Patent Application
20170044516
The present application is based on, and claims priority from, Taiwan Application Serial Number 104126050, filed on Aug. 11, 2015, and China Application Serial Number 201510815421.4, filed on Nov. 23, 2015, the disclosure of which are hereby incorporated by reference herein in its entirety.
A sequence listing submitted as a text file via EFS-Web is incorporated herein by reference. The text file containing the sequence listing is named “0965-A24497-US_Seq_Listing.txt”; its date of creation was Dec. 29, 2015; and its size is 11,648 bytes.
The technical field relates to a biochemistry reactive material and device for eliminating electronegative low-density lipoprotein (LDL) and a method for treating blood or plasma ex vivo to eliminate electronegative low-density lipoprotein therein
Low-density lipoprotein (LDL) is a kind of lipoprotein that is a product of lipoprotein lipase action. Lipoproteins play a role in lipid transportation. It has long been known that the level of cholesterol carried by low-density lipoprotein is associated with the occurrence and presence of cardiovascular diseases.
At present, in the medical field, plasma LDL cholesterol (LDL-C) is still used as an indicator for estimating cardiovascular diseases. However, the low-density lipoprotein level in the plasma of patients with acute myocardial infarction has no tendency to increase.
Due to external factors, such as excess oxidation pressure, etc., low-density lipoprotein will be post-translation modified, and presents higher electronegativity to become electronegative LDL or L5.
Electronegative LDL (L5) electronegative low-density lipoprotein is a major factor for causing cardiovascular disease. L5 is almost undetectable in a normal human body. In addition, it has been in vitro and in vivo verified that L5 will damages vascular endothelial cells and activate monocytes and platelets, and result in systemic inflammation, atherosclerosis and myocardial infraction.
Therefore, a novel material, device and/or method for eliminating an electronegative low-density lipoprotein is/are needed.
The present disclosure provides a biochemistry reactive material, comprising a substrate and an enzyme composition immobilized on the substrate. The enzyme composition is selected from a group consisting of a first enzyme, a second enzyme, and a combination thereof. The first enzyme eliminates a glycan residue of an electronegative low-density lipoprotein (LDL). The second enzyme eliminates ceramide carried by an electronegative LDL. The biochemistry reactive material is capable of eliminating electronegative low-density lipoprotein.
The present disclosure also provides a biochemistry reactive device, comprising: the biochemistry reactive material as mentioned above, and a container for containing the biochemistry reactive material. The container has at least one inlet and at least one outlet, wherein a liquid sample enters into the biochemistry reactive device from the at least one inlet, and flows through the biochemistry reactive material to react with the biochemistry reactive material, and then flows out through the at least one outlet.
The present disclosure further provides a method for ex vivo treating blood or plasma, comprising (a) ex vivo contacting a blood or plasma with an enzyme composition to react the enzyme composition with the blood or plasma, wherein the enzyme composition is capable of eliminating low-density lipoprotein. The enzyme composition is selected from a group consisting of a first enzyme, a second enzyme, and a combination thereof. The first enzyme is for eliminating a glycan residue of an electronegative LDL. The second enzyme is for eliminating ceramide carried by an electronegative low-density lipoprotein. The method also comprises (b) terminating the contact between the blood or plasma and the enzyme composition to terminate the reaction of the enzyme composition with the blood or plasma.
A detailed description is given in the following embodiments with reference to the accompanying drawings.
The present invention can be more fully understood by reading the subsequent detailed description and examples with references made to the accompanying drawings, wherein:
In the following detailed description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the disclosed embodiments. It will be apparent, however, that one or more embodiments may be practiced without these specific details. In other instances, well-known structures and devices are shown schematically in order to simplify the drawing.
In one embodiment of the present disclosure, the present disclosure provides a biochemistry reactive material which is capable of eliminating electronegative low-density lipoprotein (electronegative LDL). Examples of the electronegative low-density lipoprotein mentioned above may comprise, but are not limited to, low-density lipoproteins L1, L2, L3, L4, L5, etc. In one embodiment, the electronegative low-density lipoprotein mentioned above may be low-density lipoprotein L5. Moreover, L5 is the most electronegative and most harmful low-density lipoprotein.
The biochemistry reactive material of the present disclosure may comprise, but is not limited to, a substrate and an enzyme composition, wherein the enzyme composition is immobilized on the substrate.
Examples of suitable substrate may comprise silica gel, cellulose, diethylaminoethyl cellulose (DEAE cellulose), chitosan, polystyrene, polysulfone, polyethersulfone, acrylate resin, polysaccharide, etc., but they are not limited thereto. The substrate may have a particle structure or a hollow-tube structure, etc. In one embodiment, the substrate may be a cellulose bead. In another embodiment, the substrate may be a chitosan bead. Moreover, the substrate may be a cellulose hollow fiber, a polysulfone hollow fiber, epoxy acrylic resin or a polyethersulfone hollow fiber, etc.
The preceding enzyme composition may comprise a first enzyme for eliminating a glycan residue of an electronegative LDL, a second enzyme for eliminating ceramide carried by an electronegative LDL, or a combination thereof, but it is not limited thereto. Source organisms of the first enzyme and the second enzyme mentioned above have no particular limitation. In one embodiment, the first enzyme and the second enzyme are bioengineered enzymes from human genome and also possibly from animal genome.
The preceding first enzyme may be sialidase or glycosidase.
The sialidase may comprise neuraminidase 1 (NEU1), neuraminidase 2 (NEU2), neuraminidase 3 (NEU3), neuraminidase 4 (NEU4) and O-sialidase bioengineered from human genome, one of the foregoing enzymes obtained through gene transformation, expression and purification, sialidase from a virus or bacterium (alias, acetylneuraminyl hydrolase), etc., but it is not limited thereto.
Examples of the glycosidase may comprise alpha- and beta-glucosidase bioengineered from human or animal genome, maltase-glucoamylase and sucrase-isomaltase, one of the foregoing enzymes obtained through gene transformation, expression and purification, N-glycosidase F (PNGase F) and glucosidase from a virus, a bacterium or other organism, etc., but they are not limited thereto.
