Identification of structural motifs critical for human G6PC2 function informed by sequence analysis and an...

Area of Science:

• Biochemistry
• Molecular Biology
• Genetics

Background:

• G6PC2 (glucose-6-phosphatase catalytic subunit 2) is expressed in pancreatic islet beta cells and regulates fasting blood glucose (FBG) by modulating insulin secretion sensitivity.
• Understanding G6PC2's structure-function relationship is key to developing therapeutic strategies for glucose metabolism disorders.

Purpose of the Study:

• To validate the AlphaFold2-predicted structure of human G6PC2.
• To elucidate the functional roles of specific G6PC2 motifs and a common SNP (rs492594) in protein expression and enzyme activity.
• To investigate the influence of cholesterol on G6PC2 variant activity.

Main Methods:

• Mutational analysis of G6PC2, including residues forming disulfide bonds, a PAP2 motif, a substrate cavity, and a CRAC motif.
• Solubilization and purification of human G6PC2 from a heterologous expression system.
• Enzyme activity assays using microsomal preparations and purified G6PC2, with and without cholesteryl hemi-succinate.

Main Results:

• Disulfide bond residues are essential for G6PC2 expression; PAP2 motif residues are critical for enzyme activity.
• Substrate cavity residues modulate enzyme activity and specificity; CRAC motif residues affect expression or activity.
• The common Val219Leu SNP (rs492594) showed differential activity in membranes, but this diminished upon purification, suggesting cholesterol interaction influences variant function. Cholesterol addition reduced Vmax for both variants.

Conclusions:

• Structural and functional characterization of G6PC2 provides insights into its catalytic mechanism and regulation.
• The findings highlight the role of cholesterol in modulating G6PC2 variant activity, particularly concerning the Val219Leu SNP.
• This study lays the groundwork for designing targeted G6PC2 inhibitors to lower FBG.