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<p>Asparagine-linked glycans are essential for the maturation and function of most eukaryotic secretory proteins. The biosynthesis and transfer of dolichylpyrophosphate-anchored GlcNAc<sub>2</sub>Man<sub>9</sub>Glc<sub>3</sub> glycan is a highly conserved process occurring in the endoplasmic reticulum (ER) membrane and involving over a dozen membrane proteins whose dysfunction is linked to congenital disorders of glycosylation (CDGs). Three membrane-integral mannosyltransferases, ALG3, ALG9 and ALG12, mediate four consecutive mannosylation reactions that convert GlcNAc<sub>2</sub>Man<sub>5</sub> to GlcNAc<sub>2</sub>Man<sub>9</sub>. Here, using chemoenzymatically synthesized lipid-linked glycan donor and acceptor analogs, we recapitulated this biosynthetic pathway in vitro. High-resolution cryo-electron microscopy structures of pseudo-Michaelis complexes of each step revealed how the branched glycan is accurately synthesized and unwanted side products are averted. Molecular dynamics simulations and mutagenesis studies uncovered a subtle but precise mechanism selecting the dolichylphosphomannose donor substrate over dolichylphosphoglucose, which is also present in the ER membrane. Our results also provide mechanistic explanations for enzyme dysfunction in CDGs and offer opportunities for <i>N</i>-glycan engineering.</p><p></p>

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Structures of ALG3/9/12 reveal the assembly logic of the N-glycan oligomannose core

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2026年3月10日

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  • J. Andrew N. Alexander,
  • Shu-Yu Chen,
  • Somnath Mukherjee,
  • Mario de Capitani,
  • Rossitza N. Irobalieva,
  • Lorenzo Rossi,
  • Parth Agrawal,
  • Julia Kowal,
  • Matheus A. Meirelles,
  • Markus Aebi,
  • Jean-Louis Reymond,
  • Anthony A. Kossiakoff,
  • Sereina Riniker,
  • Kaspar P. Locher

摘要

Asparagine-linked glycans are essential for the maturation and function of most eukaryotic secretory proteins. The biosynthesis and transfer of dolichylpyrophosphate-anchored GlcNAc2Man9Glc3 glycan is a highly conserved process occurring in the endoplasmic reticulum (ER) membrane and involving over a dozen membrane proteins whose dysfunction is linked to congenital disorders of glycosylation (CDGs). Three membrane-integral mannosyltransferases, ALG3, ALG9 and ALG12, mediate four consecutive mannosylation reactions that convert GlcNAc2Man5 to GlcNAc2Man9. Here, using chemoenzymatically synthesized lipid-linked glycan donor and acceptor analogs, we recapitulated this biosynthetic pathway in vitro. High-resolution cryo-electron microscopy structures of pseudo-Michaelis complexes of each step revealed how the branched glycan is accurately synthesized and unwanted side products are averted. Molecular dynamics simulations and mutagenesis studies uncovered a subtle but precise mechanism selecting the dolichylphosphomannose donor substrate over dolichylphosphoglucose, which is also present in the ER membrane. Our results also provide mechanistic explanations for enzyme dysfunction in CDGs and offer opportunities for N-glycan engineering.