STRUCTURAL ELUCIDATION OF GASTRIC H+, K+ ATPASE

胃 H , K ATP 酶的结构解析

• 批准号: 6308891
• 负责人: ROBERT REID TOWNSEND
• 金额: $ 0.99万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-03-01 至 2002-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6308891
• 关键词: Mammalia; biomedical resource; drug /agent; proteins; spectrometry; stomach; structural biology

The gastric H+,K+-ATPase is a cation antiporter which consists of a and b subunits with M1 = 94,000 subunits with M1 = 60-80,000, respectively (1). The cloned rat a subunit consists of 1033 amino acids (MW = 114,012) and 5 potential glycosylation sites (2), which have been proposed not to be glycosylated on the basis of their suspected location on the cytoplasmic side of the plasma membrane, the inability to bind to lectin Sepharose columns, and the non-specific staining of the a-subunit with dansyl hydrazine after periodate treatment of gel bands (3). We have recently confirmed that the a-subunit is definitively not glycosylated on the basis of monosaccharide analysis of the electro-blotted band (4). The cloned rat b subunit consists of 294 amino acids (MW = 33,689) with 7 potential N-glycosylation sites (5). The rabbit b subunit stained positively for carbohydrate on SDS gels, bound to wheat germ agglutinin and Ricin A Sepharose columns, changed mobility after treatment with PNGase and was found to contain hexose and hexosamines (3). We found that the b-subunit from rabbit contained a ratio of Gal and GlcNAc to Man which suggested the presence of lactosamine-type oligosaccharides (4). Further, the detection of GalN on the protein after removal of all N-linked oligosaccharides indicated the presence of O-linked structures. The b subunit was found to be devoid of sialic acids, a noteworthy finding since sialyl transferase activity is regio-specific in gastrointestinal tissues (6). We propose to extend these initial studies on the structure of this membrane glycoprotein with the following specific aims. (1) Determine the precise molecular weight of the glycoforms of the b-subunit using matrix-assisted laser desorption MS. (2) Locate the sites of glycosylation through analyses using LC/electrospray MS. (3) Elucidate the oligosaccharide structures and eventually define the ensemble at each eptide locus. Due to glycosylation, the b subunit migrates as a smear by SDS PAGE with an estimated molecular weight of 60-80 kDa. Precise molecular weight determination of the individual glycoforms may enable an estimation of the number of glycosylation sites which are present and provide "global" measurements to direct strategies for the additional structural studies in specific aims2 and 3. The number of the 7 potential N-linked sites which are glysoylated or whether somesites are variably glycosylated is not known. LC/electrospray MS will be used to ascertain which N-linked sites are glycosylated and to locate the O-glycosylated loci. Our third specific aim is to elucidate the oligosaccharide structures. Previous studies, using an HPLC mapping approach, indicated that the enzymatically-released oligosaccharides on the b subunit do not co-elute with any of the commonly found lactosamine or high-mannose oligosaccharides. Using HPLCsingular oligosaccharides will be prepared and their molecular weight determined using MALD-MS. Structures will be deduced using a combination of chemical and enzymatic cleavages and appropriate mass spectrometric analyses available at the Center. 1. Forte, J.G. and Lee, H.C. (1977) Gastroenterology, 73, 921-2. 2. Shull, G.E. and Lingrel, J.B. (1986) J. Biol. Chem. 261, 16788-16791. 3. Okamoto, C.T., Karpilow, J.M., Smolka, A. and Forte, J.G. (1990) Biochim. Biophys. Acta 1037, 360-372. 4. Weitzhandler, M., Kadlecik, D., Avdalovic, N., Forte, J., Chow, D. and Townsend, R.R. accepted J. Biol. Chem. (Feb/Mar) 5. Canfield, V.A., Okamoto, C.T., Chow, D., Dortman, J., Gros, P., Forte, J.G. and Levenson, R. (1990) J. Biol. Chem. 265, 19878-19884. 6. Taatjes, D.J., Roth, J., Weinstein, J., Paulson, J.C. (1988) J. Biol. Chem. 263, 6302-6309.

胃H+，K+-ATP酶是一种阳离子逆向转运蛋白， a和B亚基，M1 = 94，000亚基，M1 = 60- 80，000， 分别（1）。 克隆的大鼠a亚基由1033个氨基酸组成 酸（MW = 114，012）和5个潜在的糖基化位点（2）， 已经提出不被糖基化，基于它们的 在质膜的细胞质侧上的可疑位置， 不能与凝集素琼脂糖柱结合， 高碘酸盐后用丹酰肼对a亚基染色 凝胶带的处理（3）。 我们最近证实， a-亚基基于以下确定地不被糖基化： 电印迹条带的单糖分析（4）。 克隆的 大鼠B亚基由294个氨基酸组成（MW = 33，689），其中7个氨基酸是 潜在的N-糖基化位点（5）。 兔B亚单位染色 SDS凝胶上碳水化合物阳性，与小麦胚芽结合 凝集素和蓖麻毒素A琼脂糖凝胶柱，改变迁移率后， 用PNGase处理，发现含有己糖和己糖胺 （三）、 我们发现兔的b亚基含有一定比例的Gal， 和GlcNAc的人，这表明存在乳糖胺型 寡糖（4）。 此外，蛋白质上GalN的检测 去除所有N-连接低聚糖后表明存在 O-连接结构。 发现B亚基缺乏 唾液酸，一个值得注意的发现，因为唾液酸转移酶活性 在胃肠道组织中具有区域特异性（6）。 我们建议 将这些关于这种膜结构的初步研究 糖蛋白具有以下特定目的。 (1)确定 b亚基糖型的精确分子量， 基质辅助激光解吸质谱（matrixassisted laser desorption MS）。（2） 通过使用LC/电喷雾MS分析糖基化。（3） 阐明寡糖结构，并最终确定 在每个肽基因座处的整体。 由于糖基化，B亚基 通过SDS PAGE以涂片形式迁移，估计分子量为 60-80 kDa。 精确测定单个分子量 糖型可以使得能够估计糖基化的数量 存在并提供“全球”测量的站点， 在具体目标中进行额外结构研究的战略2， 3. 发生糖基化的7个潜在N-连接位点的数量 或者某些位点是否被非糖基化尚不清楚。 LC/电喷雾MS将用于确定哪些N-连接位点是 糖基化和定位O-糖基化位点。 我们的第三 具体目的是阐明寡糖结构。 先前 使用HPLC图谱方法的研究表明， B亚基上酶促释放的寡糖不 与任何常见的乳糖胺或高甘露糖共同作用 低聚糖。 使用HPLC将单一寡糖 制备并使用MALD-MS测定它们的分子量。 结构将使用化学和 酶裂解和适当的质谱分析 在中心可用。 1. Forte，J.G.和Lee，H.C.（一九七七年） Gastroenterology，73，921-2. 2. Shull，G.E.和Lingrel，J.B.（一九八六年） J. 261，16788-16791。 3. 冈本，CT，Karpilow，J.M.， Smolka，A.和Forte，J.G.（1990）生物化学。 Biophys. Acta 1037， 360-372. 4. Weitzhandler，M.，Kadlecik，D.，Avdalovic，N.，福特， J.，Chow，D.和汤森，R.R.接受J. Biol. Chem.（2月/3月）5。 弗吉尼亚州坎菲尔德，冈本，CT，周，D。，Dortman，J.，Gros，P.，福特， J.G.和Levenson，R。（1990）J.Biol.Chem.265，19878-19884。 6. Taatjes，D.J.，Roth，J.，Weinstein，J.，保尔森，J.C.等人（1988）J. Biol. Chem. 263，6302-6309。