Phlorizin Binding Research Articles

Soy protein–phlorizin conjugate prepared by tyrosinase catalysis: Identification of covalent binding sites and alterations in protein structure and functionality

Tyrosinase-catalyzed synthesis of soy 7S/11S–phlorizin conjugates was performed, and the reaction sites, conformation alterations and functional properties of complexes were evaluated using proteomic, in combination with multispectral technologies. Phlorizin was conjugated to 7S/11S primarily via residues of Lys, Cys, His and Arg residues. The phlorizin binding equivalents and decreased contents of free and total sulfhydryl groups and free amino groups confirmed the covalent interaction in the 7S/11S–phlorizin complexes. Conjugation with phlorizin promoted the conversion of α-helix to β-sheet and β-turn, with simultaneous transformation of the microenvironments around Trp and Tyr residues to hydrophilic and hydrophobic microenvironments, respectively, and lowering of the surface hydrophobicity of 7S/11S. The DPPH and ABTS radical scavenging abilities and α-glucosidase inhibitory activities of 7S/11S were increased by three–, two– and three–fold after the covalent binding of phlorizin. The study provided an ideal tyrosinase-catalyzed approach to fabricate custom–tailored nutritional soy protein–polyphenol products.

Comparative study of binding interactions between different dietary flavonoids and soybean β-conglycinin and glycinin: Impact on structure and function of the proteins

The mechanisms underlying the interaction between different dietary flavonoids and soybean β-conglycinin (7S) and glycinin (11S) were comparatively investigated, and the alterations in conformation and function of the complexes were further evaluated. Among the 23 flavonoids studied, 3 flavonoids with glycosides with the top three ranked T Scores in molecular docking analysis—phlorizin, luteoloside, and vitexin-4′-O-glucoside—with the highest binding affinity to 7S and 11S and quenched their intrinsic fluorescence in a static manner. The binding interactions of these flavonoids to 7S and 11S were structure dependent. Hydrophobic forces played important roles in interactions between 7S and both luteoloside and vitexin-4′-O-glucoside, whereas the binding of phlorizin to 7S, and of the three flavonoids to 11S, was driven by hydrogen bonding and van der Waals forces. The binding of these flavonoids interfered with the microenvironment around tyrosine and tryptophan, thereby altering the secondary structures of 7S and 11S. The binding of the three flavonoids enhanced solubility, emulsifying properties, thermal stability, and antioxidant capacity of 7S and 11S. The use of flavonoids could facilitate the design of soybean protein-based products with desirable functional properties.

Different phlorizin binding properties to porcine mucosa of the jejunum and ileum in relation to SGLT1 activity

Different phlorizin binding properties to porcine mucosa of the jejunum and ileum in relation to SGLT1 activity

Humanization of the Substrate Binding Site in vSGLT

Secondary active transporters utilize the energy of electrochemical gradients to drive the uphill transport of a second substrate. Sodium Glucose Cotransporters (SGLT) constitute a large family of symporters that use Na+ ions to cotransport glucose or galactose as well as a variety of other substrates. SGLTs are responsible for the absorption of dietary sugars in the intestine and reabsorption from primary urine in the kidney. Recently, inhibitors of SGLTs came into focus as novel drugs to treat diabetes. Phlorizin-derivatives competitively inhibit reabsorption of sugar from the glomerular filtrate, thereby increasing its excretion in the urine and ultimately lowering blood sugar concentrations.However, to this date, no structural information on human SGLTs is available. Only the 3D structure for the bacterial homologue vSGLT from Vibrio parahaemolyticus which does not bind phlorizin, has been solved. Therefore the binding mechanism of these inhibitors still remains not very well understood. To gain further insights into the determinants for phlorizin binding, we designed several point mutants to mimic the presumed binding site in human SGLT. Subsequently we determined sugar and phlorizin binding to the purified mutant proteins by microscale thermophoresis. This information will increase the value of vSGLT as a model system sodium-dependent sugar transport in humans and might ultimately help to design new and more specific inhibitors.

