Functional and Biochemical Characterization of Three Recombinant Human Glucose-6-Phosphate Dehydrogenase Mutants: Zacatecas, Vanua-Lava and Viangchan

Abstract

Glucose-6-phosphate dehydrogenase (G6PD) deficiency in humans causes severe disease, varying from mostly asymptomatic individuals to patients showing neonatal jaundice, acute hemolysis episodes or chronic nonspherocytic hemolytic anemia. In order to understand the effect of the mutations in G6PD gene function and its relation with G6PD deficiency severity, we report the construction, cloning and expression as well as the detailed kinetic and stability characterization of three purified clinical variants of G6PD that present in the Mexican population: G6PD Zacatecas (Class I), Vanua-Lava (Class II) and Viangchan (Class II). For all the G6PD mutants, we obtained low purification yield and altered kinetic parameters compared with Wild Type (WT). Our results show that the mutations, regardless of the distance from the active site where they are located, affect the catalytic properties and structural parameters and that these changes could be associated with the clinical presentation of the deficiency. Specifically, the structural characterization of the G6PD Zacatecas mutant suggests that the R257L mutation have a strong effect on the global stability of G6PD favoring an unstable active site. Using computational analysis, we offer a molecular explanation of the effects of these mutations on the active site.

Functional and Biochemical Analysis of Glucose-6-Phosphate Dehydrogenase (G6PD) Variants: Elucidating the Molecular Basis of G6PD Deficiency

“…Specifically, Gómez-Manzo et al [19][20][21], found that soluble G6PD levels were highest at 25 • C for the G6PD Yucatan, Valladolid, Mexico City, Nashville, Durham, Santa-Maria, A+, Zacatecas, Viangchan and Vanua-Lava variants. Furthermore, they also found that the optimal G6PD expression was time (18 h) and isopropyl-β-D-thiogalactopyranoside (IPTG) concentration dependent; however, the use of these conditions does not avoid the formation of inactive inclusion bodies [19][20][21]. On the other hand, Boonyuen et al [22] expressed G6PD Mahidol and Viangchan variants at 20 • C, inducing with 1 mM IPTG at 20 h, while the G6PD Plymouth and Mahidol were expressed at 37 • C for 24 h IPTG induction [18].…”

“…Several systems have been implemented to achieve expression and purification of both the wild-type (WT) and G6PD variants. Heterologous expression of human G6PD based on Escherichia coli strains and compatible plasmids have been widely used to produce high protein quantities for structural and functional studies [12,14,16,17,[19][20][21][23][24][25][26]. During the first characterization studies, the G6PD gene was widely fused to different expression vectors such as pKK223-3, pKK233-2, pMPM-A4Ω and pPLcmu299 [15].…”

“…They observed that both strategies significantly increased the soluble expression of recombinant G6PD Plymouth and Mahidol variants. Other successful expression vectors such as pET30b and peT3a were also frequently employed to produce large quantities of G6PD variants [9,[19][20][21]27]. More recently, the WT G6PD protein was purified using His-tagged systems such as the pET-HisTEVP vector containing the His-tag and a tobacco etch virus protease (TEVP) recognition site at the N-terminal of the protein [26], and the pET28a His-tag [22] containing the His-tag in the N-terminal where the additional His-tag does not affect the catalysis, structure or stability of the WT G6PD enzyme [22,26], making these systems a good option for obtaining sufficient protein for structural and functional studies of human G6PD and its variants.…”

“…At low temperatures, the recombinant protein can reach a proper folding. Several groups have performed the expression of both WT and mutants of G6PD at different temperatures from 15 to 37 • C [14][15][16][17][18][19][20][21]24,25]. Specifically, Gómez-Manzo et al [19][20][21], found that soluble G6PD levels were highest at 25 • C for the G6PD Yucatan, Valladolid, Mexico City, Nashville, Durham, Santa-Maria, A+, Zacatecas, Viangchan and Vanua-Lava variants.…”

“…Several groups have performed the expression of both WT and mutants of G6PD at different temperatures from 15 to 37 • C [14][15][16][17][18][19][20][21]24,25]. Specifically, Gómez-Manzo et al [19][20][21], found that soluble G6PD levels were highest at 25 • C for the G6PD Yucatan, Valladolid, Mexico City, Nashville, Durham, Santa-Maria, A+, Zacatecas, Viangchan and Vanua-Lava variants. Furthermore, they also found that the optimal G6PD expression was time (18 h) and isopropyl-β-D-thiogalactopyranoside (IPTG) concentration dependent; however, the use of these conditions does not avoid the formation of inactive inclusion bodies [19][20][21].…”