Cell-specific basolateral membrane sorting of the human liver Na(+)-dependent bile acid cotransporter.

The human Na(+)-taurocholate cotransporting polypeptide (Ntcp) is located exclusively on the basolateral membrane of hepatocyte, but the mechanisms underlying its membrane sorting domain have not been fully elucidated. In the present study, a green fluorescent protein-fused human NTCP (NTCP-GFP) was constructed using the polymerase chain reaction and was stably transfected into Madin-Darby canine kidney (MDCK) and Caco-2 cells. Taurocholate uptake studies and confocal microscopy demonstrated that the polarity of basolateral surface expression of NTCP-GFP was maintained in MDCK cells but was lost in Caco-2 cells. Nocodazole (33 microM), an agent that causes microtubular depolymerization, partially disrupted the basolateral localization of NTCP-GFP by increasing apical surface expression to 33.5% compared with untreated cells (P < 0.05). Brefeldin A (BFA; 1-2 microM) disrupted the polarized basolateral localization of NTCP, but monensin (1.4 microM) had no affect on NTCP-GFP localization. In addition, low-temperature shift (20 degrees C) did not affect the polarized basolateral surface sorting of NTCP-GFP and repolarization of this protein after BFA interruption. In summary, these data suggest that the polarized basolateral localization of human NTCP is cell specific and is mediated by a novel sorting pathway that is BFA sensitive and monensin and low-temperature shift insensitive. The process may also involve microtubule motors.

The rat liver Na(+)/bile acid cotransporter. Importance of the cytoplasmic tail to function and plasma membrane targeting.

To understand the potential functions of the cytoplasmic tail of Na(+)/taurocholate cotransporter (Ntcp) and to determine the basolateral sorting mechanisms for this transporter, green fluorescent protein-fused wild type and mutant rat Ntcps were constructed and the transport properties and cellular localization were assessed in transfected COS 7 and Madin-Darby canine kidney (MDCK) cells. Truncation of the 56-amino acid cytoplasmic tail demonstrates that the cytoplasmic tail of rat Ntcp is involved membrane delivery of this protein in nonpolarized and polarized cells and removal of the tail does not affect the bile acid transport function of Ntcp. Using site-directed mutagenesis, two tyrosine residues, Tyr-321 and Tyr-307, in the cytoplasmic tail of Ntcp have been identified as important for the basolateral sorting of rat Ntcp in transfected MDCK cells. Tyr-321 appears to be the major basolateral-sorting determinant, and Tyr-307 acts as a supporting determinant to ensure delivery of the transporter to the basolateral surface, especially at high levels of protein expression. When the two Tyr-based basolateral sorting motifs have been removed, the N-linked carbohydrate groups direct the tyrosine to alanine mutants to the apical surface of transfected MDCK cells. The major basolateral sorting determinant Tyr-321 is within a novel beta-turn unfavorable tetrapeptide Y(321)KAA, which has not been found in any naturally occurring basolateral sorting motifs. Two-dimensional NMR spectroscopy of a 24-mer peptide corresponding to the sequence from Tyr-307 to Thr-330 on the cytoplasmic tail of Ntcp confirms that both the Tyr-321 and Tyr-307 regions do not adopt any turn structure. Since the major motif YKAA contains a beta-turn unfavorable structure, the Ntcp basolateral sorting may not be related to the clathrin-adaptor complex pathway, as is the case for many basolateral proteins.

Sorting of rat liver and ileal sodium-dependent bile acid transporters in polarized epithelial cells.

The rat ileal apical Na+-dependent bile acid transporter (ASBT) and the liver Na+-taurocholate cotransporting polypeptide (Ntcp) are members of a new family of anion transporters. These transport proteins share limited sequence homology and almost identical predicted secondary structures but are localized to the apical surface of ileal enterocytes and the sinusoidal surface of hepatocytes, respectively. Stably transfected Madin-Darby canine kidney (MDCK) cells appropriately localized wild-type ASBT and Ntcp apically and basolaterally as assessed by functional activity and immunocytochemical localization studies. Truncated and chimeric transporters were used to determine the functional importance of the cytoplasmic tail in bile acid transport activity and membrane localization. Two cDNAs were created encoding a truncated transporter in which the 56-amino-acid COOH-terminal tail of Ntcp was removed or substituted with an eight-amino-acid epitope FLAG. For both mutants there was some loss of fidelity in basolateral sorting in that approximately 75% of each protein was delivered to the basolateral surface compared with approximately 90% of the wild-type Ntcp protein. In contrast, deletion of the cytoplasmic tail of ASBT led to complete loss of transport activity and sorting to the apical membrane. An Ntcp chimera in which the 56-amino-acid COOH-terminal tail of Ntcp was replaced with the 40-amino-acid cytoplasmic tail of ASBT was largely redirected (82.4 +/- 3.9%) to the apical domain of stably transfected MDCK cells, based on polarity of bile acid transport activity and localization by confocal immunofluorescence microscopy. These results indicate that a predominant signal for sorting of the Ntcp protein to the basolateral domain is located in a region outside of the cytoplasmic tail. These studies have further shown that a novel apical sorting signal is localized to the cytoplasmic tail of ASBT and that it is transferable and capable of redirecting a protein normally sorted to the basolateral surface to the apical domain of MDCK cells.

