Blood—Brain Barrier Genomics and Cloning of a Novel Organic Anion Transporter

Materials

The PCR-Select^TM cDNA subtraction kit was obtained from BD Biosciences Clontech (Palo Alto, CA, USA). [α³²P]dCTP (3,000 Ci/mmol; 1Ci = 37 GBq), [³H]estradiol 17 β-d-glucuronide (44 Ci/mmol), [¹⁴C]sucrose (0.6 Ci/mmol), [¹²⁵I]thyroxine, (T4) (165 Ci/μg), [¹²⁵I]-3,5,3′-triiodothyronine (T3) (165 %mUCi/μg) and GeneScreen Plus membrane were purchased from Perkin Elmer Life Science Products (Boston, MA, USA). Oligodeoxynucleotides were custom synthesized at Biosource International (Camarillo, CA, USA). The pCR2.1 vector, the TA Cloning kit and the INVαF′ super competent cells were obtained from Invitrogen (San Diego, CA, USA). Human embryonic kidney 293 and rat glioma C6 cells were obtained from the America Type Culture Collection (Rockville, MD, USA). The λDNA/HindIII and φX174 RF DNA/HaeIII DNA standard fragments and EcoRI were purchased from Promega (Madison, WI, USA). Megaprime DNA labeling kit was obtained from Amersham Pharmacia Biotech (Arlington Heights, IL, USA). Qiafilter^TM Maxi Kit Qiaquick and PCR purification kit were obtained from Qiagen (Valencia, CA, USA). The pSPT19 vector and DIG RNA labeling kit for in situ hybridization (ISH) were purchased from Roche Molecular Biochemicals (Indianapolis, IN, USA). NotI, SalI, and XbaI were obtained from New England Biolabs (Beverly, MA, USA). mMessage mMachine kit was purchased from Ambion (Houston, TX, USA). Tris—HCl ready gel of 12%, biotinylated sodium dodecyl sulfate-polyacrylamide gel electrophoresis molecular weight standards, mini-protein electrophoresis, and mini-transblot apparatuses were obtained from Bio-Rad Labs (Hercules, CA, USA). Biotinylated goat anti-rabbit IgG (H + L) and Vectastain ABC Elite peroxidase kit were obtained from Vector Labs Inc. (Burlingame, CA, USA). Ziduvudine (AZT) was obtained from Moravek (Brea, CA, USA). Bicinchoninic acid protein assay kit was purchased from Pierce (Rockford, IL, USA). Unlabeled estradiol 17 β-d-glucuronide and other molecular biology grade reagents were obtained from Sigma Chemical (St Louis, MO, USA). Female oocyte-positive Xenopus frogs (50 to 70 g) were purchased from Nasco (Fort Atkinson, WI, USA). Mouse actin and rat type 1 large neutral amino acid transporter (LAT1) cDNAs were prepared as described previously (Boado et al, 2003a).

Isolation of Poly(A+) RNA

Poly(A+) RNA was isolated from rat brain capillaries, C6 and RG2 implanted rat brain tumor, and rat brain and peripheral tissues using a single step method described previously (Boado and Pardridge, 1991). RG2 and C6 brain tumors were produced in Fischer rats as described previously (Suzuki et al, 2004). The yield of poly(A+) RNA from rat brain capillaries isolated from a pool of 21 cerebral rat brain hemispheres was 3.2 μg, whereas the yield averaged 20 μg/g tissue of total brain and peripheral organs.

