Controlled conductivity at low pH in Protein L chromatography enables separation of bispecific and other antibody formats by their binding valency.

The complex molecular formats of recent therapeutic antibodies, including bispecific antibodies, antibody fragments, and other fusion proteins, makes the task of purifying the desired molecules in a limited number of purification steps more and more challenging. Manufacturing these complicated biologics can be substantially improved in the affinity capture stage if the simple bind-and-elute mode is accompanied by targeted removal of the impurities, such as mis-paired antibodies and oligomers or aggregates. Here, we report a method, based on the binding valency to Protein L resin, of separating proteins during the elution step by simply controlling the conductivity at low pH. We show that the method efficiently separated targeted antibodies from mis-paired and aggregated species. Notably, the number of Protein L binding sites can be built into the molecule by design to facilitate the purification. This method may be useful for purifying various antibody formats at laboratory and manufacturing scales.

Transport of carnitine and acetylcarnitine by carnitine/organic cation transporter (OCTN) 2 and OCTN3 into epididymal spermatozoa.

Carnitine and acetylcarnitine are important for the acquisition of motility and maturation of spermatozoa in the epididymis. In this study, we examined the involvement of carnitine/organic cation transporter (OCTN) in carnitine and acetylcarnitine transport in epididymal spermatozoa of mice. Uptake of both compounds by epididymal spermatozoa was time-dependent and partially Na(+)-dependent. Kinetic analyses revealed the presence of a high-affinity transport system in the spermatozoa, with K(m) values of 23.6 and 6.57 muM for carnitine and acetylcarnitine respectively in the presence of Na(+). Expression of OCTN2 and OCTN3 in epididymal spermatozoa was confirmed by immunofluorescence analysis. The involvement of these two transporters in carnitine and acetylcarnitine transport was supported by a selective inhibition study. We conclude that both Na(+)-dependent and -independent carnitine transporters, OCTN2 and OCTN3, mediate the supply of carnitine and acetylcarnitine to epididymal spermatozoa in mice.

Viral envelope protein gp64 transgenic mouse facilitates the generation of monoclonal antibodies against exogenous membrane proteins displayed on baculovirus.

We have been investigating the functional display of multipass membrane protein such as transporter or G-protein coupled receptor on the budded baculovirus (BV). We tested the use of a viral envelope protein gp64 transgenic mouse for the direct immunization of these membrane proteins displayed on BVs. The gp64 transgenic mice showed only a weak response to virus compared to wild type BALB/c mice. Immunizing gp64 transgenic mice with the BV expressing peptide transporter PepT1, we obtained 47 monoclonal antibodies (mAbs). These mAbs were specific to the PepT1 on the pancreatic cancer cells AsPC-1 by fluorocytometric analysis and exhibited antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity to AsPC-1. We also generated 7 mAbs by immunizing gp64 transgenic mice on a CCR2-deficient background with the BV expressing chemokine receptor CCR2 together with partially purified CCR2. These mAbs possessed specific binding to CCR2 in CHO cells on fluorocytometric analysis, and exhibited neutralizing activities for ligand-dependent inhibition of cyclic AMP production. This method provides a powerful tool for the generation of therapeutic/diagnostic mAbs against membrane proteins.

Organic anion transporting polypeptide-C mediates arsenic uptake in HEK-293 cells.

Arsenic is an established human carcinogen. The role of aquaglyroporins (AQPs) in arsenic disposition was recently identified. In order to examine whether organic anion transporting polypeptide-C (OATP-C) also plays a role in arsenic transport, OATP-C cDNA was transfected into cells of a human embryonic kidney cell line (HEK-293). Transfection increased uptake of the model OATP-C substrate, estradiol-17beta-D-glucuronide, by 10-fold. In addition, we measured uptake and cytotoxicity of arsenate, arsenite, monomethylarsonate(MMA(V)), and dimethylarsinate (DMA(V)). Transfection of OATP-C increased uptake and cytotoxicity of arsenate and arsenite, but not of MMA(V) or DMA(V). Rifampin and taurocholic acid (a substrate of OATP-C) reversed the increased toxicity of arsenate and arsenite seen in OATP-C-transfected cells. The increase in uptake of inorganic arsenic was not as great as that of estradiol-17beta-D-glucuronide. Our results suggest that OATP-C can transport inorganic arsenic in a (GSH)-dependent manner. However, this may not be the major pathway for arsenic transport.

Recovery of functional peptide transporter PepT1 in budded baculovirus fraction.

