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It is well known that several of the swainsonine-containing plant species found widespread around the world have a negative economic impact in each country. In Argentina, most of the information on the poisonous plant species that produce α-mannosidosis is published in Spanish and thus not available to most English speaking researchers interested in toxic plants. Therefore, the aim of this review is to summarize the information about swainsonine-containing plants in Argentina, which are extensively distributed throughout different ecoregions of the country. To date, five species from three genera have been shown to induce α-mannosidosis in livestock in Argentina: Ipomoea carnea subsp. fi stulosa, Ipomoea hieronymi subsp. calchaquina (Convolvulaceae), Astragalus garbancillo, Astragalus pehuenches (Fabaceae), and Sida rodrigoi (Malvaceae). These species contain the indolizidine alkaloid swainsonine, which inhibits the lysosomal enzyme α-mannosidase and consequently affects glycoprotein metabolism, resulting in partially metabolized sugars. The prolonged consumption of these poisonous plants produces progressive weight loss and clinical signs related to a nervous disorder, characterized by tremors of head and neck, abnormalities of gait, difficulty in standing, ataxia and wide-based stance. Histological lesions are mainly characterized by vacuolation of different cells, especially neurons of the central nervous system. The main animal model used to study α-mannosidosis is the guinea pig because, when experimentally poisoned, it exhibits many of the characteristics of naturally intoxicated livestock. Estación Experimental Agropecuaria Bariloche Fil: Cholich, Luciana Andrea. Universidad Nacional del Nordeste. Facultad de Ciencias Veterinarias; Argentina Fil: Cholich, Luciana Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Martinez, Agustin. Instituto Nacional de Tecnologia Agropecuaria (INTA). Estacion Experimental Agropecuaria Bariloche. Grupo de Sanidad Animal; Argentina Fil: Micheloud, Juan Francisco. Universidad Católica de Salta. Facultad de Ciencias Veterinarias y Agronomía; Argentina Fil: Micheloud, Juan Francisco. Instituto Nacional de Tecnologia Agropecuaria (INTA). Instituto de Investigación Animal del Chaco Semiárido. Área de Sanidad Animal; Argentina Fil: Pistán, Maria Elena. Universidad Nacional del Nordeste. Facultad de Ciencias Veterinarias; Argentina Fil: Garcia, Enrique Nicolas. Universidad Nacional del Nordeste. Facultad de Ciencias Veterinarias; Argentina Fil: Robles, Carlos Alejandro. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Bariloche. Grupo de Sanidad Animal; Argentina Fil: Ortega, Hugo Hector. Universidad Nacional del Litoral. Instituto de Ciencias Veterinarias del Litoral; Argentina Fil: Ortega, Hugo Hector. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Gimeno, Eduardo Juan. Universidad Nacional de la Plata. Facultad de Ciencias Veterinarias; Argentina Fil: Gimeno, Eduardo Juan. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Alpha-Mannosidosis (AM) is an ultra-rare storage disorder caused by a deficiency of lysosomal alpha-mannosidase encoded by the MAN2B1 gene. Clinical presentation of AM includes mental retardation, recurrent infections, hearing loss, dysmorphic features, and motor dysfunctions. AM has never been reported in Tunisia. We report here the clinical and genetic study of six patients from two Tunisian families with AM. The AM diagnosis was confirmed by an enzymatic activity assay. Genetic investigation was conducted by Sanger sequencing of the mutational hotspots for the first family and by ES analysis for the second one. In the first family, a frameshift duplication p.(Ser802GlnfsTer129) was identified in the MAN2B1 gene. For the second family, ES analysis led to the identification of a missense mutation p.(Arg229Trp) in the MAN2B1 gene in four affected family members. The p.(Ser802GlnfsTer129) mutation induces a premature termination codon which may trigger RNA degradation by the NMD system. The decrease in the levels of MAN2B1 synthesis could explain the severe phenotype observed in the index case. According to the literature, the p.(Arg229Trp) missense variant does not have an impact on MAN2B1 maturation and transportation, which correlates with a moderate clinical sub-type. To explain the intra-familial variability of cognitive impairment, exome analysis allowed the identification of two likely pathogenic variants in GHR and SLC19A3 genes potentially associated to cognitive decline. The present study raises awareness about underdiagnosis of AM in the region that deprives patients from accessing adequate care. Indeed, early diagnosis is critical in order to prevent disease progression and to propose enzyme replacement therapy.

Multiplex tandem mass spectrometry (MS/MS)-based enzyme activity assays for newborn screening (NBS) and diagnosis of lysosomal storage diseases (LSDs) in newborns, using dried blood spots (DBS) on newborn screening cards, have garnered much attention due to its sensitivity, high precision, and the capability to screen for an unprecedented number of diseases in a single assay. Herein we report the development of MS/MS-based enzyme assays for the diagnosis of α-mannosidosis and fucosidosis. These new protocols are able to distinguish untreated patients from random newborns, carriers and a post-bone marrow transplant patient. We have successfully multiplexed the α-mannosidosis assay with a multiplex MS/MS assay for the screening and diagnosis of other LSDs, namely Fabry, Pompe, MPS I, Gaucher, Niemann-Pick-A/B, and Krabbe diseases. Additionally, we also multiplexed the fucosidosis NBS assay with a 5-plex assay that tests for MPS-II, MPS-IIIB, MPS-IVA, MPS-VI and MPS-VII.

Inflammatory bowel disease (IBD) is a refractory disease of the gastrointestinal tract that is believed to develop in genetically susceptible individuals. Glycosylation, a type of post-translational modification, is involved in the development of a wide range of diseases, including IBD, by modulating the function of various glycoproteins. To identify novel genes contributing to the development of IBD, we analyzed single nucleotide polymorphisms (SNPs) of glycosylation-related genes in IBD patients and identified MAN2A1, encoding alpha-mannosidase II (α-MII), as a candidate gene. α-MII plays a crucial, but not exclusive, role in the maturation of N-glycans. We also observed that intestinal epithelial cells (IECs), which establish the first-line barrier and regulate gut immunity, selectively expressed α-MII with minimal expression of its isozyme, alpha-mannosidase IIx (α-MIIx). This led us to hypothesize that IEC-intrinsic α-MII is implicated in the pathogenesis of IBD. To test this hypothesis, we generated IEC-specific α-MII-deficient (α-MIIΔIEC) mice. Although α-MII deficiency has been shown to have a minimal effect on N-glycan maturation in most cell types due to the compensation by α-MIIx, ablation of α-MII impaired the maturation of N-glycans in IECs. α-MIIΔIEC mice were less susceptible to dextran sulfate sodium-induced colitis compared with control littermates. In accordance with this, neutrophil infiltration in the colonic mucosa was attenuated in α-MIIΔIEC mice. Furthermore, gene expression levels of neutrophil-attracting chemokines were downregulated in the colonic tissue. These results suggest that IEC-intrinsic α-MII promotes intestinal inflammation by facilitating chemokine expression. We propose SNPs in MAN2A1 as a novel genetic factor for IBD.Key words: inflammatory bowel disease, alpha-mannosidase II, intestinal epithelial cell, N-glycosylation.