Furthermore, the second enzyme may be ceramidase.
The ceramidase may comprise N-acylsphingosine amidohydrolase 1 (ASAH1), N-acylsphingosine amidohydrolase 2 (ASAH2), N-acylsphingosine amidohydrolase 2B (ASAH2B), N-acylsphingosine amidohydrolase 2C (ASAH2C), N-acylethanolamine acid amidase, alkaline ceramidase 1, alkaline ceramidase 2, alkaline ceramidase 3, but it is not limited thereto.
In one embodiment, the enzyme composition in the biochemistry reactive material of the present disclosure is the first enzyme. In this embodiment, the first enzyme mentioned above may be sialidase, but it is not limited thereto.
In another embodiment, the enzyme composition in the biochemistry reactive material of the present disclosure is the second enzyme. In this embodiment, the second enzyme mentioned above may be N-acylsphingosine amidohydrolase 2, but it is not limited thereto.
In another embodiment, the enzyme composition in the biochemistry reactive material of the present disclosure is a combination of the first enzyme and the second enzyme. In this embodiment, the first enzyme mentioned above may be sialidase, but it is not limited thereto, and the second enzyme mentioned above may be N-acylsphingosine amidohydrolase 2, but it is not limited thereto.
In another embodiment of the present disclosure, the present disclosure provides a biochemistry reactive device, and the device can be used for eliminating electronegative low-density lipoprotein in a liquid sample.
Examples of the foregoing liquid sample may comprise an aqueous solution, a buffer, blood, plasma, etc., but they are not limited thereto.
Examples of the foregoing electronegative low-density lipoprotein may comprise electronegative low-density lipoprotein L1, L2, L3, L4 and/or L5, etc., but they are not limited thereto. In one embodiment, the electronegative low-density lipoprotein mentioned above may be electronegative low-density lipoprotein L5.
A cross-sectional view of a structure of the biochemistry reactive device of the present disclosure is shown in
Refer to
The biochemistry reactive material 101 may comprise, but is not limited to a substrate and an enzyme composition, wherein the enzyme composition is immobilized on the substrate.
The substrate mentioned above may comprise, but is not limited to, silica gel, cellulose, diethylaminoethyl cellulose, chitosan, polystyrene, polysulfone, polyethersulfone, acrylate resin, polysaccharide, etc. The substrate may have a particle structure or a hollow-tube structure, etc., but it is not limited thereto.
The enzyme composition may comprise, but is not limited to, a first enzyme for eliminating a glycan residue of an electronegative LDL, a second enzyme for eliminating ceramide carried by an electronegative LDL or a combination thereof. Source organisms of the first enzyme and the second enzyme mentioned above have no particular limitation. In one embodiment, the first enzyme and the second enzyme are human.
The preceding first enzyme may be sialidase or glycosidase.
The sialidase may comprise, but is not limited to, neuraminidase 1 (NEU1), neuraminidase 2 (NEU2), neuraminidase 3 (NEU3), neuraminidase 4 (NEU4) and 0-sialidase bioengineered from human genome, one of the foregoing enzymes obtained through gene transformation, expression and purification, sialidase from a virus, a bacterium or other organism, etc.
The glycosidase may comprise, but is not limited to, alpha- and beta-glucosidase bioengineered from human or animal genome, maltase-glucoamylase and sucrase-isomahase, one of the foregoing enzymes obtained through gene transformation, expression and purification, N-glycosidase F (PNGase F) and glucosidase from a virus, a bacterium or other organism, etc.
In addition, the second enzyme may be ceramidase. The ceramidase may comprise, but is not limited to, N-acylsphingosine amidohydrolase 1 (ASAH1), N-acylsphingosine amidohydrolase 2 (ASAH2), N-acylsphingosine amidohydrolase 2B (ASAH2B), N-acylsphingosine amidohydrolase 2C (ASAH2C), N-acylethanolamine acid amidase, alkaline ceramidase 1, alkaline ceramidase 2, alkaline ceramidase 3.
In one embodiment, the enzyme composition in the biochemistry reactive material 101 mentioned above is the first enzyme. In this embodiment, the first enzyme mentioned above may be sialidase, but it is not limited thereto.
In another embodiment, the enzyme composition in the biochemistry reactive material 101 mentioned above is the second enzyme. In this embodiment, the second enzyme mentioned above may be N-acylsphingosine amidohydrolase 2, but it is not limited thereto.
In another embodiment, the enzyme composition in the biochemistry reactive material 101 mentioned above is a combination of the first enzyme and the second enzyme. In this embodiment, the first enzyme mentioned above may be sialidase, but it is not limited thereto, and the second enzyme mentioned above may be N-acylsphingosine amidohydrolase 2, but it is not limited thereto.
Furthermore, a material of the container 103 of the biochemistry reactive device 100 of the present disclosure may comprise glass, acrylic, polypropylene, polyethylene, stainless steel, titanium alloy, etc., but it is not limited thereto. In one embodiment, a material of the container 103 of the biochemistry reactive device 100 of the present disclosure may be polypropylene. In addition, a shape of the container 103 has no particular limitation, and in one embodiment, the container 103 is a hollow column.
In one embodiment, as shown in
When the substrate of the biochemistry reactive material 101 is a hollow-tube structure, polyurethane (PU) can be used to package the device without using the filtering material 109.
In one embodiment, the container 103 may be a hollow column, and two ends of the hollow column of the container 103 have a first inlet 1051 of the inlet mentioned above and a first outlet 1071 of the outlet mentioned above, respectively. In this embodiment, the substrate of the biochemistry reactive material 101 may have a particle structure or a hollow-tube structure.
In another embodiment, as shown in
In this embodiment, the substrate of the biochemistry reactive material 101 may have a particle structure or a hollow-tube structure. In one specific embodiment, the substrate of the biochemistry reactive material 101 has a hollow-tube structure, and in this specific embodiment, the substrate of the biochemistry reactive material 101 may be cellulose hollow fiber, but it is not limited thereto.
