Shuji Miyagawa1, Akira Maeda2, Hiroshi Eguchi2, Takuji Kawamura2, Noriyuki Kashiyama2, Takehisa Ueno2, Noriaki Usui2, Sachiko Kondo3, Kazuaki Nakano4, Hitomi Matsunari4, Masahito Watanabe4, Hiroshi Nagashima4. 1. Division of Organ Transplantation (E9), Department of Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address: miyagawa@orgtrp.med.osaka-u.ac.jp. 2. Division of Organ Transplantation (E9), Department of Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. 3. Medical & Biological Laboratories Co., Ltd., Sakae 4-5-3, Naka-ku, Nagoya, Aichi 460-0008, Japan. 4. Department of Life Science, Meiji University, Higashisanda 1-1-1, Tama-ku, Kawasaki, Kanagawa 214-8571, Japan.
Abstract
BACKGROUND: N-glycans isolated from neonatal porcine islet-like cell clusters (NPCCs) were analyzed by a mapping technique, to examine the differences in glycosylation and antigenicity between adult pig islets (APIs) and NPCCs. METHODS: NPCCs were isolated from 1-to-3 day-old neonatal wild-type pigs and cultured for 9 days, using the technique described by Korbutt et al. The extract was proteolyzed by treatment with a chymotrypsin and trypsin mixture and further digested with glycoamidase A to release the N-glycans. After the removal of the peptide materials, the reducing ends of the N-glycans were derivatized with 2-aminopyridine. This mixture was applied to DEAE, amide and ODS columns. PA-oligosaccharides were also subjected to MALDI TOF-MS analysis. RESULTS: The NPCC glycans were comprised of 14 neutral, 5 mono-sialyl and 5 di-sialyl glycans. As a feature of the N-glycans of NPCC, NPCC contained large amounts of high mannose structures. On the other hand, all of the hybrid and complex types contained a Fucα1-6GlcNAc structure, but were not modified with sulfate residues. Among them, the NPCC preparation contained five neutral and two mono-sialyl glycans and two di-sialyl glycans that were not typically found in adult islets, and seven of these nine were not detected in human islets. Moreover, most of the structures could be clearly identified in this study. CONCLUSIONS: The data herein will be helpful for future studies of the glycoantigen associated with NPCC.
BACKGROUND:N-glycans isolated from neonatal porcine islet-like cell clusters (NPCCs) were analyzed by a mapping technique, to examine the differences in glycosylation and antigenicity between adult pig islets (APIs) and NPCCs. METHODS: NPCCs were isolated from 1-to-3 day-old neonatal wild-type pigs and cultured for 9 days, using the technique described by Korbutt et al. The extract was proteolyzed by treatment with a chymotrypsin and trypsin mixture and further digested with glycoamidase A to release the N-glycans. After the removal of the peptide materials, the reducing ends of the N-glycans were derivatized with 2-aminopyridine. This mixture was applied to DEAE, amide and ODS columns. PA-oligosaccharides were also subjected to MALDI TOF-MS analysis. RESULTS: The NPCC glycans were comprised of 14 neutral, 5 mono-sialyl and 5 di-sialyl glycans. As a feature of the N-glycans of NPCC, NPCC contained large amounts of high mannose structures. On the other hand, all of the hybrid and complex types contained a Fucα1-6GlcNAc structure, but were not modified with sulfate residues. Among them, the NPCC preparation contained five neutral and two mono-sialyl glycans and two di-sialyl glycans that were not typically found in adult islets, and seven of these nine were not detected in human islets. Moreover, most of the structures could be clearly identified in this study. CONCLUSIONS: The data herein will be helpful for future studies of the glycoantigen associated with NPCC.