Self-association of human beta-galactocerebrosidase: Dependence on pH, salt, and surfactant.

Krabbe disease, also known as globoid cell leukodystrophy, is a rare genetic neurodegenerative disease caused by a deficiency of the galactocerebrosidase enzyme. To understand the association status of human beta-galactocerebrosidase (hGALC) in solution, we employed analytical ultracentrifugation (AUC). Our AUC results show that hGALC has a tendency for reversible self-association. Self-association decreases as the concentration of sodium chloride increases from 50 to 500 mM. This indicates that ionic interactions are involved in the association. The association is also dependent on pH, and high order oligomerization decreases as the pH increases from 4.5 to 7.5. Taken together, our results indicate that hGALC has the highest tendency for oligomerization at physiological ionic strength and pH (lysosomal lumen). This is the first report describing the self-associating property of hGALC in solution.

Structure of an endogalactosylceramidase from Rhodococcus hoagii 103S reveals the molecular basis of its substrate specificity.

Endoglycoceramidases (EGCs) are family 5 glycoside hydrolases that catalyze hydrolysis of the glycosidic linkages between the oligosaccharide and ceramide moieties of glycosphingolipids. Three orthologs of EGCs, each with distinct substrate specificity, have been identified to date, including EGC-I, EGC-II, and EGALC. Although the structures of EGC-I and EGC-II have been reported, the substrate preference mechanism of EGC enzymes remains unclear. Here, we determined the crystal structure of EGALC from Rhodococcus hoagii 103S at a resolution of 1.20 Å. Distinct from EGC-I and EGC-II, which both have a tunnel-like substrate binding site, the structure of EGALC accommodates substrates in a long groove. Further, the oligosaccharide-binding region of groove could be divided into two small pockets that separately bind to the Gal1 and to the Gal3/Gla3 present in 6-gala series substrates. Structural and sequence comparisons of EGC enzymes revealed that the conformation and length of their Nß8-Lα1 regions are crucial in determining the architectures of their specific substrate binding sites. Importantly, molecular docking analyses indicate that the substrate specificity of each EGC is mainly derived from the complementarity of its active site groove/tunnel with substrates adopting particular conformations. Our study provide insights for understanding the catalytic mechanism of EGALC, which will help protein engineering for improving the substrate preference and catalytic efficiency of EGC enzymes toward important glycosphingolipid substrates.

Cell-based high-throughput screening identifies galactocerebrosidase enhancers as potential small-molecule therapies for Krabbe's disease.

Krabbe's disease, also known as globoid cell leukodystrophy (GLD), is a lysosomal storage disease caused by the deficiency of the lysosomal enzyme ß-galactocerebrosidase (GALC), resulting in severe neurological manifestations related to demyelination secondary to elevated galactosylsphingosine (psychosine) with its subsequent cytotoxicity. The only available treatment is hematopoietic stem cell transplantation, which delays disease onset but does not prevent long-term neurological manifestations. This article describes the identification of small molecules that enhance mutant GALC activity, identified by quantitative cell-based high-throughput screening (qHTS). Using a specific neurologically relevant murine cell line (145M-Twi) modified to express common human hGALC-G270D mutant, we were able to detect GALC activity in a 1,536-well microplate format. The qHTS of approximately 46,000 compounds identified three small molecules that showed significant enhancements of residual mutant GALC activity in primary cell lines from GLD patients. These compounds were shown to increase the levels of GALC-G270D mutant in the lysosomal compartment. In kinetic assessments, these small molecules failed to disturb the GALC kinetic profile under acidic conditions, which is highly desirable for folding-assisting molecules operating in the endoplasmic reticulum and not affecting GALC catalytic properties in the lysosomal compartment. In addition, these small molecules rescued the decreased GALC activity at neutral pH and partially stabilized GALC under heat-denaturating conditions. These drug-like compounds can be used as the starting point to develop novel small-molecule agents to treat the progressive neurodegenerative course of GLD. © 2016 Wiley Periodicals, Inc.

Molecular Mechanisms of Disease Pathogenesis Differ in Krabbe Disease Variants.