Identification of phlorizin binding domains in sodium-glucose cotransporter family: SGLT1 as a unique model system.

The sodium glucose cotransporter SGLT1 expressed mainly in the intestine and kidney has been explored extensively for understanding the mechanism of sugar cotransport and its inhibition by a classical competitive inhibitor, phlorizin (Pz). It has been shown that inhibition of SGLT1 by Pz involves its interaction followed by major conformational changes in the Pz binding domain (PBD) in C-terminal loop 13. However, the mechanism of Pz inhibition and its interaction with other members of SGLT is not known. In this hypothesis, we performed molecular modeling of SGLT1-loop 13 with Pz and carried out primary sequence analyses and secondary structure predictions to determine qualitatively similar PBDs in C-termini of human SGLT2-4, except for vSGLT, which contains an unstructured short C-terminus. The ranking of predictions of Pz interaction strongly agrees with the following ranking of previously reported Pz inhibition: SGLT2>SGLT1>SGLT4>SGLT3>>vSGLT. In addition, the sugar binding residues were found to be quite conserved among all SGLT members investigated here. Based on these preliminary analyses, we propose that other Pz-sensitive SGLTs are also inhibited via mechanism similar to SGLT1 where an aglucone of Pz, phloretin, interacts with PBD and glucoside moiety with sugar binding residues. Our hypothesis sets the stage for future analyses on investigation of Pz interaction with SGLT family and further suggests that Pz modeling may be explored to design novel inhibitors targeting several SGLT members.

Bridging the gap between structure and kinetics of human SGLT1

The Na(+)-glucose cotransporter hSGLT1 is a member of a class of membrane proteins that harness Na(+) electrochemical gradients to drive uphill solute transport. Although hSGLT1 belongs to one gene family (SLC5), recent structural studies of bacterial Na(+) cotransporters have shown that Na(+) transporters in different gene families have the same structural fold. We have constructed homology models of hSGLT1 in two conformations, the inward-facing occluded (based on vSGLT) and the outward open conformations (based on Mhp1), mutated in turn each of the conserved gates and ligand binding residues, expressed the SGLT1 mutants in Xenopus oocytes, and determined the functional consequences using biophysical and biochemical assays. The results establish that mutating the ligand binding residues produces profound changes in the ligand affinity (the half-saturation concentration, K(0.5)); e.g., mutating sugar binding residues increases the glucose K(0.5) by up to three orders of magnitude. Mutation of the external gate residues increases the Na(+) to sugar transport stoichiometry, demonstrating that these residues are critical for efficient cotransport. The changes in phlorizin inhibition constant (K(i)) are proportional to the changes in sugar K(0.5), except in the case of F101C, where phlorizin K(i) increases by orders of magnitude without a change in glucose K(0.5). We conclude that glucose and phlorizin occupy the same binding site and that F101 is involved in binding to the phloretin group of the inhibitor. Substituted-cysteine accessibility methods show that the cysteine residues at the position of the gates and sugar binding site are largely accessible only to external hydrophilic methanethiosulfonate reagents in the presence of external Na(+), demonstrating that the external sugar (and phlorizin) binding vestibule is opened by the presence of external Na(+) and closes after the binding of sugar and phlorizin. Overall, the present results provide a bridge between kinetics and structural studies of cotransporters.

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

To investigate whether the C-terminal loop 13 of rabbit sodium/glucose cotransporter SGLT1 is involved in the recognition of the substrate d-glucose, isolated loop 13 (amino acids (aa) 541-638) was immobilized to a lipid bilayer. Interactions were investigated by surface plasmon resonance spectroscopy using an antibody directed against the late part of the loop (aa 606-631) or the glucoside transport inhibitor phlorizin. Specific binding of the antibody to the loop could be detected. The number of bound antibodies decreased upon the addition of d-glucose but not upon the addition of l-glucose. Phlorizin also significantly lowered the number of bound antibodies. Binding of phlorizin to the loop could also be demonstrated directly. Binding of phlorizin was, however, reduced to a similar extent upon the addition of either d-glucose or l-glucose, indicating their unspecific competition with the inhibitor's sugar moiety. Thus, the presence of a stereospecific glucose interaction site in the late part of the loop and a second, but non-stereospecific, sugar binding site on the same loop was assumed. To investigate whether the early part of loop 13 contains this non-stereospecific sugar binding site, peptides containing aa 541-598 were expressed in Escherichia coli and purified. Both d-glucose and l-glucose quenched the peptides tryptophan fluorescence and reduced the Trp accessibility to acrylamide to a similar degree. In view of the recently proposed transmembrane orientation of loop 13, the two binding sites may be part of the extracellular (stereospecific) and intracellular (non-stereospecific) sugar interaction sites of SGLT1.