Suppression Subtractive Hybridization

Suppression subtractive hybridization was performed using the PCR-Select cDNA subtraction kit as reported previously (Li et al, 2001a). The SSH procedure was performed using rat brain capillary mRNA to produce tester cDNA and rat liver/kidney mRNA to produce driver cDNA. Double-stranded cDNA was synthesized using 1 μg of either tester or driver poly(A+) RNA. cDNA size was normalized by digestion with RsaI, and specific oligodeoxynucleotide adaptors were ligated to the RsaI-digested tester cDNA. After hybridization of tester and driver cDNAs, the rat brain capillary-specific sequences were amplified by polymerase chain reaction (PCR) (Li et al, 2001a). The PCR products were cloned into the pCR2.1 vector, and INVαF′ super competent cells were transformed to form a subtracted rat brain capillary cDNA library (Li et al, 2001a). For the differential hybridization, randomly selected colonies were blotted onto GeneScreen Plus membranes using a 96-well dot-blot apparatus (Bio-Rad). The hybridization was performed with ³²P-labeled subtracted or unsubtracted tester cDNA as described previously (Li et al, 2001a). Clones that produced stronger hybridization signal with the subtracted probe compared with that of the unsubtracted cDNA were selected for further characterization. The insert size was determined by EcoRI digestion and agarose gel electrophoresis.

Cloning of Full-Length cDNA

To isolate full-length cDNAs corresponding to clones identified in the SSH cloning, a rat brain capillary cDNA library in the pSPORT vector was produced as described previously (Li et al, 2001b). This library was screened with ³²P-partial cDNAs isolated by SSH using colony hybridization (Boado et al, 2003a). Clones K-2 and LK-3 were isolated in the SSH cloning, and the DNA sequence of the partial clone indicated that the clone was novel and warranted further examination. The partial insert was released and labeled with [³²P] and was used to screen 2.4 × 10⁵ recombinants of the rat brain capillary pSPORT cDNA library. Forty positive clones were identified, and 25 of them were isolated for further characterization. The cDNA inserts were released by double digestion with NotI and SalI, and samples were characterized by ethidium bromide staining of agarose gels after electrophoresis. Clones were confirmed by Southern blot analysis performed with ³²P-labeled LK-3 cDNA under high-stringency conditions as described previously (Boado et al, 1999).

DNA Sequence Analysis

DNA sequencing of isolated clones was performed in both directions as described previously (Boado et al, 2003a). Initial DNA sequencing was performed with M13 forward and reverse primers, which are located in pSPORT at the 5′- and 3′-flanking regions of NotI and SalI sites, respectively. Full-length cDNAs were entirely sequenced in both directions by primer walking using custom synthesized oligodeoxynucleotides as described previously (Boado et al, 2003a). Primers were designed using the program Oligos 4.0 with T_m > 60°C and absence of either stable stem loops or secondary structures. Similarities with other transporters in GenBank were investigated using the BLAST program (NCBI, NIH, USA). DNA sequencing indicated the partial clone, LK-3, isolated in the SSH procedure, corresponded to a 2.6 kb full-length cDNA with novel sequence (AF306546) with distant homology to organic anion transporters. Since subsequent Northern blotting showed the gene was only expressed at the BBB, the gene was named BBB-specific organic anion transporter type 1 or BSAT1.

Northern Blot Analysis

Two micrograms of poly(A+) RNA were resolved on 1.5% agarose/2.2 mol/L formaldehyde gels, and blotted onto GeneScreen Plus membranes. Northern blots were hybridized with ³²P-labeled LAT1, 4F2hc, BSAT1, or β-actin cDNAs as described previously (Boado et al, 1999). cDNAs were labeled with [³²P]dCTP by the random priming technique using the Megaprimer DNA labeling kit (Boado et al, 2003a). Autoradiograms were performed with Biomax MS film and exposed at −70°C for various periods. Films were scanned with an Umax PowerLook III scanner.

