Proteome and Glycoproteome Analyses Reveal the Protein N-Linked Glycosylation Specificity of STT3A and STT3B

Abstract

STT3A and STT3B are the main catalytic subunits of the oligosaccharyltransferase complex (OST-A and OST-B in mammalian cells), which primarily mediate cotranslational and post-translocational N-linked glycosylation, respectively. To determine the specificity of STT3A and STT3B, we performed proteomic and glycoproteomic analyses in the gene knock-out (KO) and wild-type HEK293 cells. In total, 3961 proteins, 4265 unique N-linked intact glycopeptides and 629 glycosites representing 349 glycoproteins were identified from all these cells. Deletion of the STT3A gene had a greater impact on the protein expression than deletion of STT3B, especially on glycoproteins. In addition, total mannosylated N-glycans were reduced and fucosylated N-glycans were increased in STT3A-KO cells, which were caused by the differential expression of glycan-related enzymes. Interestingly, hyperglycosylated proteins were identified in KO cells, and the hyperglycosylation of ENPL was caused by the endoplasmic reticulum (ER) stress due to the STT3A deletion. Furthermore, the increased expression of the ATF6 and PERK indicated that the unfolded protein response also happened in STT3A-KO cells. Overall, the specificity of STT3A and STT3B revealed that defects in the OST subunit not only broadly affect N-linked glycosylation of the protein but also affect protein expression.

Terminal Unfolded Protein Responses-related genes predict prognosis and associate with proliferation and apoptosis in clear cell renal cell carcinoma

“…According to the report by Wen et al [47] , although there were different preferences for glycosylation sites between the OST-A complex and the OST-B complex, in the absence of one of them, the other complex can act as a compensatory role to complement the intracellular glycosylation process, which may explain why the silencing of STT3B did not cause lethal ER stress.…”

High-Throughput Mass Spectrometry Analysis of N-Glycans and Protein Markers after FUT8 Knockdown in the Syngeneic SW480/SW620 Colorectal Cancer Cell Model

“…Aberrant glycosylation is ever-present in oncogenic transformation, with a capital influence on tumor progression, immune evasion, invasiveness, metastatic potential, and (radio)­chemoresistance. − Consequently, numerous studies have addressed the importance of glycosylation in several types of cancer, including CRC. , However, the impact of aberrant glycosylation is complex because it encompasses the glycosylation pattern and the proteome composition, not exclusively the glycoenzymes . Furthermore, changes in the glycosylation machinery affect the biological activity and interactome of glycoproteins, magnifying the map of alterations due to impaired glycosylation.…”

“… 42 , 43 However, the impact of aberrant glycosylation is complex because it encompasses the glycosylation pattern and the proteome composition, not exclusively the glycoenzymes. 44 Furthermore, changes in the glycosylation machinery affect the biological activity and interactome of glycoproteins, magnifying the map of alterations due to impaired glycosylation. This critical consideration has not always been addressed with the degree of attention it deserves, 45 , 46 so our study has leveraged the multiomic proteomic and glycomic approach to assess more closely the extent of glycosylation changes in CRC.…”

Terminal Unfolded Protein Responses-related genes predict prognosis and associate with proliferation and apoptosis in clear cell renal cell carcinoma

“…46 Current studies have shown that the OST complex is involved in the maintenance of the ER homeostasis, and the loss or abnormal function of its composition affects the ER stress state in cells. 15 Wen et al 47 found that the lack of STT3A in human embryonic kidney cells HEK293 caused ER stress and activated the UPR pathway, and Ding et al 48 found that the loss of RPN1, a non-catalytic subunit of the OST complex, caused ER stress-mediated apoptosis in breast cancer cells.…”