A robust bacterial high-throughput screening system to evaluate single nucleotide polymorphisms of human homogentisate 1,2-dioxygenase in the context of alkaptonuria
Abstract Alkaptonuria (AKU) is a rare inborn error of metabolism caused by a defective homogentisate 1,2-dioxygenase (HGD), an enzyme involved in the tyrosine degradation pathway. Loss of HGD function leads to the accumulation of homogentisic acid (HGA) in connective body tissues in a process called...
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Nature Portfolio
2022-11-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-022-23702-y |
| _version_ | 1811217726692130816 |
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| author | Sien Lequeue Jessie Neuckermans Ine Nulmans Ulrich Schwaneberg Tamara Vanhaecke Joery De Kock |
| author_facet | Sien Lequeue Jessie Neuckermans Ine Nulmans Ulrich Schwaneberg Tamara Vanhaecke Joery De Kock |
| author_sort | Sien Lequeue |
| collection | DOAJ |
| description | Abstract Alkaptonuria (AKU) is a rare inborn error of metabolism caused by a defective homogentisate 1,2-dioxygenase (HGD), an enzyme involved in the tyrosine degradation pathway. Loss of HGD function leads to the accumulation of homogentisic acid (HGA) in connective body tissues in a process called ochronosis, which results on the long term in an early-onset and severe osteoarthropathy. HGD’s quaternary structure is known to be easily disrupted by missense mutations, which makes them an interesting target for novel treatment strategies that aim to rescue enzyme activity. However, only prediction models are available providing information on a structural basis. Therefore, an E. coli based whole-cell screening was developed to evaluate HGD missense variants in 96-well microtiter plates. The screening principle is based on HGD’s ability to convert the oxidation sensitive HGA into maleylacetoacetate. More precisely, catalytic activity could be deduced from pyomelanin absorbance measurements, derived from the auto-oxidation of remaining HGA. Optimized screening conditions comprised several E. coli expression strains, varied expression temperatures and varied substrate concentrations. In addition, plate uniformity, signal variability and spatial uniformity were investigated and optimized. Finally, eight HGD missense variants were generated via site-directed mutagenesis and evaluated with the developed high-throughput screening (HTS) assay. For the HTS assay, quality parameters passed the minimum acceptance criterion for Z’ values > 0.4 and single window values > 2. We found that activity percentages versus wildtype HGD were 70.37 ± 3.08% (for M368V), 68.78 ± 6.40% (for E42A), 58.15 ± 1.16% (for A122V), 69.07 ± 2.26% (for Y62C), 35.26 ± 1.90% (for G161R), 35.86 ± 1.14% (for P230S), 23.43 ± 4.63% (for G115R) and 19.57 ± 11.00% (for G361R). To conclude, a robust, simple, and cost-effective HTS system was developed to reliably evaluate and distinguish human HGD missense variants by their HGA consumption ability. This HGA quantification assay may lay the foundation for the development of novel treatment strategies for missense variants in AKU. |
| first_indexed | 2024-04-12T06:59:30Z |
| format | Article |
| id | doaj.art-b238b82c35804e088b81a5edcf5920f8 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-04-12T06:59:30Z |
| publishDate | 2022-11-01 |
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| spelling | doaj.art-b238b82c35804e088b81a5edcf5920f82022-12-22T03:43:03ZengNature PortfolioScientific Reports2045-23222022-11-0112111510.1038/s41598-022-23702-yA robust bacterial high-throughput screening system to evaluate single nucleotide polymorphisms of human homogentisate 1,2-dioxygenase in the context of alkaptonuriaSien Lequeue0Jessie Neuckermans1Ine Nulmans2Ulrich Schwaneberg3Tamara Vanhaecke4Joery De Kock5Liver Therapy & Evolution Team, In Vitro Toxicology and Dermato-Cosmetology (IVTD) Research Group, Faculty of Medicine and Pharmacy, Vrije Universiteit BrusselLiver Therapy & Evolution Team, In Vitro Toxicology and Dermato-Cosmetology (IVTD) Research Group, Faculty of Medicine and Pharmacy, Vrije Universiteit BrusselLiver Therapy & Evolution Team, In Vitro