Cloning and characterization of a novel peptidase from rat and human ileum.

A novel 100-kDa ileal brush border membrane protein (I100) has been purified by anionic glycocholate affinity chromatography. Polyclonal antibodies raised against this protein were utilized to clone and characterize I100 in rats. A partial length human I100 cDNA was identified by hybridization screening. In the rat, the I100 protein is a 746-amino acid glycosylated (calculated core molecular mass of 80 kDa) type II integral membrane protein found on the apical surface of ileal villus enterocytes. Its 2.6-kilobase mRNA is expressed in distal small intestine in rats and in humans. The I100 cDNA is homologous to but distinct from human prostate-specific membrane antigen and rat brain N-acetylaspartylglutamate peptidase. It is expressed on both the basolateral and apical surfaces of stably transfected Madin Darby canine kidney cells. Analysis of these stably transfected Madin Darby canine kidney cells and I100 immunoprecipitates of rat ileal brush border membrane vesicles reveals that it has dipeptidyl peptidase IV activity. Future invesitgations will need to determine the exact substrate specificity of this novel peptidase.

Multiple factors regulate the rat liver basolateral sodium-dependent bile acid cotransporter gene promoter.

The hepatic uptake of bile acids from the portal circulation is primarily dependent upon a sodium-dependent basolateral membrane transporter. In order to begin to investigate the factors controlling rat liver sodium-dependent bile acid cotransporter (ntcp) gene expression, we isolated approximately 30 kilobase pairs of rat genomic DNA in three overlapping lambdaphage clones. The rat ntcp gene is distributed over 16.5 kilobase pairs as five exons. Primer extension analysis revealed two closely spaced transcription initiation sites, 27 and 41 nucleotides downstream of a TATA sequence. Regulation of transcription was investigated first by transfection of primary rat hepatocytes by a series of 5'-deleted rat ntcp promoter-driven luciferase constructs (from approximately -6 kilobase pairs to -59 base pairs of upstream sequences, terminating at nucleotide +47), identifying a minimal promoter element: nucleotide -158 to +47. This minimal promoter was active in transfected HepG2, but inactive in NIH3T3, Caco-2, and Madin-Darby canine kidney cells, indicating that the determinants of hepatocyte-specific expression reside within this region. The individual elements within the minimal promoter were investigated via transfection of HepG2 cells by a series of 20 mutant plasmids, each containing a 10-base pair sequential block mutation. Eight mutant constructs profoundly suppressed promoter activity; encompassing sequences from -66 to +4 nt, and +15 to +24 nucleotides, while no other 10-base pair mutation significantly interfered with minimal promoter activity. Deoxyribonuclease I footprint analysis of the minimal promoter revealed three bound regions; -92 to -74 (footprint C), -50 to -37 (footprint B), and -17 to +12 (footprint A). Gel mobility shift assays provided evidence for hepatocyte nuclear factor 1 binding within footprint A and a liver-enriched factor(s) that binds within a novel palindrome in footprint B. These studies indicate that three elements direct the basal and tissue-restricted expression of the rat ntcp promoter; a TATA element, the liver-enriched transcription factor hepatocyte nuclear factor 1, and an unknown liver-enriched factor that binds within a novel palindrome in footprint B.

Terminal marking of triosephosphate isomerase: consequences of deamidation.

