Epitopic structure of Tn glycophorin A for an anti-Tn antibody (MLS 128).
Glycophorin A was digested with glycoprotease (Pasteurella haemolytica) and the digest was fractionated by a combination of high-pressure column chromatographies to produce the glycopeptides GPA-1 to GPA-6. Sequence analysis of the glycopeptides revealed that two serine residues (Ser-14 and Ser-15) are not glycosylated, Thr-17 and Ser-19 being glycosylated instead, in disagreement with the accepted structure. The glycopeptides thus obtained were treated with sialidase and beta-galactosidase. The Tn antigenicity, as assayed by the binding to a monoclonal anti-Tn antibody (MLS 128), was found exclusively in the glycopeptides including three (cluster I) or four (cluster II) consecutive residues of GalNAc-Ser/Thr, whereas the glycopeptide (GPA-2) containing two nonconsecutive GalNAc-Ser/Thr residues had practically no Tn antigenicity. The immunoreactivities of GPA-1 and GPA-3, containing both clusters I and II, and GPA-4, containing cluster II, were 63% (calcd. 67%), 81% (calcd. 86%), and 50% (calcd. 50%), respectively, of the immunoreactivity of GPA-5 or GPA-6, containing cluster I (the average being taken as the basis), based on the reactivity per GalNAc residue. These results indicate that clusters I and II react with the antibody to the same extent. The structure consisting of three consecutive glycosylated Ser/Thr residues may be essential for Tn antigenicity in the light of previous results for ovine submaxillary mucin.
Nakada
H
H
Department of Biotechnology, Faculty of Engineering, Kyoto Sangyo University, Japan.
Inoue
M
M
Numata
Y
Y
Tanaka
N
N
Funakoshi
I
I
Fukui
S
S
Mellors
A
A
Yamashina
I
I
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000737:chemistry / Q000276:immunology*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D000939:Epitopes / Q000032:analysis* / Q000737:chemistry; D006020:Glycopeptides / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D006021:Glycophorins / Q000737:chemistry* / Q000276:immunology; D006801:Humans; D016978:Mannheimia haemolytica / Q000201:enzymology; D008666:Metalloendopeptidases; D008969:Molecular Sequence Data; D009077:Mucins / Q000502:physiology; D012756:Sheep
D000911:Antibodies, Monoclonal; D015295:Antigens, Tumor-Associated, Carbohydrate; D000939:Epitopes; D006020:Glycopeptides; D006021:Glycophorins; D009077:Mucins; C040286:Tn antigen; D008666:Metalloendopeptidases; C070738:O-sialoglycoprotein endopeptidase