Protein O-mannosyltransferases (PMTs) initiate O-mannosylation of proteins in the ER. Trichoderma reesei strains displayed a single representative of each PMT subfamily, Trpmt1, Trpmt2 and Trpmt4. In this work, two knockout strains ΔTrpmt1 and ΔTrpmt4 were obtained. Both mutants showed retarded growth, defective cell walls, reduced conidiation and decreased protein secretion. Additionally, the ΔTrpmt1 strain displayed a thermosensitive growth phenotype, while the ΔTrpmt4 strain showed abnormal polarity. Meanwhile, OETrpmt2 strain, in which the Trpmt2 was over-expressed, exhibited increased conidiation, enhanced protein secretion and abnormal polarity. Using a lectin enrichment method and MS/MS analysis, 173 O-glycoproteins, 295 O-glycopeptides and 649 O-mannosylation sites were identified as the targets of PMTs in T. reesei. These identified O-mannoproteins are involved in various physiological processes such as protein folding, sorting, transport, quality control and secretion, as well as cell wall integrity and polarity. By comparing proteins identified in the mutants and its parent strain, the potential specific protein substrates of PMTs were identified. Based on our results, TrPMT1 is specifically involved in O-mannosylation of intracellular soluble proteins and secreted proteins, specially glycosidases. TrPMT2 is involved in O-mannosylation of secreted proteins and GPI-anchor proteins, and TrPMT4 mainly modifies multiple transmembrane proteins. The TrPMT1-TrPMT4 complex is responsible for O-mannosylation of proteins involved in cell wall integrity. Overexpression of TrPMT2 enhances protein secretion, which might be a new strategy to improve expression efficiency in T. reesei.

蛋白质O-甘露糖基转移酶 (PMT) 启动ER 中蛋白质的O-甘露糖基化。里氏木霉菌株展示了每个 PMT 亚科的单一代表，Tr pmt1、Tr pmt2和 Tr pmt4。在这项工作中，双基因敲除株ΔTR PMT1 和ΔTR PMT4 获得。两种突变体均表现出生长迟缓、细胞壁缺陷、分生孢子减少和蛋白质分泌减少。此外，该ΔTR PMT1 菌株显示的热敏生长表型，而ΔTR PMT4菌株显示异常的极性。同时，OETr pmt 2 菌株，其中 Trpmt2过度表达，表现出增加的分生孢子，增强的蛋白质分泌和异常极性。使用凝集素富集方法和MS / MS分析，173个ö -glycoproteins，295个ö -glycopeptides和649个ö -mannosylation位点被确定为在光电倍增管的目标里氏木霉。这些经鉴定的O-甘露糖蛋白参与各种生理过程，如蛋白质折叠、分选、运输、质量控制和分泌，以及细胞壁完整性和极性。通过比较突变体及其亲本菌株中鉴定的蛋白质，鉴定了 PMT 的潜在特异性蛋白质底物。根据我们的结果，TrPMT1 特别参与 细胞内可溶性蛋白和分泌蛋白，特别是糖苷酶的O-甘露糖基化。Tr PMT2参与 分泌蛋白和GPI-锚蛋白的O-甘露糖基化，Tr PMT4主要修饰多种跨膜蛋白。的Tr的PMT1- Tr的PMT4复负责ø参与细胞壁的完整性的蛋白-mannosylation。Tr PMT2 的过表达增强了蛋白质分泌，这可能是提高里氏木霉表达效率的新策略。