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Method Article

In Vitro Enzyme Measurement to Test Pharmacological Chaperone Responsiveness in Fabry and Pompe Disease

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DOI:

10.3791/56550

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December 20th, 2017

 ,  ,  ,  ,  , 
1Albrecht-Kossel-Institute, University Rostock Medical Center, 2Department of Biology, University Federico II, 3Centogene AG

In This Article

Summary

There is a demand to make pre-clinical testing for a novel class of "orphan" drugs called pharmacological chaperones reproducible, fast, and efficient. We developed a simple, highly standardized, and versatile cell culture-based assay to screen for eligible patients as well as novel pharmacological chaperone drugs.

Abstract

The use of personalized medicine to treat rare monogenic diseases like lysosomal storage disorders (LSDs) is challenged by complex clinical trial designs, high costs, and low patient numbers. Hundreds of mutant alleles are implicated in most of the LSDs. The diseases are typically classified into 2 to 3 different clinical types according to severity. Moreover, molecular characterization of the genotype can help predict clinical outcomes and inform patient care. Therefore, we developed a simple cell culture assay based on HEK293H cells heterologously over-expressing the mutations identified in Fabry and Pompe disease. A similar assay has recently been introduced as a preclinical test to identify amenable mutations for Pharmacological Chaperone Therapy (PCT) in Fabry disease. This manuscript describes an amended cell culture assay which enables rapid phenotypic assessment of allelic variants in Fabry and Pompe disease to identify eligible patients for PCT and may aid in the development of novel pharmacochaperones.

Introduction

There are over a dozen lysosomal storage disorders (LSDs) related to glycosidase dysfunction as a result of primary gene mutations. In Fabry (OMIM #301500) and Pompe (OMIM #232300) disease, more than 500 and 200 missense mutations1,2,3 have been reported, respectively, which corresponds to about 60% of the total mutation count. Numerous new gene variants are still being identified, many of which have unknown significance. Extensive biochemical studies revealed that certain genotypes do not lead to a complete loss-of-function of the GLA gene (OMIM *300644) in Fabry disease, but cause the corresponding enzyme to fail to reach a thermodynamically favored folding state4. This results in ER retention and premature degradation of the otherwise functional enzyme. Similar conclusions have been drawn in other LSDs including Pompe disease5. Moreover, molecular characterization of enzyme variants can facilitate clinical interpretation of the mutations at the time of diagnosis6, suggesting that LSD progression is an individual process based on the nature of the mutation. Therefore, the conventional classification into typically 2 to 3 different clinical types should be reassessed in order to streamline clinical counselling and therapeutic decisions.

Enzyme Replacement Therapy (ERT) is available for both diseases. ERT, however, has limited efficacy in affected tissues/organs such as the brain and skeletal muscle. Furthermore, ERT can elicit an immunogenic response that jeopardizes its therapeutic benefits. Pharmacological Chaperones (PCs) are an attractive treatment alternative for patients with so-called responsive mutations. PCs serve as a molecular scaffold for correct protein folding and stabilization which in turn prevents endoplasmic reticulum (ER) retention and ER-associated degradation of the enzyme. Moreover, PCs can be administered orally and are potentially able to cross the blood brain barrier. Therefore, PCT might be a more viable option for treating patients with certain genotypes. For an extensive review on PC application in LSDs, refer to the excellent review by Parenti7.

The discovery of hundreds of disease causing mutant alleles challenges pre-clinical drug testing and necessitates a simple, fast, and highly standardized assessment of amenable patients for a personalized medicine approach. In order to assess the detrimental effects of LSD gene mutations and to test candidate mutations to predict amenable patients for PCT, a highly standardized over-expression system in HEK293H cells that allows for fast and reliable enzyme activity measurement was developed. Similar over-expression systems have been previously described for Fabry and Pompe disease using either COS-78,9,10,11, HeLa cells12, or HEK29313,14,15,16 cells for the glycosidase gene.

A very similar method has even been patented as a "Method to predict response to Pharmacological Chaperone treatment of diseases"17 indicating the relevance of a cell culture system capable of being integrated into clinical practice.

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Protocol

1. Preparation of Mutant pcDNA3.1/GLA and pcDNA3.1/GAA Constructs

NOTE: The cloning strategies for the GLA and GAA coding sequences (cds) have been reported earlier15,18.

