Topology and glycosylation of the gastric h.k-atpase

The gastric H,K-ATPase is the protein responsible for acid secretion in the stomach Like the Na.K-ATPase, the H,K-ATPase is a heterodimenc P-type ATPase consisting of an alpha-subunit that traverses the membrane several times and a heavily glycosylated beta-subumt with one transmembrane segment. The number of reported transmembrane segments for the alpha subunit differs, varying from 7-12. Matrix-assisted laser desorption mass spectrometry (MAI.DI-MS) of protease-released peptides from H,K-ATPase enriched vesicles indicated the presence of six cytoplasmic regions of the H,K-ATPase, Ala4-Argl05, Serl65-Lys22, Arg361Lys793, Ala838-Lys851, Thr950-Arg964 and Lysl018-Tyrl035 The presence of6 cyloplasmic regions support an even number of transmembrane domains and favor a topological model with 10 transmembrane domains for the a-subunit Initial analysis of the masses of peptides from the transmembrane segments support this topology Only peptides from the N-terminus of the bctasubunit were observed confirming the single transmembrane domain model with most of the bela-subunit being lumenally oriented to the harsh, acidic environment of the stomach The location and structural features of the bela-subunit oligosaccharides \vas investigated using a combination of HPLC separation of tryptic peptides, MALDI-MS and glycosidase digestions The studies revealed that all 7 N-Iinked sequons on the rabbit H,K-ATPase 99, 103, 130, 146, 161, 193 and 222 are fully glycosylated. While two sites, Asn99 and Asnl 93, were glycosylaled with oligomannosidic oligosaccharides, all the other sites contained lactosamine-typc structures Analysis of the total oligosaccharide pool revealed 24 lactosamine species (hi-, tri- and tetraantennary structures), with all branches terminated in alpha-linked Gal residues, and most possessing a single Fuc residue. Nine novel oligosaccharides contained multiple alpha-linked Gal residues per branch Bi- and tri-antennary structures, with and without lactosamine repeals were observed at Asnl46 and Asnlol. Tetra-antennary structures with lactosamine repeats were found only at Asn 130, and this site also contained most of the structures with multiple alphalinked Gal residues per branch. Cross-species glycosylation studies are under investigation (The mass spectrometry was performed at the UCSF Mass Spectromctrv. supported bv NTH NCRR-BRTP01614.