Elucidation of an essential structure recognized by an anti-GalNAc alpha-Ser(Thr) monoclonal antibody (MLS 128).
To determine the epitopic structure for an anti-GalNAc alpha-Ser(Thr) (anti-Tn) monoclonal antibody, MLS 128, asialo-ovine submaxillary mucin was digested with various proteases, and the digests were fractionated by immunoaffinity column chromatography and high performance liquid chromatography. From the tryptic digest, a glycopeptide, GP-I, and five other glycopeptides, GP-1-5, were obtained as bound and unbound fractions, respectively, of the immunoaffinity column. By solid phase radioimmunoassaying, it was found that GP-I was strongly immunoreactive, whereas GP-1-5 were poorly immunoreactive. On treatment with V8 protease, GP-I was converted to two glycopeptides, one with poor reactivity and the other with intermediate reactivity. From the thermolysin digest, the smallest fragment, GP-II, was isolated, which was as strongly immunoreactive as GP-I. GP-II corresponded to a part of GP-I, its sequence being Leu-Ser*-Glu-Ser*-Thr*-Thr*-Gln-Leu-Pro-Gly, where asterisks denote amino acids to which an alpha-GalNAc residue is attached. Other anti-Tn monoclonal antibodies, NCC-LU-35 and CA 3239, showed essentially the same reactivity to these glycopeptides as MLS 128 did. The glycopeptides (GP-1-5), which exhibited poor immunoreactivity, contained various GalNAc-containing structures, such as GalNAc-Ser, GalNAc-Thr, GalNAc-Ser-(GalNAc)-Ser, and GalNAc-Thr-(GalNAc)-Thr. These results indicate that a glycopeptide including a cluster structure, Ser*-Thr*-Thr*, is an essential part of the epitope recognized by anti-Tn antibodies.
Nakada
H
H
Department of Biotechnology, Faculty of Engineering, Kyoto Sangyo University, Japan.
Numata
Y
Y
Inoue
M
M
Tanaka
N
N
Kitagawa
H
H
Funakoshi
I
I
Fukui
S
S
Yamashina
I
I
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001212:Asialoglycoproteins; D002241:Carbohydrates / Q000276:immunology; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D000939:Epitopes / Q000276:immunology; D006020:Glycopeptides / Q000276:immunology; D008969:Molecular Sequence Data; D009077:Mucins / Q000276:immunology; D011487:Protein Conformation; D011863:Radioimmunoassay; D012756:Sheep; D013363:Submandibular Gland / Q000378:metabolism; D013820:Thermolysin / Q000737:chemistry; D014357:Trypsin
D000911:Antibodies, Monoclonal; D001212:Asialoglycoproteins; D002241:Carbohydrates; D000939:Epitopes; D006020:Glycopeptides; D009077:Mucins; D014357:Trypsin; D013820:Thermolysin