Enzyme kinetics and inhibition parameters of human leukocyte glucosylceramidase

Karataş, Mesut; Dogan, Senol; Spahiu, Emrulla; Ašić, Adna; Bešić, Larisa; Turan, Yusuf

doi:10.1016/j.heliyon.2020.e05191

Cited by 4 publications

(4 citation statements)

References 66 publications

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“…Similar to the K + conductance, the human variants M393T and Q65P also exhibited reduced and enhanced proton conductance, respectively, and the loss-of-function M393T mutation is predicted to result in lysosomal overacidification. Consistently, the PD-associated lysosomal enzyme GBA has an optimal pH range of 4.7 to 5.5 for activity ( 17 ), which prompts us to speculate that M393T potentiates PD by dropping the lysosomal pH below this range. Conversely, the Q65P mutant, which protects against PD, drives larger proton currents and may provide optimal lysosomal function.…”

Section: Discussionmentioning

confidence: 79%

“…Therefore, the PD-associated, loss-of-function mutation M393T may also result in lysosomal overacidification and jeopardize the function of lysosomal enzymes such as GBA. Because GBA requires a narrow pH range of 4.7 to 5.5 for optimal activity ( 17 ), overacidification may lead to reduced GBA activity, which, in turn, may result in the accumulation of α-synuclein and PD pathogenesis.…”

Section: Resultsmentioning

confidence: 99%

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pH regulates potassium conductance and drives a constitutive proton current in human TMEM175

et al. 2022

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Human TMEM175, a noncanonical potassium (K + ) channel in endolysosomes, contributes to their pH stability and is implicated in the pathogenesis of Parkinson’s disease (PD). Structurally, the TMEM175 family exhibits an architecture distinct from canonical potassium channels, as it lacks the typical TVGYG selectivity filter. Here, we show that human TMEM175 not only exhibits pH-dependent structural changes that reduce K + permeation at acidic pH but also displays proton permeation. TMEM175 constitutively conducts K + at pH 7.4 but displays reduced K + permeation at lower pH. In contrast, proton current through TMEM175 increases with decreasing pH because of the increased proton gradient. Molecular dynamics simulation, structure-based mutagenesis, and electrophysiological analysis suggest that K + ions and protons share the same permeation pathway. The M393T variant of human TMEM175 associated with PD shows reduced function in both K + and proton permeation. Together, our structural and electrophysiological analysis reveals a mechanism of TMEM175 regulation by pH.

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Section: Discussionmentioning

confidence: 79%

Section: Resultsmentioning

confidence: 99%

pH regulates potassium conductance and drives a constitutive proton current in human TMEM175

et al. 2022

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“…The concentration of sphingosine released from C18:1 ceramide was obtained by measuring the area under the curve for sphingosine relative to an unnatural sphingosine (C17:1 sphingosine, 100 pmol) used as an internal standard in the assay, using the LC–MS MRM-HR method reported by us . The glucosylceramidase assay was performed using C17:0 glucosylceramide (substrate, 20 μM) for 120 min. The concentration of C17:0 glucosylceramide consumed in this assay was obtained by measuring the area under the curve for C17:0 glucosylceramide in the enzymatic assay relative to the controls (heat-denatured proteomes and substrate only reaction) used in the assay, using the LC–MS MRM-HR method described in this study (see supplementary Table S1).…”

