Design of a stable human acid-β-glucosidase: towards improved Gaucher disease therapy and mutation classification

Sarka Pokorna; Olga Khersonsky; Rosalie Lipsh-Sokolik; Adi Goldenzweig; Rebekka Nielsen; Yacov Ashani; Yoav Peleg; Tamar Unger; Shira Albeck; Orly Dym; Asa Tirosh; Rana Tarayra; Michaël Hocquemiller; Ralph Laufer; Shifra Ben-Dor; Israel Silman; Joel L Sussman; Sarel J Fleishman; Anthony H Futerman

doi:10.1111/febs.16758

Design of a stable human acid-β-glucosidase: towards improved Gaucher disease therapy and mutation classification

Sarka Pokorna, Olga Khersonsky, Rosalie Lipsh-Sokolik, Adi Goldenzweig, Rebekka Nielsen, Yacov Ashani, Yoav Peleg, Tamar Unger, Shira Albeck, Orly Dym, Asa Tirosh, Rana Tarayra, Michaël Hocquemiller, Ralph Laufer, Shifra Ben-Dor, Israel Silman, Joel L Sussman, Sarel J Fleishman, Anthony H Futerman^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

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Abstract

Acid-β-glucosidase (GCase, EC 3.2.1.45), the lysosomal enzyme which hydrolyzes the simple glycosphingolipid, glucosylceramide (GlcCer), is encoded by the GBA1 gene. Biallelic mutations in GBA1 cause the human inherited metabolic disorder, Gaucher disease (GD), in which GlcCer accumulates, while heterozygous GBA1 mutations are the highest genetic risk factor for Parkinson's disease (PD). Recombinant GCase (e.g., Cerezyme®) is produced for use in enzyme replacement therapy for GD and is largely successful in relieving disease symptoms, except for the neurological symptoms observed in a subset of patients. As a first step towards developing an alternative to the recombinant human enzymes used to treat GD, we applied the PROSS stability-design algorithm to generate GCase variants with enhanced stability. One of the designs, containing 55 mutations compared to wild type human GCase, exhibits improved secretion and thermal stability. Furthermore, the design has higher enzymatic activity than the clinically used human enzyme when incorporated into an AAV vector, resulting in a larger decrease in the accumulation of lipid substrates in cultured cells. Based on stability-design calculations, we also developed a machine-learning based approach to distinguish benign from deleterious (i.e., disease-causing) GBA1 mutations. This approach gave remarkably accurate predictions of the enzymatic activity of single nucleotide polymorphisms in the GBA1 gene that are not currently associated with GD or PD. This latter approach could be applied to other diseases to determine risk factors in patients carrying rare mutations.

Original language	English
Pages (from-to)	3383-3399
Number of pages	17
Journal	FEBS Journal
Volume	290
Issue number	13
Early online date	21 Feb 2023
DOIs	https://doi.org/10.1111/febs.16758
Publication status	Published - Jul 2023

Bibliographical note

Funding Information:
We thank Drs Maxim Itkin and Sergey Malitsky (Lipidomics) and Dr Yael Fridman‐Sirkis (DSF measurements) from the Life Sciences Core Facilities at the Weizmann Institute of Science, Dr Ron Rotkopf and Shani Blumenreich‐Kashani for help with statistical analysis, Yochai Maytal for help with collating data for Table S1 and Chen Yaacobi for creating the GBA cell lines. Research in the Futerman laboratory was supported by a Sponsored Research Agreement between the Weizmann Institute of Science (via Yeda, its technology transfer office) and Lysogene. Research in the Fleishman laboratory was supported by a Consolidator Award from the European Research Council (815379), the Israel Science Foundation (1844), the Dr Barry Sherman Institute for Medicinal Chemistry and by a charitable donation in memory of Sam Switzer. SP was partially supported by the Czech Academy of Sciences (Czech/Israel scientific program). AHF is the Joseph Meyerhoff Professor of Biochemistry at the Weizmann Institute of Science.