Inhibitor Binding in the Human Renal Low- and High-Affinity Na+/Glucose Cotransporters

The kidney contains two Na(+)/glucose cotransporters, called SGLT2 and SGLT1, arranged in series along the length of the proximal tubule. The low-affinity transporter, SGLT2, is responsible for the reabsorption of most of the glucose in the kidney. There is recent interest in SGLT2 as a target for the treatment of type II diabetes using selective inhibitors based on the structure of the phenylglucoside, phlorizin (phloretin-2'-beta-glucoside). In this study, we examined the inhibition of alpha-methyl-d-glucopyranose transport by phlorizin and a new candidate drug, sergliflozin-A [(2-[4-methoxyphenyl]methyl)phenyl beta-d-glucopyranoside], in COS-7 cells expressing hSGLT1 and hSGLT2. Inhibition by phlorizin was competitive, with K(i) values of 0.3 muM in hSGLT1 and 39 nM in hSGLT2. Inhibition by sergliflozin-A was also competitive, with K(i) values of 1 muM in hSGLT1 and 20 nM in hSGLT2. Phloretin [3-(4-hydroxyphenyl)-1-(2,4,6-trihydroxyphenyl)-1-propanone; the aglucone of phlorizin] was a less potent inhibitor, with IC(50) values of 142 muM in hSGLT1 and 25 muM in hSGLT2. Site-directed mutagenesis of residues believed to be in the phlorizin binding site showed that only Cys610 is involved in inhibitor binding in the human transporters. Mutation of Cys610 in hSGLT1 to lysine resulted in an increased IC(50) for all inhibitors. In contrast, mutagenesis of the analogous Cys615 in hSGLT2 produced the opposite effect, a decrease in IC(50) for phlorizin and sergliflozin-A. The differences in the effects of the mutations between hSGLT1 and hSGLT2 suggest that this cysteine holds key residues in place rather than participating directly in inhibitor binding.

D-Glucose-Recognition and Phlorizin-Binding Sites in Human Sodium/d-Glucose Cotransporter 1 (hSGLT1): A Tryptophan Scanning Study

In order to gain a better understanding of the structure-function relation in hSGLT1, single Trp residues were introduced into a functional hSGLT1 mutant devoid of Trps at positions that previously had been postulated to be involved in sugar recognition/translocation and/or phlorizin binding. The mutant proteins were expressed in Pichia pastoris, purified, and reconstituted into liposomes. In transport experiments the putative sugar binding site mutants W457hSGLT1 and W460hSGLT1 showed a drastic decrease in affinity toward alpha-methyl-d-glucopyranoside with Km values of 13.3 and 5.26 mM compared to 0.4 mM of the Trp-less hSGLT1. In addition, a strong decrease in the inhibitory effect of phlorizin was observed. In Trp fluorescence studies the position of the emission maxima of the mutants, their sensitivity to N-bromosuccinimide oxidation, and their interaction with water soluble quenchers demonstrate that Trp457 and Trp460 are in contact with the hydrophilic extravesicular environment. In both mutants Trp fluorescence was quenched significantly, but differently, by various glucose analogues. They also show significant protection by d-glucose and phlorizin against acrylamide, KI, or TCE quenching. W602hSGLT1 and W609hSGLT1, the putative aglucone binding site mutants, exhibit normal sugar and phlorizin affinity, and show fluorescence properties which indicate that these residues are located in a very hydrophilic environment. Phlorizin and phloretin, but not d-glucose, protect both mutants against collisional quenchers. Depth-calculations using the parallax method suggest a location of Trp457 and Trp460 at an average distance of 10.8 A and 7.4 A from the center of the bilayer, while Trp602 and Trp609 are located outside the membrane. These results suggest that in the native carrier residues Gln at position 457 and Thr at position 460 reside in a hydrophilic access pathway extending 5-7 A into the membrane to which sugars as well as the sugar moiety of inhibitory glucosides bind. Residues Phe602 and Phe609 contribute by their hydrophobic aromatic residues toward binding of the aglucone part of phlorizin. Thereby in the phlorizin-carrier complex a close vicinity between these two subdomains of the transporter is established creating a phlorizin binding pocket with the previously estimated dimensions of 10 x 17 x 7 A.