In Situ Hybridization

In situ hybridization was performed on cytocentrifuged isolated rat brain capillaries. An ∼0.6 kb BSAT1 cDNA fragment was obtained from the partial BSAT1 clone by double digestion of the pCR2.1 vector with SalI and XbaI and subcloned into the pSPT19 vector at the same restriction endonuclease sites (Li et al, 2001a). Antisense and sense RNA probes were generated using SalI and XbaI linearized pSPT19-BSAT1 plasmid and T7 or SP6 polymerases, respectively, in the presence of digoxigenin-11-uridine triphosphate (DIG-11-UTP). Freshly prepared rat brain capillaries were cytocentrifuged onto glass slides, and the capillaries were fixed with 10% formalin and stored at −70°C (Li et al, 2001a). The slides were hybridized with 12 ng digoxigenin-labeled probes in 30 μL of hybridization solution overnight at 42°C, and control sections were hybridized with equal amount of sense cRNA as reported previously (Li et al, 2001a).

Real-Time Polymerase Chain Reaction Measurement of Rat Brain Capillary and Whole Brain mRNA

Poly(A+) RNA was isolated from rat brain capillaries or rat whole brain using a single step method as previously described (Boado and Pardridge, 1991). Reverse transcription (RT) was performed according to the Invitrogen SuperScript III First-Strand Synthesis System for PCR using 0.4 μg of poly(A+) RNA as the template and oliogodeoxythymidine as primer in a total volume of 21 μL per reaction. The RT product was diluted by 20-fold before real-time PCR assays. Real-time PCR was performed using the BioRad iQ SYBR Green Supermix according to the manufacturer's instruction. In each PCR reaction in 25 μL, 2 μL of the diluted RT product was used as the template. The primer sequences for rat β-actin (GenBank accession no 42475962) are 5′-GGGACGATATGGAGAAGATTTGG-3′ and 5′-TGAAGGTCTCAAACATGATCTGG-3′. The primer sequences for rat BSAT1 are 5′-AACCTCACTCCCAACATCTCTC-3′ and 5′- GTATCCACTTCACACTCATCGTTTA-3′.

Each primer pair was selected to amplify the sequence shared by contiguous exons of a target gene, to eliminate the possible interference of genomic DNA. Polymerase chain reaction was initiated by a 3-min incubation at 95°C, followed by 40 cycles consisting of 10 secs annealing at 58.3°C, 10 secs extension at 72°C, and 10 secs denaturing at 95°C. Melting curve of each PCR product was determined by measuring the ratio of single-strand DNA versus double-strand DNA at each 0.5°C of temperature increase from 55 to 94°C. Single peak in the melting curve with a signature T_m suggests pure PCR product and specificity of the reaction. The threshold cycle (C_t) value represents the relative amount of the target mRNA in the RNA pool.

Western Blotting

A rabbit polyclonal antiserum against the C-terminus of rat BSAT1 was prepared using the synthetic peptide LQPKYWPGKETRL corresponding to amino acids 706 to 718 of the deduced amino acid sequence of BSAT1. The synthetic peptide was used to screen for antibody titer by enzyme-linked immunosorbent assay. Anti-BSAT1 antibodies were affinity purified after production of an affinity column by coupling the synthetic peptide to cyanogen bromide-activated Sepharose 4B. Rat brain capillary protein was resolved in a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions for Western blotting. Proteins on the gel were electroblotted onto a nitrocellulose membrane. Immunoblot analysis was performed with the affinity-purified anti-BSAT1 antibodies either without or with absorption of the antibody by the synthetic peptide. Biotinylated goat anti-rabbit IgG was used as secondary antibody. Results were visualized with the ABC Elite detection kit as described previously (Pardridge et al, 1990). Western blot analysis with the affinity-purified anti-rat BSAT1 antibody showed that the antibody reacted with a single 55-kDa immunoreactive protein in rat brain capillaries, and that this signal was completely removed by absorption of the anti-BSAT1 antibody with the BSAT1 C-terminal synthetic peptide. However, it is believed that this represents a sequence-specific artifactual cross-reaction of this C-terminal peptide antibody with a non-BSAT1 protein, because the same 55-kDa immunoreactive species was detected in multiple tissues, which do not express the BSAT1 mRNA.