  1. Site-directed Mutagenesis Using Site-Directed Mutagenesis
    1. Use the reference sequences NM_000169.2 and NM_000152.4 as templates for the mutagenesis of GLA and GAA genes, respectively. Have a set of high purity salt free primers (25-37-mers) synthesized by a commercial provider, with sense and antisense primers carrying one of the respective sequence modifications central to their length to individually introduce the mutation. Use the free primer design tool to support the primer design19.
    2. For the reaction mixture, use the standard conditions for the reaction solution and PCR conditions provided by the manufacturer.
      1. Mix 5 µL of 10x reaction buffer, 10 ng of double-stranded template plasmid DNA (pcDNA3.1/GLA or pcDNA3.1/GAA), 125 ng of each primer, 1 µL of the provided dNTP mixture, 3 µL of DMSO reagent in an appropriate volume of deionized water (final reaction volume: 50 µL). Finally, add 2.5 U of DNA polymerase and mix by pipetting up and down. As a negative control, carry along a no-primer sample.
    3. Start the PCR using the following program: step 1: 95 °C for 1 min, step 2: 95 °C for 50 s, step 3: 60 °C for 50 s, step 4: 68 °C for 8 min, repeat step 2-4 18 times, and step 5: 68 °C for 10 min.
      1. Following PCR, add 1 µL of the DpnI restriction enzyme (10 U/µL) and further incubate the reaction vial at 37 °C for 1 h.
  2. Transformation and Screening for the Desired Clone
    1. Transform an aliquot of ultracompetent cells in accordance with the manufacturer's recommendations. Use SOC medium (tryptone 2% (w/v), yeast extract 0.5% (w/v), NaCl 10 mM, KCl 2.5 mM, sterilize at 121 °C, and then add sterile-filtered solutions of MgCl2 and glucose up to final concentrations of 10 and 20 mM, respectively) instead of the manufacturer's medium. After the procedure, plate 250 µL of the sample mutagenesis on an LB plate containing 100 µg/mL ampicillin and incubate at 37 °C for 18 h.
    2. Assure that the number of transformants is >10 and the reaction yields at least three times as many colonies as the no-primer control reaction, e.g., using a luminous plate to facilitate colony counting. Then pick 3 colonies and prepare 3 mL overnight cultures in LB Broth medium.
    3. The next day, carry out plasmid preparation with a standard kit and analyze whole sequence using T7 (5ʹ-TAA TAC GAC TCA CTA TAG GG-3ʹ) and BGHr (5ʹ-TAG AAG GCA CAG TCG AGG-3ʹ) primers via standard Sanger sequencing.
    4. Use a suitable molecular biology tool to analyze the sequence. When the desired mutation is detected and no further sequence abnormality compared to NM_000169.2 (α-galactosidase A) or NM_000152.4 (acid α-glucosidase) is seen, select the clone for transfection-grade plasmid purification.
    5. Determine the purity of the DNA by measuring the absorbance in a spectrophotometer.
      NOTE: Allow only preparations that yield a plasmid purity with a 260/280 absorbance ratio of >1.8 for cell culture experiments.

2. Cultivation of HEK293H Cells

  1. Maintain HEK293H cells in high glucose (4.5 g/L) Dulbecco´s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Keep the cells in a water-jacket incubator at 37 °C under a 5% CO2 atmosphere.
  2. Cultivate the cells to a density of 80 - 90%.
  3. Aspirate the medium and wash once using phosphate buffered saline (PBS) without Ca2+ and Mg2+.
  4. Passage by adding 0.05% Trypsin-EDTA and incubate for 5 min at 37 °C and 5% CO2.
  5. Split the cells 1:15 in fresh medium and seed into a new T75 flask to maintain the permanent culture. Do not use cells with more than 25 passages.

3. pcDNA3.1/GLA and pcDNA3.1/GAA Plasmid Transfection and Treatment of HEK293H

  1. 24 h prior to the transfection, wash the HEK293H cells in a T75 cell culture flask once with PBS with Ca2+, Mg2+. Harvest the cells with 0.05% Trypsin-EDTA as stated above and seed 1.5 x 105 cells in the cavities of a 24 well culture plate using 500 µL DMEM medium supplemented with 10% FBS without antibiotics.
  2. Carry out a transfection protocol according to the manufacturer's manual. Typically, use a mixture of 1 µg of plasmid DNA and 2.5 µL of transfection reagent in 100 µL of serum-free DMEM. Incubate for 20 min at room temperature and add to the cells in a drop-wise manner thereafter.
  3. Remove the medium containing the transfection reagent after a period of 4 h at 37 °C/5% CO2 and add 500 µL of fresh DMEM with 10% FBS/ 1% penicillin/streptomycin.
    NOTE: During this step, 1-Deoxygalactonojirimycin Hydrochloride (DGJ) or 1-Deoxynojirimycin Hydrochloride (DNJ) might be added to the culture medium where intended (use an aqueous stock solution of 10 mM in order to obtain a final concentration of 20 µM DGJ and DNJ). Fresh DGJ or DNJ was added 42 h after plasmid transfection.