There are numerous poisonous plants that can induce intralysosomal accumulation of glycoproteins and neurologic syndromes. Here we describe for the first time, a disease caused by ingesting Sida rodrigoi Monteiro in goats in North-western Argentina. The animals showed weight loss, indifference to the environment, unsteady gait and ataxia. Histopathologic studies showed vacuolization in cells of various organs, mainly in the CNS. The material deposited in the cells was positive for LCA (Lens culinaris agglutinin), WGA (Triticum vulgaris agglutinin), sWGA (succinyl-Triticum vulgaris agglutinin) and Con-A (Concanavalia ensiformis agglutinin) lectins. Finally, toxic levels of swansonine were identified in the plant. The present investigation allowed to recognize S. rodrigoi Monteiro poisoning as a plant induced α-mannosidosis. Fil: Micheloud, Juan Francisco. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Investigación Animal del Chaco Semiárido; Argentina. Universidad Católica de Salta; Argentina Fil: Marin, Raúl. Universidad Nacional de Jujuy; Argentina Fil: Colque Caro, Luis Adrián. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Investigación Animal del Chaco Semiárido; Argentina. Universidad Católica de Salta; Argentina Fil: Martinez, Olga Gladys. Universidad Nacional de Salta; Argentina Fil: Gardner, Dale. United States Department of Agriculture. Agriculture Research Service; Estados Unidos Fil: Gimeno, Eduardo Juan. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Cátedra de Patología General Veterinaria; Argentina

Lysosomal alpha-mannosidase with acidic pH optimum is ubiquitous in human tissues where is expressed in two major forms, A and B that are the product of a single gene located on chromosome 19. Mutations in the gene encoding for alpha-mannosidase cause alpha- mannosidosis, an autosomal recessive disease, resulting in the accumulation of unprocessed mannose containing oligosaccharide material. This rare disease has an estimated incidence of 1/500.0.00 live births and clinically is divided into three subgroups. Today the most promising therapy for this disease is the enzyme replacement therapy. To develop this strategy a mouse model for alpha-mannosidosis has been generated and a recombinant human alpha-mannosidase has been produced from Chinese-hamster ovary cells. Interestingly it has been shown that the recombinant enzyme, used in high dose, can cross the blood brain barrier. This recombinant enzyme has been tested in the first randomized study investigating the efficacy of enzyme replacement therapy in patients with alpha-mannosidosis. This review contains the scientific progresses on lysosomal alpha-mannosidase from the cloning to the beginning of the therapy.

Lysosomal storage diseases (LSDs) are a group of 70 monogenic disorders characterized by the lysosomal accumulation of a substrate. As a group, LSDs affect ~1 in 5000 live births; however, each individual storage disease is rare, limiting the ability to perform natural history studies or to perform clinical trials. Perhaps in no other biomedical field have naturally occurring large animal (canine, feline, ovine, caprine, and bovine) models been so essential for understanding the fundamentals of disease pathogenesis and for developing safe and effective therapies. These models were critical for the development of hematopoietic stem cell transplantation in α- and β- mannosidosis, fucosidosis, and the mucopolysaccharidoses; enzyme replacement therapy for fucosidosis, the mucopolysaccharidoses, and neuronal ceroid lipofuscinosis; and small molecule therapy in Niemann–Pick type C disease. However, their most notable contributions to the biomedical field are in the development of gene therapy for LSDs. Adeno-associated viral vectors to treat nervous system disease have been evaluated in the large animal models of α-mannosidosis, globoid cell leukodystrophy, GM1 and GM2 gangliosidosis, the mucopolysaccharidoses, and neuronal ceroid lipofuscinosis. This review article will summarize the large animal models available for study as well as their contributions to the development of central and peripheral nervous system dysfunction in LSDs.

α-Mannosidosis (AM) results from deficient lysosomal α-mannosidase (LAMAN) activity and subsequent substrate accumulation in the lysosome, leading to severe pathology. Many of the AM-causative mutations compromise enzyme folding and could be rescued with purpose-designed pharmacological chaperones (PCs). We found that PCs combining a LAMAN glycone-binding motif based on the 5N,6O-oxomethylidenemannojirimycin (OMJ) glycomimetic core and different aglycones, in either mono- or multivalent displays, elicit binding modes involving glycone and nonglycone enzyme regions that reinforce the protein folding and stabilization potential. Multivalent derivatives exhibited potent enzyme inhibition that generally prevailed over the chaperone effect. On the contrary, monovalent OMJ derivatives with LAMAN aglycone binding area-fitting substituents proved effective as activity enhancers for several mutant LAMAN forms in AM patient fibroblasts and/or transfected MAN2B1-KO cells. This translated into a significant improvement in endosomal/lysosomal function, reverting not only the primary LAMAN substrate accumulation but also the additional downstream consequences such as cholesterol accumulation. Ministerio de Economía y Competitividad SAF2016-76083-R, CTQ2015-64425-C2-1-R Junta de Andalucía FQM2012-1467 Japan Society for the Promotion of Science 17K10051

OBJECTIVE: The lysosomal storage disease alpha-mannosidosis is caused by the deficiency of the lysosomal acid hydrolase alpha-mannosidase (LAMAN) leading to lysosomal accumulation of neutral mannose-linked oligosaccharides throughout the body, including the brain. Clinical findings in alpha-mannosidosis include skeletal malformations, intellectual disabilities and hearing impairment. To date, no curative treatment is available. We previously developed a beneficial enzyme replacement therapy (ERT) regimen for alpha-mannosidase knockout mice, a valid mouse model for the human disease. However, humoral immune responses against the injected recombinant human alpha-mannosidase (rhLAMAN) precluded long-term studies and chronic treatment. METHODS: Here, we describe the generation of an immune-tolerant alpha-mannosidosis mouse model that allowed chronic injection of rhLAMAN by transgenic expression of a catalytically inactive variant of human LAMAN in the knockout background. RESULTS: Chronic ERT of rhLAMAN revealed pronounced effects on primary substrate storage throughout the brain, normalization of lysosomal enzyme activities and morphology as well as a decrease in microglia activation. The positive effect of long-term ERT on neuronal lysosomal function was reflected by an improvement of cognitive deficits and exploratory activity. in vivo and in vitro uptake measurements indicate rapid clearance of rhLAMAN from circulation and a broad uptake into different cell types of the nervous system. INTERPRETATION: Our data contribute to the understanding of neurological disorders treatment by demonstrating that lysosomal enzymes such as rhLAMAN can penetrate into the brain and is able to ameliorate neuropathology. ispartof: ANNALS OF CLINICAL AND TRANSLATIONAL NEUROLOGY vol:2 issue:11 pages:987-1001 ispartof: location:United States status: published

Alpha-mannosidosis (AM) (OMIM 248500) is a rare lysosomal storage disease. The understanding of the central nervous system (CNS) pathology is limited. This study is the first describing the CNS pathology and the correlation between the CNS pathology and intellectual disabilities in human AM. Thirty-four patients, aged 6-35 years, with AM were included. Data from 13 healthy controls were included in the analysis of the magnetic resonance spectroscopy (MRS). Measurements of CNS neurodegeneration biomarkers in cerebrospinal fluid (CSF), CSF-oligosaccharides, and performance of cerebral magnetic resonance imaging (MRI) and MRS were carried out. On MRI, 5 of 10 patients had occipital white matter (WM) signal abnormalities, and 6 of 10 patients had age-inappropriate myelination. MRS demonstrated significantly elevated mannose complex in gray matter and WM. We found elevated concentrations of tau-protein, glial fibrillary acidic protein and neurofilament light protein in 97 patients, 74% and 41% of CSF samples, respectively. A negative correlation between CSF-biomarkers and cognitive function and CSF-oligosaccharides and cognitive function was found. The combination of MRS/MRI changes, elevated concentrations of CSF-biomarkers and CSF-oligosaccharides suggests gliosis and reduced myelination, as part of the CNS pathology in AM. Our data demonstrate early neuropathological changes, which may be taken into consideration when planning initiation of treatment.

