Difference between revisions of "Part:BBa K2865001:Design"

Revision as of 03:01, 4 October 2018

AR185-T2A-EGFP, nanobody inhibiting RyR2 phosphorylation

Design Notes

RyR2-specific nanobodies were isolated from a phage display library of variable domains of camellidae heavy chain-only antibodies (VHH). AR185, one of the isolated nanobodies, inhibiting RyR2 phosphorylation in an in vitro assay was then chosen.

Cell Culture Neonatal rat ventricular myocytes were isolated from 1-2 day old Sprague-Dawley neonatal rats. Briefly, neonatal rats were sacrificed by decapitation and whole hearts were removed. Ventricles were then isolated and transferred to 1x Hank’s Buffered Saline Solution (HBSS) with 0.06% trypsin and incubated overnight at 4°C. The following day, 10mg/mL Collagenase II (Worthington Biochemical) in 1x HBSS was added to isolate individual cardiomyocytes. Resulting suspensions were pre-plated to remove contaminating fibroblasts, and plated at a density of 1 x 106 cells/well in 6-well dishes in Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen) containing 10% fetal bovine serum. All tissue culture plates that were seeded with Myocytes were pre-treated with 0.1% gelatin for at least 1 h prior to seeding. After 24 h in culture, myocytes were washed with 1x PBS and cells were placed in DMEM with 0.5x Nutridoma-SP (Roche Applied Sciences). Purification of RyR2 by GST-FKBP12 The expression and purification of GST-FKBP12 were reported previously. Briefly, the cDNAs encoding rat FKBP12 were cloned into pGEX-4T-2 vector with a C-terminal 6×His tag. The plasmids were transformed into BL21 (DE3) strain for protein overexpression. When OD600 reached 1.0, the temperature was adjusted to 16°C, and 0.4 mM IPTG was added for induction overnight. Cells were collected, lysed and centrifuged, recombinant protein were purified using Ni2+-NTA resin (Qiagen) and eluted by imidazole containing buffer. Anion exchange chromatography (SOURCE 15Q, GE Healthcare) was performed for further purification. For preparation of sarcoplasmic reticulum membrane from rat heart, A single rat heart was diced into small pieces, and re-suspended in 5 volumes of homogenization buffer contain-ing 25 mM Tris, pH 7.5, 150 mM NaCl, 5mM EDTA, and the protease inhibitor cocktail (0.2 mM PMSF, 1.3 μg/ml apro-tinin, 0.7 μg/ml pepstatin, and 5 μg/ml leupeptin). Fifteen cycles of homogenization were performed with a blender. The debris was removed by low speed centrifugation (6,000 g) for 6 min. The supernatant was then centrifuged at high speed (20,000 g) for 1 hour. The pellet was resuspended in 2 volumes of homogenization solution and flash frozen in liquid nitrogen. The RyR2 was purified following the same strategy for the purification of RyR1(15)and the RyR2 (16). The rat sarcoplasmic reticulum membrane (1/4 of total membrane from a single heart) was solubilized at 4°C for 2 hours in the homogenization buffer plus 2% CHAPS, 1% soybean lecithin, and 2 mM DTT. Approximately 10 mg of GST-FKBP12 was added to the extraction system. After ultra-high speed centrifugation (200,000 g), the supernatant was applied to GS4B (GE Healthcare) column. The resin was washed with buffer similar to the homogenization buffer, except that the NaCl concentration was changed to 1 M, and 2 mM DTT and 0.1% Digitonin were supplemented. The complex was eluted by 75 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 mM GSH, 0.1% Digitonin, 2 mM DTT, 5 mM EDTA, and pro-tease inhibitors. The eluted protein was concentrated before size exclusion chromatography (SEC, Superose 6 10/300 GL, GE Healthcare) in the buffer containing 25 mM Tris, pH 7.5, 300 mM NaCl, 0.1% Digitonin, 2 mM DTT, 5 mM EDTA, and protease inhibitors.

