Lostao MP et al. (2021), The Molecular Basis of Glucose Galactose Malabs...

XB-ART-58433

Function (Oxf) 2021 Jan 01;25:zqab040. doi: 10.1093/function/zqab040.

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The Molecular Basis of Glucose Galactose Malabsorption in a Large Swedish Pedigree.

Lostao MP , Loo DD , Hernell O , Meeuwisse G , Martin MG , Wright EM .

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Glucose-galactose malabsorption (GGM) is due to mutations in the gene coding for the intestinal sodium glucose cotransporter SGLT1 (SLC5A1). Here we identify the rare variant Gln457Arg (Q457R) in a large pedigree of patients in the Västerbotten County in Northern Sweden with the clinical phenotype of GGM. The functional effect of the Q457R mutation was determined in protein expressed in Xenopus laevis oocytes using biophysical and biochemical methods. The mutant failed to transport the specific SGLT1 sugar analog α-methyl-D-glucopyranoside (αMDG). Q457R SGLT1 was synthesized in amounts comparable to the wild-type (WT) transporter. SGLT1 charge measurements and freeze-fracture electron microscopy demonstrated that the mutant protein was inserted into the plasma membrane. Electrophysiological experiments, both steady-state and presteady-state, demonstrated that the mutant bound sugar with an affinity lower than the WT transporter. Together with our previous studies on Q457C and Q457E mutants, we established that the positive charge on Q457R prevented the translocation of sugar from the outward-facing to inward-facing conformation. This is contrary to other GGM cases where missense mutations caused defects in trafficking SGLT1 to the plasma membrane. Thirteen GGM patients are now added to the pedigree traced back to the late 17th century. The frequency of the Q457R variant in Västerbotten County genomes, 0.0067, is higher than in the general Swedish population, 0.0015, and higher than the general European population, 0.000067. This explains the high number of GGM cases in this region of Sweden.

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Species referenced: Xenopus laevis
Genes referenced: ran slc5a1.2
GO keywords: glucose transmembrane transporter activity

???displayArticle.disOnts??? glucose metabolism disease [+]
???displayArticle.omims??? GLUCOSE/GALACTOSE MALABSORPTION; GGM