Stable Transfection of Human Embryonic Kidney 293 Cells

The full-length (fl) BSAT1 cDNA was released from the pSPORT vector by double digestion with NotI and EcoRI and cloned into the pcDNA3.1 vector (Invitrogen) at the same restriction endonuclease sites, and this plasmid is designated pcDNA/BSAT1(fl). Human 293 cells transfected with this plasmid are designated 293/BSAT1(fl) cells. Human embryonic kidney 293 cells were plated in minimal essential medium/10% horse serum in six-well dishes a day before the transfection. Four μg of pcDNA3.1-BSAT1(fl) or pcDNA 3.1 control (no insert) were used to transfect human embryonic kidney 293 cells using 8 μL, of lipofectamine 2000 as per the manufacturer's instructions (Invitrogen). Forty-eight hours after transfection, cells were selected in minimal essential medium/10% horse serum containing 600 to 750 μg/mL of G418 for 14 days. Cells transfected with the BSAT1 (fl) cDNA are designated 293-BSAT1 (fl) cells.

A second human embryonic kidney 293 stably transfected cell line was established with a rat BSAT1 expression construct lacking both the 5′- and 3′-UTR of the rBSAT1 transcript. The 2.0-kb BSAT1 orf was obtained from pSPORT-BSAT1 by PCR cloning with Stratagene PfuUltra Hotstart DNA polymerase, forward primer (5′-phosphate-AATGGACACTTCATCCAAAGAAAATGC) and a reverse primer (5′-phosphate-CTTAAAGTCGGGTCTCCTTGCCTG). The PCR-generated BSAT1 orf cDNA was subcloned in the pcDNA3.1(+) vector at the EcoR V site to form the pcDNA-BSAT1(orf) plasmid named clone 994. Positive clones with the correct orientation of the BSAT1(orf) were identified by restriction endonuclease mapping (i.e., XbaI), and verified by DNA sequencing. Insertion of the full Kozak site immediately preceding the ATG initiation codon (i.e., GCCGCCACC) and single nucleotide errors introduced in the PCR cloning were corrected by site-directed mutagenesis using the Stratagene QuikChange II XL Kit as described previously (Boado et al, 2003a). Cells transfected with the BSAT1(orf) cDNA are designated 293-BSAT1(orf) cells.

Influx Assays in Stably Transfected 293 Cells

293-BSAT1(orf) or 293-BSAT1(fl) cells were washed with physiological buffer (PB: 118 mmol/L NaCl, 25 mmol/L HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), pH 7.4, 4.9 mmol/L KCl, 1.4 mmol/L K₂HPO₄, 1.2 mmol/L MgCl₂, 1 mmol/L CaCl₂, and 11 mmol/L d-glucose) and incubated in PB containing 1 to 2 μCi/mL of ³H-estradial 17 β-D-glucuronide (E2G), and 0.1 to 0.2 μCi/mL of ¹⁴C-sucrose for 2.5 to 40 mins at 37°C in the presence or absence of competitors. Alternatively, the tracer was [¹²⁵I]T4 or [¹²⁵I]triiodothyronine (T3). Wells were washed three times with 3 mL of ice-cold PB and cells were dissolved in 200 μL of 1N NaOH. Aliquots of cell lysates were used to determine the uptake of the radioactive substrate with double isotope (³H and ¹⁴C) liquid scintillation counting. The volume of distribution (VD) of the E2G and sucrose were computed from the ratio of the DPM/μg cell protein divided by the DPM/μL of medium. The VD of sucrose, which ranged from 0.5 to 1.5 μL/mg_p, was subtracted from the VD of E2G. The VD was divided by the incubation time to calculate the cell clearance (μL/min per mg protein) of the labeled substrate. The clearance data were fit to the Michaelis—Menten equation by nonlinear regression analysis to estimate the half-saturation constant, Km (μmol/L), the maximal transport velocity, V_max (pmol/min per mg protein), and the constant of nonsaturable transport, K_D (μL/min per mg protein), or the half-inhibition constant, K_i (μmol/L), as described previously (Li et al, 2001c). The sodium dependency of the transporter was assessed by stoichiometric replacement of NaCl in the medium with choline chloride.

