Glycan degradation promotes macroautophagy.

Macroautophagy promotes cellular homeostasis by delivering cytoplasmic constituents to lysosomes for degradation [Mizushima, Nat. Cell Biol. 20, 521-527 (2018)]. However, while most studies have focused on the mechanisms of protein degradation during this process, we report here that macroautophagy also depends on glycan degradation via the glycosidase, α-l-fucosidase 1 (FUCA1), which removes fucose from glycans. We show that cells lacking FUCA1 accumulate lysosomal glycans, which is associated with impaired autophagic flux. Moreover, in a mouse model of fucosidosis-a disease characterized by inactivating mutations in FUCA1 [Stepien et al., Genes (Basel) 11, E1383 (2020)]-glycan and autophagosome/autolysosome accumulation accompanies tissue destruction. Mechanistically, using lectin capture and mass spectrometry, we identified several lysosomal enzymes with altered fucosylation in FUCA1-null cells. Moreover, we show that the activity of some of these enzymes in the absence of FUCA1 can no longer be induced upon autophagy stimulation, causing retardation of autophagic flux, which involves impaired autophagosome-lysosome fusion. These findings therefore show that dysregulated glycan degradation leads to defective autophagy, which is likely a contributing factor in the etiology of fucosidosis.

A mouse model for fucosidosis recapitulates storage pathology and neurological features of the milder form of the human disease.

Fucosidosis is a rare lysosomal storage disorder caused by the inherited deficiency of the lysosomal hydrolase α-L-fucosidase, which leads to an impaired degradation of fucosylated glycoconjugates. Here, we report the generation of a fucosidosis mouse model, in which the gene for lysosomal α-L-fucosidase (Fuca1) was disrupted by gene targeting. Homozygous knockout mice completely lack α-L-fucosidase activity in all tested organs leading to highly elevated amounts of the core-fucosylated glycoasparagine Fuc(α1,6)-GlcNAc(ß1-N)-Asn and, to a lesser extent, other fucosylated glycoasparagines, which all were also partially excreted in urine. Lysosomal storage pathology was observed in many visceral organs, such as in the liver, kidney, spleen and bladder, as well as in the central nervous system (CNS). On the cellular level, storage was characterized by membrane-limited cytoplasmic vacuoles primarily containing water-soluble storage material. In the CNS, cellular alterations included enlargement of the lysosomal compartment in various cell types, accumulation of secondary storage material and neuroinflammation, as well as a progressive loss of Purkinje cells combined with astrogliosis leading to psychomotor and memory deficits. Our results demonstrate that this new fucosidosis mouse model resembles the human disease and thus will help to unravel underlying pathological processes. Moreover, this model could be utilized to establish diagnostic and therapeutic strategies for fucosidosis.

Associations between neurologic dysfunction and lesions in canine fucosidosis.

Canine fucosidosis in English Springer spaniels is the only animal model of the neurovisceral lysosomal storage disease fucosidosis available for preclinical therapeutic trials. For this reason, it is crucial to identify critical time points in disease progression, and if there are particular lesions associated with specific aspects of neurologic dysfunction. Historical records of 53 canine fucosidosis cases from 1979 to 2009 containing a neurologic dysfunction score assessing motor, behavioral and sensory dysfunction were interrogated by statistical analysis. Motor and behavioral dysfunction scores assessing gait deficits and apprehensive behavior first significantly increased at 12-17 months, and increased at each 6-month interval thereafter. Sensory dysfunction scores, assessing hearing loss, balance and vision deterioration, did not significantly increase until 18-23 months, and coincided with a rapid decline in neurologic function. Regression analysis incorporating published neuropathology data, measured by image analysis, identified neuroinflammation and apoptotic cell death as significant informative predictors of increasing neurologic dysfunction. These findings indicate that the level of neuropathology required to induce consistent and conspicuous clinical signs in canine fucosidosis is reached by approximately 12 months of age in the absence of other disease processes. Significant association between neuroinflammation and apoptotic cell death also suggests that specifically targeting these lesions combined with enzyme replacement in future studies may reduce disease burden in fucosidosis. Overall, examining this historical clinical data to identify associations between the extent of neuropathology and degree of clinical dysfunction provides a useful reference tool for monitoring disease and evaluating therapeutic trials conducted in canine fucosidosis.

