High-Yield Functional Expression of Human Sodium/d-Glucose Cotransporter1 inPichia pastorisand Characterization of Ligand-Induced Conformational Changes as Studied by Tryptophan Fluorescence

Abstract

Studies on the structure-function relationship of transporters require the availability of sufficient amounts of the protein in a functional state. In this paper, we report the functional expression, purification, and reconstitution of the human sodium/d-glucose cotransporter1 (hSGLT1) in Pichia pastoris and ligand-induced conformational changes of hSGLT1 in solution as studied by intrinsic tryptophan fluorescence. hSGLT1 gene containing FLAG tag at position 574 was cloned into pPICZB plasmid, and the resulting expression vector pPICZB-hSGLT1 was introduced into P. pastoris strain GS115 by electroporation. Purification of recombinant hSGLT1 by nickel-affinity chromatography yields about 3 mg of purified recombinant hSGLT1 per 1-liter of cultured Pichia cells. Purified hSGLT1 migrates on SDS-PAGE with an apparent mass of 55 kDa. Kinetic analysis of hSGLT1 in proteoliposomes revealed sodium-dependent, secondary active, phlorizin-sensitive, and stereospecific alpha-methyl-d-glucopyranoside transport, demonstrating its full catalytic activity. The position of the maximum intrinsic tryptophan fluorescence and titration with hydrophilic collisional quenchers KI, acrylamide, and trichloroethanol suggested that most of Trps in hSGLT1 in solution are in a hydrophobic environment. In the presence of sodium, sugars that have been identified earlier as substrate for the transporter increase intrinsic fluorescence in a saturable manner by a maximum of 15%. alpha-Methyl-d-glucopyranoside had the highest affinity (K(d) = 0.71 mM), followed by d-glucose, d-galactose, d-mannose, and d-allose which showed a much lower affinity. l-Glucose was without effect. d-Glucose also increased the accessibility of the Trps to hydrophilic collisional quenchers. On the contrary phlorizin, the well-established inhibitor of SGLT1, decreased intrinsic fluorescence by a maximum of 50%, and induced a blue shift of maximum (5 nm). Again, the effects were sodium-dependent and saturable and a high affinity K(d) of 5 muM was observed. In addition the surface of hSGLT1 was labeled with 1-anilinonaphthalene-8-sulfonic acid, a reporter molecule for the surface hydrophobicity. In the presence of sodium, addition of d-glucose decreased ANS fluorescence whereas phlorizin increased ANS fluorescence. Thus three conformational states of SGLT1 could be defined which differ in their packing density and hydrophobicity of their surface. They reflect properties of the empty carrier, the d-glucose loaded carrier facing the outside of membrane and the complex of the outside-orientated carrier with phlorizin.

Three Surface Subdomains Form the Vestibule of the Na+/Glucose Cotransporter SGLT1

“…Furthermore, depending on the conformation of the carrier (see for example Ref. 43), different disulfide linkages may be formed.…”

Role of β-Adrenoceptors in Memory Consolidation: β3-Adrenoceptors Act on Glucose Uptake and β2-Adrenoceptors on Glycogenolysis

“…All of these GLUTs as well as the SGLT transporter on endothelial cells are inhibited by the nonselective drug cytochalasin B. The effects of this nonspecific GLUT inhibition is contrasted by the effect of two more selective inhibitors, phloretin (Takakura et al, 1991;Duelli and Kuschinsky, 2001) and phlorizin (Ehrenkranz et al, 2005;Tyagi et al, 2005), that inhibit GLUT-1 and SGLT, respectively. The effect of different doses of the GLUT inhibitors was examined by injection 30 min after strongly reinforced training, a time at which 2-DG inhibits memory (Table 1).…”

Ligand-mediated conformational changes and positioning of tryptophans in reconstituted human sodium/d-glucose cotransporter1 (hSGLT1) probed by tryptophan fluorescence

“…hSGLT1 was expressed in Pichia pastoris and purified to homogeneity by nickel-affinity chromatography, and reconstituted into liposomes according to the method describe in Ref [7].…”

“…All fluorescence studies on hSGLT1 reported so far were either carried out in Xenopus oocytes [6] or in solution [7]. However in solution some transporter proteins can bind ligands from both sides as reported for UhpT transporter of E. coli [8] so we decided to perform fluorescence studies of hSGLT1 in a reconstituted form which is more relevant for membrane proteins.…”