The N-linked oligosaccharides of the β-subunit of rabbit gastric H,K- ATPase: Site localization and identification of novel structures

The gastric H,K-ATPase is responsible for acid secretion by parietal cells. Its β-subunit is a glycoprotein which is exposed to the harsh, acidic environment of the stomach. The location and structural features of the N- linked oligosaccharides were determined using matrix-assisted laser desorption ionization mass spectrometry (MALDI/MS) (in conjunction with mass' composition analysis and exoglycosidase digestions), Edman degradation, and monosaccharide composition analysis. All seven N-linked sequons at positions 99, 103, 130, 146, 161,193, and 222 were fully glycosylated. An unusual restricted array of oligosaccharides was observed at individual Asn residues. Asn⁹⁹ was modified exclusively with oligomannosidic-type structures (Man₆GlcNAc₂-Man₈GlcNAc₂). Asn¹⁹³ contained both oligomannosidic (Man₅GlcNAc₂-Man₈GlcNAc₂) 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 dissociation was required to demonstrate that sequons separated by a single residue (⁹⁹Asn-Ile-Ser-Asp-Asn-Arg-Thr¹⁰⁵) were modified with only oligomannose and lactosamine structures, respectively. Analysis of the total oligosaccharide pool using MALDI/MS and exoglycosidase analysis revealed 24 lactosamine species (bi-, tri-, and tetraantennary structures), with all branches terminated in α-linked Gal residues, most possessing a single Fuc residue. Nine novel oligosaccharides contained multiple α-linked Gal residues per branch. Bi- and triantennary structures, with and without lactosamine repeats, were observed at Asn¹⁴⁶ and Ash¹⁶¹. Tetraantennary structures with lactosamine repeats were found only at Asn¹³⁰, and this site also contained most of the structures with multiple α-linked Gal residues per branch.