Nicotiana benthamianaα-galactosidase A1.1 can functionally complement human α-galactosidase A deficiency associated with Fabry disease

Kassiani Kytidou, Jules Beekwilder, Marta Artola, Eline van Meel, Ruud H.P. Wilbers, Geri F. Moolenaar, Nora Goosen, Maria J. Ferraz, Rebecca Katzy, Patrick Voskamp, Bogdan I. Florea, Cornelis H. Hokke, Herman S. Overkleeft, Arjen Schots, Dirk Bosch, Navraj Pannu, Johannes M.F.G. Aerts

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Abstract

α-Galactosidases (EC 3.2.1.22) are retaining glycosidases that cleave terminal α-linked galactose residues from glycoconjugate substrates. α-Galactosidases take part in the turnover of cell wall-associated galactomannans in plants and in the lysosomal degradation of glycosphingolipids in animals. Deficiency of human α-galactosidase A (α-Gal A) causes Fabry disease (FD), a heritable, X-linked lysosomal storage disorder, characterized by accumulation of globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3). Current management of FD involves enzyme-replacement therapy (ERT). An activitybased probe (ABP) covalently labeling the catalytic nucleophile of α-Gal A has been previously designed to study α-galactosidases for use in FD therapy. Here, we report that this ABP labels proteins in Nicotiana benthamiana leaf extracts, enabling the identification and biochemical characterization of an N. benthamiana α-galactosidase we name here A1.1 (gene accession ID GJZM-1660). The transiently overexpressed and purified enzyme was a monomer lacking N-glycans and was active toward 4-methylumbelliferyl-α-D-galactopyranoside substrate (Km = 0.17 mM) over a broad pH range. A1.1 structural analysis by X-ray crystallography revealed marked similarities with human α-Gal A, even including A1.1's ability to hydrolyze Gb3 and lyso-Gb3, which are not endogenous in plants. Of note, A1.1 uptake into FD fibroblasts reduced the elevated lyso-Gb3 levels in these cells, consistent with A1.1 delivery to lysosomes as revealed by confocal microscopy. The ease of production and the features of A1.1, such as stability over a broad pH range, combined with its capacity to degrade glycosphingolipid substrates, warrant further examination of its value as a potential therapeutic agent for ERT-based FD management.

LanguageEnglish
Pages10042-10058
JournalJournal of Biological Chemistry
Volume293
Issue number26
DOIs
Publication statusPublished - 29 Jun 2018

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Galactosidases
alpha-Galactosidase
Fabry Disease
Tobacco
Enzyme Replacement Therapy
Glycosphingolipids
Substrates
Enzymes
Nucleophiles
Glycoconjugates
Confocal microscopy
Glycoside Hydrolases
X ray crystallography
X Ray Crystallography
Fibroblasts
Disease Management
Lysosomes
Galactose
Confocal Microscopy
Structural analysis