In another embodiment of the present disclosure, the present disclosure provides a method for ex vivo treating blood or plasma. By the method for ex vivo treating blood or plasma, an electronegative low-density lipoprotein in blood or plasma can be eliminated. The foregoing electronegative low-density lipoprotein may comprise, but is not limited to, electronegative low-density lipoprotein L1, L2, L3, L4 and/or L5, etc. In one embodiment, the electronegative low-density lipoprotein mentioned above is electronegative low-density lipoprotein L5.
The method for ex vivo treating blood or plasma may comprise the following steps, but it is not limited thereto.
First, a blood or plasma ex vivo contacts with an enzyme composition to react the enzyme composition with the blood or plasma, wherein the enzyme composition is capable of eliminating electronegative low-density lipoprotein.
The preceding enzyme composition may comprise a first enzyme for eliminating a glycan residue of an electronegative LDL, a second enzyme for eliminating ceramide carried by an electronegative LDL or a combination thereof, but it is not limited thereto. Source organisms of the first enzyme and the second enzyme mentioned above have no particular limitation. In one embodiment, the first enzyme and the second enzyme are human.
The preceding first enzyme may be sialidase or glycosidase.
The sialidase may comprise neuraminidase 1 (NEU1), neuraminidase 2 (NEU2), neuraminidase 3 (NEU3), neuraminidase 4 (NEU4) and O-sialidase from a human, or one of the foregoing enzymes obtained through gene transformation, expression and purification, sialidase from a virus, a bacterium or other organisms, etc., but it is not limited thereto.
Examples of the glycosidase may comprise alpha- and beta-glucosidase bioengineered from human animal genome, maltase-glucoamylase and sucrase-isomaltase, one of the foregoing enzymes obtained through gene transformation, expression and purification, N-glycosidase F (PNGase F) and glucosidase from a virus, a bacterium or other organism, etc., but they are not limited thereto.
Furthermore, the second enzyme mentioned above may be ceramidase.
The ceramidase may comprise N-acylsphingosine amidohydrolase 1 (ASAH1), N-acylsphingosine amidohydrolase 2 (ASAH2), N-acylsphingosine amidohydrolase 2B (ASAH2B), N-acylsphingosine amidohydrolase 2C (ASAH2C), N-acylethanolamine acid amidase, alkaline ceramidase 1, alkaline ceramidase 2, alkaline ceramidase 3, but it is not limited thereto.
In one embodiment, the enzyme composition used in the method for ex vivo treating blood or plasma of the present disclosure is the first enzyme. In this embodiment, the first enzyme mentioned above may be sialidase, but it is not limited thereto.
In another embodiment, the enzyme composition used in the method for ex vivo treating blood or plasma of the present disclosure is the second enzyme. In this embodiment, the second enzyme mentioned above may be N-acylsphingosine amidohydrolase 2, but it is not limited thereto.
In another embodiment, the enzyme composition used in the method for ex vivo treating blood or plasma of the present disclosure is a combination of the first enzyme and the second enzyme. In this embodiment, the first enzyme mentioned above may be sialidase, but it is not limited thereto, and the second enzyme mentioned above may be N-acylsphingosine amidohydrolase 2, but it is not limited thereto.
Furthermore, in one embodiment, the enzyme composition used in the method for ex vivo treating blood or plasma of the present disclosure can be immobilized on a substrate. Examples of the substrate may comprise silica gel, cellulose, diethylaminoethyl cellulose, chitosan, polystyrene, polysulfone, polyethersulfone, resin, polysaccharide, but they are not limited thereto. Moreover, the substrate may have a particle structure or a hollow-tube structure.
In the method for ex vivo treating blood or plasma of the present disclosure, time for ex vivo contacting the blood or plasma with the enzyme composition may be about 0.25-8 hours. In one embodiment, time for ex vivo contacting the blood or plasma with the enzyme composition may be about 2 hours.
Furthermore, in the method for ex vivo treating blood or plasma of the present disclosure, temperature for ex vivo contacting the blood or plasma with the enzyme composition may be about 4-40° C. In one embodiment, temperature for ex vivo contacting the blood or plasma with the enzyme composition may be about 37° C.
In addition, in the method for ex vivo treating blood or plasma of the present disclosure, the blood or plasma may ex vivo contact with the enzyme composition at about pH 5-10. In one embodiment, the blood or plasma may ex vivo contact with the enzyme composition at about pH 7.4.
Afterward, contact between the blood or plasma and the enzyme composition is terminated to terminate the reaction of the enzyme composition with the blood or plasma.
A manner for terminating the contact between the blood or plasma and the enzyme composition has no particular limitation, for example, for terminating the contact between the blood or plasma and the enzyme composition, the blood or plasma can be separated from the enzyme composition, or the enzyme composition can be deactivated, etc.
1. Obtainment of Electronegative Low-Density Lipoprotein (Electronegative LDL)
(1) Purifications for Electronegative Low-Density Lipoprotein
Blood samples to be used for LDL isolation were obtained from subjects. After the initial screening, blood samples were removed from the subjects with precaution against coagulation and ex vivo oxidation. The plasma was treated with Complete Protease Inhibitor Cocktail (Roche; Cat. No. 05056489001; 1 tablet/100 mL) to prevent protein degradation.
Lipoprotein Preparation from a Human
The plasma was overlaid with 2 mL Milli-Q water and spun at 20,000 rpm for 2 hours. The upper white fraction and chylomicrons were removed, and the remnant layer which contains VLDL, IDL, LDL and HDL was saved for a series of isolation steps.