[125I]Thyroxine Efflux in Stably Transfected 293 Cells with Prealbumin Trap Technique

The isolation of the efflux kinetics in cells preloaded with [¹²⁵I]T4 is complicated by the rapid re-entry of the isotope into the cell. The re-entry of T4 was eliminated by the addition of human prealbumin to the medium. Human plasma prealbumin is a high-affinity binder of T4, and this rapid binding prevents any further influx of T4 into the cell. 293-BSAT1(orf) cells were plated in Biocoat collagen I-coated 24-well cluster dishes and grown to confluency. The cells were washed with warm physiological buffer (118 mmol/L NaCl, 25 mmol/L HEPES, 4.9 mmol/L KCl, 1.4 mmol/L K₂HPO₄, 1.2 mmol/L MgCl₂, 1 mmol/L CaCl₂, 11 mmol/L glucose, pH 7.4) and incubated with 200 μL of physiological buffer containing 3.26 μCi/mL ¹²⁵I-T4 (34 nmol/L) at 37°C for 15 mins. Efflux was initiated by adding 10 μL of 2 μg/μL of prealbumin (2 μmol/L final concentration) to the medium, and was terminated by the rapid addition of 2.5 mL of ice-cold physiological buffer. After three washes with ice-cold physiological buffer, the cells in each well were solubilized with 200 μL 1N NaOH by overnight incubation at room temperature. Uptake result was determined by γ-counting and protein assay of the lysates.

In Vitro Transcription and Cross-Linking of BSAT1 RNA to Polysome Proteins

The full-length rat BSAT1 cDNA was obtained by NruI/NotI double digestion of the pCD-BSAT1(fl) plasmid, followed by Qiagen gel purification. ³²P-labeled BSAT1(fl) cloned RNA (cRNA) was synthesized by in vitro transcription in a 20 μL volume using 0.3 μg DNA template and T7 polymerase with the MAXIscript in vitro transcription kit for 1 h in the presence of [³²P]UTP, as described previously (Tsukamoto et al, 1996). Purity and activity of the ³²P-labeled cRNA was determined by trichloroacetic acid precipitation of the cRNAs before and after filtration through a G-25 column. The purity of the BSAT1(fl) cRNA was 89%. The activity the BSAT1(fl) cRNA was 2.0 × 10⁶ c.p.m./μL. The size of the BSAT1(fl) cRNA was estimated to be 2.7 kb by electrophoresis in a 1.5% agarose/formaldehyde gel. To perform ultraviolet cross-linking of the ³²P-cRNA to brain capillary endothelial polysome or cytosolic proteins, 2.0 × 10⁵ c.p.m. of freshly labeled cRNA was incubated with 2 μg protein from either the P100 polysomic extracts or the S100 cytosolic extracts of the bovine brain capillary endothelial cells for 10 mins at 30°C in 20 μL of binding buffer, as described previously (Boado et al, 2003b). The binding buffer is 12 mmol/L HEPES, pH 7.5, 0.25 mmol/L EDTA (ethylenediaminetetraacetic acid), 10 mmol/L potassium chloride, 5 mmol/L magnesium chloride, 0.5 mmol/L dithiothreitol, 0.2 mg/mL yeast tRNA, 5% glycerol, and 0.5 U/μL of RNasin RNase inhibitor. RNase T1 (5 U/μL) was then added to reaction mixtures for a 30 mins incubation at 37°C. Reaction mixtures were cross-linked using a Stratalinker 1800 UV oven (Stratagene, San Diego, CA, USA) at 254 nm for 5 mins. Binding reactions were transferred to 1% sodium dodecyl sulfate sample buffer and boiled for 3 mins. RNA/protein complexes were separated by electrophoresis using a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gels were dried and exposed to Kodak BioMax MS-1 film for 2 days at −70°C using intensifying screens.