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	Figure 1. The Northern Swedish Pedigree of GGM patients. The original pedigree, containing cases 1–6, filled symbols (square male, circle female), were traced back to a couple in the late 17th century.7,8 Four suspected cases were also identified in a family with 12 children (hatched symbol) of a distant relative to case 3. Six additional GGM patients: 7, 9–12, and 14, have now been placed on this pedigree, but DNA was only obtained for 6 identified cases in all: 1, 9–12, and 3 others not placed in the pedigree (15–17). The known GGM cases were traced to 4 of the 13 siblings born between 1719 and 1724.
	Figure 2. Sanger sequencing of child with GGM and his parents. The proband has a CGG-R457 mutation, while the mother and father are heterozygotes.
	Figure 3. αMDG uptake by WT and Q457R mutant SGLT1. Oocytes were injected with 50 nl (1 µg/µl) of cRNA coding for WT or Q457R mutant SGLT1. After 3 days, uptake of 50 µM 14C-αMDG was measured. Values represent the mean of 8 oocytes and the error bars indicate the standard error. Q457R mutant uptake was not different from uptake by NI oocytes, 1.7% of the WT uptake (3 vs. 180 pmoles/h/oocyte).
	Figure 4. Western blot analysis of WT and Q457R mutant SGLT1 expressed in oocytes. Seven days after injection with WT or Q457R mutant cRNA, protein was extracted from 2 oocytes each. The equivalent to 1/3 oocyte was run in a 12% SDS-PAGE gel and after transfer to a nitrocellulose membrane, and was probed with an SGLT1 antipeptide antibody raised to residues 602–613, at 1:1000 dilution. Both WT and Q457R mutant proteins ran as 2 broad bands, one 70 kDa band which corresponds to the complex glycosylated form, less intense in the mutant, and another of ∼60 kDa which corresponds to core glycosylated form
	Figure 5. (Freeze-fracture) P fracture face of the plasma membrane of a NI oocyte and oocytes injected with Q457R or WT cRNA. NI oocyte (A) showed 7-nm-diameter particles in the P-face at a density of 200/mm2, whereas in WT injected oocyte (B), the density of particles increased to 1000/mm2. Similar density was obtained in Q457R cRNA injected oocytes (C). Q457R injected oocyte is the same as the one in Figures 7B and 8A. The 25 mM Na+/glucose current was −800 nA at –50 mV for the WT oocyte and 0 for Q457R (Figure 7); the SGLT1 charge movements were 8.5 and 3.7 nC (Figure 8A). Scale bar 200 nanometers.
	Figure 6. Immunolocalization of SGLT1 protein in human small intestine of a normal (control) (A) and a GGM patient (9) with the Q457R missense mutation (B). Double staining procedures shows SGLT1 (red) as a sharp line localized to the brush border of the enterocytes and the basal nucleus stained in blue. Goblet cells between the enterocytes were unstained for SGLT1. There were no differences in location of SGLT1 in enterocytes from the normal and the GGM (magnification x125). In a separate case of GGM, C292Y, the mutant protein did not reach the brush border membrane and was trapped just above the nuclei.14 Scale bar 30 micrometers.
	Figure 7. Steady-state currents induced by αMDG and phlorizin in WT and Q457R mutant proteins. Seven days after injection with cRNA, oocytes expressing WT and mutant SGLT1 were perfused with 100 mM NaCl buffer and currents were measured using a 2-electrode voltage clamp. The difference in steady-state currents measured in the absence and in the presence of αMDG or phlorizin (Pz) is plotted at each test potential from −150 to +50 mV. A. In WT, 25 mM αMDG induced Na+ inward current (1500 nA at −150 mV) whereas 0.1 mM Pz blocked the Na+ leak current (+143 nA at −150 mV). B. In Q457R mutant, both 100 mM αMDG and 0.5 mM Pz inhibited the Na+ leak current (+180 and + 360 nA at −150 mV, respectively). Similar results were obtained on oocytes from 3 different frog donors. The oocyte expressing Q457R-cRNA is the same one as shown in the oocyte in Figure 8B (Q/V with and without sugar) and Figure 5 (freeze-fracture)
	Figure 8. A. Charge-voltage (Q/V) relationships for WT and Q457R SGLT1. A. Charge was obtained by integration of SGLT1 current transients in the absence of sugar (see methods). The symbols correspond to the experimental data after the subtraction of the NI oocyte values, and normalized to WT charge at + 50 mV. The dotted lines are drawn according to the Boltzmann relation. Mutant Qmax (maximal charge transfer) was 40% of the WT value (3.7 ± 0.2 vs. 8.8 ± 0.3 nC). The V0.5 values were −47 and −50 mV, and z 1.4. B. Q/V relationships of Q457R SGLT1 in the absence and in the presence of the 50 mM αMDG. Q was obtained by the integration of current transients in the absence and in the presence of 50 mM αMDG. The symbols correspond to the experimental data and the curves are drawn according to the Boltzmann relation. The addition of 50 mM αMDG induced an increase of Qmax, from 11.5 to 15.5 nC, and a shift of V0.5 from −27 to + 7 mV, but no change in z (1.1).
	Figure 9. A. Charge-voltage (Q/V) relationships for WT and Q457R SGLT1. A. Charge was obtained by integration of SGLT1 current transients in the absence of sugar (see methods). The symbols correspond to the experimental data after the subtraction of the NI oocyte values, and normalized to WT charge at + 50 mV. The dotted lines are drawn according to the Boltzmann relation. Mutant Qmax (maximal charge transfer) was 40% of the WT value (3.7 ± 0.2 vs. 8.8 ± 0.3 nC). The V0.5 values were −47 and −50 mV, and z 1.4. B. Q/V relationships of Q457R SGLT1 in the absence and in the presence of the 50 mM αMDG. Q was obtained by the integration of current transients in the absence and in the presence of 50 mM αMDG. The symbols correspond to the experimental data and the curves are drawn according to the Boltzmann relation. The addition of 50 mM αMDG induced an increase of Qmax, from 11.5 to 15.5 nC, and a shift of V0.5 from −27 to + 7 mV, but no change in z (1.1).
	Figure 10. Molecular model showing sugar-binding site of WT hSGLT1 and mutant Q457R. The molecular models of the outward and inward-facing conformations of hSGLT1 were based on the crystal structures of the sodium sialic acid symporter SiaT from Proteus mirabilis (PDB ID 5NV9) and the sodium glucose cotransporter vSGLT from Vibrio parahaemolyticus (PDB ID 3DH4).34 Computational and experimental approaches were used to validate the homology models. A sugar-binding site in outward open conformation. The sugar coordinating residues Q457, H83, Y290, N78, and E102 are not shown. Note that Q457R does not interact with sugar in this conformation. B. The sugar-binding site in the inward conformation. Note that the inward tilt of TM10 positions Q457 to interact with the pyranose oxygen and the C#6 –OH of glucose. C. Sugar binding site for mutant Q457R in the inward conformation. Note that in this conformation the side-chain of Arg457 encounters steric hindrance to binding with glucose.

References [+] :

Ameur, SweGen: a whole-genome data resource of genetic variability in a cross-section of the Swedish population. 2017, Pubmed