Elucidation of binding specificity of Jacalin toward O-glycosylated peptides: quantitative analysis by frontal affinity chromatography

• Published in: Glycobiology (Oxford) 2006-01, Vol.16 (1), p.46-53
• Main Authors: Tachibana, Kouichi, Nakamura, Sachiko, Wang, Han, Iwasaki, Hiroko, Tachibana, Kahori, Maebara, Kanako, Cheng, Lamei, Hirabayashi, J, Narimatsu, H
• Format: Article
• Language: English
• Subjects: 5-carboxyfluorescein succinimidyl ester; Alexa Fluor 488; Antigens, Tumor-Associated, Carbohydrate - chemistry; Antigens, Tumor-Associated, Carbohydrate - metabolism; benzylp; Chromatography, Affinity; FAC; FAM; frontal affinity chromatography; GalNac; Glucosyltransferases - chemistry; glycopeptide; high-performance liquid chromatography; hinge peptide; HPLC; human IgA1 hinge domain peptide; Humans; Jacalin; mucin; Mucins - chemistry; Mucins - metabolism; N-acetylgalactosamine; O-glycan; Oligopeptides - chemistry; Oligopeptides - metabolism; p-nitrophenyl; Plant Lectins - chemistry; Plant Lectins - metabolism; pNP; pp-GalNAc-T; Protein Binding; sialyl-T; sialyl-Tn; STn; Substrate Specificity; TBS; Tris-buffered saline; UDP; UDP-GalNAc : polypeptide N-acetylgalactosaminyltransferase; uridine diphosphate
• ISSN: 0959-6658; 1460-2423
• DOI: 10.1093/glycob/cwj038

Jacalin, a lectin from the jackfruit Artocarpus integrifolia, has been known as a valuable tool for specific capturing of O-glycoproteins such as mucins and IgA1. Though its sugar-binding preference for T/Tn-antigens is well established, its detailed specificity has not been elucidated. In this study, we prepared a series of mucin-type glycopeptides using human glycosyltransferases, that is, ST6GalNAc1, Core1Gal-T1 and -T2, {szligbeta}3Gn-T6, and Core2GnT1, and investigated their binding to immobilized Jacalin by frontal affinity chromatography (FAC). As a result, consistent with the previous observation, Jacalin showed high affinity for T-antigen (Core1) and Tn-antigen (alpha N-acetylgalactosamine)-attached peptides. Furthermore, we here show as novel findings that (1) Jacalin also showed significant affinity for Core3 and sialyl-T (ST)-attached peptides, but (2) Jacalin could not bind to Core2, Core6, and sialyl-Tn (STn)-attached peptides. The results were also confirmed by FAC using p-nitrophenyl (pNP)-derivatized saccharides. In conclusion, Jacalin binds to a GalNAc[alpha]1-peptide, in which C6-OH of [alpha]GalNAc is free (i.e., Core1, Tn, Core3, and ST), whereas it cannot recognize a GalNAc[alpha]1-peptide with a substitution at the C6 position (i.e., Core2, Core6, and STn). These findings provide useful information when applying jacalin for functional analysis of mucin-type glycoproteins and glycopeptides.