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.