To progressively separate VLDL (d=0.93-1.006), IDL (d=1.006-1.019), LDL (1.019-1.063 g/dL) and HDL (1.063-1.210 g/dL) from one another, the remnant sample was sequentially adjusted to d=1.006, d=1.019, d=1.063, d=1.210, respectively, by adding potassium bromide, and then the remnant samples sequentially adjusted to d=1.006, d=1.019 and d=1.063 were centrifuged at 45,000 rpm for 24 hours at 4° C., and the remnant sample sequentially adjusted to d=1.210 was centrifuged at 45,000 rpm for 48 hours at 4° C. After centrifugation at each isolation step, IDL was discarded while VLDL, LDL and HDL were collected. Isolated VLDL, LDL and HDL samples were treated with 5 mM EDTA and nitrogen to avoid ex vivo oxidation. After that, VLDL, LDL and HDL samples were dialyzed against buffer A (20 M, pH 8.0, 0.5 M EDTA) for 24 hours (×3 times) to remove excessive potassium bromide, and were filtrated through 0.22-μm filter (Sartorius; Minisart®) to sterilize the samples.
(2) LDL Subfractions
Approximately 30 mg of LDL material was injected onto a UnoQ12 anion-exchange column (BioRad) by using the ÄKTA fast-protein liquid chromatography (FPLC) pump (GE Healthcare Life Sciences, Pittsburgh, Pa.). LDL was eluted according to electronegativity by the use of a multistep gradient of buffer B (1 mol/L NaCl in buffer A) at a flow rate of 2 mL/minute. In short, samples were equilibrated with buffer A for 10 minutes, followed by being linearly increased to 15% buffer B in 10 minutes (fraction 1), linearly increased to 20% buffer B in 30 minutes (fraction 2, 3), kept at 20% buffer B for 10 minutes (fraction 4) and linearly increased to 100% buffer B in 20 minutes (fraction 5). Lastly, the effluents were monitored at 280 nm.
(3) Purification of Fractionated LDL
Based on the gradient profile, each of the LDL fractions were pooled. The volume of each subfraction was constant. Dilution of LDL during chromatography depended on the injection volume. The respective fractions were concentrated with Centriprep® filters (YM-30; EMD Millipore Corp., Billerica, Mass.), dialyzed against buffer A (20 M, pH8.0, 0.5 M EDTA) for 24 hours (3 days) and sterilized by passing through 0.22-μm filters (Sartorius; Minisart®). The isolated fractions were quantified at their protein concentrations by the Lowry method and then stored at 4° C.
2. Screening of NEU2 or NEU4
(1) Transformation (Gene Cloning for pCMV6 Vector with NEU2 and NEU4 Genes)
NEU2 (neuraminidase 2) and NEU4 (neuraminidase 4) were purchased from Origene, RC219858 and RC203948. Genes were amplified by ECOS™ 101 DH5α Competent Cells (Yeastern, FYE608) according to the manufacturer's directions.
In short, 1 vial of competent cells with 5 μL plasmid was vortexed for 1 second and then incubated on ice for 5 minutes. After 45 second heat-shock at 42° C., the mixture was plate on LB agar with Kanamycin.
Colonies were checked with PCR by VP1.5 and XL39 primers. Procedures of the PCR comprises: 95° C. for 1 minute for pre-PCR denaturation; 2 cycles of 95° C. for 10 seconds, 62° C. for 20 seconds, 72° C. for 4 minutes; 2 cycles of 95° C. for 10 seconds, 60° C. for 20 seconds, 72° C. for 4 minutes; 2 cycles of 95° C. for 10 seconds, 58° C. for 20 seconds, 72° C. for 4 minutes; 15 cycles of 95° C. for 10 seconds, 56° C. for 20 seconds, 72° C. for 4 minutes; 72° C. for 10 minutes for post-PCR incubation and holding on 4° C.
(2) Plasmid Extraction
After confirming the insertion of transformed colonies, transformed cells were plate-out into 5 ml LB broth with 25 mg/ml kanamycin, and then incubated at 37° C. overnight.
Plasmid DNA was extracted according to the protocol of Plasmid Miniprep Plus Purification Kit (GeneMark, DP01P). In short, the bacteria were centrifuged for 1 minute at 14,000×g, and the media was removed. The pellet was re-suspended in 200 μL Solution I by pipetting, then 200 μL Solution II was added therein and mixed by inverting the tube. 200 μL Solution III was added to the tube and mixed by inverting the tube 5 times. The lysate was centrifuged at top speed for 5 minutes and a compact white pellet formed along the side of the tube. The spin column was inserted into a collection tube, and the clear lysate was moved to spin column and spun at top speed for 1 minute. The flow-through was discarded, and 500 μL Endotoxin Removal Wash Solution was loaded to the spin column and kept for 2 minutes to equilibrate the membrane, then spun at top speed for 1 minute. The filtrate was discarded, and 700 μL Washing Solution was added to the spin column and spun at top speed for 1 minute, and then this step was repeated. The filtrate was discarded and the spin column was centrifuged for 5 minutes at top speed to remove residual traces of ethanol. The spin column was transferred into a new tube and 35 μL H2O was added to the spin column and kept for 1-2 minutes and the tube was centrifuged at top speed for 2 minutes to elute the DNA. The DNA quantified by microplate spectrophotometer (Epoch, BioTek).
(3) Transfection on HEK Cells and Protein Purification
One day before transfection, 1.25*105 HEK293T cells were placed in 500 μL DMEM medium in 24-well plate. For each well of cells to be transfected, 1 μg of DNA was diluted in 100 μL serum-free medium, and 1.5 μL of Lipofectamine 2000 Transfection Reagent (Invitrogen) was add thereto and mixed gently and incubated for 30 minutes at room temperature. After incubation, the complex was added to each well containing cells and mixed gently. The cells were incubated at 37° C. in a CO2 incubator for 20 hours. The transfected cells were lysed by RIPA which containing protease inhibitor to prepare to purify the proteins.
In short, 80 μL ANTI-FLAG M2 Magnetic Beads (Sigma-Aldrich) were equilibrated for one-well cell lysate purification. After protein-resin binding at 4° C. overnight, the bound FLAG fusion protein was eluted by competitive elution with 150 μg/ml 3× FLAG peptide for 2 times, the eluate was collected, and the protein checked by western blot.