4. Cell Harvest and α-galactosidase A or Acid α-glucosidase Activity Measurement

  1. Cell harvest
    1. On the day of the harvest, remove the cells from the incubator and aspirate the medium. Carefully wash the cells 2 times with PBS with Ca2+ and Mg2+.
      NOTE: This step is critical because DGJ and DNJ are potent inhibitors of α-galactosidase and α-glucosidase, respectively, and any leftover would invalidate the test.
    2. Add 200 µL of deionized water directly on top of the cells. Rinse the cells from the plate and transfer them to a 1.5 mL reaction tube.
  2. Homogenization by freezing and thawing
    1. Put the samples in an appropriate foam rack and vortex for 5 s to make the lysis more efficient. Put the samples alternating in liquid nitrogen for 10 s and in a room temperature water bath until the thawing was complete (5 min).
    2. Repeat this procedure 5 times and then spin the samples for 5 min at 10,000 x g. Retain the supernatant and pipette in a new reaction tube.
  3. Protein concentration determination using bicinchoninic acid (BCA) assay
    1. Prepare a fresh tube for each sample containing 40 µL of deionized H2O and add 10 µL of sample. Mix solution by vortexing briefly and transfer 10 µL into a cavity of a 96 well plate (each sample in triplicate). Dilute a 2 mg/mL bovine serum albumin (BSA) stock solution in deionized H2O as follows to obtain a standard curve: 50 µL H2O/50 µL BSA; 60 µL H2O/40 µL BSA; 70 µL H2O/30 µL BSA; 80 µL H2O/20 µL BSA; 90 µL H2O/10 µL BSA; 100 µL H2O.
    2. Start the reaction by adding 200 µL of BCA reagent (Reagent A and reagent B mixed at a 50:1 ratio) and incubate for 1 h in the dark at 37 °C under slight agitation on an orbital shaker (300 rpm). Measure the absorbance at 560 nm in a plate reader.
      NOTE: The samples typically contain between 1 and 1.5 µg protein per µL.
  4. Enzyme activity measurement with artificial 4-Methylumbelliferyl substrates (4-MUG)
    1. Dilute the calculated amount of each sample and pipet into fresh 1.5 mL reaction tubes to obtain 0.05 (for α-galactosidase A) or 0.5 (for acid α-glucosidase) µg protein/µL solutions. Vortex the samples for 5 s again and pipet 10 µL of this dilution into a 96 well plate (each sample in duplicate).
    2. Start the reaction by adding 20 µL of the respective substrate solution:
      For α-galactosidase A: 2 mM 4-Methylumbelliferyl-α-D-galactopyranoside (4-MU-gal) in 0.06 M phosphate citrate buffer, pH 4.7.
      For acid α-glucosidase: 2 mM 4-methylumbelliferyl α-D-glucopyranoside (4-MU-glu) in 0.025 M sodium acetate, pH 4.0.
    3. Incubate the enzyme reactions for 1 h in the dark at 37 °C under slight agitation on an orbital shaker (300 rpm). Terminate the reaction by the addition of 200 µL of 1.0 M, pH 10.5 adjusted glycine-NaOH buffer.
    4. Prepare a standard curve of 4-methylumbelliferone (4-MU) from a 0.01 mg/mL stock as follows:
      100 µL H2O/ no 4-MU; 80 µL H2O/ 20 µL 4-MU; 60 µL H2O/ 40 µL 4-MU; 40 µL H2O/ 60 µL 4-MU; 20 µL H2O/ 80 µL 4-MU; no H2O/ 100 µL 4-MU, pipet 10 µL of each dilution into the 96 well plate (in duplicates) and add 200 µL of the 1.0 M glycine-NaOH buffer to each well in order to adjust the volume and pH.
    5. Measure the enzyme activity in a fluorescence reader equipped with the appropriate filter set and analyze the data using the appropriate software for the fluorescence reader device.
      NOTE: Both 4-MUG substrates are reduced to 4-MU during exposure to α-galactosidase A or acid α-glucosidase. Released 4-MU is a fluorochrome, which can be measured at 360 and 465 nm as the excitation and emission wavelengths, respectively, using a microplate fluorescence reader.