Oligodendrocyte loss during the disease course in a canine model of the lysosomal storage disease fucosidosis.

Hypomyelination is a poorly understood feature of many neurodegenerative lysosomal storage diseases, including fucosidosis in children and animals. To gain insight into hypomyelination in fucosidosis, we investigated lysosomal storage, oligodendrocyte death, and axonal and neuron loss in CNS tissues of fucosidosis-affected dogs aged 3 weeks to 42 months using immunohistochemistry, electron microscopy, and gene expression assays. Vacuole accumulation in fucosidosis oligodendrocytes commenced by 5 weeks of age; all oligodendrocytes were affected by 16 weeks. Despite progressive vacuolation, mature oligodendrocyte loss by apoptosis (caspase-6 positive) in the corpus callosum and cerebellar white matter stabilized by 16 weeks, with no further subsequent loss. Axonal neurofilament loss progressed only in late disease, suggesting that disturbed axon-oligodendrocyte interactions are unlikely to be the primary cause of hypomyelination. A 67% decline in the number of Purkinje cell layer oligodendrocytes coincided with a 67% increase in the number of caspase-6-positive Purkinje cells at 16 weeks, suggesting that early oligodendrocyte loss contributes to Purkinje cell apoptosis. Fucosidosis hypomyelination appeared to follow normal spatiotemporal patterns of myelination, with greater loss of oligodendrocytes and larger downregulation of CNP, MAL, and PLP1 genes at 16 weeks in the cerebellum versus the frontal cortex. These studies suggest that survival of oligodendrocytes in fucosidosis is limited during active myelination, although the mechanisms remain unknown.

Towards a selected reaction monitoring mass spectrometry fingerprint approach for the screening of oligosaccharidoses.

The oligosaccharidoses are a group of metabolic disorders resulting from a deficiency in enzymes responsible for the catabolism of protein bound oligosaccharides and are typified by the accumulation of corresponding sugars in the urine. Screening is typically accomplished using thin layer chromatography. However, analyte specificity can be a problem and thus complicate interpretation of results. For this reason we developed a mixed mode liquid chromatography tandem mass spectrometry assay for the screening of the oligosaccharidoses which potentially mitigates many of the problems associated with thin layer chromatography. Samples from patients previously diagnosed with I-Cell disease, mannosidosis, Pompe, galactosialidosis, and fucosidosis were derivatized with 3-methyl-1-phenyl-2-pyrazolin-5-one and subjected to analysis by liquid chromatography tandem mass spectrometry. Results were compared to normal control samples. Preliminary results suggest that each oligosaccharidoses produces a unique selected reaction monitoring fingerprint and that the developed method may be an effective screening and diagnostic tool for these disorders.

Fucosidosis: MRI and MRS findings.

Fucosidosis is a rare, autosomal recessive lysosomal storage disease in which fucose-containing glycolipids, glycoproteins, and oligosaccharides accumulate in tissues as a consequence of alpha-L: -fucosidase enzyme deficiency. We present the MR imaging findings of diffuse white-matter hyperintensity and pallidal curvilinear streak hyperintensity in a 6-year-old Caucasian girl with a diagnosis of fucosidosis based on cDNA isolated from skin fibroblasts. This report also includes the MRS findings of a decreased N-acetylaspartate/choline ratio together with an abnormal peak at 3.8 ppm which expand the knowledge of the neuroradiological spectrum of this rare disease.

Crystal structure of Thermotoga maritima alpha-L-fucosidase. Insights into the catalytic mechanism and the molecular basis for fucosidosis.

Fucosylated glycoconjugates are involved in numerous biological events, and alpha-l-fucosidases, the enzymes responsible for their processing, are therefore of crucial importance. Deficiency in alpha-l-fucosidase activity is associated with fucosidosis, a lysosomal storage disorder characterized by rapid neurodegeneration, resulting in severe mental and motor deterioration. To gain insight into alpha-l-fucosidase function at the molecular level, we have determined the crystal structure of Thermotoga maritima alpha-l-fucosidase. This enzyme assembles as a hexamer and displays a two-domain fold, composed of a catalytic (beta/alpha)(8)-like domain and a C-terminal beta-sandwich domain. The structures of an enzyme-product complex and of a covalent glycosyl-enzyme intermediate, coupled with kinetic and mutagenesis studies, allowed us to identify the catalytic nucleophile, Asp(244), and the Brønsted acid/base, Glu(266). Because T. maritima alpha-l-fucosidase occupies a unique evolutionary position, being far more closely related to the mammalian enzymes than to any other prokaryotic homolog, a structural model of the human enzyme was built to document the structural consequences of the genetic mutations associated with fucosidosis.