3. Efficacy Test for NEU2 or NEU4
(1) Protein Quantification
Pierce BCA Protein Assay Kit (Thermo) was used for protein quantification according to the manufacturer's directions.
In short, 25 μL serial diluted BSA standard and 5 μL sample in 20 μL sample diluent were pipetted into a 96-well microplate. To prepare BCA working reagent, 50 parts of BCA Reagent A was mixed with 1 part of BCA Reagent B and placed on ice until use. 200 μL of the BCA working reagent was added to each well and mixed thoroughly, and the plate was covered and incubated at 37° C. for 30 minutes. The absorbance at 562 nm was measured by spectrophotometer (Epoch, BioTek).
(2) Apoptosis Measurements
Endothelial cells were used after 3 or 4 passages and maintained in DMEM (Invitrogen™, Thermo Fisher Scientific) containing 10% FBS. During treatment, FBS was reduced to 5% in DMEM. 1×104 cells were seeded in 96-well plate for 24 hours for subconfluent cultures, and the cultured cells were exposed to PBS (lipoprotein-free, negative control) or graded (25, 50, and 100 μg/mL) LDL subfractions, unfractionated normolipidemic LDL, and LDL/L1/L5 incubated with sialidase for 24 hours. Apoptosis was assessed with visualization by a Zeiss Axiovert 200 fluorescence microscope and filters to capture digital images based on Hoechst 33342, propidium iodide (red), and calcein AM (green) staining of nuclear, apoptotic DNA membrane integrity and cytoplasm respectively according to the protocol of the manufacturer (Invitrogen™, Thermo Fisher Scientific).
(3) LC/MSE Analysis for Protein Composition
LDL subfractions were quantified the protein contents by use of quantitative proteomics techniques utilizing serially coupled liquid chromatography data-independent parallel-fragmentation mass spectrometry (LC/MSE). Such analysis has been shown to be highly quantitative with respect to both relative and/or absolute (when incorporating spiked internal peptide standards in the data collection/analysis procedures) protein abundance in complex protein mixtures. Quantitative analysis was performed essentially as previously described (PMCID: PMC3816395; Pure Appl Chem. 2011; 83(9): 10.1351/PAC-CON-10-12-07. Chemical composition—oriented receptor selectivity of L5, a naturally occurring atherogenic low-density lipoprotein), except on a Waters nanoACQUITY UPLC System and Xevo® G2-XS QTof mass spectrometer (Waters Corporation, MA, USA).
In brief, total proteins isolated from each LDL subfraction were first digested with trypsin, and the resulting tryptic peptides were chromatographically separated on a Nano-Acquity separations module (Waters Corporation, MA, USA) incorporating a 50 fmol-on-column tryptic digest of yeast alcohol dehydrogenase as the internally spiked protein quantification standard. Peptide elution will be executed through a 75 im×25 cm BEH C-18 column under gradient conditions at a flow rate of 300 nL/minute over 30 minutes at 35° C. The mobile phase was composed of acetonitrile as the organic modifier and formic acid (0.1% v/v) for molecule protonation. Mass spectrometry was performed on a Xevo® G2-XS QTof instrument equipped with a nano-electrospray ionization interface and operated in the data-independent collection mode (MSE). Parallel ion fragmentation was programmed to switch between low (4 eV) and high (15-45 eV) energies in the collision cell, and data was collected from 50 to 2000 m/z utilizing glu-fibrinopeptide B as the separate data channel lock mass calibrant. Data was processed with ProteinLynx GlobalServer v2.4 (Waters). Deisotoped results were searched for protein association from the Uniprot (www.uniprot.org) human protein database.
4. Screening of ASAH2
(1) Transformation (Gene Cloning for pCMV6 Vector with ASAH2 Genes):
ASAH2 (N-acylsphingosine amidohydrolase 2) was purchased from Origene, RC203706. Genes were amplified by ECOS™ 101 DH5α Competent Cells (Yeastern, FYE608) according to the manufacturer's directions.
In short, 1 vial of competent cells with 5 μL plasmid was vortexed for 1 second and then incubated on ice for 5 minutes. After 45 second heat-shock at 42° C., the mixture was plate on LB agar with Kanamycin.
Colonies were checked with PCR by VP1.5 and XL39 primers. Procedures of the PCR comprise: 95° C. for 1 minute for pre-PCR denaturation; 2 cycles of 95° C. for 10 seconds, 62° C. for 20 seconds, 72° C. for 4 minutes; 2 cycles of 95° C. for 10 seconds, 60° C. for 20 seconds, 72° C. for 4 minutes; 2 cycles of 95° C. for 10 seconds, 58° C. for 20 seconds, 72° C. for 4 minutes; 15 cycles of 95° C. for 10 seconds, 56° C. for 20 seconds, 72° C. for 4 minutes; 72° C. for 10 minutes for post-PCR incubation and holding on 4° C.
(2) Plasmid Extraction
After confirming the insertion of transformed colonies, transformed cells were plate-out into 5 ml LB broth with 25 mg/ml kanamycin, and then incubated at 37° C. overnight.
Plasmid DNA was extracted according to the protocol of Plasmid Miniprep Plus Purification Kit (GeneMark, DP01P). In short, the bacteria were centrifuged for 1 minute at 14,000× g, and the media was removed. The pellet was re-suspended in 200 μL Solution I by pipetting, then 200 μL Solution II was added therein and mixed by inverting the tube. 200 μL Solution III was added to the tube and mixed by inverting the tube 5 times. The lysate was centrifuged at top speed for 5 minutes and a compact white pellet formed along the side of the tube. The spin column was inserted into a collection tube, and the clear lysate was removed to spin column and spun at top speed for 1 minute. The flow-through was discarded, and 500 μL Endotoxin Removal Wash Solution was loaded to the spin column and kept for 2 minutes to equilibrate the membrane, then spun at top speed for 1 minute. The filtrate was discarded, and 700 μL Washing Solution was added to the spin column and spun at top speed for 1 minute, and then this step was repeated. The filtrate was discarded and the spin column was centrifuged for 5 minutes at top speed to remove residual traces of ethanol. The spin column was transferred into a new tube and 35 μL H2O was added to the spin column and kept for 1-2 minutes and the tube was centrifuged at top speed for 2 minutes to elute the DNA. The DNA quantified by microplate spectrophotometer (Epoch, BioTek).
