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.

Keywords: ALG glycosyltransferases; ALG13 isoforms; ALG13/14 UDP-N-acetylglucosamine transferase; N-glycosylation; congenital disorders of glycosylation (CDG); lipid-linked oligosaccharide (LLO).

FIGURE 1

Preparation and feasibility test of lipid-linked acceptor GlcNAc-PP-Dol (Gn-PDol, C95). (A) The semisynthetic strategy of lipid-linked acceptor Gn-PDol (C95); (B) Work flow of an in vitro quantitative GnTase assay for ALG13/14 complex; (C) The UPLC chromatogram of glycans released from ALG13/14 reaction mixture. Reaction was performed using HEK293 cells membrane fraction with the standard reaction mixture and incubated for 1 h. Peaks eluted earlier represent Gn and eluted later represent Gn2; (D) ESI-MS spectra of peaks eluted at 7.39 and 7.79 min in UPLC correspond to Gn2 ([Gn2+Na]+).

FIGURE 2

Recombinant ALG13/14 complex catalyzes the formation of Gn2-PDol. (A) Western blot and co-immunoprecipitation analysis of recombinant co-expressed ALG13 and ALG14 in E. coli. The crude extract prepared from E. coli cells was analyzed by western blot with anti-FLAG antibody or with anti-His antibody (left panel). The co-immunoprecipitation performed using Ni-NTA His binding resin or anti-FLAG Affinity Matrix, and immunoblotted with anti-FLAG antibody or anti-His antibody (right panel); (B) The UPLC chromatogram of glycans released from different reaction mixture. Reaction was performed using crude extract from co-expressed strain (5 μg total protein) or two different single expressed strain (5 μg total protein from each strain); (C) The UPLC chromatogram of glycans released from the recombinant ALG13/14 reaction mixture which add recombinant yAlg1 for additional 1 h. Peaks eluted at 10.41 min and 10.71 min represent Man-Gn2 (M1Gn2); (D) ESI-MS spectra of peaks eluted at 10.41 min and 10.71 min in UPLC correspond to M1Gn2 ([M1Gn2+H]⁺).

FIGURE 3

Enzymatic properties of ALG13/14 GnTase. (A) Native PAGE and SDS-PAGE analysis of purified heteromeric ALG13/14 complex; (B) Optimal pH was examined using various buffers, including sodium acetate-acetic acid buffer (pH 4.0, 5.0 and 6.0); Tris/HCl buffer (pH 6.0, 7.0, 8.0 and 9.0); glycine/NaOH buffer (pH 9.0 and 10.0); (C) Optimal temperature was evaluated at indicated temperatures (10°C, 20°C, 30°C, 37°C, 50°C and 60°C); (D) The specificity of the nucleotide sugar donor was studied by performing the reaction with 2 mM GDP-Man, UDP-Glc, or UDP-GlcNAc; (E) Divalent cation dependency was examined using 10 mM ions (Co²⁺, Mn²⁺ and Mg²⁺, respectively) or 10 mM EDTA for depletion conditions; (F) The K _m (94.1 μM ± 9.4 μM) and k _cat (919.5 min⁻¹ ± 54.3 min⁻¹) values for the substrate Gn-PDol whose concentration ranged from 20 μM to 100 μM, were calculated by nonlinear regression with a constant concentration of 2 mM UDP-GlcNAc. Each data point represents the mean ± SD value calculated from three independent experiments.

FIGURE 4

Human ALG13 isoform 1 fails to form GnTase complex with ALG14. (A) The gene map of ALG13-iso1 and ALG13-iso2. The two isoforms transcript from ALG13 gene and share a same N-terminal while the C-terminal substantially different; (B) The UPLC chromatogram of glycans released from the reaction of ALG13 isoforms. HA-tagged ALG13 isoforms were expressed in HEK293 cell and enriched by using anti-HA agarose; (C) Co-immunoprecipitation analysis of ALG13 isoforms and ALG14. HA-tagged ALG13 isoforms and FLAG-tagged ALG14 were co-transfected in HEK293 cells, the co-immunoprecipitation performed using anti-HA agarose, and immunoblotted with anti-FLAG antibody; (D) Yeast complementary assay of ALG13 isoforms. An ALG13/14 deficient yeast strain XGY186 was grown under Gal promoter driving yAlg13 and yAlg14. ALG13 isoform and ALG14 were transfected and culture in YPA plate with galactose or glucose and incubated at 30°C for 2 days. iso: isoform.

FIGURE 5

The semi-quantitative assay for ALG13- and ALG14-CDG mutants. (A) ALG13- and ALG14-CDG missense mutations distribution diagram. Six mutations in ALG13-iso2 and five mutations in ALG14 (labeled in red) were constructed; (B) Schematic diagram of ALG13/14 quantification. E. coli cells was broke by sonication and centrifuged to observe the membrane fraction, after washing 3 times by lysis buffer, the membrane fraction was solubilized and the detergent extract was used for GnTase activity assay, and the active ALG13/14 complex amount was semi-quantified using FLAG-ALG13 amount by anti-FLAG antibody; (C) The UPLC chromatogram of glycans released from the reaction of wild type ALG13/14, ALG13 N107S/ALG14 and ALG13/ALG14 P65L. Reaction included ALG13/ALG14 (0.1 ng/μl FLAG-ALG13 semi-quantified by western-blot) with the standard reaction mixture for 15 min.