Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom;
Institute of Sciences of Food Production, Consiglio Nazionale delle Ricerche (CNR) Unit of Lecce, I-73100 Lecce, Italy.
Proc Natl Acad Sci U S A. 2017 Aug 8;114(32):8544-8549. doi: 10.1073/pnas.1703682114. Epub 2017 Jul 24.
Glycoproteins traversing the eukaryotic secretory pathway begin life in the endoplasmic reticulum (ER), where their folding is surveyed by the 170-kDa UDP-glucose:glycoprotein glucosyltransferase (UGGT). The enzyme acts as the single glycoprotein folding quality control checkpoint: it selectively reglucosylates misfolded glycoproteins, promotes their association with ER lectins and associated chaperones, and prevents premature secretion from the ER. UGGT has long resisted structural determination and sequence-based domain boundary prediction. Questions remain on how this single enzyme can flag misfolded glycoproteins of different sizes and shapes for ER retention and how it can span variable distances between the site of misfold and a glucose-accepting N-linked glycan on the same glycoprotein. Here, crystal structures of a full-length eukaryotic UGGT reveal four thioredoxin-like (TRXL) domains arranged in a long arc that terminates in two β-sandwiches tightly clasping the glucosyltransferase domain. The fold of the molecule is topologically complex, with the first β-sandwich and the fourth TRXL domain being encoded by nonconsecutive stretches of sequence. In addition to the crystal structures, a 15-Å cryo-EM reconstruction reveals interdomain flexibility of the TRXL domains. Double cysteine point mutants that engineer extra interdomain disulfide bridges rigidify the UGGT structure and exhibit impaired activity. The intrinsic flexibility of the TRXL domains of UGGT may therefore endow the enzyme with the promiscuity needed to recognize and reglucosylate its many different substrates and/or enable reglucosylation of N-linked glycans situated at variable distances from the site of misfold.
糖蛋白穿越真核分泌途径始于内质网 (ER)，在那里，其折叠由 170kDaUDP-葡萄糖：糖蛋白葡萄糖基转移酶 (UGGT) 进行调查。该酶作为单一糖蛋白折叠质量控制检查点：它选择性地重新糖基化错误折叠的糖蛋白，促进它们与 ER 凝集素和相关伴侣的结合，并防止从 ER 过早分泌。UGGT 长期以来一直抵制结构测定和基于序列的结构域边界预测。仍有疑问的是，这种单一酶如何能够标记不同大小和形状的错误折叠糖蛋白以保留在 ER 中，以及如何跨越同一糖蛋白上错误折叠部位和接受葡萄糖的 N-连接聚糖之间的可变距离。在这里，全长真核 UGGT 的晶体结构揭示了四个硫氧还蛋白样 (TRXL) 结构域排列在一个长弧中，该弧终止于两个紧密夹住葡萄糖基转移酶结构域的β-三明治中。该分子的折叠拓扑结构复杂，第一个β-三明治和第四个 TRXL 结构域由不连续的序列片段编码。除了晶体结构外，15-Å 冷冻电镜重建揭示了 TRXL 结构域的结构域间灵活性。设计额外的结构域间二硫键的双半胱氨酸点突变使 UGGT 结构刚性化，并表现出活性受损。因此，UGGT 的 TRXL 结构域的固有灵活性可能赋予该酶识别和重新糖基化其许多不同底物所需的混杂性，或者使与错误折叠部位的可变距离处的 N-连接聚糖重新糖基化。
