Phlorizin Recognition in a C-terminal Fragment of SGLT1 Studied by Tryptophan Scanning and Affinity Labeling

Abstract

SGLT1 as a sodium/glucose cotransporter is strongly inhibited by phlorizin, a phloretin 2-glucoside that has strong interactions with the C-terminal loop 13. We have examined phlorizin recognition by the protein by sitedirected single Trp scanning mutagenesis experiments. Six mutants (Q581W, E591W, R601W, D611W, E621W, and L630W) of truncated loop 13 (amino acids 564 -638) were expressed in Escherichia coli and purified to homogeneity. Changes in Trp quenching and positions of the emission maxima were determined after addition of phlorizin. D611W displayed the largest quenching of 80%, followed by R601W (67%). It also exhibited the maximum red shift in Trp fluorescence (ϳ14 nm), indicating an exposure of this region to a more hydrophilic environment. Titration experiments performed for each mutant showed a similar affinity for all mutants, except for D611W, which exhibited a significantly lower affinity (K d Ϸ 54 M). Also the maximum change in the collisional quenching constant by acrylamide was noted for D611W (K SV ‫؍‬ 11 M ؊1 in the absence of phlorizin and 55 M ؊1 in its presence). Similar results were obtained with phloretin. CD measurements and computer modeling revealed that D611W is positioned in a random coil situated between two ␣-helical segments. By combining gel electrophoresis, enzymatic fragmentation, and matrix-assisted laser desorption ionization mass spectrometry, we also analyzed truncated loop 13 photolabeled with 3-azidophlorizin. The attachment site of the ortho-position of aromatic ring B of phlorizin was localized to Arg-602. Taken together, these data indicate that phlorizin binding elicits changes in conformation leading to a less ordered state of loop 13. Modeling suggests an interaction of the 4-and 6-OH groups of aromatic ring A of phlorizin with the region between amino acids 606 and 611 and an interaction of ring B at or around amino acid 602. Phloretin seems to interact with the same region of the protein.In mammals, transepithelial transport of D-glucose is mediated by SGLT1 (sodium/glucose cotransporter-1), which can be found in the brush-border membranes of the small intestine and kidney. The transporter facilitates the effective uptake of glucose into cells driven by the electrochemical potential difference of sodium. Coupling and translocation are supposed to be accompanied by conformational changes in the protein. Such changes could be induced, for example, by Na ϩ , which increases the affinity of the cotransporter for sugar (1, 2). Extensive mutagenesis studies revealed that the N-terminal half of the protein contains the Na ϩ -binding sites, whereas glucose binds and permeates through the C-terminal half of the cotransporter (3, 4). Amino acids 162-173 apparently constitute part of an external Na ϩ pore in the SGLT1 protein, whereas sugar binding is controlled by Gln-457 and Thr-460 (5, 6).The cotransport system is inhibited by glucosides with either aromatic or aliphatic aglucon residues. Phlorizin, a ␤-glucoside of the aromatic compound phloretin, is the most potent com...

C-terminal Loop 13 of Na+/Glucose Cotransporter 1 Contains Both Stereospecific and Non-stereospecific Sugar Interaction Sites

“…In previous studies binding of the aglucone moiety of phlorizin could be shown to occur around aa 602 (ring B) and between aa 606 and aa 611 (ring A) (6). These interactions would bring the phlorizin molecule into a position where the glucose moiety points to a region downstream to the C terminus.…”

“…MALDI Mass Spectrometry-Mass spectra were acquired in the positive ion linear mode on a Voyager DE-PRO MALDI system (PE Biosystems) as described previously (6). Briefly, after mixing the solubilized protein with 2,5-dihydroxybenzoic acid matrix (saturated 2,5-dihydroxybenzoic acid solution in 0.1% trifluoroacetic acid and 50% acetonitrile), 0.5-l aliquots of the mixture were dispensed onto the sample support followed by 0.5 l of ␣-cyano-4-hydroxycinnamic acid matrix solution (solution of ␣-cyano-4-hydroxycinnamic acid in 0.1% trifluoroacetic acid and 50% acetonitrile).…”

“…Phlorizin, a ␤-glucoside of the aromatic compound phloretin, is the most potent inhibitor with an apparent K i value of 1 M (11). In previous studies we have shown that phlorizin binds to loop 13 of rabbit SGLT1 (aa 541-638) via the aromatic aglucone moiety inducing conformational changes of the loop, predominantly in the region between aa 600 and aa 621 (4,6) of the C terminus. Mutagenesis experiments provided also data on individual amino acids critically important for the interaction with the inhibitors (6,13,14).…”

Functional characterization of a putative disaccharide membrane transporter in crustacean intestine

“…Phloridzin is a competitive inhibitor of SGLT1 that acts in a twostep process. Initially, instead of glucose binding, phloridzin binds with SGLT1, followed by a slow isomerization that results in phloridzin bound to the receptor-site of the SGLT (Raja et al, 2003). With phloridzin blocking the receptor, glucose cannot bind to the symporter and uptake is blocked on the apical side of the intestinal cell (Zheng et al, 2012).…”

Forces and Dynamics of Glucose and Inhibitor Binding to Sodium Glucose Co-transporter SGLT1 Studied by Single Molecule Force Spectroscopy

“…To this end various methods have been used such as kinetic studies (7), electrophysiology methods (8), tryptophan scanning studies (9,10), mutagenesis studies (11,12), x-ray crystallography (13), and plasmon resonance spectroscopy (14) to name a few. Crystallographic data are available for Vibrio parahemeolyticus sodium/galactose symporter (vSGLT) (13) in the sodium-and galactose-bound state.…”