Tyagarajan K, Lipniunas P H, Townsend R R, Forte J G
Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3200, USA.
Biochemistry. 1997 Aug 19;36(33):10200-12. doi: 10.1021/bi9706125.
The gastric H,K-ATPase is responsible for acid secretion by parietal cells. Its beta-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 alpha-linked Gal residues, most possessing a single Fuc residue. Nine novel oligosaccharides contained multiple alpha-linked Gal residues per branch. Bi- and triantennary structures, with and without lactosamine repeats, were observed at Asn146 and Asn161. Tetraantennary structures with lactosamine repeats were found only at Asn130, and this site also contained most of the structures with multiple alpha-linked Gal residues per branch.
胃H⁺，K⁺-ATP酶负责壁细胞分泌胃酸。其β亚基是一种糖蛋白，暴露于胃部恶劣的酸性环境中。利用基质辅助激光解吸电离质谱法（MALDI/MS）（结合质量组成分析和外切糖苷酶消化）、埃德曼降解法和单糖组成分析确定了N-连接寡糖的位置和结构特征。99、103、130、146、161、193和222位的所有7个N-连接糖基化位点均被完全糖基化。在各个天冬酰胺残基处观察到一组不寻常的受限寡糖。天冬酰胺99仅被低聚甘露糖型结构（Man6GlcNAc2-Man8GlcNAc2）修饰。天冬酰胺193同时含有低聚甘露糖型（Man5GlcNAc2-Man8GlcNAc2）和乳糖胺型结构，表明在单个糖基化位点将低聚甘露糖转化为乳糖胺型的途径存在显著的“渗漏”。需要使用碰撞诱导解离的MALDI/MS来证明被单个残基隔开的糖基化位点（99位天冬酰胺-异亮氨酸-丝氨酸-天冬氨酸-天冬酰胺-精氨酸-105位苏氨酸）分别仅被低聚甘露糖和乳糖胺结构修饰。使用MALDI/MS和外切糖苷酶分析对总寡糖库进行分析，发现了24种乳糖胺种类（双天线、三天线和四天线结构），所有分支均以α连接的半乳糖残基结尾，大多数含有单个岩藻糖残基。9种新型寡糖每个分支含有多个α连接的半乳糖残基。在天冬酰胺146和天冬酰胺161处观察到有和没有乳糖胺重复序列的双天线和三天线结构。仅在天冬酰胺130处发现有乳糖胺重复序列的四天线结构，并且该位点还包含大多数每个分支含有多个α连接半乳糖残基的结构。
