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Development of a mouse monoclonal antibody against the chondroitin sulfate-protein linkage region derived from shark cartilage

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Abstract

Glycosaminoglycans (GAGs) like chondroitin sulfate (CS) and heparan sulfate (HS) are synthesized on the tetrasaccharide linkage region, GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser, of proteoglycans. The Xyl can be modified by 2-O-phosphate in both CS and HS, whereas the Gal residues can be sulfated at C-4 and/or C-6 in CS but not in HS. To study the roles of these modifications, monoclonal antibodies were developed against linkage glycopeptides of shark cartilage CS proteoglycans, and one was characterized in detail. This antibody bound hexa- and pentasaccharide-peptides more strongly than unsaturated tetrasaccharide-peptides with the unnatural fourth sugar residue (unsaturated hexuronic acid), suggesting the importance of the fifth and/or fourth saccharide residue GalNAc-5 and/or GlcA-4. Its reactivity was not affected by treatment with chondro-4-sulfatase or alkaline phosphatase, suggesting that 4-O-sulfate on the Gal residues and 2-O-phosphate on the Xyl residue were not recognized. Treatment with weak alkali to cleave the Xyl-Ser linkage completely abolished the binding activity, suggesting the importance of the peptide moiety of the hexasaccharide-peptide for the binding. Based on the amino acid composition and matrix-assisted laser desorption ionization time-of-flight mass spectrometry analyses, it was revealed that the peptide moiety is composed of four amino acids, Ser, Pro, Gly, and Glu. Furthermore, the antibody stained wild-type CHO cells significantly, but much weakly mutant cells deficient in xylosyl- or galactosyltransferase-I required for the biosynthesis of the linkage region. These results suggest that the antibody recognizes the structure GalNAc(±6-O-sulfate)-GlcA-Gal-Gal-Xyl-Ser-(Pro, Gly, Glu). The antibody will be a useful tool for investigating the significance of the linkage region in the biosynthesis and/or intracellular transport of different GAG chains especially since such tools to study the linkage region are lacking.

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Abbreviations

2AB:

2-aminobenzamide

BSA:

bovine serum albumin

CS:

chondroitin sulfate

DS:

dermatan sulfate

ELISA:

enzyme-linked immunosorbent assay

GAG:

glycosaminoglycan

Gal:

D-galactose

GalNAc:

N-acetyl-D-galactosamine

GlcA:

D-glucuronic acid

GlcN:

D-glucosamine

GlcNAc:

N-acetyl-D-glucosamine

Hep:

heparin

∆HexA:

4,5-unsaturated hexuronic acid or 4-deoxy-α-L-threo-hex-4-enepyranosyluronic acid

HPLC:

high performance liquid chromatography

HS:

heparan sulfate

IdoA:

L-iduronic acid

PG:

proteoglycan

Xyl:

D-xylose

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Acknowledgements

This work was supported in part by Grants-in-aid for Scientific Research (B) (20390019) (to K.S.) and Scientific Research (C) (21590057) (to S.Y.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT), and for Regional R&D Proposal-Based Program from Northern Advancement Center for Science & Technology of Hokkaido, Japan (to K.S.).

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Authors and Affiliations

  1. Laboratory of Proteoglycan Signaling and Therapeutics, Graduate School of Life Science, Hokkaido University, Frontier Research Center for Post-Genomic Science and Technology, Nishi 11-choume, Kita 21-jo, Kita-ku, Sapporo, Hokkaido, 001-0021, Japan

    Chizuru Akatsu, Duriya Fongmoon, Shuji Mizumoto, Shuhei Yamada & Kazuyuki Sugahara

  2. Thailand Excellence Center for Tissue Engineering, Department of Biochemistry, Faculty of Medicine, Chiang Mai University, Chiang Mai, 50200, Thailand

    Duriya Fongmoon & Prachya Kongtawelert

  3. Lampang Cancer Center, Department of Medicine, Ministry of Public Health, Lampang, 52000, Thailand

    Duriya Fongmoon

  4. Institut de Chimie Organique et Analytique—CNRS UMR 6005, UFR Sciences, Université d’Orléans, BP 6759, 45067, Orléans Cedex, France

    Jean-Claude Jacquinet

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  1. Chizuru Akatsu
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  2. Duriya Fongmoon
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  3. Shuji Mizumoto
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  4. Jean-Claude Jacquinet
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  6. Shuhei Yamada
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  7. Kazuyuki Sugahara
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Correspondence to Shuhei Yamada.

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Akatsu, C., Fongmoon, D., Mizumoto, S. et al. Development of a mouse monoclonal antibody against the chondroitin sulfate-protein linkage region derived from shark cartilage. Glycoconj J 27, 387–399 (2010). https://doi.org/10.1007/s10719-010-9286-1

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  • DOI: https://doi.org/10.1007/s10719-010-9286-1

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