α-Mannosidosis is an autosomal recessive lysosomal storage disorder caused by mutations in the MAN2B1 gene, encoding lysosomal α-mannosidase. The disorder is characterized by a range of clinical phenotypes of which the major manifestations are mental impairment, hearing impairment, skeletal changes, and immunodeficiency. Here, we report an α-mannosidosis mutation database, amamutdb.no, which has been constructed as a publicly accessible online resource for recording and analyzing MAN2B1 variants (http://amamutdb.no). Our aim has been to offer structured and relational information on MAN2B1 mutations and genotypes along with associated clinical phenotypes. Classifying missense mutations, as pathogenic or benign, is a challenge. Therefore, they have been given special attention as we have compiled all available data that relate to their biochemical, functional, and structural properties. The α-mannosidosis mutation database is comprehensive and relational in the sense that information can be retrieved and compiled across datasets; hence, it will facilitate diagnostics and increase our understanding of the clinical and molecular aspects of α-mannosidosis. We believe that the amamutdb.no structure and architecture will be applicable for the development of databases for any monogenic disorder.

A disease of the nervous system is reported in goats in the semiarid region of northeastern Brazil. Histological examination showed diffuse vacuolation of neurons and epithelial cells of the pancreas, thyroid, renal tubules, and liver. The swainsonine-containing plant Ipomoea verbascoidea was found on both farms where the goats originated. This plant was experimentally administered to 3 goats, inducing clinical signs and histologic lesions similar to those observed in spontaneous cases. On the lectin histochemical analysis, cerebellar cells and pancreatic acinar cells gave positive reactions to Triticum vulgaris agglutinin (WGA), succinylated Triticum vulgaris agglutinin (sWGA), Lens culinaris agglutinin (LCA), Canavalia ensiformis agglutinin (ConA), Pisum sativum agglutinin (PSA), Ricinus communis agglutinin (RCA120), Arachis hypogaea agglutinin (PNA), and Phaseolus vulgaris erythroagglutinin (PHA-E) suggesting storage of α-fucose, α-D-mannose, α-D-glucose, β-D-N-acetyl-glucosamine, N-acetyl-galactosamine, and acetyl-neuraminic acid. This pattern of lectin staining partially agrees with results previously reported for poisoning by swainsonine-containing plants. The chemical analysis of dried leaves of I. verbascoidea detected swainsonine (0.017%), calystegine B1 (0.16%), calystegine B2 (0.05%), and calystegine C1 (0.34%). It is concluded that I. verbascoidea causes α-mannosidosis in goats.

In Alpha-mannosidosis (MIM 248500) the patients accumulate mainly unbranched oligosaccharide chains in the lysosomes in all body tissues, including the brain. With ensuing therapeutic modalities in man (BMT and ERT) non-invasive methods of monitoring the effect of treatment are needed. Paramount is the possible effect of the treatment on the brain, since this organ is regarded as difficult to reach because of the blood-brain barrier. We therefore performed proton nuclear magnetic resonance spectroscopy (MRS) of the brain in two untreated patients, and a 16-year-old patient treated with BMT at the age of 10 to assess whether this non-invasive method could be applied in the monitoring of the accumulation of abnormal chemicals in the brain of patients. We found an abnormal peak that was not present in the treated patient. A similar pattern was also found in MRS of urine from patients, reflecting the concentration of oligosaccharides in serum and tissues. We therefore conclude that MRS can be a useful method to monitor the effect of treatment for Alpha-Mannosidosis.

Plasma levels of beta-mannosidase activity were measured in the phenotypically normal members of a family of goats in which 12 cases of beta-mannosidosis have been reported. Normal or carrier genotypes were assigned on the basis of these results, and mating tables drawn up. Similar data were collected on a number of other small families unrelated to the one with beta-mannosidosis. The results indicate that the plasma beta-mannosidase level is a reasonably good indicator of carrier status and confirm that the disease beta-mannosidosis is inherited in a recessive manner and is not X-linked.

Clinical and pathologic findings for the spontaneous poisoning by Sida carpinifolia in cattle are described in this study. A survey on field cases of S. carpinifolia in cattle was carried out on farms of Alto Vale do Itajai, State of Santa Catarina, southern Brazil. Sixteen affected animals were clinically evaluated and 9 were subjected to postmortem examination. The main clinical signs consisted of marching gait, alert gaze, head tremors, and poor growth. Histologic and ultrastructural lesions consisted of vacuolization and distension of neuronal perikarya, mainly from Purkinje cells, and of the cytoplasm of acinar pancreatic and thyroid follicular cells. Clinical signs and lesions varied from mild to severe. Improvement of the clinical signs was observed in cattle after a period of up to 90 days without consuming the plant; however, residual lesions, mainly characterized by axonal spheroids and absence of Purkinje neurons in some areas of the cerebellum, were observed in these cases. It is concluded that the natural chronic consumption of S. carpinifolia was the etiologic cause of storage disease in cattle in this study.

Despite the progress in the treatment of lysosomal storage disorders (LSDs) mainly by enzyme replacement therapy, only limited success was reported in targeting the appropriate lysosomal enzyme into the brain. This prevents efficient clearance of neuronal storage, which is present in many of these disorders including alpha-mannosidosis. Here we show that the neuropathology of a mouse model for alpha-mannosidosis can be efficiently treated using recombinant human alpha-mannosidase (rhLAMAN). After intravenous administration of different doses (25-500 U/kg), rhLAMAN was widely distributed among tissues, and immunohistochemistry revealed lysosomal delivery of the injected enzyme. Whereas low doses (25 U/kg) led to a significant clearance (

Descrevem-se os achados clínicos e patológicos da intoxicação experimental por Sida carpinifolia em bovinos. Para a reprodução experimental da doença, folhas verdes da planta foram coletadas semanalmente na região do Alto Vale do Itajaí e fornecidas in natura diariamente para cinco bovinos nas doses de 10 e 20g/kg por 120 dias, 40g/kg por 30 dias, e 30 e 40g/kg de peso animal por 150 dias. Um bovino morreu e os outros foram eutanasiados ao final do período de consumo da planta. Os principais sinais clínicos consistiam de andar em marcha, olhar atento e tremores de cabeça e foram de intensidade leve a acentuado conforme a dose de planta e tempo de consumo. A lesão histológica predominante caracterizava-se por vacuolização e tumefação de neurônios (principalmente os de Purkinje), das células acinares pancreáticas e células foliculares da tireóide. Ultra-estruturalmente verificou-se vacúolos, por vezes, contendo material finamente granular em neurônios, células acinares pancreáticas e células foliculares da tireóide. S. carpinifolia causa doença de depósito lisossomal em bovinos quando consumida por período prolongado, mesmo em pequenas doses.