(3) Transfection on HEK Cells and Protein Purification
One day before transfection, 1.25*105 HEK293T cells were placed in 500 μL DMEM medium in 24-well plate. For each well of cells to be transfected, 1 μg of DNA was diluted in 100 μL serum-free medium, and 1.5 μl, of Lipofectamine 2000 Transfection Reagent (Invitrogen) was add thereto and mixed gently and incubated for 30 minutes at room temperature. After incubation, the complex was added to each well containing cells and mixed gently. The cells were incubated at 37° C. in a CO2 incubator for 20 hours. The transfected cells were lysed by RIPA which containing protease inhibitor to prepare to purify the proteins.
In short, 80 μL ANTI-FLAG M2 Magnetic Beads (Sigma-Aldrich) were equilibrated for one-well cell lysate purification.
After protein-resin binding at 4° C. overnight, the bound FLAG fusion protein was eluted by competitive elution with 150 μg/ml 3× FLAG peptide for 2 times, the eluate was collected, and the protein checked by western blot.
5. Efficacy Test for ASAH2
(1) Protein Quantification
Pierce BCA Protein Assay Kit (Thermo) was used for protein quantification according to the manufacturer's directions.
In short, 25 μL serial diluted BSA standard and 5 μL sample in 20 μL sample diluent were pipetted into a 96-well microplate. To prepare BCA working reagent, 50 parts of BCA Reagent A was mixed with 1 part of BCA Reagent B and placed on ice until use. 200 μL of the BCA working reagent was added to each well and mixed thoroughly, and the plate was covered and incubated at 37° C. for 30 minutes. The absorbance at 562 nm was measured by spectrophotometer (Epoch, BioTek).
(2) Lipid Extraction
30 μg LDL/L1/L5 were incubated with 5 μg ASAH2 in ASAH2 buffer (200 mM Tris-HCl at pH 8.4, 1.5 M NaCl, 25 mM CaCl2) at 37° C. After 2 or 24 hours incubation, samples were transferred to a glass tube. 1 mL H2O, 2.5 mL methanol and 1.25 mL CHCl3 were added to samples, and vortexed for 15 seconds. Then, additional 0.9 mL H2O and 1.25 mL CHCl3 were applied to samples, vortexed for 15 seconds, and centrifuged at 3000 rpm for 10 minutes. Bottom layer organic solvents were transferred to a 2.0 mL glass tube using a glass syringe. Each sample was flushed with nitrogen until dry pallets, and dissolved with 0.25 mL sample solution (isopropanol/acetonitrile/H2O=2:1:1).
(3) LC/MSE Analysis for Lipid Composition
Total lipids, phospholipids, neutral lipids and free fatty acid from each subfractions of LDL were quantified the lipid contents by use of liquid chromatography data-independent parallel-fragmentation mass spectrometry (LC/MSE). Quantitative analysis was performed essentially as previously described.
In brief, lipids were chromatographically separated on a ACQUITY UPLC System (Waters Corporation, MA, USA) incorporating a CSH™ 1.7 μm, 2.1 mm×10 cm C-18 column under gradient conditions at a flow rate of 400 μL/minute over 18 minutes at 55° C. The mobile phase A will be composed of 10 mM NH4HCO2 in ACN/H2O (60/40) and 0.1% formic acid (0.1% v/v), mobile phase B will be composed of 10 mM NH4HCO2 in IPA/ACN (90/10) and 0.1% formic acid (0.1% v/v) for molecule protonation. Mass spectrometry was performed on a Xevo® G2-XS QT of instrument equipped with an electrospray ionization interface and operated in the data-independent collection mode (MSE). Parallel ion fragmentation was programmed to switch between low (4 eV) and high (35-55 eV) energies in the collision cell, and data was collected from 50 to 1600 m/z utilizing leucin as the separate data channel lock mass calibrant. Data was processed with MarkerLynx (Waters).
1. Transformation
(1) NEU2
Transformation result for NEU2 is shown in
According to
(2) ASAH2
Transformation result for ASAH2 is shown in
Colony 7 was selected to be amplified, and plasmid of ASAH2 was stocked.
2. Transfection
Transfection of NEU4/ASAH2 genes was confirmed by western blot, and the result is shown in
Conditions for the gene transfection are shown in the following:
HEK293T 1.25×105 cells in 24 well
Plasmid: NEU4 and ASAH2
DNA amount: 1 μg
Transfected by Lipofectamine
SDS-PAGE: using 5 μl sample
Primary antibody: anti-DDK (1:2000)
3. Protein Purification
(1) NEU2 Purification
The result for NEU2 purification is shown in
(2) ASAH2 Purification
The result for ASAH2 purification is shown in
Method 1
0.4454 g heat-activated silica gel was placed in 7 mL CHCl3, and APTS was added therein by a weight of 1/5 weight of heat-activated silica gel to form a mixture. After stirring at room temperature for 24 hours, the mixture was filtered. The obtained solid was drained in vacuum at 50° C. After the solid was drained, 5% glutaraldehyde (phosphate Buffer, pH=8, IX TBS) was added to the solid, and stirred for 21 hours to form a solution. The solid in the solution was filtered out and washed with water and a solid substance was obtained. NEU2 (1/100-10000 wt %) was added to the solid substance and diluted with phosphate buffer, 1×TBS pH=8 to a volume of 2 mL, and reacted with the solid substance at room temperature for 24 hours. Finally, the solid substance was filtered out and washed with phosphate buffer (pH=8) and an enzyme immobilized product (ITRI-Siw-Nu-01) was obtained.
