Ziegler F D, Maley F, Trimble R B
Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany 12201.
J Biol Chem. 1988 May 25;263(15):6986-92.
There are 14 potential Asn-X-Thr/Ser glycosylation sites, or sequons, in the yeast external invertase sequence. Of these, 13 are wholly or partially glycosylated to give an average of 9-10 oligosaccharides/subunit (Reddy, V. A., Johnson, R. S., Biemann, K., Williams, R. S., Ziegler, F. D., Trimble, R. B., and Maley, F. (1988) J. Biol. Chem. 263, 6978-6985). On digestion of native holoenzyme by endo-beta-N-acetylglucosaminidase H (Endo H) an average of about seven oligosaccharides per subunit are released without affecting enzyme activity (Trimble, R. B., and Maley, F. (1977) J. Biol. Chem. 252, 4409-4412). To determine whether the remaining Endo H-resistant chains were restricted to a limited number of unique sequons or were randomly distributed on all 13, Endo H-treated native invertase was digested with either thermolysin or trypsin and the resultant glycopeptides isolated by reversed-phase high pressure liquid chromatography and gel filtration. It was found that the oligosaccharides attached to Asn92, Asn247, and Asn350 were partially resistant to Endo H, while those at Asn45 and Asn337) were completely resistant. Bio-Gel P-4 analysis revealed the Endo H-resistant oligosaccharides at Asn45, Asn92, Asn247, and Asn337 to be Man8-14GlcNAc, while the minor residual carbohydrate at Asn350 was Man greater than 50GlcNAc. The Endo H-susceptible oligosaccharides at Asn4, Asn146, and Asn256 were Man greater than 50GlcNAc while all other glycosylation sites contained Man8-14GlcNAc. Based on a hydropathic analysis of invertase, the two most Endo H-resistant oligosaccharides at Asn45 and Asn337 were located in the more hydrophobic regions of the molecule. These may form part of the folded protein structure or interacting subunit surfaces, thus restricting their accessibility to Endo H.
酵母外切转化酶序列中有14个潜在的天冬酰胺- X -苏氨酸/丝氨酸糖基化位点，即糖基化序列。其中，13个位点被全部或部分糖基化，每个亚基平均有9 - 10个寡糖（雷迪，V. A.，约翰逊，R. S.，比曼，K.，威廉姆斯，R. S.，齐格勒，F. D.，特里姆布尔，R. B.，和马利，F.（1988年）《生物化学杂志》263，6978 - 6985）。用内切β - N -乙酰葡糖胺糖苷酶H（内切H）消化天然全酶时，每个亚基平均释放出约7个寡糖，且不影响酶活性（特里姆布尔，R. B.，和马利，F.（1977年）《生物化学杂志》252，4409 - 4412）。为了确定剩余的对内切H有抗性的糖链是局限于有限数量的独特糖基化序列，还是随机分布在所有13个位点上，用嗜热菌蛋白酶或胰蛋白酶消化经内切H处理的天然转化酶，然后通过反相高压液相色谱和凝胶过滤分离得到的糖肽。结果发现，与天冬酰胺92、天冬酰胺247和天冬酰胺350相连的寡糖对内切H有部分抗性，而与天冬酰胺45和天冬酰胺337相连的寡糖则完全抗性。生物凝胶P - 4分析表明，天冬酰胺45、天冬酰胺92、天冬酰胺247和天冬酰胺337处对内切H有抗性的寡糖为甘露糖8 - 14 N -乙酰葡糖胺，而天冬酰胺350处少量残留的碳水化合物为甘露糖大于50 N -乙酰葡糖胺。天冬酰胺4、天冬酰胺146和天冬酰胺256处对内切H敏感的寡糖为甘露糖大于50 N -乙酰葡糖胺，而所有其他糖基化位点均含有甘露糖8 - 14 N -乙酰葡糖胺。基于对转化酶的亲水性分析，天冬酰胺45和天冬酰胺337处两个对内切H抗性最强的寡糖位于分子中疏水性更强的区域。这些寡糖可能构成折叠蛋白结构或相互作用亚基表面的一部分，从而限制了它们对内切H的可及性。