Phage Display and Biopanning Camel library construction were reported previously. Peripheral blood mononuclear cells (PBMCs) were isolated from a total of 300ml blood sample, taken via Leucosep® tubes (Greiner Bio-One, Frickenhausen, Germany). Total RNA was extracted and VHH genes were cloned by nested PCR as described previously. The final PCR products (~ 400bp) were cloned into the phagemid vector pCANTAB5E (GE Healthcare Life Science, Pittsburgh, USA) and transformed into freshly prepared electro-competent E. coli TG1 cells. Cells were plated on LB agar plates supplemented with ampicillin and glucose. After culturing overnight at 37℃, colonies were scraped from the plates and stored at -80℃ in LB supplemented with 20% glycerol. Biopanning was performed as described previously. The 96-well Maxisorp plate was coated with 100 μg/ml RyR2 overnight at 4°C. Both the plate and 1011– 1012 pfu of phage were blocked with 1% skimmed milk for 1 hour at room temperature. Then pre-blocked phage supernatant was added to each well to allow binding. After 1 hour of incubation at room temperature, the unbound and nonspecifically bound phages were removed using 5 washes. The specifically bound phage was eluted with 100 μl pH 2.0 elution buffer for 10 minutes at room temperature. The eluate was neutralized with 30 μl of 1 M Tris-HCl buffer (pH 8.5) and was used to infect freshly prepared E. coli TG1 cells. After four rounds of panning, 300 randomly picked clones were analyzed for RyR2 binding by phage ELISA.

ELISA The phage ELISA was performed as previously described Briefly, Nunc MaxiSorp 96- well flat-bottomed plates were coated with 50 μl of 5 μg/ml RyR2 overnight at 4°C. Both the plate and phage were blocked with 1% skimmed milk for 1 hour at room temperature. Pre-blocked phage supernatant was then added to the plate. Binding was detected with a horseradish peroxidase (HRP)-conjugated mouse anti-M13 antibody (GE Healthcare). Expression and purification of anti-RyR2 Nanobody fragments Nanobody-encoding gene segments were re-cloned in vector pUR5850. Expression of recombinant Nanobody fragments in the periplasm of E. coli and purification by means of immobilized metal ion affinity chromatography (IMAC) were performed as has been described for previously. RyR2 Phosphorylation RyR2 was immunoprecipitated by incubating 500 μg of rat ventricular homogenate with anti-RyR antibody in 0.5 ml of a modified radioimmunoprecipitation assay buffer [RIPA; 50 mM Tris HCl buffer (pH 7.4)/0.9% NaCl/5.0 mM NaF/1.0 mM Na3VO4/0.5% Triton X-100/protease inhibitors] as described. After incubation with protein A-Sepharose beads for 1 h, samples were transferred into 10 μl of 1.5× phosphorylation buffer (8 mM MgCl2/10 mM EGTA/50 mM Tris/Pipes, pH 6.8) containing either PKA catalytic subunit (Sigma, St.Louis, MO), or inhibitor PKI5–24 (500 nM, Calbiochem, San Diego) with or without different nanobody fragments (10 ug/ml). Samples were pretreated with alkaline phosphatase (AP) to maximally dephosphorylate PKA sites before PKA phosphorylation. The stoichiometry of RyR2 PKA phosphorylation was determined by using [32P]-ATP standards. The [32P]/RyR2 ratio was calculated by dividing [32P]-phosphorylation of RyR2 by the amount of high-affinity [3H]-ryanodine binding, as described.

AAV9 vector production The construct of the intracellular antibody fragment, AR185 or AR117, was engineered to express an upstream EGFP reporter separated from the nanobody sequence by a T2A sequence. The sequence encoding the whole fragments construct was subcloned into the pAAV vector resulting in AAV.AR185 and AAV.AR177. For the production of AAV9 pseudotyped vectors, the plasmids were used for cotransfection in 293T cells together with pAAV-RC9, encoding the AAV-9 cap sequence, and rAAV2-retro helper vector, containing the AAV-2 rep gene as well as adenoviral helper sequences. AAV vectors were produced, purified, and titrated using standard procedures.