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Kytidou, Kassiani ; Beekwilder, Jules ; Artola, Marta ; van Meel, Eline ; Wilbers, Ruud H.P. ; Moolenaar, Geri F. ; Goosen, Nora ; Ferraz, Maria J. ; Katzy, Rebecca ; Voskamp, Patrick ; Florea, Bogdan I. ; Hokke, Cornelis H. ; Overkleeft, Herman S. ; Schots, Arjen ; Bosch, Dirk ; Pannu, Navraj ; Aerts, Johannes M.F.G. / Nicotiana benthamianaα-galactosidase A1.1 can functionally complement human α-galactosidase A deficiency associated with Fabry disease. In: Journal of Biological Chemistry. 2018 ; Vol. 293, No. 26. pp. 10042-10058.
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title = "Nicotiana benthamianaα-galactosidase A1.1 can functionally complement human α-galactosidase A deficiency associated with Fabry disease",
abstract = "α-Galactosidases (EC 3.2.1.22) are retaining glycosidases that cleave terminal α-linked galactose residues from glycoconjugate substrates. α-Galactosidases take part in the turnover of cell wall-associated galactomannans in plants and in the lysosomal degradation of glycosphingolipids in animals. Deficiency of human α-galactosidase A (α-Gal A) causes Fabry disease (FD), a heritable, X-linked lysosomal storage disorder, characterized by accumulation of globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3). Current management of FD involves enzyme-replacement therapy (ERT). An activitybased probe (ABP) covalently labeling the catalytic nucleophile of α-Gal A has been previously designed to study α-galactosidases for use in FD therapy. Here, we report that this ABP labels proteins in Nicotiana benthamiana leaf extracts, enabling the identification and biochemical characterization of an N. benthamiana α-galactosidase we name here A1.1 (gene accession ID GJZM-1660). The transiently overexpressed and purified enzyme was a monomer lacking N-glycans and was active toward 4-methylumbelliferyl-α-D-galactopyranoside substrate (Km = 0.17 mM) over a broad pH range. A1.1 structural analysis by X-ray crystallography revealed marked similarities with human α-Gal A, even including A1.1's ability to hydrolyze Gb3 and lyso-Gb3, which are not endogenous in plants. Of note, A1.1 uptake into FD fibroblasts reduced the elevated lyso-Gb3 levels in these cells, consistent with A1.1 delivery to lysosomes as revealed by confocal microscopy. The ease of production and the features of A1.1, such as stability over a broad pH range, combined with its capacity to degrade glycosphingolipid substrates, warrant further examination of its value as a potential therapeutic agent for ERT-based FD management.",
author = "Kassiani Kytidou and Jules Beekwilder and Marta Artola and {van Meel}, Eline and Wilbers, {Ruud H.P.} and Moolenaar, {Geri F.} and Nora Goosen and Ferraz, {Maria J.} and Rebecca Katzy and Patrick Voskamp and Florea, {Bogdan I.} and Hokke, {Cornelis H.} and Overkleeft, {Herman S.} and Arjen Schots and Dirk Bosch and Navraj Pannu and Aerts, {Johannes M.F.G.}",
year = "2018",
month = "6",
day = "29",
doi = "10.1074/jbc.RA118.001774",
language = "English",
volume = "293",
pages = "10042--10058",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology",
number = "26",

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Kytidou, K, Beekwilder, J, Artola, M, van Meel, E, Wilbers, RHP, Moolenaar, GF, Goosen, N, Ferraz, MJ, Katzy, R, Voskamp, P, Florea, BI, Hokke, CH, Overkleeft, HS, Schots, A, Bosch, D, Pannu, N & Aerts, JMFG 2018, 'Nicotiana benthamianaα-galactosidase A1.1 can functionally complement human α-galactosidase A deficiency associated with Fabry disease', Journal of Biological Chemistry, vol. 293, no. 26, pp. 10042-10058. https://doi.org/10.1074/jbc.RA118.001774

Nicotiana benthamianaα-galactosidase A1.1 can functionally complement human α-galactosidase A deficiency associated with Fabry disease. / Kytidou, Kassiani; Beekwilder, Jules; Artola, Marta; van Meel, Eline; Wilbers, Ruud H.P.; Moolenaar, Geri F.; Goosen, Nora; Ferraz, Maria J.; Katzy, Rebecca; Voskamp, Patrick; Florea, Bogdan I.; Hokke, Cornelis H.; Overkleeft, Herman S.; Schots, Arjen; Bosch, Dirk; Pannu, Navraj; Aerts, Johannes M.F.G.

In: Journal of Biological Chemistry, Vol. 293, No. 26, 29.06.2018, p. 10042-10058.

Research output: Contribution to journalArticleAcademicpeer-review

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T1 - Nicotiana benthamianaα-galactosidase A1.1 can functionally complement human α-galactosidase A deficiency associated with Fabry disease

AU - Kytidou, Kassiani

AU - Beekwilder, Jules

AU - Artola, Marta

AU - van Meel, Eline

AU - Wilbers, Ruud H.P.

AU - Moolenaar, Geri F.