Mammalian triosephosphate isomerase spontaneously deamidates at Asn71 and Asn15 located at the subunit interface of the isologous dimer. These deamidations have been proposed to constitute the terminal marking event in the degradation of the enzyme. A series of physical and chemical studies detailed here reveals that the overall structure of the enzyme is substantially altered by these deamidations. The far-uv CD spectra show a 30% lower secondary structure with a blue shifted ellipticity minimum and increased fluorescence (10-22%) with a red-shifted emission maximum (8.7-15.6 nm) indicates exposure of tryptophans to a more polar environment. Increased binding of the fluorescent hydrophobic probe 1,1'-bis(4-anilino)-naphthalene-5,5'-disulfonic acid to the deamidated enzyme corroborates these spectral observations and also suggests that the hydrophobic residues at the subunit interface are exposed as a result of the deamidation. Decreased subunit cross-linking (80 vs 20%) of the deamidated enzyme by the bifunctional reagent ethylene glycolbis (succinimidylsuccinate) also indicates a loosening of the two subunits at the interface. These structural changes are accompanied by a decreased thermal stability (3.1 degrees C lower Tm) and an increased susceptibility to dissociation in urea. The terminal marking also results in the generation of new proteolytic sites and increases the susceptibility to proteolysis. Hybrid dimers from rabbit and yeast (lacking Asn71) showed that deamidation of the rabbit Asn71-yeast Asn15 pair does not accelerate deamidation of the remaining rabbit Asn15 site, indicating that deamidation of Asn71 is a prerequisite for deamidation of Asn15. These studies are consistent with the proposal that the specific deamidations at the subunit interface cause significant structural changes which lead to degradation of the protein.

The hinged lid of yeast triose-phosphate isomerase. Determination of the energy barrier between the two conformations.

Covalent modification of Glu165 in the catalytic center of triose-phosphate isomerase with the substrate analogue 3-chloroacetol phosphate traps the complex in two conformations. The two resulting 31P NMR resonances at 6.9 and 5.7 ppm appear to reflect conformations in which the hinged lid (residues 167-176) is in the open and closed positions. The conformation represented by the 5.7-ppm resonance is more stable, and unfolding and refolding in guanidine converts all of the molecules to the 5.7-ppm conformation. The complete conformational transition from 6.9 to 5.7 ppm also takes place as a function of time and temperature. Under these conditions the native enzyme retains more than 80% of the catalytic activity, indicating that this conversion is not due to thermal denaturation of the enzyme. Circular dichroic and fluorescence spectroscopy indicate that the 3-chloroacetol phosphate-modified enzyme does not undergo major structural changes. From the temperature dependence of this transition, an energy barrier of 144 kJ/mol (34.4 kcal/mol) was calculated for this conversion.

Limited proteolysis of triose-phosphate isomerase and characterization of the catalytically active peptide complex.

Limited proteolysis of the triose-phosphate isomerase (EC 5.3.1.1) by subtilisin generates peptides that remain noncovalently attached and catalytically active. Edman degradation of the peptides showed that the primary proteolytic sites for yeast triose-phosphate isomerase are the Leu174-Ala175 bond followed by Ser52-Leu53. The Leu174-Ala175 site is of particular interest, since it forms part of the hinged lid that closes over the catalytic center, and this bond is only 12.2 A (open) or 9.8 A (closed) from the catalytic residue Glu165. The higher Km, kcat, and kcat/Km values exhibited by the catalytically active peptide complex suggest that the substrate is not bound as tightly to the catalytic center. In addition, increased methylglyoxal formation by the cleaved enzyme indicates that the enzyme-substrate complex is less protected from the solvent. Circular dichroic and fluorescence spectra show that the overall structure of the peptide complex is similar to the native enzyme but with local structural perturbations around the tryptophans. Also, the peptide complex is more susceptible to denaturation by guanidine and exhibits lower Tm values, indicating a loose interaction between the fragments. Unfolding, dissociation, and refolding experiments suggest that the fragments have strong inherent secondary structural features and can reassociate into catalytically active structures.

Interactions between the catalytic centers and subunit interface of triosephosphate isomerase probed by refolding, active site modification, and subunit exchange.

The effects of unfolding, refolding, and hybridization of triosephosphate isomerase (TPI) subunits from different species and subunits which have been specifically modified at the active site have been examined. These effects have been evaluated in terms of changes in catalytic parameters, CD spectra, and susceptibility to denaturation. Dissociation followed by reassociation yields an active dimer but with increased Km, reduced kcat, and increased susceptibility to inactivation and unfolding in denaturants. These data suggest that while the general structure of the refolded dimer is similar to the native enzyme, its complete original structure is not restored. Covalent reaction of the active site Glu165 with the substrate analogue 3-chloroacetol phosphate (CAP) results in dimers with increased susceptibility to unfolding and inactivation by denaturants (i.e. the rates of inactivation and unfolding are (TPICAP)2 greater than (TPI-TPICAP) greater than (TPI)2). These data point to the interactions between the catalytic center and the subunit interface. Subunits of TPI from different species, in spite of structural differences at the subunit interface, hybridized to active heterodimers. Subunit hybridization was random among monomers from different mammals, preferential between yeast and mammalian or avian monomers. Hybridization did not occur between avian and mammalian monomers under these conditions. These data provide information on the elements in the interface of the dimer and the relationship of the catalytic center with the subunit interface.