Recombinant canine alpha-l-fucosidase: expression, purification, and characterization.

Canine fucosidosis has proven to be an excellent large animal model both for the equivalent human disorder and, in more general terms, for the central nervous system pathology found in many of the lysosomal storage disorders. Most importantly studies in this animal model were among the first to convincingly show that bone marrow transplantation could successfully modify the course of clinical central nervous system disease and to define some of the important parameters for successful treatment. In order to evaluate other, more generally applicable routes to treatment of central nervous system disease in the lysosomal storage disorders we have expressed recombinant canine alpha-l-fucosidase (rcFUC) in Chinese hamster ovary and Madin-Darby canine kidney cells to levels of between 2 and 13 mg/liter of culture medium and purified the enzyme to apparent homogeneity by affinity chromatography on fucosylamine-linked agarose. rcFUC is composed of subunits of M(r) 50 kDa and the native enzyme is a homotrimer of M(r) 156 kDa. Kinetic properties of rcFUC were similar to those of FUC isolated from both human and dog liver. rcFUC was shown to be effective in correcting the storage phenotype of human fucosidosis cells after endocytosis via the mannose-6-phosphate-receptor-mediated pathway. It was also shown to degrade fucosylated storage products isolated from affected dog brain. The availability of large amounts of rcFUC will allow us to explore ways of extending the proven efficacy of enzyme replacement therapy to the treatment of central nervous system pathology using the fucosidosis dog as a model system.

Degradation of blood group A glycolipid A-6-2 by normal and mutant human skin fibroblasts.

The degradation of blood group glycolipid A-6-2 (GalNAc(alpha1-->3)[Fuc alpha1-->2]Gal(beta1-->4)GlcNAc(beta1-->3)Gal(beta1-->4)Glc(beta1-->1')C er, IV2-alpha-fucosyl-IV3-alpha-N-acetylgalactosaminylneolact otetraosylceramide), tritium-labeled in its ceramide moiety, was studied in situ, in skin fibroblast cultures from normal controls, from patients with defects of lysosomal alpha-N-acetylgalactosaminidase, and from patients with other lysosomal storage diseases. Uptake of the glycolipid with apolipoprotein E-coated liposomes was linear with time and with the amount of glycolipid added. In normal cells, the expected array of less polar products and some lipids resulting from re-using the liberated sphingosine, mainly sphingomyelin and phosphatidylcholine, were formed. In alpha-N-acetylgalactosaminidase-deficient cells, the glycolipid was virtually not degraded; product formation was less than 2% of the normal control rate, suggesting that blood group A-active glycolipids contribute as storage compounds to the pathogenesis of this disease. The expected accumulation of degradation intermediates was seen in fucosidosis, and in Sandhoff, Gaucher, and Farber disease cells, whereas normal turnover rates were found in Tay-Sachs disease cells, G(M2) activator-deficient (variant AB of G(M2) gangliosidosis) and in sulfatide activator- (sap-B-) deficient cells. In G(M1) gangliosidosis and in sap precursor-deficient cells, the lysosomal glycolipid catabolism was found to be strongly retarded; accumulation of individual products could not be seen. Skin fibroblasts from patients with alpha-N-acetylgalactosaminidase deficiency (Schindler disease) cannot degrade the major blood group A glycolipid.

Fucosidosis: genetic and biochemical analysis of eight cases.

