A Liquid Chromatography-Quadrupole-Time-of-Flight Mass Spectrometric Assay for the Quantification of Fabry Disease Biomarker Globotriaosylceramide (GB3) in Fabry Model Mouse

Shin, Seok‐Ho; Park, Min‐Ho; Byeon, Jin‐Ju; Lee, Byeong Ill; Park, Yuri; Ko, Ah-Ra; Seong, Mi-ran; Lee, So‐Yeon; Kim, Mi Ra; Seo, Jinwook; Jung, Myung Eun; Jin, Dong‐Kyu; Shin, Young Geun

doi:10.3390/pharmaceutics10020069

Cited by 7 publications

(5 citation statements)

References 30 publications

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“…This may be a result of limited biodistribution of the enzyme to the kidney, but it may also relate to the hydroxylated Gb3 species that are expressed in the kidney and seemingly impervious to ERT. Several studies have explored Gb3 glycolipid profiles in Fabry disease mouse models and the effects of ERTs on these, but the coverage of Gb3 glycolipid species were inconsistent and often not comprehensive (Togawa et al, 2010;Durant et al, 2011;Shin et al, 2018). The most comprehensive study of Gb3 profiles performed on organs from Fabry disease mice found that the di-hydroxylated Gb3 isoforms are quite prevalent in kidney (Ishii et al, 2020).…”

Section: Discussionmentioning

confidence: 99%

A universal GlycoDesign for lysosomal replacement enzymes to improve circulation time and biodistribution

Chen

Tian²,

Yasuda³

et al. 2023

Front. Bioeng. Biotechnol.

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Currently available enzyme replacement therapies for lysosomal storage diseases are limited in their effectiveness due in part to short circulation times and suboptimal biodistribution of the therapeutic enzymes. We previously engineered Chinese hamster ovary (CHO) cells to produce α-galactosidase A (GLA) with various N-glycan structures and demonstrated that elimination of mannose-6-phosphate (M6P) and conversion to homogeneous sialylated N-glycans prolonged circulation time and improved biodistribution of the enzyme following a single-dose infusion into Fabry mice. Here, we confirmed these findings using repeated infusions of the glycoengineered GLA into Fabry mice and further tested whether this glycoengineering approach, Long-Acting-GlycoDesign (LAGD), could be implemented on other lysosomal enzymes. LAGD-engineered CHO cells stably expressing a panel of lysosomal enzymes [aspartylglucosamine (AGA), beta-glucuronidase (GUSB), cathepsin D (CTSD), tripeptidyl peptidase (TPP1), alpha-glucosidase (GAA) or iduronate 2-sulfatase (IDS)] successfully converted all M6P-containing N-glycans to complex sialylated N-glycans. The resulting homogenous glycodesigns enabled glycoprotein profiling by native mass spectrometry. Notably, LAGD extended the plasma half-life of all three enzymes tested (GLA, GUSB, AGA) in wildtype mice. LAGD may be widely applicable to lysosomal replacement enzymes to improve their circulatory stability and therapeutic efficacy.

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Section: Discussionmentioning

confidence: 99%

A universal GlycoDesign for lysosomal replacement enzymes to improve circulation time and biodistribution

Chen

Tian²,

Yasuda³

et al. 2023

Front. Bioeng. Biotechnol.

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“…The concentration of Gb3 was determined by LC‐MS/MS according to a published procedure with modifications 27 …”

Section: Methodsmentioning

confidence: 99%

Defective lysosomal storage in Fabry disease modifies mitochondrial structure, metabolism and turnover in renal epithelial cells