The lysosomal storage disease alpha-mannosidosis is due to absence or defective function of lysosomal alpha-mannosidase, resulting in primary storage of undegraded mannose-rich oligosaccharides. Disease has been described in humans, cattle, cats, mice, and guinea pigs and is characterized in all species by progressive neurologic deterioration and premature death. We analyzed the neurodegenerative processes relative to clinical disease in alpha-mannosidosis guinea pigs as a human disease model, from birth to end-stage disease. Before the onset of obvious neurologic abnormalities at 2 months, we observed widespread neuronal lysosomal vacuolation including secondary accumulation of GM3 ganglioside, widespread axonal spheroids, and reduced myelination of white matter. Histopathologic changes subsequently showed rapid progression in severity in a pattern common to a number of different lysosomal storage disorders, with additional abnormalities including accumulation of GM2 ganglioside and cholesterol, astrogliosis, neuron loss particularly in the cerebellum, and activation and infiltration of the CNS with microglia/macrophages. End-stage clinical disease was seen at 10 to 14 months of age. Our findings show that complex neuropathologic changes in alpha-mannosidosis guinea pigs are already present at birth, before clinical changes are evident, and similar events are likely to occur in patients with this disorder.

Lysosomal storage diseases (LSDs) are debilitating neurometabolic disorders for most of which long-term effective therapies have not been developed. Gene therapy is a potential treatment but a critical barrier to treating the brain is the need for global correction. We tested the efficacy of cisterna magna infusion of adeno-associated virus type 1 (AAV1) expressing feline alpha-mannosidase gene in the postsymptomatic alpha-mannosidosis (AMD) cat, a homologue of the human disease. Lysosomal alpha-mannosidase (MANB) activity in the cerebrospinal fluid (CSF) and serum were increased above the control values in untreated AMD cats. Clinical neurological signs were delayed in onset and reduced in severity. The lifespan of the treated cats was significantly extended. Postmortem histopathology showed resolution of lysosomal storage lesions throughout the brain. MANB activity in brain tissue was significantly above the levels of untreated tissues. The results demonstrate that a single cisterna magna injection of AAV1 into the CSF can mediate widespread neuronal transduction of the brain and meaningful clinical improvement. Thus, cisterna magna gene delivery by AAV1 appears to be a viable strategy for treatment of the whole brain in AMD and should be applicable to many of the neurotropic LSDs as well as other neurogenetic disorders.

Background Alpha-mannosidosis is caused by mutations in MAN2B1, leading to loss of lysosomal alpha-mannosidase activity. Symptoms include intellectual disabilities, hearing impairment, motor function disturbances, facial coarsening and musculoskeletal abnormalities. Methods To study the genotype-phenotype relationship for alpha-mannosidosis 66 patients were included. Based on the predicted effect of the mutations and the subcellular localisation of mutant MAN2B1 in cultured cells, the patients were divided into three subgroups. Clinical and biochemical data were collected. Correlation analyses between each of the three subgroups of genotype/subcellular localisation and the clinical and biochemical data were done to investigate the potential relationship between genotype and phenotype in alpha-mannosidosis. Statistical analyses were performed using the SPSS software. Analyses of covariance were performed to describe the genotype-phenotype correlations. The phenotype parameters were modelled by the mutation group and age as a covariate. P values of

Beta-mannosidase, a lysosomal enzyme which acts exclusively at the last step of oligosaccharide catabolism in glycoprotein degradation, functions to cleave the unique beta-linked mannose sugar found in all N-linked oligosaccharides of glycoproteins. Deficiency of this enzyme results in beta-mannosidosis, a lysosomal storage disease characterized by the cellular accumulation of small oligosaccharides. In human beta-mannosidosis, the clinical presentation is variable and can be mild, even when caused by functionally null mutations. In contrast, two existing ruminant animal models have disease that is consistent and severe. To further explore the molecular pathology of this disease and to investigate potential treatment strategies, we produced a beta-mannosidase knockout mouse. Homozygous mutant mice have undetectable beta-mannosidase activity. General appearance and growth of the knockout mice are similar to the wild-type littermates. At >1 year of age, these mice exhibit no dysmorphology or overt neurological problems. The mutant animals have consistent cytoplasmic vacuolation in the central nervous system and minimal vacuolation in most visceral organs. Thin-layer chromatography demonstrated an accumulation of disaccharide in epididymis and brain. This mouse model closely resembles human beta-mannosidosis and provides a useful tool for studying the phenotypic variation in different species and will facilitate the study of potential therapies for lysosomal storage diseases.

Alpha-mannosidosis is a lysosomal storage disorder characterised by the lysosomal accumulation of mannose-containing oligosaccharides and a range of pathological consequences, caused by a deficiency of the lysosomal enzyme alpha-mannosidase. One of the major features of alpha-mannosidosis is progressive neurological decline, for which there is no safe and effective treatment. Implantation of stem cells into the central nervous system has been proposed as a potential therapy for these disorders. We report the construction and characterisation of mouse embryonic stem cell lines for the sustained over-expression of recombinant human lysosomal alpha-mannosidase (rhalphaM). Two vectors (involving recombinant human alpha-mannosidase expression driven by either the chicken beta-actin promoter/CMV enhancer or by the elongation factor 1-alpha promoter) were constructed and used to transfect mouse D3 embryonic stem cells. Selected clonal cell lines were isolated and tested to evaluate their expression of recombinant human alpha-mannosidase. Stem cell clones transfected with the chicken beta-actin promoter/CMV enhancer maintained rhalphaM expression levels throughout differentiation. This expression was not markedly elevated above background. In contrast, the vector incorporating the elongation factor 1-alpha promoter facilitated substantial over-expression of alpha-mannosidase when analysed out to 21 days of differentiation in stably transfected cell lines. The highest expressing cell line was found to qualitatively retain a similar differentiation potential to untransfected cells, and to secrete alpha-mannosidase that could mediate a reduction in the level of oligosaccharides stored by human alpha-mannosidosis skin fibroblasts. These results suggest potential for the use of this cell line for investigation of a stem cell therapy approach to treat alpha-mannosidosis.

