Binding characteristics of an anti-Siaalpha2-6GalNAcalpha-Ser/Thr (sialyl Tn) monoclonal antibody (MLS 132).
To determine the epitopic structure for an anti-Siaalpha2-6GalNAcalpha-Ser/Thr (anti-sialyl Tn) monoclonal antibody, MLS 132, ovine submaxillary mucin (OSM) was digested with the combination of trypsin and thermolysin and the digest fractionated by immunoaffinity column chromatography and HPLC. From tryptic digest, a major glycopeptide designated as T3 was obtained as an immunoaffinity column-bound fraction. On solid-phase radioimmunoassay, it was found that T3 exhibited strong immunoreactivity with MLS 132. On treatment with thermolysin, T3 was converted into about 50 fragments, as found on fractionation by HPLC. Several of them were strongly immunoreactive and had the same amino acid sequence, i.e. Phe-Ser*-Gly-Glu-Thr*-Ser*-Thr*-Thr*-Val-Ile-Ser*-Gly-Thr*-Asn-Val, where asterisks denote the sites of attachment of carbohydrate. Of these, one was fully sialylated, the others having one Ser or Thr with unsialylated GalNAc attached. Results of analyses of the carbohydrate attached in these glycopeptides led us to postulate that a cluster composed of four sialyl Tn antigens is the essential epitopic structure for MLS 132.
Tanaka
N
N
Department of Biotechnology, Faculty of Engineering, Kyto Sangyo, University, Japan.
Nakada
H
H
Inoue
M
M
Yamashina
I
I
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000737:chemistry* / Q000276:immunology*; D002846:Chromatography, Affinity; D000939:Epitopes / Q000737:chemistry; D006020:Glycopeptides / Q000737:chemistry / Q000276:immunology; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D012756:Sheep
D000911:Antibodies, Monoclonal; D015295:Antigens, Tumor-Associated, Carbohydrate; D000939:Epitopes; D006020:Glycopeptides; C055491:sialosyl-Tn antigen