THE N-LINKED OLIGOSACCHARIDES OF THE BETA-SUBUNIT OF RABBIT GASTRIC H,K-ATPASE - SITE LOCALIZATION AND IDENTIFICATION OF NOVEL STRUCTURES

The gastric PI,K-ATPase is responsible for acid secretion by parietal cells, Its beta-subunit is a glycoprotein which is exposed To the hars h, acidic environment of the stomach. The location and structural feat ures of the N-linked oligosaccharides were determined using matrix-ass isted laser desorption ionization mass spectrometry (MALDI/MS) (in con junction with mass composition analysis and exoglycosidase digestions) , Edman degradation, and monosaccharide composition analysis. All seve n N-linked sequons at positions 99, 103, 130, 146, 161, 193, and 222 w ere fully glycosylated. An unusual restricted array of oligosaccharide s was observed at individual Asn residues, Asn(99) was modified exclus ively with oligomannosidic-type structures (Man(6)GlcNAc(2)-Man(8)GlcN Ac(2)). Asn(193) contained both oligomannosidic (Man(5)GlcNAc(2)-Man(8 )GlcNAc(2)) and lactosamine-type structures, indicating significant '' leakiness'' in the pathway which converts oligomannose to lactosamine- type at a single glycosylation site. MALDI/MS with collision-induced d issociation was required To demonstrate that sequons separated by a si ngle residue ((99)Asn-Ile-Ser-Asp-Asn-Arg-Thr(105)) were modified with only oligomannose and lactosamine structures, respectively, Analysis of the total oligosaccharide pool using MALDI/MS and exoglycosidase an alysis revealed 24 lactosamine species (bi-, tri, and tetraantennary s tructures), with all branches terminated in alpha-linked Gal residues, most possessing a single Fuc residue, Nine novel oligosaccharides con tained multiple alpha-linked Gal residues per branch, Bi-and triantenn ary structures, with and without lactosamine repeats, were observed at Asn(146) and Asn(161). Tetraantennary structures with lactosamine rep eats were found only at Asn(130), and this site also contained most of the structures with multiple alpha-linked Gal residues per branch.