Genetic diseases affecting the brain typically have widespread lesions that require global correction. Lysosomal storage diseases are good candidates for central nervous system gene therapy, because active enzyme from genetically corrected cells can be secreted and taken up by surrounding diseased cells, and only small amounts of enzyme (

Este trabalho inclui os estudos clínicos e patológicos da doença de armazenamento lisossomal induzida pelo consumo espontâneo de Sida carpinifolia. A enfermidade foi observada em três rebanhos, que juntos eram compostos por 51 caprinos, dos quais, 25 foram afetados e 11 necropsiados. Nos três surtos, S. carpinifolia era a vegetação predominante nos piquetes ocupados pelos animais. Clinicamente, a doença caracterizou-se por distúrbios neurológicos que consistiam de ataxia, hipermetria, posturas anormais, tremores musculares afetando principalmente as regiões da cabeça e pescoço, dificuldade para ingestão de alimentos e quedas freqüentes. Estes sinais clínicos eram exacerbados pela movimentação. Em alguns animais, embora com um quadro clínico estabilizado, as alterações neurológicas persistiram durante 24 meses após sua retirada dos piquetes infestados por S. carpinifolia. A doença foi reproduzida administrando-se S. carpinifolia, in natura ou seca à sombra, para 3 caprinos. Um caprino recebeu Sida rhombifolia, ad libidum, por 40 dias e não desenvolveu alterações clínicas ou patológicas. Na necropsia não havia alterações. Microscopicamente, as principais alterações foram distensão e vacuolização citoplasmáticas em neurônios e, em menor intensidade, em células da glia do sistema nervoso central. Alterações similares foram observadas em células acinares pancreáticas, hepatócitos, células tubulares renais, células foliculares epiteliais da tireóide e macrófagos de órgãos linfóides. Nos animais que não mais ingeriam S. carpinifolia por períodos de um mês ou mais, observou-se uma diminuição da vacuolização citoplasmática de neurônios, que apresentavam citoplasma eosinofílico e aspecto enrugado. Nestes casos, notou-se também desaparecimento neuronal especialmente em células de Purkinje e gliose local. Em cortes cerebelares, esta doença de armazenamento foi caracterizada como ?-manosidose pelo estudo histoquímico por lectinas. Os vacúolos nas células de Purkinje reagiram fortemente com as lectinas Concanavalia ensiformis, Triticum vulgaris e Triticum vulgaris succinilado. O padrão obtido neste estudo é similar ao encontrado em intoxicação por plantas que apresentam swainsoniana como princípio tóxico. This report includes the clinical and pathological studies of a lysosomal storage disease which spontaneously occurred in three flocs of goats e after consumption of Sida carpinifolia, the predominant plant in the paddocks where the animals were grazing. In the outbreaks a total of 25 out of 51 animals were affected. Post-mortem examination was performed on 11 goats. The disease was experimentally induced by dosing goats with Sida carpinifolia. The plant was administered in natura or dried to 3 animals. No clinical or pathological changes were observed in one goat dosed with Sida rhombifolia ad libidum during 40 days. Clinical signs of the poisoning were ataxia, hypermetria, muscle tremors in the head and neck and disorders of deglutition. The clinical signs were exacerbated by movement. After the surviving animals had been moved to other pastures and stopped eating the plant, clinical signs were still observed during 24 months. At necropsy, no significant gross lesions were observed. Microscopic lesions included various degrees of vacuolization in the cytoplasm of neurons and glial cells. Similar lesions were observed in the acinar pancreatic cells, hepatocytes, proximal convoluted tubular cells, follicular epithelial cells of the thyroid gland and macrophages of lymph nodes. In the surviving animals, mild neuronal cytoplasmic vacuolization was observed, and few cells were eosinophilic and shrunken. In these cases neurons, especially Purkinje cells, had disappeared. Through the histochemical study of the cerebellar sections, the lysosomal storage disease was characterized as an alpha-mannosidosis. The vacuoles within the Purkinje cells strongly reacted with lectins of Concanavalia ensiformis, Triticum vulgaris and succinylated Triticum vulgaris. The pattern observed in this investigation is similar to those seen in other poisonings by swainsonine-containing plants.

α-Mannosidosis storage disease is a rare auto- somal recessive disease that is caused by a deficiency of the lysosomal enzyme α-mannosidase. In this arti- cle, a proband in China was preliminarily diagnosed as having α-mannosidosis by clinical symptoms, imaging examination, and enzyme assay. Definitive diagno- sis was performed by directly sequencing the MAN2B1 gDNA and cDNA of the peripheral blood leukocyte from the patient. Finally, denaturing high-performance liq- uid chromatography screening, conservative analysis, and protein secondary structure prediction were used to identify the novel mutation. The results showed that the patient has compound heterozygous mutations in the MAN2B1 gene, c.856G > A (p.E286K, novel) and c.788C > T (p.P263L). Her parents are heterozygote that carry one of these two mutations respectively. Patho- genicity identification of the novel mutation showed that the p.E286K mutation is a disease-causing muta- tion. Our work enriches the human MAN2B1 gene muta- tion database. As far as we know, this research is thus far the first gene diagnosis case of a Chinese patient with α-mannosidosis.

Lysosomal alpha-mannosidase (EC 3.2.1.24) is required in the degradation of the asparagine-linked carbohydrates of glycoproteins. Deficiency of this enzyme leads to the lysosomal storage disorder alpha-mannosidosis. As an initial step toward enzyme replacement therapy for alpha-mannosidosis, the human lysosomal alpha-mannosidase cDNA was cloned into the pcDNA 3.1 vector and expressed in Chinese hamster ovary cells. Dimethyl sulfoxide (DMSO) added to the cell culture media to induce growth arrest led to a 4-fold increase in the enzyme production, with an average yield of 3.2 mg L(-1) day(-1). alpha-Mannosidase was secreted as an active homodimer of a 130-kDa precursor that was proteolyzed into two polypeptides of 55 and 72 kDa during the subsequent purification of the enzyme. N-terminal sequence analysis of the purified enzyme revealed that the proteolysis occurred close to a cleavage site previously identified in the intracellular form of lysosomal alpha-mannosidase. Generation of monoclonal antibodies against the recombinant enzyme made it possible to develop a single-step immunoaffinity purification procedure for alpha-mannosidase. The immunoaffinity-purified enzyme which mainly consisted of the 130-kDa precursor, displayed specific activity and kinetics similar to those of the processed form. Recombinant alpha-mannosidase was taken up by cultured alpha-mannosidosis fibroblasts and was trafficked to the lysosomes via the mannose 6-phosphate pathway where it reduced the amounts of stored mannose-containing oligosaccharides.

Beta-mannosidase deficiency results in beta-mannosidosis, a severe neurodegenerative lysosomal storage disease identified in cattle, goats, and humans. To more fully understand the molecular pathology of this disease, the mutation associated with bovine beta-mannosidosis was identified by sequence analysis of cDNA from an affected calf. A transition mutation of G to A at position 2574 of the cDNA coding sequence creates a premature stop codon near the 3' end of the protein coding region. To aid commercial breeders of Salers cattle, a PCR-based test was developed to detect the mutation for beta-mannosidosis carrier screening. Application of this test also revealed the presence of two beta-mannosidase pseudogenes. Portions of the pseudogenes were amplified with allele-specific primers and then sequenced. One pseudogene was highly homologous (99% sequence identity) to the expressed cDNA sequence over the 1292 bp that were sequenced, while the other showed more divergence (83% sequence identity) in the 477 bp that were sequenced. Both are processed pseudogenes that are not expressed. The severity of the bovine beta-mannosidosis phenotype suggests that the 22 C-terminal amino acids of beta-mannosidase play an important role in the function of this enzyme.

