C-terminal loop 13 of Na+/glucose cotransporter 1 contains both stereospecific and non-stereospecific sugar interaction sites

Abstract

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-glu-cose 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. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.