A proteomic study of sodium/D-glucose cotransporter 1 (SGLT1): topology of loop 13 and coverage of other functionally important domains.
In order to obtain further information about the structure and function of human sodium/D-glucose cotransporter 1 (hSGLT1), the recombinant protein was subjected, either after reconstitution into liposomes or in its free form, to proteolysis followed by nanoscale microcapillary liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS). The peptides released from SGLT1 proteoliposomes by trypsin bead digestion represented the early N-terminal, loop 7, and loop 9, supporting topology models that place these domains on the extracellular side of the protein. Trypsin bead digestion generated, however, also a number of peptides derived from loop 13 whose topology with regard to the membrane is hitherto a point of debate. Sequence coverage was provided from amino acids 559 to 644, suggesting that loop 13 is almost completely accessible at the extravesicular face of the proteoliposomes. These results support the notion that major parts of loop 13, essential for the interaction with transport inhibitors in vivo, are located extracellularly in intact cells. In-gel trypsin, chymotrypsin, and in particular trypsin/chymotrypsin digestion of recombinant SGLT1 in combination with LC-MS/MS provide extensive sequence coverage of the protein, including domains involved in sugar and inhibitor binding and potential phosphorylation sites. These studies demonstrate that proteomic analysis combined with mass spectrometry is a useful tool to characterize regions of SGLT1 that are important for its function and regulation.
Kumar
Azad
A
Max Planck Institute of Molecular Physiology, Otto-Hahn-Str. 11, Dortmund 44227, Germany.
Tyagi
Navneet K
NK
Arevalo
Enrique
E
Miller
Keith W
KW
Kinne
Rolf K H
RK
D000595:Amino Acid Sequence; D001665:Binding Sites; D006801:Humans; D008081:Liposomes / Q000737:chemistry; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry* / Q000235:genetics; D017434:Protein Structure, Tertiary; D040901:Proteomics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D051269:Sodium-Glucose Transporter 1 / Q000737:chemistry* / Q000235:genetics; D021241:Spectrometry, Mass, Electrospray Ionization
D008081:Liposomes; D010455:Peptides; D011994:Recombinant Proteins; C495674:SLC5A1 protein, human; D051269:Sodium-Glucose Transporter 1