Abstract
Krabbe disease is a rare and severe lysosomal disorder affecting the white matter of the central and peripheral nervous system. It is characterized by neurodegeneration, with the most common form being infantile Krabbe disease, typically diagnosed within the first year of life. This autosomal-recessive disease is caused by mutations in the GALC gene, which encodes the lysosomal enzyme β-galactocerebrosidase. This study focuses on a β-galactocerebrosidase variant, with Thr112Ala identified as a homozygous mutation in a patient with infantile Krabbe disease. To understand the structural effects of this mutation, we conducted all-atom molecular dynamics simulations of both the mutant and wild-type (wt) enzymes at cytosolic (pH 7.0) and lysosomal pH (pH 4.5), as β-galactocerebrosidase is localized in the lysosome. The results showed differences in protein flexibility, the hydrogen bond network, and the stability of secondary structure elements between the wild type and mutant enzymes. Additionally, the mutation affected the size of the substrate-binding pocket at lysosomal pH, even though the mutation site is not part of the active/binding site of the enzyme. These findings provide valuable insights into how the mutation impacts the structure of β-galactocerebrosidase in the lysosomal environment, contributing to the understanding of Krabbe disease's molecular mechanisms.
Peng H, Lam YW, Lau KF, Zhou Z, Herdt AR, Gelb MH, Lee CW. Quantification profiles of enzyme activity, secretion, and psychosine levels of Krabbe disease galactosylceramidase missense variants. J Biol Chem. 2025 Jul;301(7):110315. doi: 10.1016/j.jbc.2025.110315. Epub 2025 May 29. PMID: 40449593; PMCID: PMC12256330.
Abstract
Krabbe disease is an autosomal recessive, demyelinating disorder caused by mutations in the GALC gene. Missense mutation variants (MMVs) account for most pathogenic alleles in patients; however, their mechanistic implications and correlations to clinical phenotype remain unclear. To address these questions, we generated a GALC knockout human oligodendrocytic cell line to conduct a robust GALC-MMVs expression study using a panel of 31 GALC-MMVs. Twenty-six clinically relevant variants dramatically reduced enzyme activity (92-100%). Notably, residual GALC activity strongly correlated with the age of disease-onset in reported cases (Pearson's r > 0.94, p < 0.0001), suggesting that enzyme activity resulting from MMV expression in this model may serve as a readout for clinical prognostication. In addition, we identified p.I562T, a predominant pseudodeficiency variant in the newborn screening programs, which synergistically impairs protein function and likely triggers disease-onset when inherited co-allelic with certain MMVs. We also identified MMVs that increased protein retention intracellularly and/or decreased secretion. This quantitative analysis of misfolding characteristics could be valuable for identifying MMVs amenable to pharmacological chaperone therapy. Finally, we observed an inverse correlation between residual GALC activity and endogenous psychosine levels in the MMV panel. Given the importance of psychosine as a biomarker for diagnosis and newborn screening, the psychosine accumulation phenotype in our model highlights its potential use for drug discovery. Overall, this study provides a comprehensive overview of the functional deficits and mis-trafficking caused by GALC-MMVs, deepens our understanding of molecular genetics and genotype-phenotype correlations in Krabbe disease, and highlights the potential of our platform for genetic and therapeutic applications.
Peng H, Lam YW, Zhou Z, Herdt AR, Gelb MH, Lee CW. Expression study of Krabbe Disease GALC missense variants - Insights from quantification profiles of residual enzyme activity, secretion and psychosine levels. bioRxiv [Preprint]. 2024 Oct 17:2024.10.17.618938. doi: 10.1101/2024.10.17.618938. Update in: J Biol Chem. 2025 Jul;301(7):110315. doi: 10.1016/j.jbc.2025.110315. PMID: 39464077; PMCID: PMC11507934.
Abstract
Krabbe disease (KD) is an autosomal recessive lysosomal storage disorder caused by loss-of-function mutations in the GALC gene, which encodes for the enzyme galactosylceramidase (GALC). GALC is crucial for myelin metabolism. Functional deficiency of GALC leads to toxic accumulation of psychosine, dysfunction and death of oligodendrocytes, and eventual brain demyelination. To date, 46 clinically-relevant, pathogenic GALC missense mutations (MMs) have been identified in KD patients. These MMs are present in ∼70% of KD cases reported over 8 published studies between 1996 - 2019. However, the mechanisms by which these MMs lead to GALC functional deficiency and their correlations with clinical phenotype remain poorly understood. To address this, we generated a GALC -knockout human oligodendrocytic cell line (MO3.13/ GALC -KO) using CRISPR-Cas9 method to assess GALC function and GALC secretion. We evaluated 5 polymorphic and 31 clinically-relevant MM variants (MMVs) using transient expression assays. Our results showed that 26 MMVs, including 10 co-variants with p.I562T, reduced GALC activity by 92% - 100% compared to wild-type GALC (WT-GALC). MMVs from infantile-onset KD patients produced < 2% of WT activity, whereas those associated with juvenile- and adult-onset cases retained up to 7% of WT activity. Residual GALC activity was correlated with mature, lysosomal GALC protein levels (Pearson r = 0.93, P<0.0001). Many low-activity MMVs did not correspondingly impair GALC secretion. Twenty-one of the 26 low-activity MMVs showed a 21% - 100% reduction in sec-GALC levels, indicating varying degrees of GALC mis-trafficking among these variants. Importantly, GALC activity among MMVs strongly correlates with clinical disease severity, based on the age of symptom onset in patients with either homozygous MM (Pearson r = 0.98, P<0.0001, n = 7) or compound heterozygous (Pearson r = 0.94, P<0.0001, n = 12) MM-null mutation genotypes. Thus, our data suggests that GALC activity could serve as a prognostic disease indicator under specific experimental conditions. We further investigated the impact of pathogenic MMVs on psychosine accumulation, a key biomarker for KD. Psychosine levels were 21-fold higher in mock control cells compared to WT-GALC transfected cells (mock = 0.349 pmol/mg, WT-GALC = 0.016 pmol/mg), but negatively correlated with GALC activity among pathogenic MMVs (Pearson r = -0.63, P < 0.01, n = 15). Although psychosine levels were higher in most MMVs associated with infantile-onset KD, no significant correlations with clinical onset were detected. Overall, our study provides a comprehensive quantitative analysis of the functional deficits and mis-trafficking associated with clinically-relevant GALC MMVs, enhancing our understanding of the molecular genetics and genotype-phenotype correlations of the GALC gene in Krabbe disease.
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