laser desorption ionization time of flight mass spectrometry. The sequences were determined at low picomole amounts by a combination of enzymatic digestions with high-performance liquid chromatography, and were composed of disaccharide building units including O [GlcUAβ1-3GalNAc], C [GlcUAβ1-3GalNAc(6S)], A [GlcUAβ1-3GalNAc(4S)], and/or D [GlcUA(2S)β1-3GalNAc(6S)], where 2S, 4S, and 6S represent 2-O-, 4-O-, and 6-O-sulfate, respectively. As many as 24 different sequences including minor ones were revealed, exhibiting a high degree of structural diversity reflecting the enormous heterogeneity of the parent polysaccharides. Nineteen of them were novel, with the other four reported previously as unsaturated counterparts obtained after digestion with chondroitinase. Microarrays of these structurally defined octasaccharide fractions were prepared using low picomole amounts of their lipid-derivatives to investigate the binding specificity of four commercial anti-chondroitin sulfate antibodies CS-56, MO-225, 2H6, and LY111. The results revealed that multiple unique sequences were recognized by each antibody, which implies that the common conformation shared by the multiple primary sequences in the intact chondroitin sulfate chains is important as an epitope for each monoclonal antibody. Comparison of the specificity of the tested antibodies indicates that CS-56 and MO-225 specifically recognize octasaccharides containing an A-D tetrasaccharide sequence, whereas 2H6 and LY111 require a hexasaccharide as a minimum size for their binding, and prefer sequences with A- and C-units such as C-C-A-C (2H6) or C-C-A-O, C-C-A-A, and C-C-A-C (LY111) for strong binding but require no D-unit.",
keywords = "Antibody epitope, Chondroitin sulfate, Octasaccharides, Sugar sequencing, Sulfation",
author = "Deepa, {Sarama S.} and Shuhei Yamada and Shigeyuki Fukui and Kazuyuki Sugahara",
year = "2007",
month = "6",
doi = "10.1093/glycob/cwm021",
language = "English (US)",
volume = "17",
pages = "631--645",
journal = "Glycobiology",
issn = "0959-6658",
publisher = "Oxford University Press",
number = "6",

}

TY - JOUR

T1 - Structural determination of novel sulfated octasaccharides isolated from chondroitin sulfate of shark cartilage and their application for characterizing monoclonal antibody epitopes

AU - Deepa, Sarama S.

AU - Yamada, Shuhei

AU - Fukui, Shigeyuki

AU - Sugahara, Kazuyuki

PY - 2007/6

Y1 - 2007/6

N2 - Twelve octasaccharide fractions were obtained from chondroitin sulfate C derived from shark cartilage after hyaluronidase digestion. Their sugar and sulfate composition was assigned by matrix-assisted laser desorption ionization time of flight mass spectrometry. The sequences were determined at low picomole amounts by a combination of enzymatic digestions with high-performance liquid chromatography, and were composed of disaccharide building units including O [GlcUAβ1-3GalNAc], C [GlcUAβ1-3GalNAc(6S)], A [GlcUAβ1-3GalNAc(4S)], and/or D [GlcUA(2S)β1-3GalNAc(6S)], where 2S, 4S, and 6S represent 2-O-, 4-O-, and 6-O-sulfate, respectively. As many as 24 different sequences including minor ones were revealed, exhibiting a high degree of structural diversity reflecting the enormous heterogeneity of the parent polysaccharides. Nineteen of them were novel, with the other four reported previously as unsaturated counterparts obtained after digestion with chondroitinase. Microarrays of these structurally defined octasaccharide fractions were prepared using low picomole amounts of their lipid-derivatives to investigate the binding specificity of four commercial anti-chondroitin sulfate antibodies CS-56, MO-225, 2H6, and LY111. The results revealed that multiple unique sequences were recognized by each antibody, which implies that the common conformation shared by the multiple primary sequences in the intact chondroitin sulfate chains is important as an epitope for each monoclonal antibody. Comparison of the specificity of the tested antibodies indicates that CS-56 and MO-225 specifically recognize octasaccharides containing an A-D tetrasaccharide sequence, whereas 2H6 and LY111 require a hexasaccharide as a minimum size for their binding, and prefer sequences with A- and C-units such as C-C-A-C (2H6) or C-C-A-O, C-C-A-A, and C-C-A-C (LY111) for strong binding but require no D-unit.

AB - Twelve octasaccharide fractions were obtained from chondroitin sulfate C derived from shark cartilage after hyaluronidase digestion. Their sugar and sulfate composition was assigned by matrix-assisted laser desorption ionization time of flight mass spectrometry. The sequences were determined at low picomole amounts by a combination of enzymatic digestions with high-performance liquid chromatography, and were composed of disaccharide building units including O [GlcUAβ1-3GalNAc], C [GlcUAβ1-3GalNAc(6S)], A [GlcUAβ1-3GalNAc(4S)], and/or D [GlcUA(2S)β1-3GalNAc(6S)], where 2S, 4S, and 6S represent 2-O-, 4-O-, and 6-O-sulfate, respectively. As many as 24 different sequences including minor ones were revealed, exhibiting a high degree of structural diversity reflecting the enormous heterogeneity of the parent polysaccharides. Nineteen of them were novel, with the other four reported previously as unsaturated counterparts obtained after digestion with chondroitinase. Microarrays of these structurally defined octasaccharide fractions were prepared using low picomole amounts of their lipid-derivatives to investigate the binding specificity of four commercial anti-chondroitin sulfate antibodies CS-56, MO-225, 2H6, and LY111. The results revealed that multiple unique sequences were recognized by each antibody, which implies that the common conformation shared by the multiple primary sequences in the intact chondroitin sulfate chains is important as an epitope for each monoclonal antibody. Comparison of the specificity of the tested antibodies indicates that CS-56 and MO-225 specifically recognize octasaccharides containing an A-D tetrasaccharide sequence, whereas 2H6 and LY111 require a hexasaccharide as a minimum size for their binding, and prefer sequences with A- and C-units such as C-C-A-C (2H6) or C-C-A-O, C-C-A-A, and C-C-A-C (LY111) for strong binding but require no D-unit.

KW - Antibody epitope

KW - Chondroitin sulfate

KW - Octasaccharides

KW - Sugar sequencing

KW - Sulfation

UR - http://www.scopus.com/inward/record.url?scp=34447309579&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=34447309579&partnerID=8YFLogxK

U2 - 10.1093/glycob/cwm021

DO - 10.1093/glycob/cwm021

M3 - Article

C2 - 17317718

AN - SCOPUS:34447309579

VL - 17

SP - 631

EP - 645

JO - Glycobiology

JF - Glycobiology

SN - 0959-6658

IS - 6

ER -