An in vitro assay for enzymatic studies on human ALG13/14 heterodimeric UDP-N-acetylglucosamine transferase

Abstract

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.

“Mix and match” auto-assembly of glycosyltransferase domains delivers biocatalysts with improved substrate promiscuity

“…In addition, a few examples of genes encoding dimeric GT-B composed of 2 separated domains have been reported ( 47 , 48 , 49 ). In all cases, these genes are closely related to the enzyme involved in N -acetylglucosamine transfer from UDP-Glc N Ac to Glc N Ac-PP-Dolichol, which takes place in the protein N -glycosylation pathway in the endoplasmic reticulum in eukaryotes ( 50 , 51 ). This protein is composed of 2 subunits, Agl13 and Agl14, that can be, respectively, mapped by sequence alignment to the C - and N - terminal domains of bacterial GT MurG, as subsequently confirmed by the NMR structure of Alg13, involved in the sugar donor binding ( 52 ).…”