Experimental rat HF model All animal procedures and experiments were performed in accordance with the guidelines of Animal Care and Use Committee of the Second Military Medical University. The animal experiments were performed according to the NIH guidelines (Guide for the care and use of laboratory animals). Ten- to twelve-week-old Sprague-Dawley male rats were randomized to receive either myocardial infarction (MI) or a sham procedure. Briefly, rats were deeply anesthetized before the operation. Following intubation and placement on a respirator, a left lateral thoracotomy was performed in the 3rd and 4th intercostal space, and a ligature was placed around the left anterior descending coronary artery 2 mm below its origin. Ischemia was verified by visual inspection, the chest was closed, and the rat was extubated and returned to its cage. Sham-operated animals underwent the same procedure without ligation of the left anterior descending coronary artery(28). The rats that received MI were further randomly divided into three experimental groups as follows: heart failure group, AAV presenilin 1 treated (AAV-PSEN1) group and AAV presenilin 1 treated control group (AAV-Empty). The vector was intravenously injected into the tail vein of the rat as a 150 µL bolus using a sterile syringe and 29-gauge needle.

Ultrasound cardiographs Under the anesthesia with isoflurane, the cardiac function of the rat was examined with a Visual Sonics Ultrasound system. The chamber volume at the end of systole and diastole, fractional shortening, and ejection fraction of the left ventricle were analyzed. Transmission electron microscopy Eight weeks after preparation of the animal model, the ventricle myocardial tissues of rats were fixed with paraformaldehyde and osmium tetroxide, dehydrated through a graded ethanol series and then embedded in Epon. Thin sections were cut using a LEICA EM UC6 (Leica, Austria), stained with uranyl acetate and lead citrate. An H-7650 transmission electronic microscope (TEM, Hitachi, Japan) was used for ultrastructural analyses.

Single ventricular myocytes isolation, laser scanning confocal microscope analysis and cardiac myocyte contractility The enzymatic technique was used to isolate the ventricular myocytes. In brief, rats were anesthetized with chloral hydrate (350 mg/kg, intraperitoneal) and the aorta was rapidly cannulated after the hearts were excised and perfused with a rate of 8 ml/min in a Langendorff apparatus. Hearts were firstly perfused with Tyrode’s solution (in mM: 135 NaCl, 5.4 KCl, 0.33 NaH2PO4, 10 glucose and 5 HEPES, with pH adjusted to 7.30 with NaOH). Approximately eight minutes later, the Tyrode’s solution was replaced by a modified Tyrode’s solution in which 0.64 mg/ml collagenase typeⅡ (335 U/mg), 0.2% BSA and 57.6 µM Ca2+ were added. The heart was enzymatically digested continuously until the cardiac apex became soft. The ventricular tissue was then cut into pieces in Kraft-Bruhe solution (in mM: 50 L-glutamic acid, 30 KCl, 80 KOH, 30 NaH2PO4, 20 taurine, 10 HEPES, 10 glucose, 3 MgSO4 and 0.47 EGTA, with pH adjusted to 7.35 with KOH) and beaten gently to obtain single ventricular cardiomyocytes. Both of the Tyrode’s and Kraft-Bruhe solution used in the experiment were pre-warmed to 37°C and bubbled with 100% O2, and all the experimental processes were conducted at room temperature (22-24°C). Intra-cellular and intra-SR Ca2+, as well as Ca2+ sparks imaging were performed using a Leica TCS SP2 confocal microscope (Leica, Germany) in xy and xyt mode. To measure the intra-cellular calcium transients induced by caffeine, isolated cardiomyocytes were loaded with 10 µM Fluo-4AM (purchased from East-Chemical Technology Co., Ltd.) for 30 min at 37°C, and some isolated cardiomyocytes were loaded with 10 µM Fluo-5N (purchased from East-Chemical Technology Co., Ltd.) for 3 h at 37°C. Both Fluo-4AM and Fluo-5N were excited by the 488-nm line of an argon-ion laser, and the fluorescence was acquired at wavelengths of 500–560 nm. For quantitative studies, the temporal dynamics in fluorescence were expressed as ΔF/F0 = (F–F0)/F0, where F represents fluorescence at time t and F0 stands for baseline fluorescence. Cells were placed in a chamber mounted on the stage of an inverted microscope (Zeiss X-40; Go'ttingen, Germany) at room temperature (22–24°C) and field stimulated (10 V) at a frequency of 1 Hz for 10 ms in duration using a pair of platinum wires. The contractile kinetics of the myocytes were measured using a high-speed video edge-detection system (IonOptix, Milton, MA)

