Biomedical subjects

H Wang

Publications and source records attributed to H Wang.

At least 1,315 records · Page 73Linked to original sources

PubMed1998

Isoform specific phosphorylation of protein phosphatase 2C expressed in COS7 cells.

Of the six distinct isoforms of mouse protein phosphatase 2C (PP2C) (alpha, beta-1, beta-2, beta-3, beta-4 and beta-5), PP2C alpha was specifically phosphorylated on the serine residue(s) when expressed in COS7 cells. Analysis of phosphorylation sites using site-directed mutagenesis demonstrated that Ser-375 and/or Ser-377 were phosphorylated in vivo. These serine residues were the sites of phosphorylation by casein kinase II in vitro. Phosphorylation of PP2C alpha was enhanced two-fold by the addition of okadaic acid to the culture medium, but addition of cyclosporin A had no such effect. These results suggest that the expressed PP2C alpha is phosphorylated by a casein kinase II-like protein kinase and dephosphorylated by PP1 and/or PP2A in COS7 cells.

Animals↗

PubMed1998

Dendritic spines containing mu-opioid receptors in rat striatal patches receive asymmetric synapses from prefrontal corticostriatal afferents.

Prefrontal corticostriatal afferents to the caudate-putamen nucleus (CPN) have been implicated in motor and cognitive functions that are subject to opioid modulation through the mu-opioid receptor (MOR). We examined the cellular basis for this modulation by combining anterograde transport of biotinylated dextran amine (BDA) following injections into the rat prefrontal cortex with immunocytochemical detection of MOR in patch compartments of the CPN. The BDA-labeled neurons in deep layer V and layer VI of the dorsal part of the anterior cingulate cortex and the medial agranular cortex projected bilaterally, with an ipsilateral predominance, to MOR-enriched patches in the dorsomedial and dorsocentral CPN, respectively. BDA-labeled terminals often apposed MOR-immunoreactive dendrites and perikarya but formed exclusively asymmetric, excitatory-type synapses mainly with dendritic spines. Of the total anterogradely labeled axon terminals forming asymmetric synapses, 40% (151 of 377) were with MOR-labeled spines, and 58% (220 of 377) were with unlabeled dendritic spines. In addition, immunogold-silver particles for MOR were seen in 14% (134 of 938) of all BDA-labeled axons and axon terminals. These dually labeled axon terminals also formed asymmetric synapses with dendritic spines that contained MOR immunoreactivity. The proportions of BDA-labeled axon terminals forming asymmetric synapses with MOR-labeled or unlabeled spines were similar in the CPN ipsilateral and contralateral to the cortical injections. These results suggest that, in patch compartments of the CPN, MOR plays a critical role in postsynaptic response to, but also in presynaptic modulation of, prefrontal corticostriatal excitation.

Afferent Pathways↗

PubMed1998

Cloning and functional characterization of a potential-sensitive, polyspecific organic cation transporter (OCT3) most abundantly expressed in placenta.

We have isolated a cDNA from rat placenta which, when expressed heterologously, mediates the transport of a wide spectrum of organic cations. The cDNA codes for a protein of 551 amino acids containing 12 putative transmembrane domains. Northern blot analysis indicates that this transporter is expressed most abundantly in the placenta and moderately in the intestine, heart, and brain. The expression is comparatively low in the kidney and lung and is undetectable in the liver. This transporter is distinct from the previously cloned organic cation transporters (OCT1, OCT2, NKT, NLT, RST, and OCTN1). When expressed in HeLa cells, the cDNA induces the transport of tetraethylammonium and guanidine. Competition experiments indicate that this transport process recognizes a large number of organic cations, including the neurotoxin 1-methyl-4-phenylpyridinium, as substrates. The cDNA-induced transport is markedly influenced by extracellular pH. However, when expressed in Xenopus laevis oocytes, the cDNA-induced transport is electrogenic, associated with the transfer of positive charge into the oocytes. Under voltage clamp conditions, tetraethylammonium evokes inward currents that are concentration- and potential-dependent. This potential-sensitive organic cation transporter, designated as OCT3, represents a new member of the OCT gene family.

Amino Acid Sequence↗

PubMed1998

Mutations in the hydrophobic surface of an amphipathic groove of 14-3-3zeta disrupt its interaction with Raf-1 kinase.