Hydrophobic ligand binding properties of the human lipocalin apolipoprotein M

Apolipoprotein M (apoM) is a plasma protein associated mainly with HDL. ApoM is suggested to be important for the formation of prebeta-HDL, but its mechanism of action is unknown. Homology modeling has suggested apoM to be a lipocalin. Lipocalins share a structurally conserved beta-barrel, which in many lipocalins bind hydrophobic ligands. The aim of this study was to test the ability of apoM to bind different hydrophobic substances. ApoM was produced both in Escherichia coli and in HEK 293 cells. Characterization of both variants with electrophoretic and immunological methods suggested apoM from E. coli to be correctly folded. Intrinsic tryptophan fluorescence of both apoM variants revealed that retinol, all-trans-retinoic acid, and 9-cis-retinoic acid bound (dissociation constant = 2-3 microM), whereas other tested substances (e.g., cholesterol, vitamin K, and arachidonic acid) did not. The intrinsic fluorescence of two apoM mutants carrying single tryptophans was quenched by retinol and retinoic acid to the same extent as wild-type apoM, indicating that the environment of both tryptophans was affected by the binding. In conclusion, the binding of retinol and retinoic acid supports the hypothesis that apoM is a lipocalin. The physiological relevance of this binding has yet to be elucidated.

Na+-d-glucose cotransporter in the kidney of Leucoraja erinacea: molecular identification and intrarenal distribution

Studies on membrane vesicles from the kidney of Leucoraja erinacea suggested the sole presence of a sodium-D-glucose cotransporter type 1 involved in renal D-glucose reabsorption. For molecular characterization of this transport system, an mRNA library was screened with primers directed against conserved regions of human sglt1. A cDNA was cloned whose nucleotide and derived amino acid sequence revealed high homology to sodium glucose cotransporter 1 (SGLT1). Xenopus laevis oocytes injected with the respective cRNA showed sodium-dependent high-affinity uptake of D-glucose. Many positions considered functionally essential for sodium glucose cotransporter 1 (SGLT1) are also found in the skate protein. High conservation preferentially in transmembrane helices and small linking loops suggests early appearance and continued preservation of these regions. Larger loops, especially loop 13, which is associated with phlorizin binding, were more variable, as is the interaction with the specific inhibitor in various species. To study the intrarenal distribution of the transporter, a skate SGLT1-specific antibody was generated. In cryosections of skate kidney, various nephron segments could be differentiated by lectin staining. Immunoreaction with the antibody was observed in the proximal tubule segments PIa and PIIa, the early distal tubule, and the collecting tubule. Thus Leucoraja, in contrast to the mammalian kidney, employs only SGLT1 to reabsorb d-glucose in the early, as well as in the late segments of the proximal tubule and probably also in the late distal tubule (LDT). Thereby, it differs also partly from the kidney of the close relative Squalus acanthias, which uses SGLT2 in more distal proximal tubule segments but shows also expression in the later nephron parts.