Method 2
3-glycidoxypropyltrimethoxysilane was added to heat-activated silica gel in toluene by a weight of 1/5 weight of heat-activated silica gel, refluxed for 20 hours, and then filtered. The obtained solid was washed with acetone and then drained in vacuum. NEU2 (1/100-10000 wt %) was added to the solid and stirred in phosphate buffer for 2 hours and 15 minutes, and then the solid was filtered out. The solid was washed with deionized water and a buffer (pH 8) to obtain an enzyme immobilized product.
Method 3
1 g cellulose beads in 15 mL water were adjusted to about pH 11 by a NaOH solution, and then 1 g cyanogen bromide was added therein at room temperature. After about 30 minutes, the cellulose beads were washed in deionized water and a phosphate buffer (pH 8) in order. NEU2 in a phosphate buffer was added to the cellulose beads by a weight ratio of 1/600, and stirred overnight. After that, the cellulose beads were washed in a phosphate buffer (pH 8) to obtain an enzyme immobilized product.
Method 4
0.5 g cellulose beads in 1.5 mL water were refluxed in 10 mL toluene, and then cellulose beads were filtered out and washed with acetone and a phosphate buffer (pH 8). After that, the cellulose beads were added to 5% (w/v) glutaraldehyde (phosphate buffer, pH 8) and stirred at room temperature for 21 hours. Afterward, the cellulose beads were filtered out, and washed in a phosphate buffer (pH 8) to obtain glutaraldehyde-activated-cellulose beads. NEU2 in a phosphate buffer was added to the cellulose beads by a weight ratio of 2/1000, and stirred overnight. After that, the cellulose beads were washed in a phosphate buffer (pH 8) to obtain an enzyme immobilized product.
Method 5
Hypogel® 200NH2 were added to 5% (w/v) glutaraldehyde (phosphate buffer, pH 8) and stirred at room temperature for 21 hours. Afterward, the solid substance was filtered out, and washed with a phosphate buffer (pH 8) to obtain glutaraldehyde-activated gel. NEU2 (1/10000 wt %) was diluted with a phosphate buffer (pH=8) to a volume of 15 mL, and mixed with 1.13 g of the glutaraldehyde-activated gel at room temperature for 20 hours. Finally, the solid was filtered out and washed in a phosphate buffer (pH=8) to obtain an enzyme immobilized product.
Method 6
1 g diethylaminoethyl cellulose (DEAE cellulose) was washed in water, suspended in an NaOH solution (1 M aqueous solution), stirred for 10 minutes, and then filtered out and washed in water. The obtained solid substance was suspended in 10 mL dioxane to form a suspension. 2 g cyanuric chloride and 10 mL toluene were added to the suspension and stirred for 30 minutes and then the solid therein was filtered out. The solid was washed with dioxane, water and acetone in order and dried under reduced pressure to form an activated solid support. After that, NEU2 1/10000 (wt %) was added to the activated solid support and stirred for 18 hours. Afterward, the activated solid support was filtered out and washed in water to obtain an enzyme immobilized product.
Method 7
0.5 g chitosan beads were added to 10 mL 0.5% glutaraldehyde, and stirred at room temperature for 1 hour, and then washed with water, continuously and thoroughly to form activated beads. After that, the activated beads were reacted with NEU2 1/3500 (wt %) at room temperature for 2 hours, filtered out and then washed with deionized water to obtain an enzyme immobilized product.
Method 8
1 g cellulose hollow fiber, as per the procedures in Method 4, was activated by APTS and glutaraldehyde, and then reacted with NEU2 3/10000 (wt %) in phosphate buffer (pH=8), stirred overnight, and washed with a phosphate buffer (pH=8) to obtain an enzyme immobilized product.
Method 9
1 g cellulose hollow fiber, as per the procedures in Method 3, was activated by cyanogen bromide, and then reacted with NEU2 in phosphate buffer (pH=8), stirred overnight, washed with a phosphate buffer (pH=8) to obtain an enzyme immobilized product.
Method 10
ECR-8204F epoxy-acrylate resin was washed in deionized water, reacted with ASAH2 1/10000 (wt %), adjusted to a volume of 2 mL with a 0.2 M sodium phosphate buffer, and then stirred for 24 hours. After that, epoxy-acrylate resin was filtered out and washed in deionized water and 2M phosphate buffer (pH=8) to obtain about 52 mg of enzyme immobilized product (ITRI-EC-AS-01).
Method 11
Iontosorb MT200 cellulose beads were washed in deionized water. Next, the cellulose beads were washed with 3:7 water/dioxane, 7:3 water/dioxane, 100% dioxane in order. After that, dioxane was added to the cellulose beads, and CDI was added therein by a weight of 1/3 weight of cellulose beads, and stirred for about 0.5-1 hour to form a solution. The dioxane in the solution was removed under reduced pressure, and then NEU 2 was immediately added therein and stirred for about 2 hours and 15 minutes. After the reaction, the cellulose beads in the solution were filtered out and were washed in a buffer (pH=6.5) to obtain a wet product about 0.2 g (ITRI-CD-01).
Method 12
0.5 μg NEU2 was added to 2% w/v alginate aqueous solution to form a mixture solution. Next, the mixture solution was dropped into a stirring 2% CaCl2 (w/v) aqueous solution by a syringe needle. After that, the CaCl2 aqueous solution was continuously stirred for 30 minutes, and then particles formed in the CaCl2 aqueous solution were filtered out and washed in deionized water to obtain a wet product (ITRI-A-01).
NEU2 was immobilized by Method 2 in Example 2, and then the immobilized NEU2 was filled into a tube to form a biochemistry reactive device (immobilized-NEU2 filled device) shown in
(1) Determination of Apoptosis
Endothelial cells of blood vessel were co-cultured with electronegative low-density lipoprotein (electronegative LDL) L5 (25 μg/mL; 50 μg/mL) and L5 (1.25 μg) which was treated by the mmobilized-NEU2 filled device for 2 hours (treatment temperature 37° C., pH 7.4) for 24 hours, respectively. After that, apoptosis of the endothelial cells was determined, and the results are shown in
According to
(2) Quantitative Analysis for Electronegative Low-Density Lipoprotein (Electronegative LDL)
LDL samples were obtained from a heart disease patient. Quantitative analysis for L5 was performed on the LDL samples without treatment and those treated without enzyme at 37° C. for 2 hours or treated with NEU2 for 2 hours (treatment temperature 37° C. pH 7.4) to determine the content of L5 in the samples mentioned above. The results are shown in
According to
(3) Mass Spectrometry
Mass spectrometry analysis was performed on L5 and L5 treated with NEU2 for 2 hours (treatment temperature 37° C., pH 7.4). The results are shown in
It has been known that the feature of L5 is that serine and threonine of apolipoprotein E (apoE) are usually glycosylated.