14-3-3 proteins bind to a diverse group of regulatory molecules such as Raf-1, Cbl, and c-Bcr that are involved in signal transduction pathways. The crystal structure of 14-3-3zeta reveals a conserved amphipathic groove that may mediate the association of 14-3-3 with diverse ligands. Consistently, mutations on the charged surface of the groove (Lys-49, Arg-56, and Arg-60) decrease the binding of 14-3-3zeta to the ligands tested (Zhang, L., Wang, H., Liu, D., Liddington, R., and Fu, H. (1997) J. Biol. Chem. 272, 13717-13724). Here we report that mutations that altered the hydrophobic property of the groove, V176D, L216D, L220D, and L227D, disrupted the interaction of 14-3-3zeta with Raf-1 kinase. The reduced binding of the 14-3-3zeta mutants to Raf-1 was apparently not because of gross structural changes in the mutants as judged by their ability to form dimers, by partial proteolysis profiles, and by circular dichroism analysis. These hydrophobic residues appeared to be required for the binding of 14-3-3zeta to distinct activation states of Raf-1 because mutations V176D, L216D, L220D, and L227D reduced the interaction of 14-3-3zeta with Raf-1 from both phorbol 12-myristate 13-acetate-stimulated and unstimulated Jurkat T cells. These same mutations also disrupted the association of 14-3-3zeta with other regulatory molecules such as Cbl and c-Bcr, suggesting that the hydrophobic surface of the amphipathic groove represents part of a binding site shared by a number of 14-3-3-associated proteins. The conservation of the hydrophobic residues Val-176, Leu-216, Leu-220, and Leu-227 among known 14-3-3 family members implies their general importance in ligand binding.

14-3-3 Proteins↗

PubMed1998

Cross-lineage expression of Ig-beta (B29) in thymocytes: positive and negative gene regulation to establish T cell identity.

Developmental commitment involves activation of lineage-specific genes, stabilization of a lineage-specific gene expression program, and permanent inhibition of inappropriate characteristics. To determine how these processes are coordinated in early T cell development, the expression of T and B lineage-specific genes was assessed in staged subsets of immature thymocytes. T lineage characteristics are acquired sequentially, with germ-line T cell antigen receptor-beta transcripts detected very early, followed by CD3epsilon and terminal deoxynucleotidyl transferase, then pTalpha, and finally RAG1. Only RAG1 expression coincides with commitment. Thus, much T lineage gene expression precedes commitment and does not depend on it. Early in the course of commitment to the T lineage, thymocytes lose the ability to develop into B cells. To understand how this occurs, we also examined expression of well defined B lineage-specific genes. Although lambda5 and Ig-alpha are not expressed, the mu 0 and I mu transcripts from the unrearranged IgH locus are expressed early, in distinct patterns, then repressed just before RAG1 expression. By contrast, RNA encoding the B cell receptor component Ig-beta was found to be transcribed in all immature thymocyte subpopulations and throughout most thymocyte differentiation. Ig-beta expression is down-regulated only during positive selection of CD4(+)CD8(-) cells. Thus several key participants in the B cell developmental program are expressed in non-B lineage-committed cells, and one is maintained even through commitment to an alternative lineage, and repressed only after extensive T lineage differentiation. The results show that transcriptional activation of "lymphocyte-specific" genes can occur in uncommitted precursors, and that T lineage commitment is a composite of distinct positive and negative regulatory events.

Animals↗

PubMed1998

c-Fos expression in NMDA receptor-contained neurons in spinal cord in a rat model of inflammation: a double immunocytochemical study.

Double-labeling techniques were used to demonstrate the nociceptive activation of NMDA receptor-contained neurons in spinal dorsal horn by using c-Fos immunoreactivity as an indicator of this activation in a rat model of inflammation. About 25% and 55% of the c-Fos-immunoreactive neurons that were found in laminae I-II and lamina V showed NMDA receptor immunoreactivity, while about 4% and 11% of NMDA-receptor immunoreactive neurons in these two regions showed c-Fos immunoreactivity, respectively. The implication of the results was discussed.

Animals↗

PubMed1998

NMR solution structure of the 21 kDa chaperone protein DnaK substrate binding domain: a preview of chaperone-protein interaction.

The solution structure of the 21 kDa substrate-binding domain of the Escherichia coli Hsp70-chaperone protein DnaK (DnaK 386-561) has been determined to a precision of 1.00 A (backbone of the beta-domain) from 1075 experimental restraints obtained from multinuclear, multidimensional NMR experiments. The domain is observed to bind to its own C-terminus and offers a preview of the interaction of this chaperone with other proteins. The bound protein region is tightly held at a single amino acid position (a leucyl residue) that is buried in a deep pocket lined with conserved hydrophobic residues. A second hydrophobic binding site was identified using paramagnetically labeled peptides. It is located in a region close to the N-terminus of the domain and may constitute the allosteric region that links substrate-binding affinity with nucleotide binding in the Hsp70 chaperones.