The molecular basis of the deficiency of alpha-L-fucosidase has been investigated in eight patients who had been diagnosed clinically and enzymatically as suffering from the autosomal recessive lysosomal storage disease fucosidosis. None of the patients had a deletion or gross alteration of the alpha-L-fucosidase gene (FUCA1). Single strand conformation polymorphism (SSCP) analysis followed by direct sequencing of amplified exons and flanking regions identified putative disease causing mutations in six of the patients, who had severe forms of the disease and very low residual alpha-L-fucosidase activity and protein. They were a 10 bp deletion in exon 1 (E113fs), a 1 bp deletion at position -2 of intron 2 (S216fs), a g-->a transition at IVS5+1, point mutations W183X and N329Y in exons 3 and 6, respectively, and a compound allele consisting of a point mutation in the signal peptide in exon 1, P5R, and a 1 bp insertion in exon 6 (Y330fs). One patient in whom an SSCP change was not detected had residual alpha-L-fucosidase activity and cross reacting protein in the heterozygous range and normal metabolism of metabolites containing fucose in his fibroblasts, consistent with the low activity polymorphism. The eighth patient, who had a partial deficiency of alpha-L-fucosidase in her fibroblasts and leucocytes at a young age but normal alpha-L-fucosidase activity and protein at a later age, was homozygous for the common Q281R polymorphism in exon 5. She had no other sequence changes and Kivlin (Peters plus) syndrome has subsequently been diagnosed. The basis of her transient deficiency of alpha-L-fucosidase is not known. The detection of five novel mutations in six severely affected patients confirms the genetic heterogeneity in fucosidosis.

Histology and electron microscopy of fucosidosis of the skin. Subtle clues to diagnosis by electron microscopy.

Fucosidosis is an autosomal recessive inborn error of metabolism in which fucose-containing glycolipids, glycoproteins, and oligo- and polysaccharides accumulate in tissues as a consequence of alpha-L-fucosidase deficiency. Since the detection of this entity in 1966 several cases have been described, but until now investigations of clinically uninvolved skin have not been performed. In this study we have investigated clinically normal skin obtained from a patient with fucosidosis and his healthy sister, by light and electron microscopy, to determine whether normal skin in this condition yields clues that may have prognostic relevance. We found "empty"- appearing storage vesicles in melanocytes, endothelial cells, sweat glands, and fibroblasts in the skin.

Metabolic correction of fucosidosis lymphoid cells by galaptin-alpha-L-fucosidase conjugates.

To determine if isolated galaptin, an endogenous galactoside-binding lectin, could serve as a transport vehicle of therapeutic agents to cells, galaptin and alpha-L-fucosidase were coupled using glutaraldehyde. The conjugates were incubated with alpha-L-fucosidase-deficient, EBV-immortalized lymphoid cells from a fucosidosis patient. Conjugates were effectively bound and internalized by the cells in a lactose inhibitable manner. Internalization of conjugate resulted in the reduced accumulation of alpha-L-fucosyl-N-acetylglucosaminylasparagine, a glycopeptide that accumulates in cells of fucosidosis patients, to levels found in lymphoid cells from a healthy individual. Thus, galaptin-alpha-L-fucosidase conjugates may be useful for enzyme replacement therapy of fucosidosis. The concept of using galaptin as a transport vehicle may be applied to the delivery of other compounds to cells bearing galaptin receptors.

Distribution of saposin proteins (sphingolipid activator proteins) in lysosomal storage and other diseases.

Saposins (A, B, C, and D) are small glycoproteins required for the hydrolysis of sphingolipids by specific lysosomal hydrolases. Concentrations of these saposins in brain, liver, and spleen from normal humans as well as patients with lysosomal storage disease were determined. A quantitative HPLC method was used for saposin A, C, and D and a stimulation assay was used for saposin B. In normal tissues, saposin D was the most abundant of the four saposins. Massive accumulations of saposins, especially saposin A (about 80-fold increase over normal), were found in brain of patients with Tay-Sachs disease or infantile Sandhoff disease. In spleen of adult patients with Gaucher disease, saposin A and D accumulations (60- and 17-fold, respectively, over normal) were higher than that of saposin C (about 16-fold over normal). Similar massive accumulations of saposins A and D were found in liver of patients with fucosidosis (about 70- and 20-fold, respectively, over normal). Saposin D was the primary saposin stored in the liver of a patient with Niemann-Pick disease (about 30-fold over normal). Moderate increases of saposins B and D were found in a patient with GM1 gangliosidosis. Normal or near normal levels of all saposins were found in patients with Krabbe disease, metachromatic leukodystrophy, Fabry disease, adrenoleukodystrophy, I-cell disease, mucopolysaccharidosis types 2 and 3B, or Jansky-Bielschowsky disease. The implications of the storage of saposins in these diseases are discussed.

Characterization of SSEA-1 glycolipids from the brain of a patient with fucosidosis.

