Functional analysis of GALT variants found in classic galactosemia patients using a novel cell-free translation method

Abstract

Classic galactosemia is an autosomal recessive disorder caused by deleterious variants in the galactose-1-phosphate uridylyltransferase (GALT) gene. GALT enzyme deficiency leads to an increase in the levels of galactose and its metabolites in the blood causing neurodevelopmental and other clinical complications in affected individuals. Two GALT variants NM_000155.3:c.347T>C (p.Leu116Pro) and NM_000155.3:c.533T>G (p.Met178Arg) were previously detected in Filipino patients. Here, we determine their functional effects on the GALT enzyme through in silico analysis and a novel experimental approach using a HeLa-based cell-free protein expression system. Enzyme activity was not detected for the p.Leu116Pro protein variant, while only 4.5% of wild-type activity was detected for the p.Met178Arg protein variant. Computational analysis of the variants revealed destabilizing structural effects and suggested protein misfolding as the potential mechanism of enzymological impairment. Biochemical and computational data support the classification of p.Leu116Pro and p.Met178Arg variants as pathogenic. Moreover, the protein expression method developed has utility for future studies of GALT variants.

Keywords: GALT deficiency; HeLa‐based cell‐free expression system; classic galactosemia; galactose‐1‐phosphate uridylyltransferase; missense variants.

Figure 1

The structure of human GALT enzyme showing the affected residues. (A) Schematic view of the location of mutated amino acid residues in the dimer interface. The two subunits are shown in light gray (chain A) and dark gray (chain B). Affected residues are colored yellow and represented in ball‐and‐stick mode. (B) Leu116 is located close to the two salt bridges, Asp113^B‐Arg228^A and His114^B‐Glu220^A. Redrawn from PDB file 5IN3 using Discovery Studio Visualizer 3.5 (Accelrys Software, Inc., San Diego, CA)

Figure 2

Enzymatic activity measurement of purified wild‐type and variant GALT proteins. 100 ng of purified protein was tested in a total volume of 20 μL glycine buffer. The activity values were generated from three separate experiments. The mean and SD are indicated above the bars. Values in parentheses reflect the % of wild type activity