Significant role of cysteines in human DGAT2 activity.
(A) The location of cysteine residues in human DGAT2 was depicted by asterisks. The transmembrane domains (TMD) and the highly conserved domain are indicated by black and grey squares, respectively. Black vertical line indicates the HPHG motif. (B) The amount of newly synthesized TG in HEK293 cells overexpressing flag-tagged wild-type, mutants (C87A, C96A, C99A, C172A, C214A and C312A) with single cysteine to alanine substitution and mutant (C0) human DGAT2 with all cysteines to alanine substitution. Wild-type and mutant human DGAT2 were overexpressed in HEK293 cells for 42 hours and incubated in the presence of [14C] glycerol for additional 6 hours. Newly synthesized [14C] TG was extracted and measured by TLC analysis. The relative TG synthesis in percentage was calculated by setting the value from pcDNA3 vector-transfected cells to 100%. The mean values and standard deviations were determined from three independent experiments. (C) The immunoblots of wild-type and mutant human DGAT2. Flag-tagged wild-type and mutant human DGAT2 were overexpressed in HEK293 cells for 48 hours. Cell extracts were harvested and subjected to Western blot analysis using anti-flag antibody. Magoh protein was examined as a loading control. (D) The normalized human DGAT2 activities of wild-type and mutant human DGAT2. Relative human DGAT2 activities were determined by dividing the amount of newly synthesized TG in panel B by the relative protein amount in panel C and S2 Fig. The normalized activity of wild-type DGAT2-transfected cells was defined as 100%.