Effects of active site modification and reversible dissociation on the secondary structure of triosephosphate isomerase.

Binding of ligands to the catalytic center of mammalian triosephosphate isomerase (TPI) induces a conformational change(s) that enhances the specific deamidation of Asn71 at the subunit interface. Deamidation initiates dissociation and degradation of the enzyme in vivo and in vitro. We have utilized circular dichroism spectroscopy to examine the conformational changes in the enzyme upon ligand binding and subunit dissociation/reassociation. Native TPI from rabbit, chicken, and yeast exhibit similar spectra at pH 7.5, but are substantially different at pH 9.5. Covalent reaction of the active site Glu 165 with the substrate analogue 3-chloroacetol phosphate results in a conformational change (decrease in beta-sheet) which is similar in TPI from all three species. Reversible dissociation of the dimeric enzyme in guanidine followed by dialysis, although permitting full recovery of catalytic activity, results in refolded dimers with decreased alpha-helix. These conformational changes induced by ligand binding, pH, or reversible dissociation explain, in part, the differences in the chemical and physical properties of the enzyme from the three species at alkaline pH, the increased lability of the dissociated/reassociated enzyme, and corroborate 31P NMR data on substrate-induced conformational changes. These studies also support the concept of molecular wear and tear whereby ligand binding at the catalytic center induces conformational changes that increase the probability of covalent modification and ultimate degradation of the protein.

Relationship between the catalytic center and the primary degradation site of triosephosphate isomerase: effects of active site modification and deamidation.

Covalent modification of the active site Glu165 of triosephosphate isomerase (TPI) (EC 5.3.1.1) with the substrate analogue 3-chloroacetol phosphate (CAP) induces conformational changes similar to those observed during catalysis. We have introduced CAP into the active sites of TPI from yeast, chicken, pig, and rabbit, and assessed the effect of this modification on the structural integrity of the protein. CAP binding accelerated the specific deamidation of Asn71 in mammalian TPI. Transverse urea gradient gel electrophoretic analysis showed that the CAP-TPI dimer dissociates more readily than the native dimer. Hybrids composed of one CAP-modified subunit and one native subunit exhibited intermediate stability. The deamidated enzyme was more susceptible to proteases and denaturing conditions. Subtilisin cleaved the rabbit enzyme primarily at the Thr139-Glu140 bond. The resulting peptides remained noncovalently attached, and the enzyme retained catalytic activity. The data provide further evidence of the interactions between the catalytic center and the subunit interface and that the specific deamidation destabilizes the enzyme initiating its degradation. The enhancement of deamidation upon binding of substrate and catalysis suggest that molecular wear and tear may be involved in regulating proteolytic turnover of the enzyme.

Isolation and characterization of human glucose-6-phosphate isomerase isoforms containing two different size subunits.

Previously undetected isoforms of human glucose-6-phosphate isomerase (GPI) have been isolated utilizing substrate-induced elution of the enzyme from spherical cross-linked phosphocellulose as an affinity ligand and subjected to a series of physical and chemical studies. The two major isoforms (1, 48%, pI 9.13; 2, 36%, pI 9.00) are homodimers of subunits of 63.2 kDa (Type-A) and are charge isomers, probably representing deamidation of specific Asn-Gly sequences as in other species. Isoform 3 (13%, pI 8.84) is a heterodimer composed of the Type-A subunit and a previously unreported larger subunit of 69.8 kDa (Type-B). Isoform 4 (3%, pI 8.62) is a BB-homodimer. Structural differences in the two types of subunits are also apparent from CNBr fragmentation patterns. Carbohydrate analyses show that, even though potential N- and O-linked glycosylation sites exist, the isoforms are not due to glycosylation. Recently recognized sequence similarities between GPI and the neurotropic lymphokine, neuroleukin (NLK) suggest that GPI and NLK are either derived from the same gene or represent modifications of the same protein. The possibility of NLK-GPI dimers exists, but the new isoforms identified in this study do not appear to represent hybrids of GPI subunits with mature NLK.