Neutral glycolipids from the brain of a patient with Fucosidosis were analyzed and two complex glycolipids containing five and eight sugars were isolated from the cortical grey matter. These two glycolipids reacted with antibodies recognizing the SSEA-1 [Le(x)(X)] carbohydrate determinant. SSEA-1 glycolipids are normally expressed in human embryonic brain but are found in only small amounts in postnatal human brain. The accumulation of the two SSEA-1 glycolipids in Fucosidosis brain thus represents a defect which affects the normal developmentally regulated decrease in postnatal expression of these glycolipids, and may be a contributing factor in the abnormal brain development associated with the disease. Chemical characterization of the two isolated glycolipids by gas chromatographic and mass spectrometric analyses has identified the two glycolipids as lacto-N-fucopentaosylceramide (III) and difucosyl-neolactonorhexaosylceramide.

Canine alpha-L-fucosidase in relation to the enzymic defect and storage products in canine fucosidosis.

Canine liver alpha-L-fucosidase was purified to apparent homogeneity by affinity chromatography on agarose-epsilon-aminohexanoyl-fucopyranosylamine. It is composed of multiple forms of a common active subunit of 45-50 kDa, which can aggregate in different combinations to form polymers, predominantly dimers. Antiserum was raised against the purified enzyme. There is negligible residual alpha-L-fucosidase in the tissues of English springer spaniels with the lysosomal storage disease fucosidosis. Although no alpha-L-fucosidase protein was detected by Western blotting or by the purification procedure in the affected tissues, some enzymically inactive cross-reacting material was detected in both normal and affected tissues. This suggests that another protein without alpha-L-fucosidase activity was co-purified with the enzyme. Dog liver alpha-L-fucosidase was precipitated by goat anti-(human liver alpha-L-fucosidase) IgG, indicating homology between the enzymes in the two species. Two purified storage products isolated from the brain of a dog with fucosidosis were used as natural substrates for various preparations of canine liver alpha-L-fucosidase. Analysis of the digestion mixtures by t.l.c. and fast-atom-bombardment mass spectrometry suggests that canine alpha-L-fucosidase acts preferentially on the alpha-(1-3)-linked fucose at the non-reducing end and that removal of alpha-(1-6)-linked asparagine-linked N-acetylglucosamine is rate-limiting in the lysosomal catabolism of fucosylated N-linked glycans.

Comparison of the urinary glycoconjugates excreted by patients with type I and type II fucosidosis.

Glycoconjugates were isolated, by repeated chromatography on Biogel P-4, from urine of two patients with different phenotypes of fucosidosis, type I (acute) and type II (chronic). Studies of the isolated compounds with thin-layer chromatography, chromatography on Biogel P-4, gas-liquid chromatography, and 1H- and 13C-nuclear magnetic resonance spectroscopy showed differences in the excretion patterns of fucosyl glycoconjugates in the urine of these patients. The amount of the diglycosylasparagine, fuc alpha 1-6glcNAc-Asn, excreted was significantly lower for the type I than for the type II patient. On the other hand, the reducing hexasaccharide gal beta 1-4(fuc alpha 1-3)glcNAc beta 1-2man alpha 1-3/6man beta 1-4glcNAc was present in much greater quantities in urine from the type I patient. The differences in the excretion patterns of these glycoconjugates may be attributed to different substrate specificities for the residual alpha-L-fucosidases in the two forms of the disease. I propose that such differences may be exploited for the early laboratory diagnosis of the type II form of the disease, particularly by thin-layer chromatography.

Human and canine fucosidosis: a comparative lectin histochemistry study.

Selected formalin-fixed, paraffin-embedded tissues of human and canine fucosidosis were stained with nine different lectins. Neurons, splenic sinusoidal cells, hepatic Kupffer cells, tissue macrophages, and capillary endothelium from human patients with fucosidosis stained intensely with Ulex europaeus agglutinin-I (UEA-I), but the same cells were unstained in tissues from canine fucosidosis. Since UEA-I specifically binds to terminal fucose residues, and fucose-rich undegraded metabolites are stored in affected cells of both human and canine fucosidosis, the variable lectin staining pattern demonstrates an unexpected species-specific histochemical variability. This finding highlights the fact that although both species have decreased fucosidase activity, the precursor substrates, undegraded stored metabolites, and particular cells affected by this enzyme deficiency are different.