Amino Acid Sequence↗

PubMed1998

Valacyclovir: a substrate for the intestinal and renal peptide transporters PEPT1 and PEPT2.

Valacyclovir is a prodrug of the antiviral agent acyclovir and it does not contain a peptide bond in its structure. We studied the interaction of valacyclovir with the peptide transporters in the human intestinal cell line Caco-2 and the rat kidney proximal tubular cell line SKPT which differentially express peptide transporters PEPT1 and PEPT2. The results of the studies done with these cell lines were confirmed with the cloned peptide transporters human PEPT1 and rat PEPT2, expressed heterologously in HeLa cells. The activity of the peptide transporters was assessed by measuring the uptake of radiolabeled glycylsarcosine in the presence of a H+ gradient. Valacyclovir inhibited the uptake of glycylsarcosine with an inhibition constant (Ki) of 0.49 +/- 0.04 mM in Caco-2 cells and 0.17 +/- 0.01 mM in SKPT cells. In both cell types, the inhibition was competitive. Acyclovir, in contrast to valacyclovir, did not interact with the peptide transporters. Similar results were obtained with heterologously expressed human PEPT1 and rat PEPT2. Valacyclovir inhibited the hPEPT1-mediated glycylsarcosine transport competitively with a Ki value of 0.74 +/- 0.14 mM. The rPEPT2-mediated transport of glycylsarcosine was also inhibited by valacyclovir competitively and the Ki value for the process was 0.39 +/- 0.03 mM. Acyclovir did not interact with either of these cloned peptide transporters. We conclude that valacyclovir is a substrate for the peptide transporters PEPT1 and PEPT2 and that a peptide bond is not a prerequisite for recognition as a substrate by the peptide transporters.

Acyclovir↗

PubMed1998

Syndecans, heparan sulfate proteoglycans, maintain the proteolytic balance of acute wound fluids.

An imbalance between proteases and antiproteases is thought to play a role in the inflammatory injury that regulates wound healing. The activities of some proteases and antiproteases found in inflammatory fluids can be modified in vitro by heparin, a mast cell-derived glycosaminoglycan. Because syndecans, a family of cell surface heparan sulfate proteoglycans, are the major cellular source of heparin-like glycosaminoglycan, we asked whether syndecans modify protease activities in vivo. Syndecan-1 and syndecan-4 ectodomains are shed into acute human dermal wound fluids (Subramanian, S. V., Fitzgerald, M. L., and Bernfield, M. (1997) J. Biol. Chem. 272, 14713-14720). Moreover, purified syndecan-1 ectodomain binds cathepsin G (Kd = 56 nM) and elastase (Kd = 35 nM) tightly and reduces the affinity of these proteases for their physiological inhibitors. Purified syndecan-1 ectodomain protects cathepsin G from inhibition by alpha1-antichymotrypsin and squamous cell carcinoma antigen 2 and elastase from inhibition by alpha1-proteinase inhibitor by decreasing second order rate constants for protease-antiprotease associations (kass) by 3700-, 32-, and 60-fold, respectively. Both enzymatic degradation of heparan sulfate and immunodepletion of the syndecan-1 and -4 in wound fluid reduce these proteolytic activities in the fluid, indicating that the proteases in the wound environment are regulated by interactions with syndecan ectodomains. Thus, syndecans are shed into acute wound fluids, where they can modify the proteolytic balance of the fluid. This suggests a novel physiological role for these soluble heparan sulfate proteoglycans.

Animals↗

PubMed1998

Role of nitric oxide in the regulation of gonadotropin-releasing hormone and tyrosine hydroxylase gene expression in the male rat brain.