Publisher Copyright:
© 2023 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

All Science Journal Classification (ASJC) codes

Biochemistry
Molecular Biology
Cell Biology

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sjf_FEBSJ_DesignofaStableHuman_PV2023Final published version, 1.55 MBLicence: CC BY

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Pokorna, S., Khersonsky, O., Lipsh-Sokolik, R., Goldenzweig, A., Nielsen, R., Ashani, Y., Peleg, Y., Unger, T., Albeck, S., Dym, O., Tirosh, A., Tarayra, R., Hocquemiller, M., Laufer, R., Ben-Dor, S., Silman, I., Sussman, J. L., Fleishman, S. J., & Futerman, A. H. (2023). Design of a stable human acid-β-glucosidase: towards improved Gaucher disease therapy and mutation classification. FEBS Journal, 290(13), 3383-3399. https://doi.org/10.1111/febs.16758

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title = "Design of a stable human acid-β-glucosidase: towards improved Gaucher disease therapy and mutation classification",

abstract = "Acid-β-glucosidase (GCase, EC 3.2.1.45), the lysosomal enzyme which hydrolyzes the simple glycosphingolipid, glucosylceramide (GlcCer), is encoded by the GBA1 gene. Biallelic mutations in GBA1 cause the human inherited metabolic disorder, Gaucher disease (GD), in which GlcCer accumulates, while heterozygous GBA1 mutations are the highest genetic risk factor for Parkinson's disease (PD). Recombinant GCase (e.g., Cerezyme{\textregistered}) is produced for use in enzyme replacement therapy for GD and is largely successful in relieving disease symptoms, except for the neurological symptoms observed in a subset of patients. As a first step towards developing an alternative to the recombinant human enzymes used to treat GD, we applied the PROSS stability-design algorithm to generate GCase variants with enhanced stability. One of the designs, containing 55 mutations compared to wild type human GCase, exhibits improved secretion and thermal stability. Furthermore, the design has higher enzymatic activity than the clinically used human enzyme when incorporated into an AAV vector, resulting in a larger decrease in the accumulation of lipid substrates in cultured cells. Based on stability-design calculations, we also developed a machine-learning based approach to distinguish benign from deleterious (i.e., disease-causing) GBA1 mutations. This approach gave remarkably accurate predictions of the enzymatic activity of single nucleotide polymorphisms in the GBA1 gene that are not currently associated with GD or PD. This latter approach could be applied to other diseases to determine risk factors in patients carrying rare mutations.",

author = "Sarka Pokorna and Olga Khersonsky and Rosalie Lipsh-Sokolik and Adi Goldenzweig and Rebekka Nielsen and Yacov Ashani and Yoav Peleg and Tamar Unger and Shira Albeck and Orly Dym and Asa Tirosh and Rana Tarayra and Micha{\"e}l Hocquemiller and Ralph Laufer and Shifra Ben-Dor and Israel Silman and Sussman, {Joel L} and Fleishman, {Sarel J} and Futerman, {Anthony H}",

note = "Funding Information: We thank Drs Maxim Itkin and Sergey Malitsky (Lipidomics) and Dr Yael Fridman‐Sirkis (DSF measurements) from the Life Sciences Core Facilities at the Weizmann Institute of Science, Dr Ron Rotkopf and Shani Blumenreich‐Kashani for help with statistical analysis, Yochai Maytal for help with collating data for Table S1 and Chen Yaacobi for creating the GBA cell lines. Research in the Futerman laboratory was supported by a Sponsored Research Agreement between the Weizmann Institute of Science (via Yeda, its technology transfer office) and Lysogene. Research in the Fleishman laboratory was supported by a Consolidator Award from the European Research Council (815379), the Israel Science Foundation (1844), the Dr Barry Sherman Institute for Medicinal Chemistry and by a charitable donation in memory of Sam Switzer. SP was partially supported by the Czech Academy of Sciences (Czech/Israel scientific program). AHF is the Joseph Meyerhoff Professor of Biochemistry at the Weizmann Institute of Science. Publisher Copyright: {\textcopyright} 2023 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.",

year = "2023",

month = jul,

doi = "10.1111/febs.16758",

language = "English",

volume = "290",

pages = "3383--3399",

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Pokorna, S, Khersonsky, O, Lipsh-Sokolik, R, Goldenzweig, A, Nielsen, R, Ashani, Y , Peleg, Y , Unger, T , Albeck, S , Dym, O, Tirosh, A, Tarayra, R, Hocquemiller, M, Laufer, R, Ben-Dor, S , Silman, I , Sussman, JL , Fleishman, SJ & Futerman, AH 2023, 'Design of a stable human acid-β-glucosidase: towards improved Gaucher disease therapy and mutation classification', FEBS Journal, vol. 290, no. 13, pp. 3383-3399. https://doi.org/10.1111/febs.16758