Glycoproteinoses belong to the lysosomal storage disorders group. The common feature of these diseases is the deficiency of a lysosomal protein that is part of glycan catabolism. Most of the lysosomal enzymes involved in the hydrolysis of glycoprotein carbohydrate chains are exo-glycosidases, which stepwise remove terminal monosaccharides. Thus, the deficiency of a single enzyme causes the blockage of the entire pathway and induces a storage of incompletely degraded substances inside the lysosome. Different mutations may be observed in a single disease and in all cases account for the nonexpression of lysosomal glycosidase activity. Different clinical phenotypes generally characterize a specific disorder, which rather must be described as a continuum in severity, suggesting that other biochemical or environmental factors influence the course of the disease. This review provides details on clinical features, genotype-phenotype correlations, enzymology and biochemical storage of four human glycoprotein lysosomal storage disorders, respectively alpha- and beta-mannosidosis, fucosidosis and alpha-N-acetylgalactosaminidase deficiency. Moreover, several animal disorders of glycoprotein metabolism have been found and constitute valuable models for the understanding of their human counterparts.

Lysosomal alpha-mannosidase (EC 3.2.1.24) is an exoglycosidase in the glycoprotein degradation pathway. A deficiency of this enzyme causes the lysosomal storage disease alpha-mannosidosis. Retrovirus vector transfer of a new human alpha-mannosidase cDNA resulted in high-level expression of alpha-mannosidase enzymatic activity in deficient human and feline fibroblasts. The expressed alpha-mannosidase had the same biochemical properties (thermal stability, pH profile, inhibitor/activator sensitivity) as the native enzyme expressed in normal cells. The transferred enzyme colocalized with a control lysosomal hydrolase in cell fractionation experiments. The vector-encoded enzyme also was released at high levels from the corrected cells, and was taken up by untreated mutant cells via the mannose 6-phosphate receptor-mediated endocytic pathway (cross-correction). It is envisioned that genetic correction of a subset of cells (e.g., hematopoietic stem cells) in patients will provide a source of corrective enzyme for other affected tissues in this multisystem disease. Development of a vector expressing high levels of alpha-mannosidase that cross-corrects mutant cells will enable somatic gene transfer experiments in the cat model of human alpha-mannosidosis.

alpha-Mannosidosis is an autosomal recessive lysosomal-storage disorder caused by a deficiency of lysosomal alpha-mannosidase activity. This disease shows a wide range of clinical phenotypes, from a severe, infantile form (type I), which is fatal at

Human beta-mannosidosis is an autosomal recessive, lysosomal storage disease caused by a deficiency of the enzyme beta-mannosidase. Unlike the severe clinical manifestation of the disease in ruminants, in which it leads to neonatal death, the human disease phenotype is generally milder. In addition, the phenotypic manifestation among the reported cases of human beta-mannosidosis is variable, even among members of the same family. To understand the molecular basis of the human disease and the mechanisms for such clinical variability, we sequenced the entire coding region of the human beta-mannosidase gene using a combination of cDNA library screening, RT-PCR and 5' rapid amplification of cDNA ends (RACE). The composite cDNA is 3293 nt, consisting of an 87 nt 5'-untranslated region, 2640 nt coding region and 566 nt 3'-untranslated region. The gene was localized to human chromosome 4q22-25. Analysis of a multiple tissue northern blot demonstrated a single 3.7 kb transcript. Mutation analysis of a Czech gypsy family with two siblings differently affected with beta-mannosidosis demonstrated a homozygous A-->G transition 2 bp upstream of a splice acceptor site. The associated cryptic splice site activation and exon skipping caused by this mutation resulted in two abnormally spliced mutant mRNA species in both siblings.

alpha-Mannosidosis is a lysosomal storage disorder that is caused by the deficiency of lysosomal alpha-mannosidase. Feline alpha-mannosidosis is a well-characterized animal model used for studying pathological and therapeutic aspects of lysosomal storage disorders. We here report the purification of feline liver lysosomal alpha-mannosidase and determination of its cDNA sequence. The active enzyme consisted of three polypeptides, with molecular masses of 72, 41 and 12 kDa, joined by non-covalent forces. The cDNA sequence of feline lysosomal alpha-mannosidase was determined from reverse transcriptase PCR products obtained from skin fibroblast mRNA. The deduced amino acid sequence contained the N-terminal sequences of the 72 and 41 kDa peptides. This indicated that the enzyme is synthesized as a single-chain precursor with a putative signal peptide of 50 amino acids followed by a polypeptide chain of 957 amino acids, which is cleaved into the three polypeptides of the mature enzyme. The deduced amino acid sequence was 81.1 and 83.2% identical with the human and bovine lysosomal alpha-mannosidases sequences respectively. A 4 bp deletion was identified in an alpha-mannosidosis-affected Persian cat by DNA sequencing of reverse transcriptase PCR products. The deletion resulted in a frame shift from codon 583 and premature termination at codon 645. No lysosomal alpha-mannosidase activity could be detected in the liver of this cat. A domestic long-haired cat expressing a milder alpha-mannosidosis phenotype than the Persian cat had a lysosomal alpha-mannosidase activity of 2% of normal. This domestic long-haired cat did not possess the 4 bp deletion, proving molecular heterogeneity for feline alpha-mannosidosis.

Lysosomal storage diseases associated with early-onset pathological changes may require prenatal therapy to avert the profound effects of the metabolic error on organs, especially the central nervous system. The present investigation determined the extent of expression of beta-mannosidase deficiency in the caprine fetus at 62 days of gestation, near the end of the period of immunotolerance when donor cells can engraft in various organs without immune rejection and supply missing enzyme. Three pairs of obligate carrier goats from the beta-mannosidosis colony were mated. Out of six fetuses delivered at 62 days of gestation, one (V385) was identified by measurement of beta-mannosidase activity as the only fetus affected with beta-mannosidosis. Thin-layer chromatography and quantitation of oligosaccharides revealed the presence of tri- and disaccharides, typical of beta-mannosidosis, only in V385. Morphological analysis revealed cytoplasmic vacuolation typical of beta-mannosidosis in V385; in thyroid, spinal cord, and kidney, the pattern of vacuolation was similar to, but less severe than, that observed previously in newborn affected goats. On the basis of these results, it will be possible to determine the effects of prenatal cell transplantation therapeutic strategies performed during the period of immunotolerance by monitoring phenotypic characteristics after treatment.

alpha-Mannosidosis of Angus calves was studied both for its veterinary importance and as a model of analogous human lysosomal storage diseases. This study facilitated a similar study in Australia on Swainsona spp. intoxication of livestock in which the toxic principle was shown to be an indolizidine alkaloid, Swainsonine. These genetic and acquired alpha-mannosidoses are compared with beta-mannosidosis. Collectively the study has helped the understanding of the processes of glycosylation and catabolism of glycoproteins. An experiment of nature involving an alpha-mannosidosis chimeric calf born co-twin to a normal calf helped to define the expectations and limitations of bone marrow transplants in this type of storage disease in humans. The inherited ceroid-lipofuscinoses (Batten disease) were studied in an ovine model. Isolation and analyses of the fluorescent accumulated lipopigment denied the dogma of lipid peroxidation current in the 1970s and 1980s. It was shown that in this, and analogous diseases in humans, the dominantly accumulated species was the very hydrophobic protein, subunit c of mitochondrial ATP synthase. Contrary to the adage that this should reflect a disorder of lysosomal proteolysis, there is accumulating evidence that the primary defect resides in mitochondria. Because of its hydrophobic nature, subunit c forms paracrystaline complexes which appear resistant to proteolysis within the lysosomal apparatus.

