Mutual separation of hinge-glycopeptide isomers bearing five N-acetylgalactosamine residues from normal human serum immunoglobulin A1 by capillary electrophoresis.
Immunoglobulin A1 (IgA1) from normal human serum is known to have O-linked sugar chains, sialylated Galbeta1,3GalNAc, in the hinge portion. In order to reduce the microheterogenity of the sugar chain, the hinge glycopeptide prepared from IgA1 was sequentially treated with neuraminidase and beta-galactosidase. The asialo-, agalacto-hinge glycopeptide (HGP-SG) composed of a 33-mer peptide (HP33) and N-acetylgalactosamine (GalNAc) residues was obtained. The HGP-SG was separated into three major peaks, A, B and C, by high-performance liquid chromatography (HPLC). Each glycopeptide fraction was further separated by capillary electrophoresis (CE). Peaks A, B and C with HPLC abundantly contained HP33 bearing five and six N-acetylgalactosamine residues (HGP33-5,6GN), HGP33-4,5GN and HGP33-3,4GN, respectively. Among these glycopeptide peaks, only the HGP33-5GN peak was partly split into two peaks based on the CE analysis - HGP33-5GN-alpha and -beta. The glycopeptide, HGP25-5GN shortened by the thermolysin digest of HGP33-SG was also well separated into the alpha and beta forms by CE analysis. No differences in their mass and peptide portion were observed between HGP25-5GN-alpha and -beta. Therefore, the obtained result might indicate that HGP25-5GN-alpha was an isomer of HGP25-5GN-beta differing in its stereospecific structure of the peptide portion and/or the attachment site of the GalNAc residue.
Iwase
H
H
Department of Biochemistry, Kitasato University, Sagamihara, Kanagawa, Japan. iwaseh@med.kitasato-u.ac.jp
Tanaka
A
A
Hiki
Y
Y
Kokubo
T
T
Sano
T
T
Ishii-Karakasa
I
I
Toma
K
K
Kobayashi
Y
Y
Hotta
K
K
D000116:Acetylgalactosamine / Q000737:chemistry*; D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D019075:Electrophoresis, Capillary / Q000379:methods*; D006020:Glycopeptides / Q000737:chemistry / Q000302:isolation & purification*; D006801:Humans; D007536:Isomerism; D008969:Molecular Sequence Data; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D006020:Glycopeptides; D000116:Acetylgalactosamine