The second step of eukaryotic lipid-linked oligosaccharide (LLO) biosynthesis is catalyzed by the conserved ALG13/ALG14 heterodimeric UDP-N-acetylglucosamine transferase (GnTase). In humans, mutations in ALG13 or ALG14 lead to severe neurological disorders with a multisystem phenotype, known as ALG13/14-CDG (congenital disorders of glycosylation). How these mutations relate to disease is unknown because to date, a reliable GnTase assay for studying the ALG13/14 complex is lacking. Here we describe the development of a liquid chromatography/mass spectrometry-based quantitative GnTase assay using chemically synthesized GlcNAc-pyrophosphate-dolichol as the acceptor and purified human ALG13/14 dimeric enzyme. This assay enabled us to demonstrate that in contrast to the literature, only the shorter human ALG13 isoform 2, but not the longer isoform 1 forms a functional complex with ALG14 that participates in LLO synthesis. The longer ALG13 isoform 1 does not form a complex with ALG14 and therefore lacks GnTase activity. Importantly, we further established a quantitative assay for GnTase activities of ALG13- and ALG14-CDG variant alleles, demonstrating that GnTase deficiency is the cause of ALG13/14-CDG phenotypes.
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