Nephrotoxicity of uranyl acetate: effect on rat kidney brush border membrane vesicles

Since the Gulf war exposure to depleted uranium, a known nephrotoxic agent, there is a renewed interest in the toxic effects of uranium in general and its mechanism of nephrotoxicity which is still largely unknown in particular. In order to investigate the mechanism responsible for uranium nephrotoxicity and the therapeutic effect of urine alkalization, we utilized rat renal brush border membrane vesicles (BBMV). Uranyl acetate (UA) caused a decrease in glucose transport in BBMV. The apparent K (i) of uranyl was 139+/-30 microg uranyl/mg protein of BBMV. Uranyl at 140 microg/mg protein of BBMV reduced the maximal capacity of the system to transport glucose [V (max) 2.2+/-0.2 and 0.96+/-0.16 nmol/mg protein for control and uranyl treated BBMV (P<0.001), respectively] with no effect on the apparent K (m) (1.54+/-0.33 and 1.54+/-0.51 mM for control, and uranyl treated BBMV, respectively). This reduction in V(max) is at least partially due to a decrease in the number of sodium-coupled glucose transporters as apparent from the reduction in phlorizin binding to the uranyl treated membranes, V (max) was reduced from 247+/-13 pmol/mg protein in control BBMV to 119+/-3 pmol/mg protein in treated vesicles (P<0.001). The pH of the medium has a profound effect on the toxicity of UA on sodium-coupled glucose transport in BBMV: higher toxicity at neutral pH (around pH 7.0), and practically no toxicity at alkaline pH (7.6). This is the first report showing a direct inhibitory dose and pH dependent effect of uranyl on the glucose transport system in isolated apical membrane from kidney cortex.

Enhanced glucose absorption in the rat small intestine following repeated doses of 5-fluorouracil

Many studies demonstrated that 5-fluorouracil (5-FU) treatment of rodents caused the damage of small intestine, resulting in the malabsorption, while we recently found that repeated administration of 5-FU to rats increased Na +-dependent glucose absorption in the small intestine. This study investigated the cause of enhanced glucose absorption. 3- O-methyl- d-glucose (3-OMG) absorption was examined using the everted intestine technique. d-Glucose uptake, phlorizin binding, Western blot analysis and membrane fluidity were examined using small intestinal brush-border membrane vesicles (BBMV). Repeated oral administration of 5-FU to rats increased Na +-dependent 3-OMG absorption in the small intestine, while alkaline phosphatase activity in the small intestine decreased. Na +/K +-ATPase activity of 5-FU-treated rats was about three-fold higher than that of control rats. Although the amount of Na +-dependent glucose co-transporter (SGLT1) in 5-FU-treated rats decreased, the overshoot magnitude of d-glucose uptake in BBMV was not altered. Maximum binding of phlorizin in 5-FU-treated rats was 1.5-fold larger than that of control rats, but not altered the maximal rate of d-glucose absorption, Michaelis constant of d-glucose and dissociation constant of phlorizin. The membrane fluidity of 5-FU-treated rats increased. The enhanced d-glucose absorption in 5-FU-treated rats seems to occur secondarily due to the activation of Na +/K +-ATPase activity in basolateral membranes (BLM). Because the amounts of SGLT1 in 5-FU-treated rats decreased, the increase of turnover rate of SGLT1 and/or an expression of unknown Na +-dependent glucose co-transporter with high affinity for d-glucose and phlorizin sensitivity would contribute to the enhancement of d-glucose transport in 5-FU-treated rats.

Spray-Dried Porcine Plasma Reduces the Effects of Staphylococcal Enterotoxin B on Glucose Transport in Rat Intestine