Transporters play a critical role in many physiological and pathological states and expression of the functional transporter protein is essential in exploring its kinetics and developing effective drugs. We describe here the recovery of functional transporter protein in the baculovirus fraction. We introduced a gene encoding human peptide transporter PepT1, important for the absorption of protein hydrolytic products or peptide-mimetic drugs, into a baculovirus vector. After infection, a large amount of PepT1 appeared in the budded virus fraction compared with Sf9 cells. Uptake of [14C]glycylsarcosine was markedly increased in an acidic condition and showed a clear overshoot in PepT1-expressing virus fraction. The apparent Michaelis constant for [14C]glycylsarcosine was 0.55 +/- 0.06 mM. [14C]Glycylsarcosine uptake was inhibited by di- and tripeptides and orally active beta-lactam antibiotics. These results suggest that functional PepT1 recovers efficiently in a budded virus fraction, and, thus, this expression system will be a useful tool for characterization and screening of peptide-mimetic drugs in drug discovery.

Carnitine/xenobiotics transporters in the human mammary gland epithelia, MCF12A.

The barrier function of the human mammary gland collapses if challenged with cationic drugs, causing their accumulation in milk. However, underlying molecular mechanisms are not well understood. To gain insight into the mechanism, we characterized transport of organic cations in the MCF12A human mammary gland epithelial cells, using carnitine and tetraethylammonium (TEA) as representative nutrient and xenobiotics probes, respectively. Our results show that the mammary gland cells express mRNA and proteins of human (h) novel organic cation transporters (OCTN) 1 and hOCTN2 (a Na+-dependent carnitine carrier with Na+-independent xenobiotics transport function), which belong to the solute carrier superfamily (SLC) of transporters. Other SLC OCTs such as hOCT1 and extraneuronal monoamine transporter (EMT)/hOCT3 are also expressed at mRNA levels, but hOCT2 was undetectable. We further showed mRNA expression of ATB0+ (an amino acid transporter with a Na+/Cl(-)-dependent carnitine transport activity), and Fly-like putative transporter 2/OCT6 (a splice variant of carnitine transporter 2: a testis-specific Na+-dependent carnitine transporter). TEA uptake was pH dependent. Carnitine uptake was dependent on Na+, and partly on Cl-, compatible with hOCTN2 and ATB0+ function. Modeling analyses predicted multiplicity of the uptake mechanisms with the high-affinity systems characterized by K(m) of 5.1 microM for carnitine and 1.6 mM for TEA, apparently similar to the reported hOCTN2 parameter for carnitine, and that of EMT/hOCT3 for TEA. Verapamil, cimetidine, carbamazepine, quinidine, and desipramine inhibited the carnitine uptake but required supratherapeutic concentrations, suggesting robustness of the carnitine uptake systems against xenobiotic challenge. Our findings suggest functional roles of a network of multiple SLC organic cation/nutrient transporters in human mammary gland drug transfer.

OCTN2-mediated transport of carnitine in isolated Sertoli cells.

Carnitine is extensively accumulated in epididymis. Carnitine is also accumulated in testis at higher concentration than in the plasma and is used in spite of the presence of the blood-testis barrier. In this study, we examined the characteristics of carnitine transport in primary-cultured rat Sertoli cells, which constitute a part of the blood-testis barrier. Uptake of [3H]carnitine (11.4 nM) from the basal side of Sertoli cells was Na+-dependent and was significantly decreased in the presence of 10 microM (48.0 +/- 7.4% of control) or 100 microM unlabeled carnitine (14.6 +/- 5.7% of control). Furthermore, the uptake was significantly inhibited in the presence of 100 microM acetyl-L-carnitine, 100 microM gamma-butyrobetaine or 500 microM quinidine. In RT-PCR analysis, the high-affinity carnitine transporter OCTN2 was detected in rat whole testis tissue and primary-cultured Sertoli cells. In contrast, the low-affinity carnitine transporter ATB(0,+) was detected in rat whole testis tissue, but not in primary cultured Sertoli cells. These results demonstrate that OCTN2 mediates carnitine supply to Sertoli cells from the circulation.

A novel therapeutic approach for thrombocytopenia by minibody agonist of the thrombopoietin receptor.