It has been recently demonstrated that nitric oxide (NO), a free radical gas which may act as neurotransmitter in the brain, can stimulate the in vivo release of luteinizing hormone as well as the in vitro hypothalamic release of gonadotropin-releasing hormone (GnRH). In order to study the influence of NO on GnRH mRNA expression, two inhibitors of NO synthase (NOS) NG-monomethyl-l-arginine (NMMA) and HP-228, were microinjected into the left lateral ventricle of sham-operated and castrated male rats 4 h before sacrifice. Since the dopaminergic system can positively influence GnRH gene expression, we have also measured in the same animals tyrosine hydroxylase (TH) mRNA in tuberoinfundibular dopamine (TIDA) neurons. GnRH and TH mRNA levels were measured at the cellular level by quantitative in situ hybridization. The injection of HP-228 or NMMA induced a similar decrease (-19.5%) in GnRH mRNA. In castrated animals, the hybridization signal was 88% higher than that observed in sham-operated animals. Both HP-228 and NMMA produced in castrated animals a 39% decrease in GnRH mRNA. In contrast the injection of NOS inhibitors resulted in an increase in the amount of TH mRNA in TIDA neurons. The stimulating effect was more striking in HP-228-treated (+60%) than in NMMA-treated (+32%) animals. Castration did not induce any changes in the number of silver grains overlying TIDA neurons, while the administration of either HP-228 or NMMA induced a 43% increase in castrated animals. These results together with previous ones on GnRH release in vitro suggest that NO exerts a positive influence not only on the secretion but also on the biosynthesis of GnRH. Since NO appears to play a role in the negative regulation of dopamine, it is likely that the increase in GnRH mRNA expression is not mediated by the TIDA system.

Animals↗

PubMed1998

Immunolocalization of acyl-coenzyme A:cholesterol O-acyltransferase in macrophages.

Macrophages in atherosclerotic lesions accumulate large amounts of cholesteryl-fatty acyl esters ("foam cell" formation) through the intracellular esterification of cholesterol by acyl-coenzyme A:cholesterol O-acyltransferase (ACAT). In this study, we sought to determine the subcellular localization of ACAT in macrophages. Using mouse peritoneal macrophages and immunofluorescence microscopy, we found that a major portion of ACAT was in a dense reticular cytoplasmic network and in the nuclear membrane that colocalized with the luminal endoplasmic reticulum marker protein-disulfide isomerase (PDI) and that was in a similar distribution as the membrane-bound endoplasmic reticulum marker ribophorin. Remarkably, another portion of the macrophage ACAT pattern did not overlap with PDI or ribophorin, but was found in as yet unidentified cytoplasmic structures that were juxtaposed to the nucleus. Compartments containing labeled beta-very low density lipoprotein, an atherogenic lipoprotein, did not overlap with the ACAT label, but rather were embedded in the dense reticular network of ACAT. Furthermore, cell-surface biotinylation experiments revealed that freshly harvested, non-attached macrophages, but not those attached to tissue culture dishes, contained approximately 10-15% of ACAT on the cell surface. In summary, ACAT was found in several sites in macrophages: a cytoplasmic reticular/nuclear membrane site that overlaps with PDI and ribophorin and has the characteristics of the endoplasmic reticulum, a perinuclear cytoplasmic site that does not overlap with PDI or ribophorin and may be another cytoplasmic structure or possibly a unique subcompartment of the endoplasmic reticulum, and a cell-surface site in non-attached macrophages. Understanding possible physiological differences of ACAT in these locations may reveal an important component of ACAT regulation and macrophage foam cell formation.

Animals↗

PubMed1998

Structure and regulation of a polymorphic gene encoding folate receptor type gamma/gamma'.

The human folate receptor (hFR) type gamma and gamma' are constitutively secreted proteins that are expressed primarily in hematopoietic tissues and are potential serum markers for certain hematopoietic malignancies. hFR-gamma' is a variant of hFR-gamma with a two base deletion in its cDNA resulting in a truncated polypeptide. The gene encoding hFR-gamma' was isolated from a placental genomic library. The gene has five exons, four introns and a 5' flanking sequence which contains multiple putative regulatory elements. From RNase protection assay and RACE analysis, the major site of transcriptional initiation was identified at -56 nt. Systematically deleted fragments in the 5' region of the genomic DNA of FR-gamma' were ligated into the PGL3Basic plasmid and the reporter luciferase activity was assayed in cell lysates from transiently transfected NIH3T3 cells. From those results, putative positive and negative regulatory regions in the 5' flanking sequences were noted, and a TATA-less proximal promoter was located between -206 and -22 nt. Gel mobility shift and supershift analyses as well as mutagenesis experiments revealed that Sp1 and ets binding elements in the proximal promoter region confer transcriptional activity. From partial sequencing of genomic DNA, genomic Southern blots, RACE analysis and RNase protection assays, it appears that hFR-gamma shares the gene organization of hFR-gamma'. The results of the analysis of genomic DNA in spleen tissues from several individuals, were consistent with the interpretation that hFR-gamma and hFR-gamma' are encoded by a polymorphic gene.

