Topological and enzymatic analysis of human Alg2 mannosyltransferase reveals its role in lipid-linked oligosaccharide biosynthetic pathway.

N-glycosylation starts with the biosynthesis of lipid-linked oligosaccharide (LLO) on the endoplasmic reticulum (ER). Alg2 mannosyltransferase adds both the α1,3- and α1,6-mannose (Man) onto ManGlcNAc2-pyrophosphate-dolichol (M1Gn2-PDol) in either order to generate the branched M3Gn2-PDol product. The well-studied yeast Alg2 interacts with ER membrane through four hydrophobic domains. Unexpectedly, we show that Alg2 structure has diverged between yeast and humans. Human Alg2 (hAlg2) associates with the ER via a single membrane-binding domain and is markedly more stable in vitro. These properties were exploited to develop a liquid chromatography-mass spectrometry quantitative kinetics assay for studying purified hAlg2. Under physiological conditions, hAlg2 prefers to transfer α1,3-Man onto M1Gn2 before adding the α1,6-Man. However, this bias is altered by an excess of GDP-Man donor or an increased level of M1Gn2 substrate, both of which trigger production of the M2Gn2(α-1,6)-PDol. These results suggest that Alg2 may regulate the LLO biosynthetic pathway by controlling accumulation of M2Gn2 (α-1,6) intermediate.

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

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.

Construction of green fluorescence protein mutant to monitor STT3B-dependent N-glycosylation.

Oligosaccharyltransferases (OSTs) mediate the en bloc transfer of N-glycan intermediates onto the asparagine residue in glycosylation sequons (N-X-S/T, X≠P). These enzymes are typically heteromeric complexes composed of several membrane-associated subunits, in which STT3 is highly conserved as a catalytic core. Metazoan organisms encode two STT3 genes (STT3A and STT3B) in their genome, resulting in the formation of at least two distinct OST isoforms consisting of shared subunits and complex specific subunits. The STT3A isoform of OST primarily glycosylates substrate polypeptides cotranslationally, whereas the STT3B isoform is involved in cotranslational and post-translocational glycosylation of sequons that are skipped by the STT3A isoform. Here, we describe mutant constructs of monomeric enhanced green fluorescent protein (mEGFP), which are susceptible to STT3B-dependent N-glycosylation. The endoplasmic reticulum-localized mEGFP (ER-mEGFP) mutants contained an N-glycosylation sequon at their C-terminus and exhibited increased fluorescence in response to N-glycosylation. Isoform-specific glycosylation of the constructs was confirmed by using STT3A- or STT3B-knockout cell lines. Among the mutant constructs that we tested, the ER-mEGFP mutant containing the N185 -C186 -T187 sequon was the best substrate for the STT3B isoform in terms of glycosylation efficiency and fluorescence change. Our results suggest that the mutant ER-mEGFP is useful for monitoring STT3B-dependent post-translocational N-glycosylation in cells of interest, such as those from putative patients with a congenital disorder of glycosylation.

Stability of the recombinant anti­erbB2 scFv­Fc­interleukin­2 fusion protein and its inhibition of HER2­overexpressing tumor cells.

The anti­erbB2 scFv­Fc­IL­2 fusion protein (HFI) is the basis for development of a novel targeted anticancer drug, in particular for the treatment of HER2­positive cancer patients. HFI was fused with the anti­erbB2 antibody and human IL­2 by genetic engineering technology and by antibody targeting characteristics of HFI. IL­2 was recruited to target cells to block HER2 signaling, inhibit or kill tumor cells, improve the immune capacity, reduce the dose of antibody and IL­2 synergy. In order to analyse HFI drug ability, HFI plasmid stability was verified by HFI expression of the trend of volume changes. Additionally, HFI could easily precipitate and had progressive characteristics and thus, the buffer system of the additive phosphate­citric acid buffer, arginine, Triton X­100 or Tween­80, the establishment of a microfiltration, ion exchange, affinity chromatography and gel filtration chromatography­based purification process were explored. HFI samples were obtained according to the requirements of purity, activity and homogeneity. In vivo, HFI significantly delayed HER2 overexpression of non­small cell lung cancer (Calu­3) in human non­small cell lung cancer xenografts in nude mice, and the inhibition rate was more than 60% (P<0.05) in the group treated with 1 mg/kg the HFI dose; HFI significantly inhibited HER2 expression of breast cancer (FVB/neu) transgenic mouse tumor growth in 1 mg/kg of the HFI dose group, and in the following treatment the 400 mm3 tumors disappeared completely. Combined with other HFI test data analysis, HFI not only has good prospects, but also laid the foundation for the development of antibody­cytokine fusion protein­like drugs.