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Functional assays of human ALG9

ABSTRACT: Asparagine-linked glycans are essential for the maturation and function of most eukaryotic secretory proteins. Biosynthesis and transfer of the GlcNAc2Man9Glc3 glycan occurs on the endoplasmic reticulum (ER) membrane, in a highly conserved process involving more than a dozen membrane proteins whose dysfunction is linked to severe congenital disorders of glycosylation (CDGs). Three membrane-integral mannosyltransferases ALG3, ALG9 and ALG12 mediate four consecutive mannosylation reactions that convert GlcNAc2Man5 to GlcNAc2Man9. Using chemo-enzymatically synthesised lipid-linked glycan donor and acceptor analogues, we recapitulated this biosynthetic pathway in vitro. High-resolution cryo-EM structures of pseudo-Michaelis complexes of each step revealed the molecular basis of how the branched glycan is accurately synthesised in sequential reactions. Molecular dynamics simulations, mutagenesis, and functional assays uncovered a subtle but high precision mechanisms selecting the dolichylphosphomannose donor substrate over the also present epimer, dolichylphosphoglucose. Together, our results provide mechanistic explanations for enzyme disfunction in CDG and offer opportunities for N-glycan engineering.

INSTRUMENT(S):

ORGANISM(S): Synthetic Construct

SUBMITTER: J. Andrew Alexander

LAB HEAD: Kaspar P. Locher

PROVIDER: PXD072144 | Pride | 2026-05-30

REPOSITORIES: Pride

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			Action	DRS
	20251022_002_S1039537_No-enz_1_20_RF_20.raw	Raw
	20251022_002_S1039537_No-enz_1_20_RF_20.raw_20251217_fixed.mgf	Mgf
	20251022_002_S1039537_No-enz_1_20_RF_20.raw_20251217_fixed.mzid	Mzid
	20251022_004_S1039538_ALG9-WT_1_20_RF_20.raw	Raw
	20251022_004_S1039538_ALG9-WT_1_20_RF_20.raw_20251217_fixed.mgf	Mgf

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