The rabbit ileal lipid-binding protein. Gene cloning and functional expression of the recombinant protein.
A bile-acid-binding protein of Mr 14000 has been previously identified by photoaffinity labeling in rabbit ileal brush border membrane vesicles [Kramer et al. (1993) J. Biol. Chem. 268, 18035-18046]. This peripheral membrane-associated protein was purified and identified as an ileal lipid-binding protein. It was further shown to be identical to the cytosolic 14-kDa bile-acid-binding protein from the same tissue. Starting with sequence information from tryptic fragments, we cloned and sequenced the gene and its transcript. It has four exons (123, 176, 90, 115 bp) and three introns (1372, 2291, 3137 bp) and a similar structure as the genes from other members of the fatty-acid-binding protein family. The deduced protein has 128 amino acid residues and a calculated molecular mass of 14404 Da. It exhibits high similarity to its human (83%), mouse (77%), rat (76%) and porcine (72%) counterparts. Furthermore, the recombinant protein was produced in Escherichia coli and shown to be identical to native protein from ileal tissue. Functionality of the recombinant protein was demonstrated by labeling with various photoaffinity derivatives of bile acids. Ranking of the photolabeling efficiency of these probes towards the recombinant protein was comparable to the respective ranking towards the native protein. Polyclonal antibodies that were raised in hens against the recombinant protein, specifically recognized the ileal lipid-binding protein in the brush border membrane and cytosol from rabbit ileum. In contrast, no labeling was observed with jejunal tissue. Our results suggest a specific role of the membrane-associated ileal lipid-binding protein for the process of ileal bile acid uptake.
Stengelin
S
S
Hoechst Aktiengesellschaft-Hoechst Marion Roussel, Frankfurt am Main, Germany.
Apel
S
S
Becker
W
W
Maier
M
M
Rosenberger
J
J
Bewersdorf
U
U
Girbig
F
F
Weyland
C
C
Wess
G
G
Kramer
W
W
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001647:Bile Acids and Salts / Q000378:metabolism*; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002451:Cell Compartmentation; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000737:chemistry; D007082:Ileum / Q000737:chemistry*; D008297:Male; D008871:Microvilli / Q000737:chemistry*; D008969:Molecular Sequence Data; D029363:Organic Anion Transporters, Sodium-Dependent; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D027981:Symporters
D000345:Affinity Labels; D001647:Bile Acids and Salts; D002352:Carrier Proteins; D029363:Organic Anion Transporters, Sodium-Dependent; D011994:Recombinant Proteins; D027981:Symporters; C071908:sodium-bile acid cotransporter