The lysosomal storage disorder alpha-mannosidosis is caused by deficiency of the enzyme lysosomal alpha-mannosidase (MAN2B1). In this study, 96 disease-associated sequence variants were identified in 130 unrelated alpha-mannosidosis patients from 30 countries. Eighty-three novel variants were detected, extending the mutation spectrum from 42 to 125. In total, 256 of the 260 mutant alleles (98.5%) were identified. Most of the variants were unique to each family, however, c.2248C>T (p.Arg750Trp) was detected in 50 patients from 16 countries, and accounted for 27.3% of the disease alleles. Haplotype analysis revealed that the c.2248T variant was present on four MAN2B1 haplotype backgrounds, where one major haplotype accounted for 95% of the alleles. The distribution of the c.2248T-associated haplotypes differed remarkably from those of the control populations, suggesting that c.2248C>T has occurred on a few ancestral haplotypes where the major haplotype subsequently has spread by founder effects. The disease-associated missense mutations were introduced into the human MAN2B1 cDNA, expressed in cell culture and assayed for MAN2B1 activity. The majority of the variants were inactive, however, ten showed MAN2B1 activity above background, and more detailed studies are necessary to further evaluate the pathogenicity of these variants.

We report on two unrelated patients with different presentations of mannosidosis. One patient was affected in early childhood with a severe phenotype characteristic of type I mannosidosis. The other was diagnosed with type II mannosidosis only after the onset of progressive neurologic deterioration in late adulthood. Both were detected by non-invasive urinary screening of oligosaccharides. Lymphoblasts transformed from both patients' blood cells had markedly reduced lysosomal α-mannosidase activity. Kinetic analyses showed that α-mannosidase from the type I patient had a 400-fold reduction in affinity while that from the type II patient was reduced 40-fold. Lymphoblasts from all 4 parents had reduced α-mannosidase activity, but there were overlapping activities among these type I and type II obligate heterozygotes. We conclude that screening urinary oligosaccharides will detect mannosidosis over a wide range of phenotypes, that lymphoblasts transformed from affected heterozygotes have decreased enzymatic activity, and that the severity of clinical expression is related to the degree of enzyme impairment. © 1995 Wiley-Liss, Inc.

Deficiency of glycosaminoglycan (GAG) degradation causes a subclass of lysosomal storage disorders called mucopolysaccharidoses (MPSs), many of which present with severe neuropathology. Critical steps in the degradation of the GAG heparan sulfate remain enigmatic. Here we show that the lysosomal arylsulfatase G (ARSG) is the long-sought glucosamine-3- O -sulfatase required to complete the degradation of heparan sulfate. Arsg -deficient mice accumulate heparan sulfate in visceral organs and the central nervous system and develop neuronal cell death and behavioral deficits. This accumulated heparan sulfate exhibits unique nonreducing end structures with terminal N -sulfoglucosamine-3- O -sulfate residues, allowing diagnosis of the disorder. Recombinant human ARSG is able to cleave 3- O -sulfate groups from these residues as well as from an authentic 3- O -sulfated N -sulfoglucosamine standard. Our results demonstrate the key role of ARSG in heparan sulfate degradation and strongly suggest that ARSG deficiency represents a unique, as yet unknown form of MPS, which we term MPS IIIE.

Neuronal storage disorders are fatal neurodegenerative diseases of humans and animals that are caused by inherited deficiencies of lysosomal hydrolase activity. Affected individuals often appear normal at birth but eventually develop progressive neurologic symptoms including sensory and motor deficits, mental retardation, and seizures. We have examined efficacy of bone marrow transplantation as a means of enzyme replacement, using cats with the lysosomal storage disease alpha-mannosidosis. Treated animals showed little or no progression of neurologic signs 1-2 years after transplant, whereas untreated cats became severely impaired and reached endstage disease by 6 months of age. Increased lysosomal alpha-mannosidase activity was found in brain tissue of the treated animals, and electron microscopy revealed no evidence of lysosomal storage within most neurons. Histochemical localization of acidic alpha-D-mannoside mannohydrolase (EC 3.2. 1.24), using 5-bromo-4-chloro-3-indolyl alpha-D-mannopyranoside, showed that functional enzyme was present in neurons, glial cells, and cells associated with blood vessels. This study provides direct evidence that bone marrow transplantation as treatment for a neuronal storage disease can lead to significant levels of a missing lysosomal hydrolase within neurons of the central nervous system and to compensation for the genetic metabolic defect.

Goats affected with beta-mannosidosis, an autosomal recessive disease of glycoprotein catabolism, have deficient tissue and plasma levels of the lysosomal enzyme beta-mannosidase. Pathological characteristics include cytoplasmic vacuolation in the nervous system and viscera, and myelin deficits that demonstrate regional variation. This study was designed to determine the correlation between beta-mannosidase activity in normal animals and the severity of lesions in affected goats, and to assess the regional changes in lysosomal enzyme activity in specific regions and cell types in affected animals. Although enzyme activity in normal organs (kidney, thyroid, brain) is correlated in general with the accumulation of uncatabolized substrate and with the extent of vacuolation, this correlation does not extend to assessment of specific regions of the central nervous system (CNS). In affected goats, the activities of alpha-mannosidase, alpha-fucosidase, and beta-hexosaminidase are elevated to a greater extent in all CNS regions than in organs. The results suggest cell-specific, organ-specific, and enzyme-specific regulation of changes in lysosomal enzyme activity in the presence of metabolic perturbations, such as deficiency of beta-mannosidase activity.

α-Mannosidosis is a lysosomal storage disorder caused by mutations in the MAN2B1 gene. The clinical presentation of α-mannosidosis is variable, but typically includes mental retardation, skeletal abnormalities and immune deficiency. In order to understand the molecular aetiology of α-mannosidosis, we describe here the subcellular localization and intracellular processing of 35 MAN2B1 variants, including 29 novel missense mutations. In addition, we have analysed the impact of the individual mutations on the three-dimensional structure of the human MAN2B1. We categorize the MAN2B1 missense mutations into four different groups based on their intracellular processing, transport and secretion in cell culture. Impaired transport to the lysosomes is a frequent cause of pathogenicity and correlates with a lack of protein processing (groups 1 and 3). Mutant MAN2B1 proteins that find their way to the lysosomes are processed, but less efficiently than the wild-types (groups 2 and 4). The described four categories of missense mutations likely represent different pathogenic mechanisms. We demonstrate that the severity of individual mutations cannot be determined based only on their position in the sequence. Pathogenic mutations cluster into amino acids which have an important role on the domain interface (arginines) or on the folding of the enzyme (prolines, glycines, cysteines). Tolerated mutations generally include surface mutations and changes without drastic alteration of residue volume. The expression system and structural details presented here provide opportunities for the development of pharmacological therapy by screening or design of small molecules that might assist MAN2B1 folding and hence, transport and activity.