Immunoblotting The cells were rinsed twice with PBS to eliminate dead cells and then homogenized in cold lysis buffer containing (in mM) Tris–HCl (75), NaCl (225), EDTA (1.5), Nonidet P-40 (4.5%, w/v), sodium vanadate (5), sodium fluoride (40), sodium pyrophosphate (10), N-ethylmaleimide (10) and a protease inhibitor cocktail (Complete, Roche Diagnostics, Mannheim, Germany); the final pH was 7.4. The lysate was centrifuged at 4°C (10 min at 10,000 ×g), and the supernatant was collected. Proteins were separated by SDS-PAGE (3.5–8% gradient gels) and immunoblotted with indicated antibodies. All the assays were performed independently in triplicate.

Tissue analysis Tissue sections were processed into slides either after being formalin-fixed and paraffin-embedded (FFPE) or embedded in non-fixed optimal cutting temperature compound (OCT, Tissue-Tek); tissue samples were also lysed in the CelLytic MT Lysis reagent (Sigma). Immunohistochemistry slides were scanned using an Imagescope (Scan-Scope AT, Aperio). The H-score was defined as the sum of the values obtained by multiplying the staining intensity and proportion and ranged from 0 to 300. Ratios of pRyR2 to total RyR2 were determined by western blot and ELISA.

Statistical analysis Statistical analysis was performed using Student’s unpaired t-test to identify significant differences unless otherwise indicated. Multiple comparisons were conducted using a two-way ANOVA followed by the Bonferroni post hoc test; a P-value less than 0.05 was considered a significant difference.

results

1. Generation of Anti-RyR2 nanobodies that specifically inhibits the Phosphorylation of RyR2 S2808 We first used GST-fused FKBP12 to purify RyR2 from rat heart using published strategies. To construct the camel VHH library, blood samples of 30 non-immunized, four year-old male Bactrian camel were collected. A phage display VHH library, consisting of approximately 3 × 108 individual colonies, was constructed from B lymphocyte cDNA encoding VHHs. Over 98% of these colonies had an insert corresponding to the size of a VHH gene. Fifty randomly selected clones were sequenced, and each clone was shown to contain a distinct VHH sequence, which confirmed the heterogeneity of the individual clones from the library. 2 To select nanobodies that specifically bound to RyR2, bio-panning was performed with immobilized RyR2 protein. A strong enrichment of phage particles carrying RyR2-specific VHHs was observed after the third round of panning (Fig. 1A). Phage pools after two rounds of panning exhibited enhanced binding to RyR2, whereas no binding to BSA was found with pooled phage from any of the four rounds of panning (Fig. 1B). Total of 300 individual colonies were randomly chosen, and VHH fragments were expressed and screened for binding to RyR2 in an ELISA. From these clones, 276 antibody fragments were identified with RyR2-specific binding.. To obtain antibodies that functionally inhibit of RyR2 phosphorylation, each of the antibody fragments was tested for its effect in an ELSA based RyR2 Phosphorylation assay. 4 antibody fragments were potent inhibitors of RyR2 phosphorylation. Subsequent sequence analysis of the complementary determining regions (CDRs) revealed that only one unique clone was present in this set of antibody fragments, termed as VHH-AR185. To examine the basis of the potent inhibition of RyR2 phosphorylation by VHH-AR185, we measured the binding affinity of VHH-AR185 to RyR2 by surface plasmon resonance. For these experiments, VHHs were purified following expression from the E. coli cytosol. As shown in Fig. 1D, the affinity (KD) of VHH-AR185 to RyR2 was estimated to be 1.93 nM. The extremely slow dissociation rate of VHH-AR185 from RyR2 found through the affinity studies likely explains the inhibition of RyR2 phosphorylation by this VHH.

Figure 1. Isolation of RyR2-specific nanobody by phage display. (A) Phage-displayed nanobody fragments were selected against RyR2 after four rounds of panning. A gradual increase in phage titers was observed during each round of panning. (B) Polyclonal phage ELISA from the output phage of each round of panning. BSA was used as an irrelevant antigen. (C) Heat map generated form ELISA data of purified RyR2 channels which were PKA phosphorylated in the presence of the PKA. (D) Kinetic analysis of AR185 binding to RyR2 was performed by SPR.

Source

This nanobody was acquired through phage display and biopanning.

References