We investigated the intestinal transport of D-glucose (D-Glc) and 3 essential amino acids in a model of intestinal inflammation, and the effects of dietary supplementation with animal plasma proteins on this function. Wistar Lewis rats were fed a diet containing an isonitrogenous amount of milk protein (control group) or a diet supplemented with either spray-dried animal plasma (SDAP) or immunoglobulin concentrate (IC) from porcine plasma, from d 21 of life (weaning) until d 35. On d 30 and 33, rats were challenged intraperitoneally with Staphylococcus aureus enterotoxin B (SEB; groups SEB, SEB-SDAP, and SEB-IC) and on d 35, brush border membrane vesicles (BBMVs) were prepared and used for transport and binding studies. Administration of SEB reduced D-Glc transport across sodium glucose transporter 1 [SGLT1; 20% reduction in maximal transport rate (Vmax); P < 0.05], without affecting the Michaelis constant (Km). The results from specific phlorizin binding, Western blot, and immunohistochemistry supported the view that the effects of SEB are due to reduced expression of D-Glc transporters in the apical membrane. SEB increased the passive diffusion constant (Kd) for D-Glc 3-fold (P < 0.05). SEB did not affect mediated or passive amino acid fluxes of L-leucine, L-methionine, or L-lysine. Dietary SDAP increased the D-Glc Vmax in the SEB group without affecting the passive component. Changes in d-Glc Vmax due to SEB and to the dietary treatments were correlated with changes in the number of SGLT1 transporters present in the BBMVs (r = 0.9468; P < 0.05). Dietary IC had no observed effect. We estimate that, in rats challenged with SEB, SDAP supplementation can increase glucose absorption by 8-9% during the interdigestive periods.

Membrane topology of loop 13–14 of the Na +/glucose cotransporter (SGLT1): A SCAM and fluorescent labelling study

The accessibility of the hydrophilic loop between putative transmembrane segments XIII and XIV of the Na +/glucose cotransporter (SGLT1) was studied in Xenopus oocytes, using the substituted cysteine accessibility method (SCAM) and fluorescent labelling. Fifteen cysteine mutants between positions 565 and 664 yielded cotransport currents of similar amplitude than the wild-type SGLT1 (wtSGLT1). Extracellular, membrane-impermeant MTSES (−) and MTSET (+) had no effect on either cotransport or Na + leak currents of wtSGLT1 but 9 mutants were affected by MTSES and/or MTSET. We also performed fluorescent labelling on SGLT1 mutants, using tetramethylrhodamine-5-maleimide and showed that positions 586, 588 and 624 were accessible. As amino acids 604 to 610 in SGLT1 have been proposed to form part of a phlorizin (Pz) binding site, we measured the K i Pz and K m αMG for wtSGLT1 and for cysteine mutants at positions 588, 605–608 and 625. Although mutants A605C, Y606C and D607C had slightly higher K i Pz values than wtSGLT1 with minimal changes in K m αMG, the effects were modest and do not support the original hypothesis. We conclude that the large, hydrophilic loop near the carboxyl terminus of SGLT1 is thus accessible to the external solution but does not appear to play a major part in the binding of phlorizin.

Na-dependent D-Glucose Transport by Intestinal Brush Border Membrane Vesicles from Gilthead Sea Bream (Sparus aurata)

Brush border membrane vesicles (BBMV) enriched in sucrase, maltase and alkaline phosphatase, and impoverished in Na(+)-K(+)-ATPase, were isolated from proximal and distal intestine of the gilthead sea bream (Sparus aurata) by a MgCl(2) precipitation method. Vesicles were suitable for the study of the characteristics of D-glucose apical transport. Only one D-glucose carrier was found in vesicles from each intestinal segment. In both cases, the D-glucose transport system was sodium-dependent, phlorizin-sensitive, significantly inhibited by D-glucose, D-galactose, alpha-methyl-D-glucose, 3-O-methyl-D-glucose and 2-deoxy-D-glucose, and showed stereospecificity. Apparent affinity constants of D-glucose transport (K(t)) were 0.24 +/- 0.03 mM in proximal and 0.18 +/- 0.03 mM in distal intestine. Maximal rate of influx (Jmax) was 47.3 +/- 2.2 pmols. mg(-1) protein for proximal and 27.3 +/- 3.6 pmols. mg(-1) protein for distal intestine. Specific phlorizin binding and relative abundance of an anti-SGLT1 reactive protein were significantly higher in proximal than in distal BBMV. These results suggest the presence of the same D-glucose transporter along the intestine, with a higher density in the proximal portion. This transporter is compatible with the sodium-dependent D-glucose carrier described for other fish and with the SGLT1 of higher vertebrates.