Antibodies have brought valuable therapeutics in the clinical treatment of various diseases without serious adverse effects through their intrinsic features such as specific binding to the target antigen with high affinity, clinical safety as serum proteins, and long half-life. Agonist antibodies, furthermore, could be expected to maximize the value of therapeutic antibodies. Indeed, several IgG/IgM antibodies have been reported to induce cellular growth/differentiation and apoptosis. These agonist antibodies, however, should be further improved to exert more potent biologic activities and appropriate serum half-life depending upon the disease indications. Here, we report that IgG antibodies against the thrombopoietin receptor (Mpl), which have an absence or very weak agonist activity, can be engineered to be agonist minibodies, which include diabody or sc(Fv)2 as potent as natural ligand. Through this technological development, minibodies have been successfully constructed to bind and activate 2 types of dysfunctional mutant Mpls that cause congenital amegakaryocytic thrombocytopenia (CAMT). This drastic conversion of biologic activities by designing minibodies can be widely applicable to generate agonist minibodies for clinical application, which will constitute a new paradigm in antibody-based therapeutics.

Expression of organic cation transporter OCTN1 in hematopoietic cells during erythroid differentiation.

OBJECTIVE: Organic cation/carnitine transporter, OCTN1 (SLC22A4) shows a relatively broad tissue distribution and transports organic cations in a pH-dependent manner. However, its physiological role remains to be clarified. To understand the physiological role of OCTN1, tissue expression of OCTN1 in human and mice was characterized. METHODS: Expression of OCTN1 in various tissues and blood cells was examined by reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and flow cytometry analysis. RESULTS: Mouse OCTN1 mRNA was detected in kidney, smooth muscle, and hematopoietic tissues, such as spleen and bone marrow, by RT-PCR analysis. Further study focused on expression of OCTN1 in various types of blood cells. OCTN1 mRNA was detected in myeloid cells in mouse bone marrow, but not in lymphoid cells. Bone marrow nuclear cells positive for TER119, an erythrocyte marker, showed strong expression of OCTN1. Similarly, OCTN1 was strongly expressed in glycophorin A-positive erythroid cells obtained from human cord blood. In Western blot analysis, OCTN1 protein was detected in isolated mouse mature peripheral erythrocytes. Further analysis by RT-PCR and flow cytometry showed OCTN1 was expressed in both glycophorin A-positive and negative erythroid cells after cultivation. These findings suggested that OCTN1 transports compound(s) that are required for erythroid differentiation, maturation, and/or growth. CONCLUSION: The present study demonstrated that OCTN1 is associated with myeloid cells rather than lymphoid cells, and especially with erythroid-lineage cells at the transition stage from immature erythroid cells to peripheral mature erythrocytes.

Identification of soluble NH2-terminal fragment of glypican-3 as a serological marker for early-stage hepatocellular carcinoma.

For detection of hepatocellular carcinoma (HCC) in patients with liver cirrhosis, serum alpha-fetoprotein has been widely used, but its sensitivity has not been satisfactory, especially in small, well-differentiated HCC, and complementary serum marker has been clinically required. Glypican-3 (GPC3), a heparan sulfate proteoglycan anchored to the plasma membrane, is a good candidate marker of HCC because it is an oncofetal protein overexpressed in HCC at both the mRNA and protein levels. In this study, we demonstrated that its NH(2)-terminal portion [soluble GPC3 (sGPC3)] is cleaved between Arg(358) and Ser(359) of GPC3 and that sGPC3 can be specifically detected in the sera of patients with HCC. Serum levels of sGPC3 were 4.84 +/- 8.91 ng/ml in HCC, significantly higher than the levels seen in liver cirrhosis (1.09 +/- 0.74 ng/ml; P < 0.01) and healthy controls (0.65 +/- 0.32 ng/ml; P < 0.001). In well- or moderately-differentiated HCC, sGPC3 was superior to alpha-fetoprotein in sensitivity, and a combination measurement of both markers improved overall sensitivity from 50% to 72%. These results indicate that sGPC3 is a novel serological marker essential for the early detection of HCC.

Involvement of organic anion transporting polypeptides in the transport of troglitazone sulfate: implications for understanding troglitazone hepatotoxicity.

