Mammalian STT3A/B oligosaccharyltransferases segregate N-glycosylation at the translocon from lipid-linked oligosaccharide hydrolysis

Proc Natl Acad Sci U S A. 2018 Sep 18;115(38):9557-9562. doi: 10.1073/pnas.1806034115. Epub 2018 Sep 4.

Abstract

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.

Keywords: STT3A; STT3B; dolichol; glycosylation; oligosaccharyltransferase.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Cell Line
  • Endoplasmic Reticulum / metabolism
  • Exodeoxyribonucleases / genetics
  • Exodeoxyribonucleases / metabolism
  • Gene Knockout Techniques
  • Glycosylation
  • Hexosyltransferases / genetics
  • Hexosyltransferases / metabolism*
  • Humans
  • Hydrolysis
  • Isoenzymes
  • Lipopolysaccharides / metabolism*
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Mice
  • Oligosaccharides / metabolism*
  • Phosphoproteins / genetics
  • Phosphoproteins / metabolism
  • Protein Processing, Post-Translational / physiology*
  • Protein Transport / physiology

Substances

  • Isoenzymes
  • Lipopolysaccharides
  • Membrane Proteins
  • Oligosaccharides
  • Phosphoproteins
  • lipid-linked oligosaccharides
  • Hexosyltransferases
  • STT3A protein, human
  • STT3B protein, human
  • Exodeoxyribonucleases
  • three prime repair exonuclease 1