Binding of phlorizin to the C-terminal loop 13 of the Na +/glucose cotransporter does not depend on the [560–608] disulfide bond

The disulfide bonds of the Na +/glucose cotransporter (SGLT1) are believed to participate in the binding of the transport inhibitor phlorizin. Here, we investigated the role of the [560–608] disulfide bond on the phlorizin-binding function of the C-terminal loop 13 of SGLT1 using 3-iodoacetamidophlorizin (3-IAP) as a probe. The reactivity of 3-IAP to the fully reduced loop 13 was competitively inhibited by phlorizin, as evident from the MALDI mass spectra. It indicates that the disulfide bond is not mandatory for phlorizin binding. CD and equilibrium unfolding studies showed that the secondary structure and conformation stability of loop 13 were not affected by removing the disulfide bond. Furthermore, we generated a series of loop 13 mutants to assess the contribution of the disulfide bond to phlorizin binding. A positive correlation between the stability and phlorizin affinity of the mutant proteins was observed, implying that the protein stability, rather than the disulfide bond, is relevant to the phlorizin-binding function of loop 13.

Role of hydration in the conformational transitions between unliganded and liganded forms of loop 13 of the Na +/glucose cotransporter 1

SGLT1 as a Na +/glucose cotransporter is inhibited by phlorizin, a phloretin 2 ′-glucoside that has strong interactions with the C-terminal loop 13 (residues 541–638). Here we investigated the effect of a partial substitution of glycerol for water in the medium on the stability and phlorizin-binding function of loop 13 using fluorescence spectroscopy. Increasing the glycerol concentration promoted an increase in the stability of the protein to urea. The ability of loop 13 to expose hydrophobic surface promoted by phlorizin binding was partially lost in the presence of glycerol (20%). Glycerol also led to a decrease in the phlorizin affinity of loop 13 in solution. Approximately 15 molecules of water were taken up to cover additional surface area (137.7 ± 27.9 Å 2) upon formation of the loop 13-phlorizin complex. Together these results demonstrate quantitatively that the stability and phlorizin affinity of loop 13 are critically dependent on protein hydration.

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

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 site-directed 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 ( approximately 14 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 (Kd approximately 54 microm). Also the maximum change in the collisional quenching constant by acrylamide was noted for D611W (KSV = 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 alpha-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.

Binding of phlorizin to the isolated C-terminal extramembranous loop of the Na+/glucose cotransporter assessed by intrinsic tryptophan fluorescence.

Phlorizin, a phloretin 2'-glucoside, is a potent inhibitor of the Na(+)/glucose cotransporter (SGLT1). On the basis of transport studies in intact cells, a binding site for phlorizin was suggested in the region between amino acids 604-610 of the C-terminal loop 13. To further investigate phlorizin binding titration experiments of the intrinsic Trp fluorescence of isolated wild-type loop 13 and two mutated loops (Y604K and G609K) were carried out. Phlorizin (135 microM) produced approximately 40% quenching of the fluorescence of wild-type loop 13; quenching could also be observed with the two mutated loops. The apparent K(d) was lowest for the wild-type loop 13 (K(d) approximately 23 microM), followed by mutant G609K (57 microM) and mutant Y604K (70 microM). Binding of phlorizin was further confirmed by a decrease of the accessibility of loop 13 to the collisional quencher acrylamide. The interaction involves the aromatic moiety of the aglucone since phloretin (the aglucone of phlorizin) showed almost the same effects as phlorizin, while d-glucose did not. MALDI-TOF experiments revealed that loop 13 contained a disulfide bond between Cys 560 and Cys 608 that is very important for phlorizin-dependent fluorescence quenching. These studies provide direct evidence that loop 13 is a site (important amino acids including 604-609) for the molecular interaction between SGLT1 and phlorizin. They confirm that the aglucone part of the glucoside is responsible for this interaction.