Troglitazone is a thiazolidinedione insulin sensitizer drug that is metabolized mainly to a sulfate conjugate (M-1) in humans. It was reported to cause hepatotoxicity, although the cause has not been fully clarified. The objective of this study was to identify whether organic anion transporting polypeptide (OATP) transporters expressed at the basolateral membrane of human hepatocytes participate in troglitazone-associated hepatotoxicity. When OATP-B, OATP-C, or OATP8 was expressed in Xenopus oocytes, the transporter-mediated uptake into oocytes of troglitazone sulfate conjugate and the inhibitory effects of thiazolidinediones and the metabolites of troglitazone on estrone-3-sulfate transport were measured. M-1 was transported well by OATP-C but was not transported by OATP-B. OATP8 showed weak, but not statistically significant, transport of M-1. M-1 exhibited a strong inhibitory effect on estrone-3-sulfate transport by OATP-C and OATP8, suggesting a higher affinity than other thiazolidinediones and the metabolites of troglitazone, glucuronide conjugate and quinone metabolite. In conclusion, the sulfate conjugate of troglitazone has a higher affinity for OATPs than troglitazone itself or other metabolites. Since OATP transporters are important in the hepatic handling of bile acids, bilirubin, and other endogenous anionic compounds, M-1 may disturb the hepatic influx and efflux transport of these endogenous molecules across the basolateral membranes. Moreover, OATP-C may be involved in the hepatic toxicity of troglitazone through the inhibitory action of M-1.

Involvement of OCTN1 (SLC22A4) in pH-dependent transport of organic cations.

OCTN1 (SLC22A4) transports cationic compounds such as tetraethylammonium in a pH-sensitive and sodium-independent manner in cultured cells, and is expressed in wide variety of tissues, including kidney, muscle, placenta, heart, and others. This study focused on the clarification of its subcellular distribution in kidney and on its driving force to throw light on the pharmacological and physiological roles of OCTN1. Uptake of [14C]tetraethylammonium by membrane vesicles prepared from HEK293 cells stably transfected with human OCTN1 cDNA was osmolarity-sensitive, and the Km of tetraethylammonium was 1.28 mM at intravesicular and extravesicular pH values of 6.0 and 7.4, respectively. Tetraethylammonium uptake was pH-dependent, and overshoot uptake was observed in the presence of an outwardly directed proton gradient. A protonophore and membrane potential affected the overshoot uptake. Furthermore, preloading tetraethylammonium in the vesicles significantly increased the rate of uptake of [14C]tetraethylammonium. In mouse kidney, OCTN1 was expressed predominantly at the apical membrane of cortical proximal tubular epithelial cells. It was concluded that OCTN1 is involved in renal excretion of organic cations across the apical membrane in a pH-dependent, membrane potential-sensitive manner and is affected significantly by the organic cations on the trans side, showing counter transport activity.

Functional characterization of pH-sensitive organic anion transporting polypeptide OATP-B in human.

The pH-sensitive activity of human organic anion transporting polypeptide OATP-B, which is expressed at the apical membrane of human small intestinal epithelial cells, was functionally characterized. When initial uptake of estrone-3-sulfate, a typical substrate of OATP, was studied kinetically, we observed an increase in V(max) with decrease of pH from 7.4 to 5.0, whereas the change in K(m) was negligible. OATP-B-mediated uptake of estrone-3-sulfate was independent of sodium, chloride, bicarbonate, or glutathione, whereas the proton ionophore carbonylcyanide p-trifluoromethoxyphenylhydrazone exhibited a pH-dependent inhibitory effect, suggesting that a proton gradient is a driving force for OATP-B. When OATP-B was expressed in human embryonic kidney 293 cells, uptake activities for anionic compounds showed various kinds of pH sensitivity. Dehydroepiandrosterone-sulfate, estrone-3-sulfate, and fexofenadine were transported by OATP-B at both neutral and acidic pH, whereas estradiol-17beta-glucuronide, acetic acid, and lactic acid were not transported at all. Transport of taurocholic acid and pravastatin by OATP-B was observed only at acidic pH, demonstrating a pH-sensitive substrate specificity of OATP-B. Because the physiological pH close to the surface of intestinal epithelial cells is acidic, the roles of OATP-B in the small intestine might be different from those in other tissues, such as liver basolateral membrane. Although the driving force for OATP-B has not been fully established, the clarification of factors, such as pH, that affect the OATP-B-activity is essential for an understanding of the physiological and pharmacological relevance of the transporter in the small intestine.

Contribution of organic anion transporting polypeptide OATP-C to hepatic elimination of the opioid pentapeptide analogue [D-Ala2, D-Leu5]-enkephalin.

