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.
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