Toxicology and Dermato-Cosmetology (IVTD) Research Group, Faculty of Medicine and Pharmacy, Vrije Universiteit BrusselLehrstuhl Für Biotechnologie, RWTH Aachen UniversityIn Vitro Liver Disease Modelling Team, In Vitro Toxicology and Dermato-Cosmetology (IVTD) Research Group, Faculty of Medicine and Pharmacy, Vrije Universiteit BrusselLiver Therapy & Evolution Team, In Vitro Toxicology and Dermato-Cosmetology (IVTD) Research Group, Faculty of Medicine and Pharmacy, Vrije Universiteit BrusselAbstract Alkaptonuria (AKU) is a rare inborn error of metabolism caused by a defective homogentisate 1,2-dioxygenase (HGD), an enzyme involved in the tyrosine degradation pathway. Loss of HGD function leads to the accumulation of homogentisic acid (HGA) in connective body tissues in a process called ochronosis, which results on the long term in an early-onset and severe osteoarthropathy. HGD’s quaternary structure is known to be easily disrupted by missense mutations, which makes them an interesting target for novel treatment strategies that aim to rescue enzyme activity. However, only prediction models are available providing information on a structural basis. Therefore, an E. coli based whole-cell screening was developed to evaluate HGD missense variants in 96-well microtiter plates. The screening principle is based on HGD’s ability to convert the oxidation sensitive HGA into maleylacetoacetate. More precisely, catalytic activity could be deduced from pyomelanin absorbance measurements, derived from the auto-oxidation of remaining HGA. Optimized screening conditions comprised several E. coli expression strains, varied expression temperatures and varied substrate concentrations. In addition, plate uniformity, signal variability and spatial uniformity were investigated and optimized. Finally, eight HGD missense variants were generated via site-directed mutagenesis and evaluated with the developed high-throughput screening (HTS) assay. For the HTS assay, quality parameters passed the minimum acceptance criterion for Z’ values > 0.4 and single window values > 2. We found that activity percentages versus wildtype HGD were 70.37 ± 3.08% (for M368V), 68.78 ± 6.40% (for E42A), 58.15 ± 1.16% (for A122V), 69.07 ± 2.26% (for Y62C), 35.26 ± 1.90% (for G161R), 35.86 ± 1.14% (for P230S), 23.43 ± 4.63% (for G115R) and 19.57 ± 11.00% (for G361R). To conclude, a robust, simple, and cost-effective HTS system was developed to reliably evaluate and distinguish human HGD missense variants by their HGA consumption ability. This HGA quantification assay may lay the foundation for the development of novel treatment strategies for missense variants in AKU.https://doi.org/10.1038/s41598-022-23702-y |
| spellingShingle | Sien Lequeue Jessie Neuckermans Ine Nulmans Ulrich Schwaneberg Tamara Vanhaecke Joery De Kock A robust bacterial high-throughput screening system to evaluate single nucleotide polymorphisms of human homogentisate 1,2-dioxygenase in the context of alkaptonuria Scientific Reports |
| title | A robust bacterial high-throughput screening system to evaluate single nucleotide polymorphisms of human homogentisate 1,2-dioxygenase in the context of alkaptonuria |
| title_full | A robust bacterial high-throughput screening system to evaluate single nucleotide polymorphisms of human homogentisate 1,2-dioxygenase in the context of alkaptonuria |
| title_fullStr | A robust bacterial high-throughput screening system to evaluate single nucleotide polymorphisms of human homogentisate 1,2-dioxygenase in the context of alkaptonuria |
| title_full_unstemmed | A robust bacterial high-throughput screening system to evaluate single nucleotide polymorphisms of human homogentisate 1,2-dioxygenase in the context of alkaptonuria |
| title_short | A robust bacterial high-throughput screening system to evaluate single nucleotide polymorphisms of human homogentisate 1,2-dioxygenase in the context of alkaptonuria |
| title_sort | robust bacterial high throughput screening system to evaluate single nucleotide polymorphisms of human homogentisate 1 2 dioxygenase in the context of alkaptonuria |
| url | https://doi.org/10.1038/s41598-022-23702-y |
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