Oligosaccharyltransferases (OSTs) N-glycosylate proteins by transferring oligosaccharides from lipid-linked oligosaccharides (LLOs) to asparaginyl residues of Asn-Xaa-Ser/Thr acceptor sequons. Mammals have OST isoforms with STT3A or STT3B catalytic subunits for cotranslational or posttranslational N-glycosylation, respectively. OSTs also hydrolyze LLOs, forming free oligosaccharides (fOSs). It has been unclear whether hydrolysis is due to one or both OSTs, segregated from N-glycosylation, and/or regulated. Transfer and hydrolysis were assayed in permeabilized HEK293 kidney and Huh7.5.1 liver cells lacking STT3A or STT3B. Transfer by both STT3A-OST and STT3B-OST with synthetic acceptors was robust. LLO hydrolysis by STT3B-OST was readily detected and surprisingly modulated: Without acceptors, STT3B-OST hydrolyzed Glc₃Man₉GlcNAc₂-LLO but not Man₉GlcNAc₂-LLO, yet it hydrolyzed both LLOs with acceptors present. In contrast, LLO hydrolysis by STT3A-OST was negligible. STT3A-OST however may be regulatory, because it suppressed STT3B-OST–dependent fOSs. TREX1, a negative innate immunity factor that diminishes immunogenic fOSs derived from LLOs, acted through STT3B-OST as well. In summary, only STT3B-OST hydrolyzes LLOs, depending upon LLO quality and acceptor site occupancy. TREX1 and STT3A suppress STT3B-OST–dependent fOSs. Without strict kinetic limitations during posttranslational N-glycosylation, STT3B-OST can thus moonlight for LLO hydrolysis. In contrast, the STT3A-OST/translocon complex preserves LLOs for temporally fastidious cotranslational N-glycosylation.

寡糖基转移酶（OST）通过将寡糖从脂质连接的寡糖（LLO）转移至Asn-Xaa-Ser / Thr受体子序列的天冬酰胺残基来将N-糖基化蛋白合成。哺乳动物的OST亚型具有STT3A或STT3B催化亚基，分别用于共翻译或翻译后N-糖基化。OST还会水解LLO，形成游离的寡糖（fOS）。尚不清楚水解是否是由于一种或两种OST引起的，与N-糖基化分离和/或受调节。在缺乏STT3A或STT3B的通透性HEK293肾脏和Huh7.5.1肝细胞中测定了转移和水解。带有合成受体的STT3A-OST和STT3B-OST的传输都非常可靠。STT3B-OST的LLO水解很容易被检测到并且出人意料地受到调节：如果没有受体，STT3B-OST水解的Glc ₃Man ₉ GlcNAc ₂ -LLO但不是Man ₉ GlcNAc ₂-LLO，但在存在受体的情况下水解了两个LLO。相反，STT3A-OST对LLO的水解作用可以忽略不计。但是，STT3A-OST可能具有监管性，因为它抑制了依赖STT3B-OST的fOS。TREX1是一种负性先天免疫因子，可减少源自LLO的免疫原性fOS，也通过STT3B-OST发挥作用。总之，只有STT3B-OST才能水解LLO，具体取决于LLO的质量和受体位点的占用情况。TREX1和STT3A抑制依赖于STT3B-OST的fOS。在翻译后N-糖基化过程中，没有严格的动力学限制，STT3B-OST可以为LLO水解提供月光。相反，STT3A-OST / translocon复合物保留了LLO，用于暂时的共翻译N-糖基化。