A monoclonal antibody, PGM34, against 6-sulfated blood-group H type 2 antigen, on the carbohydrate moiety of mucin.
Mucin, a major component of mucus, is a highly O-glycosylated, high-molecular-mass glycoprotein extensively involved in the physiology of gastrointestinal mucosa. To detect and characterize mucins derived from site-specific mucous cells, we developed a monoclonal antibody, designated PGM34, by immunizing a mouse with purified pig gastric mucin. The reactivity of PGM34 with mucin was inhibited by periodate treatment of the mucin, but not by trypsin digestion. This suggests that PGM34 recognizes the carbohydrate portion of mucin. To determine the epitope, oligosaccharide-alditols obtained from pig gastric mucin were fractionated by successive gel-filtration, ion-exchange, and normal-phase HPLC, and tested for reactivity with PGM34. Two purified oligosaccharide-alditols that reacted with PGM34 were obtained. Their structures were determined by NMR spectroscopy as Fucalpha1-2Galbeta1-4GlcNAc(6SO(3)H)beta1-6(Fucalpha1-2Galbeta1-3)GalNAc-ol and Fucalpha1-2Galbeta1-4GlcNAc(6SO(3)H)beta1-6(Galbeta1-3)GalNAc-ol. None of the defucosylated or desulfated forms of these oligosaccharides reacted with PGM34. Thus, the epitope of PGM34 was determined as the Fucalpha1-2Galbeta1-4GlcNAc(6SO(3)H)beta- sequence. Immunohistochemical examination of rat gastrointestinal tract showed that PGM34 stained surface mucous cells close to the generative cell zone in the gastric fundus and goblet cells in the small intestine, but only slightly stained antral mucous cells in the stomach. These data, taken together, show that PGM34 is a very useful tool for elucidating the role of mucins with characteristic sulfated oligosaccharides.
Tsubokawa
Daigo
D
Department of Biochemistry, Kitasato University Graduate School of Medical Sciences, Sagamihara, Japan.
Goso
Yukinobu
Y
Sawaguchi
Akira
A
Kurihara
Makoto
M
Ichikawa
Takafumi
T
Sato
Noriko
N
Suganuma
Tatsuo
T
Hotta
Kyoko
K
Ishihara
Kazuhiko
K
D000017:ABO Blood-Group System / Q000276:immunology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000276:immunology*; D001894:Borohydrides / Q000737:chemistry; D002240:Carbohydrate Sequence; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis / Q000276:immunology*; D005643:Fucose / Q000276:immunology; D005752:Gastric Mucins / Q000737:chemistry / Q000276:immunology*; D005753:Gastric Mucosa / Q000737:chemistry / Q000166:cytology / Q000648:ultrastructure; D007150:Immunohistochemistry; D007413:Intestinal Mucosa / Q000737:chemistry / Q000166:cytology / Q000648:ultrastructure; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000032:analysis / Q000276:immunology* / Q000302:isolation & purification; D010504:Periodic Acid / Q000737:chemistry; D051381:Rats; D017208:Rats, Wistar; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013552:Swine; D014357:Trypsin / Q000737:chemistry
D000017:ABO Blood-Group System; D000911:Antibodies, Monoclonal; D001894:Borohydrides; D000939:Epitopes; D005752:Gastric Mucins; D009844:Oligosaccharides; D010504:Periodic Acid; D005643:Fucose; C009288:metaperiodate; D014357:Trypsin