PXD057472-1

PXD057472 is an original dataset announced via ProteomeXchange.

Title	Quantification profiles of enzyme activity, secretion and psychosine levels of Krabbe disease galactosylceramidase missense variants
Description	Krabbe disease (KD) is an autosomal recessive lysosomal storage disorder caused by loss-offunction 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 lowactivity 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.
HostingRepository	PRIDE
AnnounceDate	2025-05-19
AnnouncementXML	Submission_2025-05-19_11:59:21.345.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Ying Wai Lam
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	TMT6plex-126 reporter+balance reagent acylated residue; monohydroxylated residue; iodoacetamide derivatized residue
Instrument	Orbitrap Fusion

Revision	Datetime	Status	ChangeLog Entry
0	2024-11-03 21:50:24	ID requested
⏵ 1	2025-05-19 11:59:21	announced

Dataset with its publication pending

submitter keyword: secretion, missense mutation variant, galactosylceramidase, lysosomal storage disorder, lysosome, GALC, psychosine, crispr-cas9 knockout cell, protein trafficking, Krabbe disease, expression study, genotype-phenotype correlation, galactosylsphingosine,Globoid cell leukodystrophy

Chris Lee
contact affiliation	Biomedical Research Institute of New Jersey (BRInj) 140 East Hanover Ave Cedar Knolls New Jersey 07927
contact email	chrislee@brinj.org
lab head
Ying Wai Lam
contact affiliation	Vermont Biomedical Research Network Proteomics Facility Department of Biology University of Vermont
contact email	ylam@uvm.edu
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD057472
     1. Label: PRIDE project
     2. Name: Quantification profiles of enzyme activity, secretion and psychosine levels of Krabbe disease galactosylceramidase missense variants