Section: Methodsmentioning

confidence: 99%

Mapping Sphingolipid Metabolism Pathways during Phagosomal Maturation

2021

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Phagocytosis is an important physiological process, which, in higher organisms, is a means of fighting infections and clearing cellular debris. During phagocytosis, detrimental foreign particles (e.g. pathogens and apoptotic cells) are engulfed by phagocytes (e.g. macrophages), enclosed in membrane-bound vesicles called phagosomes, and transported to the lysosome for eventual detoxification. During this well-choreographed process, the nascent phagosome (also called early phagosome, EP) undergoes a series of spatiotemporally regulated changes in its protein and lipid composition and matures into a late phagosome (LP), which subsequently fuses with the lysosomal membrane to form the phagolysosome. While several elegant proteomic studies have identified the role of unique proteins during phagosomal maturation, the corresponding lipidomic studies are sparse. Recently, we reported a comparative lipidomic analysis between EPs and LPs and showed that ceramides are enriched on the LPs. Further, we found that this ceramide accumulation on LPs was orchestrated by ceramide synthase 2, inhibition of which hampers phagosomal maturation. Following up on this study, here, using biochemical assays, we first show that the increased ceramidase activity on EPs also significantly contributes to the accumulation of ceramides on LPs. Next, leveraging lipidomics, we show that de novo ceramide synthesis does not significantly contribute to the ceramide accumulation on LPs, while concomitant to increased ceramides, glucosylceramides are substantially elevated on LPs. We validate this interesting finding using biochemical assays and show that LPs indeed have heightened glucosylceramide synthase activity. Taken together, our studies provide interesting insights and possible new roles of sphingolipid metabolism during phagosomal maturation.

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“…The shipment of blood samples to the reference laboratory should be carried out at 4 °C [105]. The isolation of leukocytes from the whole blood should be completed within 24 h after blood collection using dextran sedimentation or the ammonium chloride lysis method [106][107][108][109]. The pellet of isolated leukocytes can be stored for at least 20 days at − 20 °C before enzyme activities are determined [108].…”

Section: In What Samples Bglu Activity Can Be Measured?mentioning

confidence: 99%

Patient centered guidelines for the laboratory diagnosis of Gaucher disease type 1

Dardis

Michelakakis

Rozenfeld

et al. 2022

Orphanet J Rare Dis

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Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder due to the deficient activity of the acid beta-glucosidase (GCase) enzyme, resulting in the progressive lysosomal accumulation of glucosylceramide (GlcCer) and its deacylated derivate, glucosylsphingosine (GlcSph). GCase is encoded by the GBA1 gene, located on chromosome 1q21 16 kb upstream from a highly homologous pseudogene. To date, more than 400 GBA1 pathogenic variants have been reported, many of them derived from recombination events between the gene and the pseudogene. In the last years, the increased access to new technologies has led to an exponential growth in the number of diagnostic laboratories offering GD testing. However, both biochemical and genetic diagnosis of GD are challenging and to date no specific evidence-based guidelines for the laboratory diagnosis of GD have been published. The objective of the guidelines presented here is to provide evidence-based recommendations for the technical implementation and interpretation of biochemical and genetic testing for the diagnosis of GD to ensure a timely and accurate diagnosis for patients with GD worldwide. The guidelines have been developed by members of the Diagnostic Working group of the International Working Group of Gaucher Disease (IWGGD), a non-profit network established to promote clinical and basic research into GD for the ultimate purpose of improving the lives of patients with this disease. One of the goals of the IWGGD is to support equitable access to diagnosis of GD and to standardize procedures to ensure an accurate diagnosis. Therefore, a guideline development group consisting of biochemists and geneticists working in the field of GD diagnosis was established and a list of topics to be discussed was selected. In these guidelines, twenty recommendations are provided based on information gathered through a systematic review of the literature and two different diagnostic algorithms are presented, considering the geographical differences in the access to diagnostic services. Besides, several gaps in the current diagnostic workflow were identified and actions to fulfill them were taken within the IWGGD. We believe that the implementation of recommendations provided in these guidelines will promote an equitable, timely and accurate diagnosis for patients with GD worldwide.

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Enzyme kinetics and inhibition parameters of human leukocyte glucosylceramidase

Cited by 4 publications

References 66 publications

pH regulates potassium conductance and drives a constitutive proton current in human TMEM175

pH regulates potassium conductance and drives a constitutive proton current in human TMEM175

Mapping Sphingolipid Metabolism Pathways during Phagosomal Maturation

Patient centered guidelines for the laboratory diagnosis of Gaucher disease type 1

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