Krabbe disease is a severe, fatal neurodegenerative disorder caused by defects in the lysosomal enzyme galactocerebrosidase (GALC). The correct targeting of GALC to the lysosome is essential for the degradation of glycosphingolipids including the primary lipid component of myelin. Over 100 different mutations have been identified in GALC that cause Krabbe disease but the mechanisms by which they cause disease remain unclear. We have generated monoclonal antibodies against full-length human GALC and used these to monitor the trafficking and processing of GALC variants in cell-based assays and by immunofluorescence microscopy. Striking differences in the secretion, processing and endosomal targeting of GALC variants allows the classification of these into distinct categories. A subset of GALC variants are not secreted by cells, not proteolytically processed, and remain trapped in the ER; these are likely to cause disease due to protein misfolding and should be targeted for pharmacological chaperone therapies. Other GALC variants can be correctly secreted by cells and cause disease due to catalytic defects in the enzyme active site, inappropriate post-translational modification or a potential inability to bind essential cofactors. The classification of disease pathogenesis presented here provides a molecular framework for appropriate targeting of future Krabbe disease therapies.

Chaperone therapy for Krabbe disease: potential for late-onset GALC mutations.

Krabbe disease is an autosomal recessive leukodystrophy caused by a deficiency of the galactocerebrosidase (GALC) enzyme. Hematopoietic stem cells transplantation is the only available treatment option for pre-symptomatic patients. We have previously reported the chaperone effect of N-octyl-4-epi-ß-valienamine (NOEV) on mutant GM1 ß-galactosidase proteins, and in a murine GM1-gangliosidosis model. In this study, we examined its chaperone effect on mutant GALC proteins. We found that NOEV strongly inhibited GALC activity in cell lysates of GALC-transfected COS1 cells. In vitro NOEV treatment stabilized GALC activity under heat denaturation conditions. We also examined the effect of NOEV on cultured COS1 cells expressing mutant GALC activity and human skin fibroblasts from Krabbe disease patients: NOEV significantly increased the enzyme activity of mutants of late-onset forms. Moreover, we confirmed that NOEV could enhance the maturation of GALC precursor to its mature active form. Model structural analysis showed NOEV binds to the active site of human GALC protein. These results, for the first time, provide clear evidence that NOEV is a chaperone with promising potential for patients with Krabbe disease resulting from the late-onset mutations.

Structural snapshots illustrate the catalytic cycle of ß-galactocerebrosidase, the defective enzyme in Krabbe disease.

Glycosphingolipids are ubiquitous components of mammalian cell membranes, and defects in their catabolism by lysosomal enzymes cause a diverse array of diseases. Deficiencies in the enzyme ß-galactocerebrosidase (GALC) cause Krabbe disease, a devastating genetic disorder characterized by widespread demyelination and rapid, fatal neurodegeneration. Here, we present a series of high-resolution crystal structures that illustrate key steps in the catalytic cycle of GALC. We have captured a snapshot of the short-lived enzyme-substrate complex illustrating how wild-type GALC binds a bona fide substrate. We have extensively characterized the enzyme kinetics of GALC with this substrate and shown that the enzyme is active in crystallo by determining the structure of the enzyme-product complex following extended soaking of the crystals with this same substrate. We have also determined the structure of a covalent intermediate that, together with the enzyme-substrate and enzyme-product complexes, reveals conformational changes accompanying the catalytic steps and provides key mechanistic insights, laying the foundation for future design of pharmacological chaperones.

Multiple rewards from a treasure trove of novel glycoside hydrolase and polysaccharide lyase structures: new folds, mechanistic details, and evolutionary relationships.

Recent progress in three-dimensional structure analyses of glycoside hydrolases (GHs) and polysaccharide lyases (PLs), the historically relevant enzyme classes involved in the cleavage of glycosidic bonds of carbohydrates and glycoconjugates, is reviewed. To date, about 80% and 95% of the GH and PL families, respectively, have a representative crystal structure. New structures have been determined for enzymes acting on plant cell wall polysaccharides, sphingolipids, blood group antigens, milk oligosaccharides, N-glycans, oral biofilms and dietary seaweeds. Some GH enzymes have very unique catalytic residues such as the Asp-His dyad. New methods such as high-speed atomic force microscopy and computational simulation have opened up a path to investigate both the dynamics and the detailed molecular interactions displayed by these enzymes.

A high-throughput screening assay using Krabbe disease patient cells.

Globoid cell leukodystrophy (GLD) or Krabbe disease is a lysosomal disease caused by ß-galactocerebrosidase (GALC) deficiency resulting in a rapidly progressive neurodegenerative disorder. Unfortunately, the only available treatment is hematopoietic bone marrow transplantation, which prevents its fulminant manifestation but without treating further neurological manifestations. Here, we describe the development of a cellular high-throughput screening (HTS) assay using GLD patient fibroblasts to screen for small molecules that enhance the residual mutant GALC enzymatic activity. Small molecules have substantial therapeutic potential in GLD because they are more prone to cross the blood-brain barrier, reaching the neuronal affected cells. The transformation of primary skin fibroblasts with SV40 large T antigen has been shown to maintain the biochemical characteristics of the GLD cells and generates sufficient cells for the HTS. Using a specific fluorescent substrate, residual GALC activity from an SV40-transformed GLD patient fibroblast was measurable in high-density microplates. The pilot quantitative HTS against a small compound collection showed robust statistics. The small molecules that showed active concentration-response curves were further studied in primary GLD fibroblasts. This cell-based HTS assay demonstrates the feasibility of employing live GLD patient cells to identify therapeutic agents that can potentially be used for the treatment of this progressive neurodegenerative disease.