Refer to
FIGS. 10A1-2 show that there is no molecule with a charge-to-mass ratio of 1700, 1884 or 2154 that is detected for L5 treated by the immobilized-NEU2 filled device for 2 hours, and that indicates that there is no glycosylation on serine and threonine of apolipoprotein E, i.e., the glycan residues of LDL have been removed.
Similarly, FIGS. 10B1-2 show that for L5 treated by the immobilized-NEU2 filled device for 2 hours, there is no glycosylation on other sites of apolipoprotein E, and that indicates that the glycan residues of LDL have been removed.
(1) LC/MSE Analysis for L5 Treated with ASAH2 for 24 Hours
L5 was treated with ASAH2 for 24 hours. LC/MSE analysis was performed on L5 without treatment and L5 with the preceding treatment to determine the ceramide content in the L5 samples mentioned above (for the detailed experimental methods, please see “5. Efficacy test for ASAH2” in “A. Method” of Example 1).
The results for LC/MSE analysis are shown in Table 1 (the four values shown in each group were obtained from determining the same sample four times). Conversion was performed to signal of each sample in Table 1 to obtain ceramide content percentage of each sample (the highest signal of the L5 without treatment was set as 100%), and the results are shown in
24-hour baseline represents ceramide content of L5 without treatment.
According to Table 1 and
(2) LC/MSE Analysis for L5 Treated with ASAH2 for 24 Hours (for the Detailed Experimental Methods, Please See “5. Efficacy Test for ASAH2” in “A. Method” of Example 1)
L5 was treated with ASAH2 for 24 hours. LC/MSE analysis was performed on L5 without treatment and L5 with the preceding treatment to determine the ceramide content in the L5 samples mentioned above.
The results for LC/MSE analysis are shown in Table 2 (the four values shown in each group were obtained from determining the same sample four times). Conversion was performed to signal of each sample in Table 2 to obtain ceramide content percentage of each sample (the highest signal of the L5 without treatment was set as 100%), and the results are shown in
24 hour baseline represents ceramide content of L5 without treatment.
According to Table 2 and
(3) LC/MSE Analysis for L5 Treated with ASAH2 in the Presence or Absence of a Buffer for 2 or 24 Hours
In the presence or absence of a buffer (200 mM Tris-HCl pH 8.4, 1.5 M NaCl, 25 mM CaCl2), L5 was treated with ASAH2 for 2 or 24 hours. LC/MSE analysis was performed on L5 without treatment and L5 with the preceding treatment to determine the ceramide content in the L5 samples mentioned above (for the detailed experimental methods, please see “5. Efficacy test for ASAH2” in “A. Method” of Example 1 except the part of mixing with the buffer or not).
Conversion was performed to signal of each sample to obtain the ceramide content percentage of each sample (the highest signal of the L5 without treatment and kept for 2 hours was set as 100%), and the results are shown in
According to
(4) LC/MSE Analysis for L5 Treated with ASAH2 in the Presence or Absence of a Buffer for 24 Hours
In the presence or absence of a buffer (200 mM Tris-HCl pH 8.4, 1.5 M NaCl, 25 mM CaCl2), L5 was treated with ASAH2 for 24 hours. LC/MSE analysis was performed on L5 without treatment and L5 with the preceding treatment to determine the ceramide content in the L5 samples mentioned above (for the detailed experimental methods, please see “5. Efficacy test for ASAH2” in “A. Method” of Example 1 except the part of mixing with the buffer or not).
The results for LC/MSE analysis are shown in Table 3 (the four values shown in each group were obtained from determining the same sample four times). Conversion was performed to signal of each sample in Table 3 to obtain ceramide content percentage of each sample (the highest signal of the L5 without treatment was set as 100%), and the results are shown in
24 hour baseline represents ceramide content of L5 without treatment.
According to Table 3 and
(5) LC/MSE Analysis for L5 Treated with ASAH2 for 24 Hours
Quantitative analysis for lipid constituents was performed on L5 and L5 treated with ASAH2 for 24 hours by mass spectrometry, and the ceramide contents of the L5 samples mentioned above were compared. The results are shown in
(6) LC/MSE Analysis for L5 Treated with ASAH2 in the Presence of a Buffer for 2 Hours
In the presence of a buffer (200 mM Tris-HCl pH 8.4, 1.5 M NaCl, 25 mM CaCl2), L5 was treated with ASAH2 for 2 hours. LC/MSE analysis was performed on L5 without treatment and L5 with the preceding treatment to determine the ceramide content in the L5 samples mentioned above (for the detailed experimental methods, please see “5. Efficacy test for ASAH2” in “A. Method” of Example 1 except the part of mixing with the buffer or not). The results for LC/MSE analysis are shown in Table 4 (the four values shown in each group were obtained from determining the same sample four times). Conversion was performed to signal of each sample in Table 4 to obtain ceramide content percentage of each sample (the highest signal of the L5 without treatment was set as 100%), and the results are shown in
According to Table 4 and
It will be apparent to those skilled in the art that various modifications and variations can be made to the disclosed embodiments. It is intended that the specification and examples be considered as exemplary only, with the true scope of the disclosure being indicated by the following claims and their equivalents.
| Number | Date | Country | Kind |
|---|---|---|---|
| 104126050 | Aug 2015 | TW | national |
| 201510815421.4 | Nov 2015 | CN | national |