The objective of this study was to examine the transport activity of the human organic anion transporter OATP-C (SLC21A6) for oligopeptides that are eliminated rapidly from the systemic circulation. We focused on an opioid peptide analogue, [D-Ala(2), D-Leu(5)]-enkephalin (DADLE), a linear pentapeptide modified to be stable. [(3)H]DADLE was taken up by rat isolated hepatocytes in a saturable manner and highly accumulated in the liver after intravenous administration to rats. The uptake of [(3)H]DADLE by the isolated hepatocytes was inhibited by several organic anions and pentapeptides, but not by tetra- or tripeptides. When OATP-C was expressed in Xenopus laevis oocytes, a significant increase in uptake of [(3)H]DADLE was observed. Moreover, the inhibitory effects of various compounds, including some peptides, on [(3)H]estrone-3-sulfate uptake by OATP-C were similar to those observed in [(3)H]DADLE uptake by rat isolated hepatocytes. In conclusion, it was demonstrated that OATP-C contributes to the rapid hepatic excretion of peptides and peptide-mimetic drugs.

Involvement of human organic anion transporting polypeptide OATP-B (SLC21A9) in pH-dependent transport across intestinal apical membrane.

Some organic anions are absorbed from the gastrointestinal tract through carrier-mediated transport mechanism(s), which may include proton-coupled transport, anion exchange transport, and others. However, the molecular identity of the organic anion transporters localized at the apical membrane of human intestinal epithelial cells has not been clearly demonstrated. In the present study, we focused on human organic anion transporting polypeptide OATP-B and examined its subcellular localization and functionality in the small intestine. Localization of OATP-B was determined by immunohistochemical analysis. Transport properties of estrone-3-sulfate and the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor pravastatin by OATP-B-transfected human embryonic kidney 293 cells were measured. OATP-B was immunohistochemically localized at the apical membrane of intestinal epithelial cells in humans. Uptake of [3H]estrone-3-sulfate and [14C]pravastatin by OATP-B at pH 5.5 was higher than that at pH 7.4. [3H]Estrone-3-sulfate transport was decreased by pravastatin, aromatic anion compounds, and the anion exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, but not by small anionic compounds, such as lactic acid and acetic acid. The inhibitory effect of pravastatin on the uptake of [3H]estrone-3-sulfate was concentration-dependent, and the IC50 value was 5.5 mM. The results suggested that OATP-B mediates absorption of anionic compounds and its activity may be optimum at the acidic surface microclimate pH of the small intestine. Accordingly, OATP-B plays a role in the absorption of anionic compounds across the apical membrane of human intestinal epithelial cells, although it cannot be decisively concluded that pH-dependent absorption of pravastatin is determined by OATP-B alone.

Interaction of activator of G-protein signaling 3 (AGS3) with LKB1, a serine/threonine kinase involved in cell polarity and cell cycle progression: phosphorylation of the G-protein regulatory (GPR) motif as a regulatory mechanism for the interaction of GPR motifs with Gi alpha.

Activator of G-protein signaling 3 (AGS3) has a modular domain structure consisting of seven tetratricopeptide repeats (TPRs) and four G-protein regulatory (GPR) motifs. Each GPR motif binds to the alpha subunit of Gi/Go (Gialpha > Goalpha) stabilizing the GDP-bound conformation of Galpha and apparently competing with Gbetagamma for GalphaGDP binding. As an initial approach to identify regulatory mechanisms for AGS3-G-protein interactions, a yeast two-hybrid screen was initiated using the TPR and linker region of AGS3 as bait. This screen identified the serine/threonine kinase LKB1, which is involved in the regulation of cell cycle progression and polarity. Protein interaction assays in mammalian systems using transfected cells or brain lysate indicated the regulated formation of a protein complex consisting of LKB1, AGS3, and G-proteins. The interaction between AGS3 and LKB1 was also observed with orthologous proteins in Drosophila where both proteins are involved in cell polarity. LKB1 immunoprecipitates from COS7 cells transfected with LKB1 phosphorylated the GPR domains of AGS3 and the related protein LGN but not the AGS3-TPR domain. GPR domain phosphorylation was completely blocked by a consensus GPR motif peptide, and placement of a phosphate moiety within a consensus GPR motif reduced the ability of the peptide to interact with G-proteins. These data suggest that phosphorylation of GPR domains may be a general mechanism regulating the interaction of GPR-containing proteins with G-proteins. Such a mechanism may be of particular note in regard to localized signal processing in the plasma membrane involving G-protein subunits and/or intracellular functions regulated by heterotrimeric G-proteins that occur independently of a typical G-protein-coupled receptor.

Studies on functional sites of organic cation/carnitine transporter OCTN2 (SLC22A5) using a Ser467Cys mutant protein.