Insights into Krabbe disease from structures of galactocerebrosidase.

Krabbe disease is a devastating neurodegenerative disease characterized by widespread demyelination that is caused by defects in the enzyme galactocerebrosidase (GALC). Disease-causing mutations have been identified throughout the GALC gene. However, a molecular understanding of the effect of these mutations has been hampered by the lack of structural data for this enzyme. Here we present the crystal structures of GALC and the GALC-product complex, revealing a novel domain architecture with a previously uncharacterized lectin domain not observed in other hydrolases. All three domains of GALC contribute residues to the substrate-binding pocket, and disease-causing mutations are widely distributed throughout the protein. Our structures provide an essential insight into the diverse effects of pathogenic mutations on GALC function in human Krabbe variants and a compelling explanation for the severity of many mutations associated with fatal infantile disease. The localization of disease-associated mutations in the structure of GALC will facilitate identification of those patients that would be responsive to pharmacological chaperone therapies. Furthermore, our structure provides the atomic framework for the design of such drugs.

Identification and characterization of 15 novel GALC gene mutations causing Krabbe disease.

The characterization of the underlying GALC gene lesions was performed in 30 unrelated patients affected by Krabbe disease, an autosomal recessive leukodystrophy caused by the deficiency of lysosomal enzyme galactocerebrosidase. The GALC mutational spectrum comprised 33 distinct mutant (including 15 previously unreported) alleles. With the exception of 4 novel missense mutations that replaced evolutionarily highly conserved residues (p.P318R, p.G323R, p.I384T, p.Y490N), most of the newly described lesions altered mRNA processing. These included 7 frameshift mutations (c.61delG, c.408delA, c.521delA, c.1171_1175delCATTCinsA, c.1405_1407delCTCinsT, c.302_308dupAAATAGG, c.1819_1826dupGTTACAGG), 3 nonsense mutations (p.R69X, p.K88X, p.R127X) one of which (p.K88X) mediated the skipping of exon 2, and a splicing mutation (c.1489+1G>A) which induced the partial skipping of exon 13. In addition, 6 previously unreported GALC polymorphisms were identified. The functional significance of the novel GALC missense mutations and polymorphisms was investigated using the MutPred analysis tool. This study, reporting one of the largest genotype-phenotype analyses of the GALC gene so far performed in a European Krabbe disease cohort, revealed that the Italian GALC mutational profile differs significantly from other populations of European origin. This is due in part to a GALC missense substitution (p.G553R) that occurs at high frequency on a common founder haplotype background in patients originating from the Naples region.

Galactocerebrosidase from human urine: purification and partial characterization.

Galactocerebrosidase (GALC, EC 3.2.1.46) was purified from human urine by a series of hydrophobic affinity column chromatography steps. The activity was enriched 176,000-fold from concentrated urine by only four columns, including octyl Sepharose, hydroxylapatite, butyl Sepharose and ethyl-agarose. The overall recovery was about 20% but only low amounts were obtained due to its low abundance. The estimated final specific activities of several batches were between 1 and 2 mmol/h per mg protein. The final purified fractions were essentially free of other lysosomal enzyme activities. The most pure fractions showed a series of bands between 50 and 53 kDa on sodium dodecylsulfate-polyacrylamide gel electrophoresis which were determined to have identical N-terminal amino acid sequence. In addition, gel filtration of partially purified GALC after disassociation showed one peak of activity estimated to have a molecular mass near 50 kDa. GALC was also purified from human brain and human placenta using the same methods demonstrating the usefulness of this procedure in obtaining GALC from solid human tissues. In addition to the bands migrating near 50 kDa from urine, there were also bands at 80 kDa and 30 kDa in some preparations. By N-terminal sequencing and the use of antipeptide antibodies, the 80 kDa band was demonstrated to have the same N-terminal amino acids as the 50-53 kDa bands. The 30 kDa band had a unique sequence. The relationship between the different molecular weight species remains to be determined. The purification of GALC and the securing of amino acid sequence information will aid in the cloning of the GALC gene. This enzyme is deficient in human patients with Krabbe disease and several animal species.