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.
Wimmer
Barbara
B
Institute for Biophysics, Johannes Kepler University Linz, Altenbergerstrasse 69, A-4040 Linz, Austria.
Raja
Mobeen
M
Hinterdorfer
Peter
P
Gruber
Hermann J
HJ
Kinne
Rolf K-H
RK
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D002942:Circular Dichroism; D015870:Gene Expression; D005947:Glucose / Q000737:chemistry* / Q000378:metabolism*; D008051:Lipid Bilayers / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011817:Rabbits; D051269:Sodium-Glucose Transporter 1 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013237:Stereoisomerism; D013379:Substrate Specificity
D008051:Lipid Bilayers; D051269:Sodium-Glucose Transporter 1; D005947:Glucose