Functional analysis of mutations in the glucose-6-phosphate transporter that cause glycogen storage disease type Ib

Abstract

The glucose-6-phosphate transporter (G6PT) deficient in glycogen storage disease type Ib is a phosphate (Pi)-linked antiporter capable of G6P:Pi and Pi:Pi exchanges. We previously characterized G6PT mutations by measuring G6P uptake activities in microsomes co-expressing G6PT and glucose-6-phosphatase-α. Here we report a new assay, based on reconstituted proteoliposomes carrying only G6PT, and characterize G6P and Pi uptake activities of 23 G6PT mutations. We show that co-expression and G6PT-only assays are equivalent in measuring G6PT activity. However, the p.Q133P mutation exhibits differential G6P and Pi transport activities, suggesting that characterizing G6P and Pi transport activities of G6PT mutations may yield insights to this genetic disorder.

The SLC37 Family of Sugar-Phosphate/Phosphate Exchangers

“…10.8) (Chen, Pan, Lee, Peng, & Chou, 2008) similar results have been seen for 22 of the 23 SLC37A4 mutations, confirming the validity of each assay system. Interestingly, the outstanding mutation, p.Q133P, identified in a patient originally classified as a nonGSD-Ia (Veiga-da-Cunha et al, 1999), is devoid of G6P transport activity regardless of the assay methods, but retains 5% wild-type P i transport activity (Chen, Pan, Lee, et al, 2008). The results suggest that further characterization of the G6P and P i transport activities of G6PT mutations may yield insights into this genetic disorder.…”

“…Preloading of proteoliposomes with P i eliminates the need for a coexpressing G6Pase, and the P i -loaded G6PT-proteoliposomes transport both G6P and P i efficiently without a coexpressing G6Pase (Fig. 10.3C) (Chen, Pan, Nandigama, et al, 2008). …”

“…Characterization of missense and codon deletion mutations that result in single amino acid alterations provides valuable information on functionally important residues in a protein. In G6PT, 31 missense and 2 codon deletion mutations have been characterized functionally via site-directed mutagenesis and transient expression assays and were shown to abolish or reduce greatly microsomal G6P uptake activity (Chen et al, 2000, 2002; Chen, Pan, Lee, et al, 2008; Hiraiwa et al, 1999). These mutations are grouped into the following three categories: helical (H), nonhelical that includes cytoplasmic (C) and luminal (L) loops, and terminal that includes N- (p.MIV) and C- (p.R415X) terminal domains (Fig.…”

“…10.9A). One SLC37A4 mutation identified in GSD-Ib/nonGSD-Ia patients, p.Q133P (Veiga-da-Cunha et al, 1999), which alters the first amino acid in this motif, was shown to be devoid of G6P transport activity but retains 5% wild-type P i transport activity (Chen, Pan, Lee, et al, 2008; Chen et al, 2002). The other SLC37A4 mutation identified in GSD-Ib patients, p.G149E (Hiraiwa et al, 1999), which alters the last amino acid in this motif, was shown to be a null mutation that completely abolished microsomal G6P and P i uptake (Chen et al, 2002).…”

Glycogen storage disease type I and G6Pase-β deficiency: etiology and therapy

“…13 of 80 identified mutations (Table 1), 31 missense and two codon deletion mutations have been functionally charac terized by site-directed mutagenesis and by both cell-based G6P transport activity assays 5,37–39 and reconstituted proteoliposome transport assays. 13,40 G6P uptake activity is completely abolished by 21 missense mutations and the two codon deletions, whereas the other mutations only partially inactivate the transporter. GSD-Ib is not restricted to any one racial or ethnic group, but the mu tations show some racial and ethnic variability (Table 2).…”