Generation of a Novel Antibody Probe to the Apical Sodium-Dependent Bile Acid Transporter That Inhibits Ileal Bile Acid Absorption
ABSTRACT Intestinal bile acid absorption is mediated by a sodium-dependent transporter located in the brush border apical membrane of ileocytes. The transmembrane topology and the role of individual amino acid residues in the bile acid transport process have been investigated by means of various experimental approaches, leading to multiple hypotheses. We raised a monoclonal antibody against a segment of the transporter comprising vicinal cysteine residues, in order to evaluate its functional role. A 14 amino acid peptide, corresponding to amino acids 104-117 of the transporter, was synthesized, and a monoclonal anti-peptide antibody was raised. In vitro uptake-inhibition studies in the presence of the monoclonal anti-peptide antibody were performed using ileal brush border membrane vesicles. Rabbit ileum was perfused in vivo with 5 mM taurocholic acid in the presence of the monoclonal antibody, and bile acid absorption inhibition was evaluated. The anti-peptide monoclonal antibody significantly reduced the in vitro uptake and in vivo absorption of taurocholic acid. The present data demonstrate the functional relevance of the 104-117 peptide segment and report the generation of a novel antibody against the apical sodium-dependent bile acid transporter (ASBT) that may be used as a therapeutic agent in hypercholesterolemia and in cholestatic pruritus.
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ABSTRACT: Anterior gradient homolog 2 (AGR2) is a functional protein with critical roles in a diverse range of biological systems, including vertebrate tissue development, inflammatory tissue injury responses, and cancer progression. Clinical studies have shown that the AGR2 protein is overexpressed in a wide range of human cancers, including carcinomas of the esophagus, pancreas, breast, prostate, and lung, making the protein as a potential cancer biomarker. However, the general biochemical functions of AGR2 in human cells remain undefined, and the signaling mechanisms that drive AGR2 to inhibit p53 are still not clearly illustrated. Therefore, it is of great interest to develop molecular probes specifically recognizing AGR2 for its detection and for the elucidation of AGR2-associated molecular mechanism. Through a bead-based and flow cytometry monitored SELEX technology, we have identified a group of DNA aptamers that can specifically bind to AGR2 with K(d) values in the nanomolar range after 14 rounds of selections. Aptamer C14B was chosen to further study, due to its high binding affinity and specificity. The optimized and shortened C14B1 has special G-rich characteristics, and the G-rich region of this binding motif was further characterized to reveal an intramolecular parallel G-quadruplex by CD spectroscopy and UV spectroscopy. Our experiments confirmed that the stability of the G-quadruplex structure was strongly dependent on the nature of the monovalent ions and the formation of G-quadruplex structure was also important for the binding capacity of C14B1 to the target. Furthermore, we have designed a kind of allosteric molecule beacon (aMB) probe for selective and sensitive detection of AGR2. In this work, we have developed new aptamer probes for specific recognition of the AGR2. Structural study have identified that the binding motif of aptamer is an intramolecular parallel G-quadruplex structure and its structure and binding affinity are strongly dependent on the nature of the monovalent ion. Furthermore, with our design of AGR2-aMB, AGR2 could be sensitively and selectively detected. This aptamer probe has great potential to serve as a useful tool for early diagnosis and prognosis of cancer and for fundamental research to elucidate the biochemical functions of AGR2.PLoS ONE 09/2012; 7(9):e46393. DOI:10.1371/journal.pone.0046393 · 3.53 Impact Factor
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ABSTRACT: The SLC10 family represents seven genes containing 1-12 exons that encode proteins in humans with sequence lengths of 348-477 amino acids. Although termed solute carriers (SLCs), only three out of seven (i.e. SLC10A1, SLC10A2, and SLC10A6) show sodium-dependent uptake of organic substrates across the cell membrane. These include the uptake of bile salts, sulfated steroids, sulfated thyroidal hormones, and certain statin drugs by SLC10A1 (Na(+)-taurocholate cotransporting polypeptide (NTCP)), the uptake of bile salts by SLC10A2 (apical sodium-dependent bile acid transporter (ASBT)), and uptake of sulfated steroids and sulfated taurolithocholate by SLC10A6 (sodium-dependent organic anion transporter (SOAT)). The other members of the family are orphan carriers not all localized in the cell membrane. The name "bile acid transporter family" arose because the first two SLC10 members (NTCP and ASBT) are carriers for bile salts that establish their enterohepatic circulation. In recent years, information has been obtained on their 2D and 3D membrane topology, structure-transport relationships, and on the ligand and sodium-binding sites. For SLC10A2, the putative 3D morphology was deduced from the crystal structure of a bacterial SLC10A2 analog, ASBT(NM). This information was used in this chapter to calculate the putative 3D structure of NTCP. This review provides first an introduction to recent knowledge about bile acid synthesis and newly found bile acid hormonal functions, and then describes step-by-step each individual member of the family in terms of expression, localization, substrate pattern, as well as protein topology with emphasis on the three functional SLC10 carrier members.Current Topics in Membranes 01/2012; 70:105-68. DOI:10.1016/B978-0-12-394316-3.00004-1 · 1.77 Impact Factor
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ABSTRACT: The solute carrier (SLC) family 10 (SLC10) comprises influx transporters of bile acids, steroidal hormones, various drugs, and several other substrates. Because the seminal transporters of this family, namely, sodium/taurocholate cotransporting polypeptide (NTCP; SLC10A1) and the apical sodium-dependent bile acid transporter (ASBT; SLC10A2), were primarily bile acid transporters, the term "sodium bile salt cotransporting family" was used for the SLC10 family. However, this notion became obsolete with the finding of other SLC10 members that do not transport bile acids. For example, the sodium-dependent organic anion transporter (SOAT; SLC10A6) transports primarily sulfated steroids. Moreover, NTCP was shown to also transport steroids and xenobiotics, including HMG-CoA inhibitors (statins). The SLC10 family contains four additional members, namely, P3 (SLC10A3; SLC10A3), P4 (SLC10A4; SLC10A4), P5 (SLC10A5; SLC10A5) and SLC10A7 (SLC10A7), several of which were unknown or considered hypothetical until approximately a decade ago. While their substrate specificity remains undetermined, great progress has been made towards their characterization in recent years. Explicitly, SLC10A4 may participate in vesicular storage or exocytosis of neurotransmitters or mastocyte mediators, whereas SLC10A5 and SLC10A7 may be involved in solute transport and SLC10A3 may have a role as a housekeeping protein. Finally, the newly found role of bile acids in glucose and energy homeostasis, via the TGR5 receptor, sheds new light on the clinical relevance of ASBT and NTCP. The present mini-review provides a brief summary of recent progress on members of the SLC10 family.Molecular Aspects of Medicine 04/2013; 34(2-3):252-269. DOI:10.1016/j.mam.2012.07.004 · 10.30 Impact Factor