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.

Rft1 catalyzes lipid-linked oligosaccharide translocation across the ER membrane

“…To rule out the possibility that Rft1 flipping activity was an artifact associated with the synthetic PP-Phy lipid carrier used, we also assayed M5GN2 linked to dolichol, its physiological lipid. M5GN2-PP-Dol was synthesized 37 for use as a substrate (Fig. 2b and Supplementary Fig.…”

“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 ).…”

Mutations in Glycosyltransferases and Glycosidases: Implications for Associated Diseases

“…In contrast, the C-terminal region of ALG13-is2 facilitates the protein's localisation to the ER by enabling its interaction with Alg14, a partnership that is vital for its glycosyltransferase function [57]. In a recent study, Wang et al [58] claimed the finding that, diverging from the previous literature, only the shorter human ALG13 isoform 2, not the longer isoform 1, forms a functional complex with ALG14 that is involved in lipidlinked oligosaccharide synthesis, as isoform 1 of ALG13 does not interact with ALG14 and consequently lacks GnTase activity. Notably, a missense mutation (p.T141L) in ALG13-is2 has been associated with focal segmental glomerulosclerosis and posterior cortical cataract disease [59], indicating its potential impact on renal filtration and eye health.…”