AU - Goosen, Nora

AU - Ferraz, Maria J.

AU - Katzy, Rebecca

AU - Voskamp, Patrick

AU - Florea, Bogdan I.

AU - Hokke, Cornelis H.

AU - Overkleeft, Herman S.

AU - Schots, Arjen

AU - Bosch, Dirk

AU - Pannu, Navraj

AU - Aerts, Johannes M.F.G.

PY - 2018/6/29

Y1 - 2018/6/29

N2 - α-Galactosidases (EC 3.2.1.22) are retaining glycosidases that cleave terminal α-linked galactose residues from glycoconjugate substrates. α-Galactosidases take part in the turnover of cell wall-associated galactomannans in plants and in the lysosomal degradation of glycosphingolipids in animals. Deficiency of human α-galactosidase A (α-Gal A) causes Fabry disease (FD), a heritable, X-linked lysosomal storage disorder, characterized by accumulation of globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3). Current management of FD involves enzyme-replacement therapy (ERT). An activitybased probe (ABP) covalently labeling the catalytic nucleophile of α-Gal A has been previously designed to study α-galactosidases for use in FD therapy. Here, we report that this ABP labels proteins in Nicotiana benthamiana leaf extracts, enabling the identification and biochemical characterization of an N. benthamiana α-galactosidase we name here A1.1 (gene accession ID GJZM-1660). The transiently overexpressed and purified enzyme was a monomer lacking N-glycans and was active toward 4-methylumbelliferyl-α-D-galactopyranoside substrate (Km = 0.17 mM) over a broad pH range. A1.1 structural analysis by X-ray crystallography revealed marked similarities with human α-Gal A, even including A1.1's ability to hydrolyze Gb3 and lyso-Gb3, which are not endogenous in plants. Of note, A1.1 uptake into FD fibroblasts reduced the elevated lyso-Gb3 levels in these cells, consistent with A1.1 delivery to lysosomes as revealed by confocal microscopy. The ease of production and the features of A1.1, such as stability over a broad pH range, combined with its capacity to degrade glycosphingolipid substrates, warrant further examination of its value as a potential therapeutic agent for ERT-based FD management.

AB - α-Galactosidases (EC 3.2.1.22) are retaining glycosidases that cleave terminal α-linked galactose residues from glycoconjugate substrates. α-Galactosidases take part in the turnover of cell wall-associated galactomannans in plants and in the lysosomal degradation of glycosphingolipids in animals. Deficiency of human α-galactosidase A (α-Gal A) causes Fabry disease (FD), a heritable, X-linked lysosomal storage disorder, characterized by accumulation of globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3). Current management of FD involves enzyme-replacement therapy (ERT). An activitybased probe (ABP) covalently labeling the catalytic nucleophile of α-Gal A has been previously designed to study α-galactosidases for use in FD therapy. Here, we report that this ABP labels proteins in Nicotiana benthamiana leaf extracts, enabling the identification and biochemical characterization of an N. benthamiana α-galactosidase we name here A1.1 (gene accession ID GJZM-1660). The transiently overexpressed and purified enzyme was a monomer lacking N-glycans and was active toward 4-methylumbelliferyl-α-D-galactopyranoside substrate (Km = 0.17 mM) over a broad pH range. A1.1 structural analysis by X-ray crystallography revealed marked similarities with human α-Gal A, even including A1.1's ability to hydrolyze Gb3 and lyso-Gb3, which are not endogenous in plants. Of note, A1.1 uptake into FD fibroblasts reduced the elevated lyso-Gb3 levels in these cells, consistent with A1.1 delivery to lysosomes as revealed by confocal microscopy. The ease of production and the features of A1.1, such as stability over a broad pH range, combined with its capacity to degrade glycosphingolipid substrates, warrant further examination of its value as a potential therapeutic agent for ERT-based FD management.

U2 - 10.1074/jbc.RA118.001774

DO - 10.1074/jbc.RA118.001774

M3 - Article

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EP - 10058

JO - Journal of Biological Chemistry

T2 - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

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