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Results

The mutagenesis procedure
To assess the efficiency of GLA gene mutagenesis, the mutations were classified into one of the following categories. This approach to generate mutations revealed that about 66.5% of the GLA mutations were obtained in the first attempt. A further 25% could be obtained after a slightly modified second PCR.

Category 1: The mutagenesis PCR was effective at first attem...

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Discussion

The protocol described herein delivers robust results for enzyme damage assessment in hereditary lysosomal diseases of metabolism. This manuscript is an amendment to the protocol published earlier15. The most crucial modifications involve stringency (i.e., in the process of mutant vector construct preparation), standardization of the cell culture protocol (i.e., HEK293H cell maintenance and transfection conditions), and the high number of experimental repetitions (at least 5), wh...

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Disclosures

The authors declare that they have no competing financial interests.

Acknowledgements

The authors would like to acknowledge Mandy Loebert and Tina Czajka for excellent technical support. We thank Flora Luo (Harvard Medical School, Boston, MA, USA) for language editing help.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Material
QuikChange II XL Site-Directed Mutagenesis Kit Stratagene, La Jolla, CA, USA#200522
Primer GLA[R301Q]-frw: 5´- CTA ATG ACC TCC AAC ACA TCA GCC C-3´MWG Eurofins Operon, Ebersberg, Germanyn.a.
Primer GLA[R301Q]-rev: 5´- GGG CTG ATG TGT TGG AGG TCA TTA G-3´MWG Eurofins Operon, Ebersberg, Germanyn.a.
Primer GLA[A156V]-frw: 5´-CTA CGA CAT TGA TGT CCA GAC CTT TGC TG-3´MWG Eurofins Operon, Ebersberg, Germanyn.a.
Primer GLA[A156V]-rev: 5´-CAG CAA AGG TCT GGA CAT CAA TGT CGT AG-3´MWG Eurofins Operon, Ebersberg, Germanyn.a.
Primer GLA[A156V]-frw: 5´-GGA AAT AAA ACC TGC ACA GGC TTC CCT GGG A-3´MWG Eurofins Operon, Ebersberg, Germanyn.a.
Primer GLA[A143T]-rev: 5´-TCC CAG GGA AGC CTG TGC AGG TTT TAT TTC C-3´MWG Eurofins Operon, Ebersberg, Germanyn.a.
Primer GAA[F455Y]-frw: 5´-CTG CCG GGA GCT TCA GGC CCT ACG A-3´MWG Eurofins Operon, Ebersberg, Germanyn.a.
Primer GAA[F455Y]-rev: 5´-TCG TAG GGC CTG AAG CTC CCG GCA G-3´MWG Eurofins Operon, Ebersberg, Germanyn.a.
Primer GAA[P545L]-frw: 5´-CAC CCT ACG TGC TTG GGG TGG TTG G-3´MWG Eurofins Operon, Ebersberg, Germanyn.a.
Primer GAA[P545L]-rev: 5´-CCA ACC ACC CCA AGC ACG TAG GGT G-3´MWG Eurofins Operon, Ebersberg, Germanyn.a.
Primer GAA[L552P]-frw: 5´-TTG GGG GGA CCC CCC AGG CGG CCA C-3´MWG Eurofins Operon, Ebersberg, Germanyn.a.
Primer GAA[P545L]-rev: 5´-GTG GCC GCC TGG GGG GTC CCC CCA A-3´MWG Eurofins Operon, Ebersberg, Germanyn.a.
Primer T7: 5´-TAA TAC GAC TCA CTA TAG GG-3´MWG Eurofins Operon, Ebersberg, Germanyn.a.
Primer BGHrev: 5´-TAG AAG GCA CAG TCG AGG-3´MWG Eurofins Operon, Ebersberg, Germanyn.a.
TryptoneCarl Roth, Karlsruhe, Germany8952.1
Yeast ExtractMerck, Darmstadt, Germany103,753
Sodium chlorideMerck, Darmstadt, Germany1,064,041,000
Potasium chlorideMerck, Darmstadt, Germany1,049,360,500
MgCl2 x 6H2OMerck, Darmstadt, Germany1,058,330,250