α-Mannosidosis is a rare lysosomal storage disease with accumulation of undegraded mannosyl-linked oligosaccharides in cells throughout the body, most notably in the CNS. This leads to a broad spectrum of neurological manifestations, including progressive intellectual impairment, disturbed motor functions, and cerebellar atrophy. To develop therapeutic outcome measures for enzyme replacement therapy that could be used for human patients, a gene knockout model of α-mannosidosis in mice was analyzed for CNS pathology and motor deficits. In the cerebellar molecular layer, α-mannosidosis mice display clusters of activated Bergman glia, infiltration of phagocytic macrophages, and accumulation of free cholesterol and gangliosides (GM1), notably in regions lacking Purkinje cells. α-Mannosidosis brain lysates also displayed increased expression of Lamp1 and hyperglycosylation of the cholesterol binding protein NPC2. Detailed assessment of motor function revealed age-dependent gait defects in the mice that resemble the disturbed motor function in human patients. Short-term enzyme replacement therapy partially reversed the observed cerebellar pathology with fewer activated macrophages and astrocytes but unchanged levels of hyperglycosylated NPC2, gangliosides, and cholesterol. The present study demonstrates cerebellar alterations in α-mannosidosis mice that relate to the motor deficits and pathological changes seen in human patients and can be used as therapeutic outcome measures.

beta-Mannosidosis is a lethal lysosomal storage disease inherited as an autosomal recessive in man, cattle and goats. Laboratory assay data of plasma beta-mannosidase activity represent a mixture of homozygous normal and carrier genotype distributions in a proportion determined by genotype frequency. A maximum likelihood approach employing data transformations for each genotype distribution and assuming a diallelic model of inheritance is described. Estimates of the transformation and genotype distribution parameters, gene frequency, genotype fitness and carrier probability were obtained simultaneously from a sample of 2,812 observations on U.S. purebred Salers cattle with enzyme activity, age, gender, month of pregnancy, month of testing, and parents identified. Transformations to normality were not required, estimated gene and carrier genotype frequencies of 0.074 and 0.148 were high, and the estimated relative fitness of heterozygotes was 1.36. The apparent overdominance in fitness may be due to a nonrandom sampling of progeny genotypes within families. The mean of plasma enzyme activity was higher for males than females, higher in winter months, lower in summer months and decreased with increased age. Estimates of carrier probabilities indicate that the test is most effective when animals are sampled as calves, although effectiveness of the plasma assay was less for males than females. Test effectiveness was enhanced through averaging repeated assays of enzyme activity on each animal. Our approach contributes to medical diagnostics in several ways. Rather than assume underlying normality for the distributions comprising the mixture, we estimate transformations to normality for each genotype distribution simultaneously with all other model parameters. This process also excludes potential biases due to data preadjustment for systematic effects. We also provide a method for partitioning phenotypic variation within each genotypic distribution which allows an assessment of the value of repeat measurements of the predictive variable for genotype assignment.

Caprine β-mannosidosis is an inherited lysosomal storage disorder due to a deficiency of β-mannosidase which cleaves β-linked mannose residues from the ends of N-asparagine linked oligosaccharides of glycoproteins. Histological and chemical examination has revealed a deficiency of compact myelin in the brains and spinal cords of affected goats. Since myelin-associated glycoprotein (MAG) is glycosylated and its metabolism could be directly affected in this disease, we investigated the possibility of a differential treatment of MAG in caprine β-mannosidosis in comparison to non-glycosylated myelin proteins. MAG, myelin basic protein (MBP), 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP), proteolipid protein (PLP) and glial fibrillary acidic protein (GFAP) were quantified by western blot analysis in whole homogenates of spinal cords and hemispheres from affected goats at 1,4 and days of age and from normal controls. The yields of isolated myelin from the spinal cords of affected goats varied from 37 to 63% of normal and were 7% or less of normal from the hemispheres. In mutant spinal cords, the deficits of MAG, CNP and PLP measured in whole homogenates corresponded reasonably well with the decreased myelin yields, but the levels of MBP were consistently much closer to control levels than those of the other myelin proteins. A greater deficiency of PLP than MBP was also apparent in the myelin fractions purified from the affected spinal cords. In homogenates of mutant hemispheres, MAG, MBP, PLP and CNP were undetectable or at trace levels in comparison to controls. There was no indication of preferential involvement of MAG in this disorder, since the MAG deficiency was comparable to that of other unglycosylated myelin proteins, and its apparent molecular weight was the same as that of controls on SDS gels suggesting normal glycosylation. GFAP was increased (115–150% of the control level) in most tissue samples from the affected goats indicative of astrocytosis.

Lysosomal beta-mannosidase was purified 160,000-fold in 24% yield from bovine kidney by a four-step purification procedure, which included concanavalin A-Sepharose, immunoaffinity, TSK-butyl and h.p.l.c. cation-exchange chromatography. When analysed by SDS/PAGE and detected by Coomassie Blue or silver staining, the purified enzyme preparation consists of two prominent peptides (100 and 110 kDa) and a third minor peptide (84 kDa). These three peptides are immunologically related and are consistently associated with beta-mannosidase activity in all chromatographic steps. Removal of N-linked carbohydrate from the 84, 100 and 110 kDa peptides decreases their molecular sizes to 75, 86 and 91 kDa respectively. Bovine kidneys lacking beta-mannosidase, activity, acquired from calves affected with beta-mannosidosis, do not contain detectable quantities of the three beta-mannosidase peptides, as judged by monoclonal- and polyclonal-antibody reactivity.

Alpha-mannosidosis is caused by the genetic defect of the lysosomal alpha-d-mannosidase (LAMAN), which is involved in the breakdown of free alpha-linked mannose-containing oligosaccharides originating from glycoproteins with N-linked glycans, and thus manifests itself in an extensive storage of mannose-containing oligosaccharides. Here we demonstrate in a model of mice with alpha-mannosidosis that native lysosomal proteins exhibit elongated N-linked oligosaccharides as shown by two-dimensional difference gel electrophoresis, deglycosylation assays, and mass spectrometry. The analysis of cathepsin B-derived oligosaccharides revealed a hypermannosylation of glycoproteins in mice with alpha-mannosidosis as indicated by the predominance of extended Man3GlcNAc2 oligosaccharides. Treatment with recombinant human alpha-mannosidase partially corrected the hyperglycosylation of lysosomal proteins in vivo and in vitro. These data clearly demonstrate that LAMAN is involved not only in the lysosomal catabolism of free oligosaccharides but also in the trimming of asparagine-linked oligosaccharides on native lysosomal proteins.

The gingival tissues of a male patient suffering from mannosidosis and presenting with gingival overgrowth have been studied. Routine histological assessment highlighted the presence of highly enlarged and vacuolated lymphocytes. The morphology of the connective tissues, fibroblasts and epithelium appeared normal. Immunohistochemical staining of the tissues for chondroitin sulfate proteoglycan demonstrated a normal distribution of this component throughout the connective tissues and intense staining associated with the vacuolated lymphocytes. In vitro studies indicated that fibroblasts isolated from the overgrown tissue did not differ from age and sex matched control fibroblasts with respect to proliferation, protein and proteoglycan synthesis. Taken together, these findings imply that the gingvial overgrowth noted in this patient was not due to a defect in the resident fibroblasts but rather reflected a secondary response to the tissues to impaired host defence mechanisms.