Schumann

Schaller

Belche

et al. 2021

J of Inher Metab Disea

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Fabry disease (FD) is an X-linked lysosomal storage disorder. Deficiency of the lysosomal enzyme alpha-galactosidase (GLA) leads to accumulation of potentially toxic globotriaosylceramide (Gb3) on a multisystem level. Cardiac and cerebrovascular abnormalities as well as progressive renal failure are severe, life-threatening long-term complications. The complete pathophysiology of chronic kidney disease (CKD) in FD and the role of tubular involvement for its progression are unclear.We established human renal tubular epithelial cell lines from the urine of male FD patients and male controls. The renal tubular system is rich in mitochondria and involved in transport processes at high-energy costs. Our studies revealed fragmented mitochondria with disrupted cristae structure in FD patient cells. Oxidative stress levels were elevated and oxidative phosphorylation was upregulated in FD pointing at enhanced energetic needs. Mitochondrial homeostasis and energy metabolism revealed major changes as evidenced by differences in mitochondrial number, energy production and fuel consumption. The changes were accompanied by activation of the autophagy machinery in FD. Sirtuin1, an important sensor of (renal) metabolic stress and modifier of different defense pathways, was highly expressed in FD. Our data show that lysosomal FD impairs mitochondrial function and results in severe disturbance of mitochondrial energy metabolism in renal cells. This insight on a tissue-specific level points to new therapeutic targets which might enhance treatment efficacy.

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“…The presence of Gb3 analogs containing lyso-Gb3 analogs has been suggested in Gla knockout mouse organs (28,29) and the plasma of patients with Fabry disease (19), but this is the first experimental data demonstrating the presence of Gb3 analogs in mouse organs. LC-MS/MS has been widely used to determine Gb3 content in the plasma of patients with Fabry disease (13) and in mouse organs (31,32), and it can detect selected Gb3 isoforms but not Gb3 analogs. In this study, Gb3 analogs constituted <2% of the standard Gb3 from Matreya LLC; however, significant amounts of Gb3 analogs were observed in the heart, kidneys, and liver (approximately 12, 30, and 16%, respectively) of our mouse model (Table 1).…”

Section: Deacylation Of Gb3 From Different Organs Of Gla Tm Tg(cag-a4mentioning

confidence: 99%

Determination of globotriaosylceramide analogs in the organs of a mouse model of Fabry disease

Ishii

Taguchi

Okino

et al. 2020

Journal of Biological Chemistry

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Fabry disease is a heritable lipid disorder caused by the low activity of α-galactosidase A and characterized by the systemic accumulation of globotriaosylceramide (Gb3). Recent studies have reported a structural heterogeneity of Gb3 in Fabry disease, including Gb3 isoforms with different fatty acids and Gb3 analogs with modifications on the sphingosine moiety. However, Gb3 assays are often performed only on the selected Gb3 isoforms. To precisely determine the total Gb3 concentration, here we established two methods for determining both Gb3 isoforms and analogs. One was the deacylation method, involving Gb3 treatment with sphingolipid ceramide N-deacylase, followed by an assay of the deacylated products, globotriaosylsphingosine (lyso-Gb3) and its analogs, by ultra-performance LC coupled to tandem MS (UPLC-MS/MS). The other method was a direct assay established in the present study for 37 Gb3 isoforms and analogs/isoforms by UPLC-MS/MS. Gb3s from the organs of symptomatic animals of a Fabry disease mouse model were mainly Gb3 isoforms and two Gb3 analogs, such as Gb3(+18) containing the lyso-Gb3(+18) moiety and Gb3(−2) containing the lyso-Gb3(−2) moiety. The total concentrations and Gb3 analog distributions determined by the two methods were comparable. Gb3(+18) levels were high in the kidneys (24% of total Gb3) and the liver (13%), and we observed Gb3(−2) in the heart (10%) and the kidneys (5%). These results indicate organ-specific expression of Gb3 analogs, insights that may lead to a deeper understanding of the pathophysiology of Fabry disease.

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A Liquid Chromatography-Quadrupole-Time-of-Flight Mass Spectrometric Assay for the Quantification of Fabry Disease Biomarker Globotriaosylceramide (GB3) in Fabry Model Mouse

Cited by 7 publications

References 30 publications

A universal GlycoDesign for lysosomal replacement enzymes to improve circulation time and biodistribution

A universal GlycoDesign for lysosomal replacement enzymes to improve circulation time and biodistribution

Defective lysosomal storage in Fabry disease modifies mitochondrial structure, metabolism and turnover in renal epithelial cells

Determination of globotriaosylceramide analogs in the organs of a mouse model of Fabry disease

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