D (+)-Glucose monohydrateMerck, Darmstadt, Germany1,083,422,500
Sodium HydroxideMerck, Darmstadt, Germany1,064,980,500
GlycineCarl Roth, Karlsruhe, Germany3908.2
Citric acidCarl Roth, Karlsruhe, GermanyX863.3
disodium hydrogen phosphateMerck, Darmstadt, Germany1,065,860,500
Sodium acetateMerck, Darmstadt, Germany1,062,640,050
Glacial acetic acidSigma Aldrich, Munich, GermanyA6283
LB AgarCarl Roth, Karlsruhe, GermanyX969.2
LB MediumCarl Roth, Karlsruhe, GermanyX968.2
Zyppy Plasmid Miniprep KitZymoResearch, Freiburg, GermanyD4020
QIAfilter Plasmid Midi KitQiagen, Hilden, Germany12245
HEK293H cell lineInvitrogen, Karlsruhe, Germany11631-017 
Dulbecco´s Modified Eagle Medium Invitrogen, Karlsruhe, Germany31966-047
HyClone fetal bovine serum GE Healthcare, South Logan, Utah, USASV30160.03
Penicillin/streptomycinInvitrogen, Karlsruhe, Germany15140-122
CELLSTAR Standard Cell Culture Flasks, Greiner Bio OneVWR International GmbH, Hannover, Germany82050-854
Cell culture plates (24 well)Sarstedt, Nümbrecht, Germany831,836
Cellstar 96 well plateGreiner bio one, Frickenhausen, Germany655185
SafeSeal reaction tubes, 1,5mL Sarstedt, Nümbrecht, Germany72,706
Lipofectamine 2000Invitrogen, Karlsruhe, Germany11668-019
1-Deoxygalactonojirimycin Hydrochloride Sigma Aldrich, Munich, GermanyD9641
1-Deoxygalactonojirimycin Hydrochloride Toronto Research Chemicals, Toronto, CanadaD236500
1-Deoxynojirimycin Sigma Aldrich, Munich, GermanyD9305
PBS Dulbecco w/o Calcium, w/o MagnesiumBiochrom, Berlin, GermanyL 1825
Trypsin-EDTA (0.05%), phenol redThermo Scientific, Braunschweig, Germany25300054
Pierce BCA Protein Assay KitThermo Scientific, Braunschweig, Germany23225
4-MethylumbelliferoneSigma Aldrich, Munich, GermanyM1381
4-Methylumbelliferyl α-D-galactopyranosideSigma Aldrich, Munich, GermanyM7633
4-Methylumbelliferyl α-D-glucopyranosideSigma Aldrich, Munich, GermanyM9766
NameCompanyCatalog NumberComments
Equipment
incubator type T6Heraeus Instruments, Hanau, Germanyn.a.
Luminous Plate Gr. 2 ECarl Roth, Karlsruhe, GermanyP265.1
3130 xl Genetic Analyzer Applied Biosystems, Darmstadt, Germanyn.a.
GFL-3032 bacterial shakerGFL, Burgwedel, Germany n.a.
Avanti J-25 centrifugeBeckman/Coulter, Krefeld, Germanyn.a.
Ultraspec 3100 pro SpectrophotometerAmersham Biosciences, Buckinghamshire, United Kingdomn.a.
water-jacket incubator Binder, Tuttlingen, Germanyn.a.
Vortex Genie 1 touch mixerScientific Industries, Bohemia, NY, USAn.a.
Z 233 MK-2 refrigerated microtube centrifugeHermle, Tuttlingen, Germany n.a.
LaboStar ultrapure water deviceSiemens, Berlin, Germanyn.a.
Thermo Shaker PST-60HL-4 orbital shakerBiosan, Riga, Latvian.a.
Tecan GENios ReaderTecan, Männedorf, Switzerlandn.a.
HI 223 Microprocessor pH MeterHANNA Instruments, Arvore, Portugaln.a.
CASY
Cell Counter + Analyser System
Model TT		Innovatis AG, Cham/Zug, Switzerland
NameCompanyCatalog NumberComments
Software
Magellan data analysis software v6.6Tecan, Männedorf, Switzerlandn.a.
GraphPad Prism 5.04GraphPad Software, Inc., La Jolla, CA; USAn.a.
Microsoft Office 2010Microsoft Corporation, Redmond, WA, USAn.a.
BioEdit Alignment Editor, V7.0.1http://www.mbio.ncsu.edu/bioedit/bioedit.htmln.a.
Abbreviations
frw = forward; rev = reverse
n.a. = not applicable

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