Amino Acid Sequence↗

PubMed1998

Chemotherapeutic drug activation of the AP24 protease in apoptosis: requirement for caspase 3-like-proteases.

AP24 is a serine protease that is activated during TNF or UV light-induced apoptosis and stimulates DNA fragmentation in isolated nuclei. The present study determined whether apoptosis induced by chemotherapeutic drugs resulted in activation of AP24 and examined the possible relationship to caspase activity. We showed that an inhibitor of AP24, DK120, could block DNA fragmentation induced in three leukemia cell lines (U937, HL-60, and CEM) by various DNA-damaging drugs including etoposide, camptothecin, chlorambucil, and the CC1065-related drug, YW201. Etoposide-induced activation of intracellular DEVD-pNa cleaving activity and apoptosis was suppressed by low micromolar concentrations of cell-permeable inhibitors of caspase-3. Furthermore, these inhibitors also suppressed activation of AP24. In contrast, DK120 did not prevent etoposide activation of DEVD-pNa cleaving activity, nor did it prevent cleavage of poly(ADP-ribose) polymerase. AP24 isolated from apoptotic cells following treatment with etoposide activated DNA fragmentation in isolated normal nuclei and was inhibited by DK120, but not by caspase inhibitors. This evidence shows that activation of caspase 3-like proteases generates signals that contribute to the activation of AP24 which may then induce nuclear DNA fragmentation in chemotherapeutic drug-induced apoptosis.

Antineoplastic Agents, Phytogenic↗

PubMed1998

Enhancement of serum-response factor-dependent transcription and DNA binding by the architectural transcription factor HMG-I(Y).

The mechanisms by which HMG-I proteins regulate cell growth are unknown, and their effects on gene expression have only been partially elucidated. We explored the potential interaction between HMG-I proteins and serum-response factor (SRF), a member of the MADS-box family of transcription factors. In cotransfection experiments, HMG-I(Y) potentiated SRF-dependent activation (by more than 5-fold) of two distinct SRF-responsive promoters, c-fos and the smooth muscle-specific gene SM22alpha. This effect was also observed with a heterologous promoter containing multiple copies of the CC(A/T)6GG (CArG) box. HMG-I proteins bound specifically to the CArG boxes of c-fos and SM22alpha in gel mobility shift analysis and enhanced binding of SRF to these CArG boxes. By chelating peptide-immobilized metal affinity chromatography, we mapped the domain of HMG-I(Y) that interacts with SRF to amino acids 50-81, a region that does not bind specifically to DNA in electrophoretic mobility shift assays even though it includes the third AT-hook DNA-binding domain. Surprisingly, HMG-I(Y) mutants that failed to bind DNA still enhanced SRF binding to DNA and SRF-dependent transcription. In contrast, deletion of the HMG-I(Y) 50-81 domain that bound SRF prevented enhancement of transcription. To our knowledge, this is the first report of an HMG-I protein interacting with a MADS-box transcription factor. Our observations suggest that members of the HMG-I family play an important role in SRF-dependent transcription and that their effect is mediated primarily by a protein-protein interaction.

Animals↗

PubMed1998

The full induction of human apoprotein A-I gene expression by the experimental nephrotic syndrome in transgenic mice depends on cis-acting elements in the proximal 256 base-pair promoter region and the trans-acting factor early growth response factor 1.

To identify molecular factors regulating apo A-I production in vivo, we induced in transgenic mice the experimental nephrotic syndrome, which results in elevated levels of HDL cholesterol (HDL-C), plasma apo A-I, and hepatic apo A-I mRNA. Human (h) apo A-I transgenic mice with different length 5' flanking sequences (5.5 or 0.256 kb, the core promoter for hepatic-specific basal expression) were injected with nephrotoxic (NTS) or control serum. With nephrosis, there were comparable (greater than twofold) increases in both lines of HDL-C, h-apo A-I, and hepatic h-apo A-I mRNA, suggesting that cis-acting elements regulating induced apo A-I gene expression were within its core promoter. Hepatic nuclear extracts from control and nephrotic mice footprinted the core promoter similarly, implying that the same elements regulated basal and induced expression. Hepatic mRNA levels for hepatocyte nuclear factor (HNF) 4 and early growth response factor (EGR) 1, trans-acting factors that bind to the core promoter, were measured: HNF4 mRNA was not affected, but that of EGR-1 was elevated approximately fivefold in the nephrotic group. EGR-1 knockout (EGR1-KO) mice or mice expressing EGR-1 were injected with either NTS or control serum. Levels of HDL-C, apo A-I, and hepatic apo A-I mRNA were lowest in nonnephrotic EGR1-KO mice and highest in nephrotic mice expressing EGR-1. Although in EGR1-KO mice HDL-C, apo A-I, and apo A-I mRNA levels also increased after NTS injection, they were approximately half of those in the nephrotic EGR-1-expressing mice. We conclude that in this model, basal and induced apo A-I gene expression in vivo are regulated by the trans-acting factor EGR-1 and require the same cis-acting elements in the core promoter.