The organic cation/carnitine transporter OCTN2 mediates transport of carnitine and organic cations in Na(+)-dependent and Na(+)-independent manners, respectively. However, the mechanism of molecular recognition of different substrates has not been clarified yet. We previously found a single amino acid change in OCTN2, Ser467Cys (S467C), in the Japanese population and observed a decreased carnitine transport but unchanged organic cation transport compared with wild type. Therefore, we conducted detailed kinetic and functional analyses of the substrate recognition sites of wild-type and S467C-mutant OCTN2. The K(m) value for carnitine of S467C-mutant was increased about 15-fold over that of the wild type. Mutual inhibition kinetics of carnitine and tetraethylammonium (TEA) were not completely competitive, suggesting that the binding sites are very close to each other, but not identical. Several organic anions such as valproate, as well as organic cations, significantly inhibited carnitine and TEA uptake by OCTN2, and valproate showed Na(+)-dependent inhibition of OCTN2-mediated TEA uptake. The Na(+)-activation kinetics of the S467C mutant was similar to that of the wild type. Furthermore, a significant decrease of the TEA uptake-inhibitory potency of valproate was observed in S467C-mutant OCTN2. These observations suggest that the decrease in affinity of S467C-mutant OCTN2 for carnitine was caused by functional alteration of the anion (carboxyl moiety of carnitine) recognition site located in trans-membrane domain 11, which is closely related to the Na(+)-binding site, on OCTN2 protein. These results demonstrate that OCTN2 has functional sites for carnitine and Na(+) and that the carnitine-binding site is involved, in part, in the recognition of organic cations.

Genetic polymorphisms of human organic anion transporters OATP-C (SLC21A6) and OATP-B (SLC21A9): allele frequencies in the Japanese population and functional analysis.

Genetic polymorphisms of human organic anion transporting polypeptides OATP-C (SLC21A6) and OATP-B (SLC21A9) in the Japanese population were analyzed. The allele frequencies of OATP-C*1a, OATP-C*1b (N130D), OATP-C*1c (R152K and D241N), and OATP-C*5 (V174A) were 35.2, 53.7, 0, and 0.7%, respectively, in 267 healthy Japanese subjects. In the OATP-C gene, we found a novel allele called OATP-C*15 possessing two single nucleotide polymorphisms (SNPs), N130D and V174A, simultaneously. The allele frequency of OATP-C*15 was 3.0%. The allele frequencies of OATP-B*1, OATP-B*2 (T392I), and OATP-B*3 (S486F) were 69.1, 0, and 30.9%, respectively. For functional analysis, each OATP-C and OATP-B allele was expressed in human embryonic kidney (HEK293) cells, and the kinetics of uptake of [(3)H]estrone-3-sulfate was determined. In the case of OATP-C alleles, no significant alteration in K(m) or V(max) values of [(3)H]estrone-3-sulfate uptake was observed, even when the V(max) values were corrected for the expression levels of OATP-C protein. In contrast, V(max), corrected with the expression level of OATP-B*3, was decreased to 42.5% of OATP-B*1, whereas the K(m) values were comparable. Since the frequency of the OATP-B*3 allele was high (30.9%) in our subjects, the SNP of S486F may affect the physiological function and/or pharmacological effects of OATP-B substrates in vivo.

Role of Lkb1, the causative gene of Peutz-Jegher's syndrome, in embryogenesis and polyposis.

Peutz-Jeghers syndrome (PJS) is a dominantly inherited human disorder characterized by gastrointestinal hamartomatous polyposis and mucocutaneous melanin pigmentation. LKB1 (STK11) serine/threonine kinase is the product of the causative gene of PJS, which has been mapped to chromosome 19p13.3. However, several studies have produced results that are not consistent with a link between LKB1 gene mutation and PJS. We constructed a knockout gene mutation of Lkb1 to determine whether it is the causative gene of PJS and to examine the biological role of the Lkb1 gene. Lkb1(-/-) mice died in utero between 8.5 and 9.5 days postcoitum. At 9.0 days postcoitum, Lkb1(-/-) embryos were generally smaller than their age-matched littermates, showed developmental retardation, and did not undergo embryonic turning. Multiple gastric adenomatous polyps were observed in 10- to 14-month-old Lkb1(+/-) mice. Our results indicate that functional Lkb1 is required for normal embryogenesis and that it is related to tumor development. The Lkb1(+/-) mouse is suitable for studying molecular mechanism underlying the development of inherited gastric tumors in PJS.