TY - JOUR

T1 - Design of a stable human acid-β-glucosidase

T2 - towards improved Gaucher disease therapy and mutation classification

AU - Pokorna, Sarka

AU - Khersonsky, Olga

AU - Lipsh-Sokolik, Rosalie

AU - Goldenzweig, Adi

AU - Nielsen, Rebekka

AU - Ashani, Yacov

AU - Peleg, Yoav

AU - Unger, Tamar

AU - Albeck, Shira

AU - Dym, Orly

AU - Tirosh, Asa

AU - Tarayra, Rana

AU - Hocquemiller, Michaël

AU - Laufer, Ralph

AU - Ben-Dor, Shifra

AU - Silman, Israel

AU - Sussman, Joel L

AU - Fleishman, Sarel J

AU - Futerman, Anthony H

N1 - Funding Information: We thank Drs Maxim Itkin and Sergey Malitsky (Lipidomics) and Dr Yael Fridman‐Sirkis (DSF measurements) from the Life Sciences Core Facilities at the Weizmann Institute of Science, Dr Ron Rotkopf and Shani Blumenreich‐Kashani for help with statistical analysis, Yochai Maytal for help with collating data for Table S1 and Chen Yaacobi for creating the GBA cell lines. Research in the Futerman laboratory was supported by a Sponsored Research Agreement between the Weizmann Institute of Science (via Yeda, its technology transfer office) and Lysogene. Research in the Fleishman laboratory was supported by a Consolidator Award from the European Research Council (815379), the Israel Science Foundation (1844), the Dr Barry Sherman Institute for Medicinal Chemistry and by a charitable donation in memory of Sam Switzer. SP was partially supported by the Czech Academy of Sciences (Czech/Israel scientific program). AHF is the Joseph Meyerhoff Professor of Biochemistry at the Weizmann Institute of Science. Publisher Copyright: © 2023 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

PY - 2023/7

Y1 - 2023/7

N2 - Acid-β-glucosidase (GCase, EC 3.2.1.45), the lysosomal enzyme which hydrolyzes the simple glycosphingolipid, glucosylceramide (GlcCer), is encoded by the GBA1 gene. Biallelic mutations in GBA1 cause the human inherited metabolic disorder, Gaucher disease (GD), in which GlcCer accumulates, while heterozygous GBA1 mutations are the highest genetic risk factor for Parkinson's disease (PD). Recombinant GCase (e.g., Cerezyme®) is produced for use in enzyme replacement therapy for GD and is largely successful in relieving disease symptoms, except for the neurological symptoms observed in a subset of patients. As a first step towards developing an alternative to the recombinant human enzymes used to treat GD, we applied the PROSS stability-design algorithm to generate GCase variants with enhanced stability. One of the designs, containing 55 mutations compared to wild type human GCase, exhibits improved secretion and thermal stability. Furthermore, the design has higher enzymatic activity than the clinically used human enzyme when incorporated into an AAV vector, resulting in a larger decrease in the accumulation of lipid substrates in cultured cells. Based on stability-design calculations, we also developed a machine-learning based approach to distinguish benign from deleterious (i.e., disease-causing) GBA1 mutations. This approach gave remarkably accurate predictions of the enzymatic activity of single nucleotide polymorphisms in the GBA1 gene that are not currently associated with GD or PD. This latter approach could be applied to other diseases to determine risk factors in patients carrying rare mutations.

AB - Acid-β-glucosidase (GCase, EC 3.2.1.45), the lysosomal enzyme which hydrolyzes the simple glycosphingolipid, glucosylceramide (GlcCer), is encoded by the GBA1 gene. Biallelic mutations in GBA1 cause the human inherited metabolic disorder, Gaucher disease (GD), in which GlcCer accumulates, while heterozygous GBA1 mutations are the highest genetic risk factor for Parkinson's disease (PD). Recombinant GCase (e.g., Cerezyme®) is produced for use in enzyme replacement therapy for GD and is largely successful in relieving disease symptoms, except for the neurological symptoms observed in a subset of patients. As a first step towards developing an alternative to the recombinant human enzymes used to treat GD, we applied the PROSS stability-design algorithm to generate GCase variants with enhanced stability. One of the designs, containing 55 mutations compared to wild type human GCase, exhibits improved secretion and thermal stability. Furthermore, the design has higher enzymatic activity than the clinically used human enzyme when incorporated into an AAV vector, resulting in a larger decrease in the accumulation of lipid substrates in cultured cells. Based on stability-design calculations, we also developed a machine-learning based approach to distinguish benign from deleterious (i.e., disease-causing) GBA1 mutations. This approach gave remarkably accurate predictions of the enzymatic activity of single nucleotide polymorphisms in the GBA1 gene that are not currently associated with GD or PD. This latter approach could be applied to other diseases to determine risk factors in patients carrying rare mutations.

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DO - 10.1111/febs.16758

M3 - Article

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SN - 1742-464X

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JO - FEBS Journal

JF - FEBS Journal

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ER -

Design of a stable human acid-β-glucosidase: towards improved Gaucher disease therapy and mutation classification

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Bibliographical note

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