Identification of the histidyl residue obligatory for the catalytic activity of the human H+/peptide cotransporters PEPT1 and PEPT2.
Histidyl residues are known to be essential for the catalytic function of the H(+)-coupled peptide transporters expressed in the intestine and the kidney, most likely participating in the binding and translocation of H+. Three histidyl residues are conserved among the intestinal and renal peptide transporters (PEPT1 and PEPT2, respectively) from different animal species. In hPEPT1, these residues are His-57, His-121, and His-260. The corresponding residues in hPEPT2 are His-87, His-142, and His-278. We have individually mutated each of these histidyl residues in hPEPT1 and in hPEPT2 and compared the catalytic function of the mutants with that of their respective wild type transporters by expressing the transporters in Xenopus laevis oocytes and also in HeLa cells. His-57 in hPEPT1 and His-87 in hPEPT2 were found to be absolutely essential for catalytic activity because the corresponding mutants had no detectable peptide transport activity. His-121 in hPEPT1 is not essential since mutation of this residue did not impair transport function. His-142 in hPEPT2 was found to play a significant role in the maintenance of transport function but was not found to be obligatory because the mutant had appreciable transport activity. The obligatory histidyl residue (His-57 in hPEPT1 and His-87 in hPEPT2) is located in an almost identical topological position in both transporters, near the extracellular surface of the second putative transmembrane domain. The second conserved histidyl residue is located in the fourth putative transmembrane domain in hPEPT1 as well as in hPEPT2. The third conserved histidyl residue is present in the cytoplasmic loop between the transmembrane domains 6 and 7 and is unlikely to play any significant role in the binding and translocation of H+ and this was supported by the findings that mutation of this histidyl residue in hPEPT1 did not interfere with transport function. The loss of transport function of hPEPT1 and hPEPT2, when His-57 in hPEPT1 and His-87 in hPEPT2 were mutated, was not due to alterations in protein expression because the expression levels of these mutants were similar to those of the respective wild type transporters in HeLa cells as assessed by immunoblot analysis. Confocal analysis of immunofluorescence in X. laevis oocytes expressing the wild type and the three histidine mutants of hPEPT1 showed that the transporter protein is expressed exclusively in the plasma membrane and that the level of expression is comparable among the wild type and the three mutants. These site-directed mutagenesis studies clearly show that His-57 in hPEPT1 and His-87 in hPEPT2 are the most critical histidyl residues necessary for the catalytic function of these transporters.
Fei
Y J
YJ
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta 30912, USA.
Liu
W
W
Prasad
P D
PD
Kekuda
R
R
Oblak
T G
TG
Ganapathy
V
V
Leibach
F H
FH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D002384:Catalysis; D002460:Cell Line; D017124:Conserved Sequence; D017931:DNA Primers; D005260:Female; D006367:HeLa Cells; D006639:Histidine; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism; D007668:Kidney / Q000378:metabolism; D008564:Membrane Potentials; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000502:physiology; D018408:Patch-Clamp Techniques; D000074011:Peptide Transporter 1; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D027981:Symporters; D014162:Transfection; D014982:Xenopus laevis
D002352:Carrier Proteins; D017931:DNA Primers; D000074011:Peptide Transporter 1; D011994:Recombinant Proteins; C082186:SLC15A1 protein, human; C489940:Slc15a1 protein, rat; D027981:Symporters; C093694:hydrogen-coupled oligopeptide transporter PepT2; D006639:Histidine