Sousa M C, Ferrero-Garcia M A, Parodi A J
Instituto de Investigaciones Bioquimicas, Fundación Campomar, Buenos Aires, Argentina.
Biochemistry. 1992 Jan 14;31(1):97-105. doi: 10.1021/bi00116a015.
It was found, in cell-free assays, that the Man8GlcNAc2 and Man7GlcNAc2 isomers having the mannose unit to which the glucose is added were glucosylated by the rat liver glucosyltransferase at 50 and 15%, respectively, of the rate of Man9GlcNAc2 glucosylation. This indicates that processing by endoplasmic reticulum mannosidases decreases the extent of glycoprotein glucosylation. All five different glycoproteins tested (bovine and porcine thyroglobulins, phytohemagglutinin, soybean agglutinin, and bovine pancreas ribonuclease B) were found to be poorly glucosylated or not glucosylated unless they were subjected to treatments that modified their native conformations. The effect of denaturation was not to expose the oligosaccharides but to make protein determinants, required for enzymatic activity, accessible to the glucosyltransferase because (a) cleavage of denatured glycoproteins by unspecific (Pronase) or specific (trypsin) proteases abolished their glucose acceptor capacities almost completely except when the tryptic peptides were held together by disulfide bonds and (b) high mannose oligosaccharides in native glycoproteins, although poorly glucosylated or not glucosylated, were accessible to macromolecular probes as concanavalin A-Sepharose, endo-beta-N-acetylglucosaminidase H, and jack bean alpha-mannosidase. In addition, denatured, endo-beta-N-acetylglucosaminidase H deglycosylated glycoproteins were found to be potent inhibitors of the glucosylation of denatured glycoproteins. It is suggested that in vivo only unfolded, partially folded, and malfolded glycoproteins are glucosylated and that glucosylation stops upon adoption of the correct conformation, a process that hides the protein determinants (possibly hydrophobic amino acids) from the glucosyltransferase.
在无细胞分析中发现，具有添加葡萄糖的甘露糖单元的Man8GlcNAc2和Man7GlcNAc2异构体，分别以Man9GlcNAc2糖基化速率的50%和15%被大鼠肝脏葡糖基转移酶糖基化。这表明内质网甘露糖苷酶的加工会降低糖蛋白糖基化的程度。所测试的所有五种不同糖蛋白（牛和猪甲状腺球蛋白、植物血凝素、大豆凝集素和牛胰腺核糖核酸酶B），除非经过改变其天然构象的处理，否则都被发现糖基化程度低或未被糖基化。变性的作用不是暴露寡糖，而是使葡糖基转移酶能够接触到酶活性所需的蛋白质决定簇，因为：(a) 用非特异性（链霉蛋白酶）或特异性（胰蛋白酶）蛋白酶切割变性糖蛋白几乎完全消除了它们的葡萄糖受体能力，除非胰蛋白酶肽通过二硫键结合在一起；(b) 天然糖蛋白中的高甘露糖寡糖，尽管糖基化程度低或未被糖基化，但可被大分子探针如伴刀豆球蛋白A - 琼脂糖、内切β - N - 乙酰葡糖胺糖苷酶H和刀豆α - 甘露糖苷酶接触到。此外，发现变性的、经内切β - N - 乙酰葡糖胺糖苷酶H去糖基化的糖蛋白是变性糖蛋白糖基化的有效抑制剂。有人提出，在体内只有未折叠、部分折叠和错误折叠的糖蛋白被糖基化，并且糖基化在采用正确构象时停止，这一过程会使蛋白质决定簇（可能是疏水氨基酸）对葡糖基转移酶隐藏起来。