Animals↗

PubMed1998

The arginine repressor of Escherichia coli K-12 makes direct contacts to minor and major groove determinants of the operators.

In order to gain further insight into the molecular mechanism of arginine-dependent operator recognition by the hexameric Escherichia coli arginine repressor we have probed protein-DNA interactions in vitro and in vivo. We have extensively applied the chemical modification-protection and premodification-interference approach to two operators, the natural operator overlapping the P2 promoter of the carAB operon and a fully symmetrical consensus sequence. Backbone contacts were revealed by hydroxyl radical footprinting and phosphate ethylation interference. Base-specific contacts to purines and pyrimidines were revealed by methylation protection and premodification interference, KMnO4 and NH2OH.HCl-specific modification of thymine and cytosine residues, base-removal (depurination and depyrimidation), and base substitution (uracil and inosine). Additional information on the groove specificity of repressor binding was obtained by small ligand binding interference (distamycin and methyl green). In vivo, we measured the effects on the repressibility of 24 single base-pair substitutions obtained by saturation mutagenesis of half an Arg box in the carAB operator. The results of these experiments point to the conclusion that a hexameric arginine repressor molecule covers four turns of the helix, makes base-specific contacts to at least one guanine (G4 or G4') and two thymine (T3, T13', or T3', T13) residues in each one of four consecutive major grooves on one face of the helix and with four A-T/T-A base-pairs, comprising the adenine residues A9, 9', 12, 12' and the thymine residues T10, 10', 11, 11', in the two outermost minor grooves of the operator, on the very same face of the DNA molecule. The hydrophobic 5-methyl groups of four thymine residues (T3, 3', 13, 13') in each Arg box contribute to major groove-specific recognition via hydrophobic and/or van der Waals interactions. The importance of minor groove contacts was further supported by the drastic effect of distamycin binding interference. In vivo, the most pronounced drops in repressibility were occasioned by mutations at positions 10 (A-->G or C), 11 (T-->A or G) and 12 (A-->G, T or C).

Arginine↗

PubMed1998

Potent cyclic urea HIV protease inhibitors with 3-aminoindazole P2/P2' groups.

Cyclic ureas containing 3-aminoindazole P2/P2' groups are extremely potent inhibitors of HIV protease. The parent 3-aminoindazole 6 showed a Ki < 0.01 nM but poor translation of enzyme activity to antiviral activity was observed. A series of 3-alkylaminoindazoles revealed that translation improved with increasing lipophilicity. An X-ray crystal structure of 6 bound to HIV protease was obtained.

Crystallography, X-Ray↗

PubMed1998

Cloning and functional expression of a cDNA encoding a mammalian sodium-dependent vitamin transporter mediating the uptake of pantothenate, biotin, and lipoate.

Previous studies have shown that a Na+-dependent transport system is responsible for the transplacental transfer of the vitamins pantothenate and biotin and the essential metabolite lipoate. We now report the isolation of a rat placental cDNA encoding a transport protein responsible for this function. The cloned cDNA, when expressed in HeLa cells, induces Na+-dependent pantothenate and biotin transport activities. The transporter is specific for pantothenate, biotin, and lipoate. The Michaelis-Menten constant (Kt) for the transport of pantothenate and biotin in cDNA-transfected cells is 4.9 +/- 1.1 and 15.1 +/- 1.2 microM, respectively. The transport of both vitamins in cDNA-transfected cells is inhibited by lipoate with an inhibition constant (Ki) of approximately 5 microM. The nucleotide sequence of the cDNA (sodium-dependent multivitamin transporter (SMVT)) predicts a protein of 68.6 kDa with 634 amino acids and 12 potential transmembrane domains. Protein data base search indicates significant sequence similarity between SMVT and known members of the Na+-dependent glucose transporter family. Northern blot analysis shows that SMVT transcripts are present in all of the tissues that were tested. The size of the principal transcript is 3.2 kilobases. SMVT represents the first Na+-dependent vitamin transporter to be cloned from a mammalian tissue.

Amino Acid Sequence↗