Defining the Topology of the N-Glycosylation Pathway in the Halophilic Archaeon Haloferax volcanii
Defining the Topology of the N-Glycosylation Pathway in the Halophilic Archaeon Haloferax volcanii
复制标题
DOI：
10.1128/jb.01200-08
复制
发表时间：
2008-12-01
影响因子：
3.2
通讯作者：
Eichler, Jerry
中科院分区：
文献类型：
--
作者：
Plavner, Noa;Eichler, Jerry
在真核生物中，N糖基化涉及内质网膜两侧的酶的作用。相反，细菌N糖基化的步骤基本上是在质膜的细胞质一侧进行的，只有组装好的多糖转移到细胞外表面的目标蛋白上。对于古生菌来说，几乎对组装这些多糖所涉及的酶的拓扑结构一无所知，这些多糖随后被N连接到细胞外表面的目标蛋白上。为了改善这种情况，亚细胞定位和拓扑预测算法、蛋白酶可及性和免疫印迹，以及定点突变后的半胱氨酸修饰，被用来定义实验证明参与N-糖基化过程的Haloferax cancanii蛋白的拓扑结构。AglJ和AglD分别参与了修饰火山杆菌S层糖蛋白的五糖组装的最早和最晚阶段，它们被证明将它们的可溶性N-末端结构域呈现给细胞质，可能包含每种酶的可能的催化部位。同样的道理也适用于ALG5-B、Dpm1-A和Mpg1-D，这些蛋白质被认为参与了这种翻译后事件。因此，结果表明，与火山杆菌S层糖蛋白的某些ASN残基相连的五糖在细胞内组装。
In Eukarya, N glycosylation involves the actions of enzymes working on both faces of the endoplasmic reticulum membrane. The steps of bacterial N glycosylation, in contrast, transpire essentially on the cytoplasmic side of the plasma membrane, with only transfer of the assembled glycan to the target protein occurring on the external surface of the cell. For Archaea, virtually nothing is known about the topology of enzymes involved in assembling those glycans that are subsequently N linked to target proteins on the external surface of the cell. To remedy this situation, subcellular localization and topology predictive algorithms, protease accessibility, and immunoblotting, together with cysteine modification following site-directed mutagenesis, were enlisted to define the topology of Haloferax volcanii proteins experimentally proven to participate in the N-glycosylation process. AglJ and AglD, involved in the earliest and latest stages, respectively, of assembly of the pentasaccharide decorating the H. volcanii S-layer glycoprotein, were shown to present their soluble N-terminal domain, likely containing the putative catalytic site of each enzyme, to the cytosol. The same holds true for Alg5-B, Dpm1-A, and Mpg1-D, proteins putatively involved in this posttranslational event. The results thus point to the assembly of the pentasaccharide linked to certain Asn residues of the H. volcanii S-layer glycoprotein as occurring within the cell.