Synthesis of lipid-linked oligosaccharides for the substrate-specificity evaluation of Alg proteins

For the enzymatic data, the authors overexpressed Alg13/14, Alg1, and Alg2 from Saccharomyces cerevisiae and human sources in Escherichia coli and used an in vitro enzymatic assay system to measure conversion of the synthetic LLO substrates: 8a–e were converted to 10a–e by Alg13/14, 10a–e were converted to 11a–e by Alg1, and 11a–e were converted to 12a–e by Alg2. As mentioned in the article, the spatial scale is mainly at the molecular/organelle level, i.e., LLO biosynthesis on the cytosolic side of the ER membrane and in the ER lumen; species sources include human, Saccharomyces cerevisiae, E. coli, plants, etc.

The temporal information is mainly reflected in the chemical reaction conditions, such as Pt/C, H2, EtOH for 2 h; in the phosphorylation step, CCl3CN/Bu4NH2PO4/DCM for 20 min, followed by NH3·H2O/THF for 45 min; and asymmetric hydrogenation with KOH, (S)-tol-binap-RuCl2(p-cymene), i-PrOH at 83 °C for 3 h.Regarding the table data, Fig. 3B is a summary table of ALG1-CDG patient mutations and relative activities.

The table uses 3 patients as column labels, namely Patient 1, Patient 2, and Patient 3, and 5 attributes as row labels, namely Age (reported), HsAlg1 mutations, Conserved ScAlg1 mutants, Relative activity of HsAlg1, and Relative activity of ScAlg1; therefore, there are 3 columns, 5 rows, and 15 data cells. Units include age in years or weeks and relative activity in percent (%). Specific data include: Patient 1 is 9 years old, with heterozygous mutations R276W and C396X, corresponding to conserved yeast mutations K266W and V385X; HsAlg1 relative activities are 43.2% and 0%, and ScAlg1 relative activities are 89% and 0%; Patient 2 died 11 weeks after birth, with heterozygous mutations S258L and Q342P, corresponding to yeast Q331P; HsAlg1 relative activities are both 0%, and ScAlg1 are NA and 0%; Patient 3 is 4 years old, with homozygous mutation M377V, corresponding to yeast M366V; HsAlg1 relative activity is 10.3%, and ScAlg1 is 40%.

The table contains missing or unavailable values; for example, for Patient 2, the Conserved ScAlg1 mutant corresponding to S258L is “--”, and the Relative activity of ScAlg1 is “NA”. Regarding error, the conversion-rate bar graph in Fig. 2 uses the standard deviation (SD) of three parallel experiments as error bars and marks significance at P<0.05, P<0.01, and P<0.001.