Mannose-rich Oligosaccharides Research Articles

Reconstitution and resonance assignments of yeast OST subunit Ost4 and its critical mutant Ost4V23D in liposomes by solid-state NMR.

N-linked glycosylation is an essential and highly conserved co- and post-translational protein modification in all domains of life. In humans, genetic defects in N-linked glycosylation pathways result in metabolic diseases collectively called Congenital Disorders of Glycosylation. In this modification reaction, a mannose rich oligosaccharide is transferred from a lipid-linked donor substrate to a specific asparagine side-chain within the -N-X-T/S- sequence (where X ≠ Proline) of the nascent protein. Oligosaccharyltransferase (OST), a multi-subunit membrane embedded enzyme catalyzes this glycosylation reaction in eukaryotes. In yeast, Ost4 is the smallest of nine subunits and bridges the interaction of the catalytic subunit, Stt3, with Ost3 (or its homolog, Ost6). Mutations of any C-terminal hydrophobic residues in Ost4 to a charged residue destabilizes the enzyme and negatively impacts its function. Specifically, the V23D mutation results in a temperature-sensitive phenotype in yeast. Here, we report the reconstitution of both purified recombinant Ost4 and Ost4V23D each in a POPC/POPE lipid bilayer and their resonance assignments using heteronuclear 2D and 3D solid-state NMR with magic-angle spinning. The chemical shifts of Ost4 changed significantly upon the V23D mutation, suggesting a dramatic change in its chemical environment.

Long-term safety and efficacy of velmanase alfa treatment in children under 6 years of age with alpha-mannosidosis: A phase 2, open label, multicenter study.

Alpha-mannosidosis (AM) is a rare, autosomal recessive, lysosomal storage disorder caused by alpha-mannosidase deficiency that leads to the accumulation of mannose-rich oligosaccharides. AM symptoms and severity vary among individuals; consequently, AM is often not diagnosed until late childhood. Velmanase alfa (VA), a recombinant human lysosomal alpha-mannosidase product, is the first enzyme replacement therapy indicated to treat non-neurological symptoms of AM in Europe. Previous studies suggested that early VA treatment in children may produce greater clinical benefit over the disease course than starting treatment in adolescents or adults; however, long-term studies in children are limited, and very few studies include children under 6 years of age. The present phase 2, multicenter, open-label study evaluated the safety and efficacy of long-term VA treatment in children under 6 years of age with AM. Five children (three males) received VA weekly for ≥24 months, and all children completed the study. Four children experienced adverse drug reactions (16 events) and two experienced infusion-related reactions (12 events). Most (99.5%) adverse events were mild or moderate, and none caused study discontinuation. Four children developed antidrug antibodies (three were neutralizing). After VA treatment, all children improved in one or more efficacy assessments of serum oligosaccharide concentrations (decreases), hearing, immunological profile, and quality of life, suggesting a beneficial effect of early treatment. Although the small study size limits conclusions, these results suggest that long-term VA treatment has an acceptable safety profile, is well tolerated, and may provide potential benefits to patients with AM under 6 years of age.

The Application of HPLC-FLD and NMR in the Monitoring of Therapy Efficacy in Alpha-Mannosidosis.

Alpha-mannosidosis is a rare lysosomal storage disorder, caused by decreased activity of α-D-mannosidase. This enzyme is involved in the hydrolysis of mannosidic linkages in N-linked oligosaccharides. Due to the mannosidase defect, undigested mannose-rich oligosaccharides (Man2GlcNAc - Man9GlcNAc) accumulating in cells are excreted in large quantities in urine. In this work, we determined the levels of urinary mannose-rich oligosaccharides in a patient subjected to novel enzyme replacement therapy. Urinary oligosaccharides were extracted using solid phase extraction (SPE), labeled by fluorescent tag 2-aminobenzamide, and quantified by high-performance liquid chromatography (HPLC) with fluorescence detector (FLD). The identity of peaks was determined by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry. In addition, the levels of urinary mannose-rich oligosaccharides were also quantified by 1H nuclear magnetic resonance (NMR) spectroscopy. The data were analyzed using one-tailed paired t-test and Pearson's correlation tests. Compared to levels before the administration of therapy, an approximately two-folds decrease in total mannose-rich oligosaccharides after one month of treatment was observed by NMR and HPLC. After four months, an approximately ten-folds significant decrease in total urinary mannose-rich oligosaccharides was detected, suggesting therapy effectiveness. A significant decrease in the levels of oligosaccharides with 7-9 mannose units was detected by HPLC. The application of both HPLC-FLD and NMR in quantification of oligosaccharide biomarkers is a suitable approach for monitoring of therapy efficacy in alpha-mannosidosis patients.

Relationship between MAN2B1 genotype/subcellular localization subgroups, antidrug antibody detection, and long-term velmanase alfa treatment outcomes in patients with alpha-mannosidosis.

Alpha-mannosidosis (AM), an autosomal recessive disorder caused by pathogenic biallelic variants in the MAN2B1 gene, leads to lysosomal alpha-mannosidase deficiency and accumulation of mannose-rich oligosaccharides. Velmanase alfa (VA), a recombinant human lysosomal alpha-mannosidase, is the first enzyme replacement therapy for non-neurological symptoms of AM. Previously, a potential relationship was identified between three MAN2B1 genotype/subcellular localization subgroups (G1, G2, and G3) and AM disease severity. In VA-treated patients with AM, it is unknown if a relationship exists between MAN2B1 genotype/subcellular localization subgroups, antidrug antibodies (ADAs), and infusion-related reactions (IRRs). This pooled analysis evaluated data from 33 VA-treated patients with AM to investigate this relationship. Overall, 10 patients were positive for ADAs, 4 of whom had treatment-emergent ADAs (G1: 3/7 [43%]; G2: 1/17 [6%]; G3: 0/9). Treatment-emergent ADA-positive patients with relatively high titers (n=2; G1: 1012 U/ml and G2: 440 U/ml) experienced mild/moderate IRRs that were well-managed; patients with lower titers (n=2) experienced no IRRs. Overall, changes from baseline in serum oligosaccharides and immunoglobulin G levels did not vary between ADA-positive and ADA-negative patients, suggesting a similar effect of VA treatment regardless of ADA status in most patients. Clinical outcomes (3MSCT and 6MWT) were also similar in most patients regardless of ADA status. While further studies are needed, these data suggest a relationship between MAN2B1 genotype/subcellular localization subgroups and ADA development, with G1 and G2 subgroups more likely to develop ADAs and IRRs. Regardless, this study suggests that ADAs have limited effect on the clinical impact of VA in most patients with AM.

Mannose-rich Oligosaccharides-functionalized selenium nanoparticles mediates Macrophage reprogramming and inflammation resolution in ulcerative colitis

Inflammatory bowel disease (IBD) is a global disease and usually classified into Crohn’s disease (CD) and ulcerative colitis (UC), in which the inflammatory response is known to play a pivotal role. Herein, a bioactive component of mannose-rich oligosaccharides (MRO) was produced by enzymatic hydrolysis with most potent anti-inflammation and employed to functionalize selenium nanoparticles (SeNPs), aming to solve of inflammation in UC by accelerating the reprogramming of macrophages. In this study, SeNPs was prepared by selenite and MRO with ascorbic acid reduction. The stability and biocompatibility of SeNPs was obviously improved by the functionalization of MRO, resulting in spheres (2 µM Se) carrying around 26 ng/ml MRO (MRO-SeNPs) with an average diameter of 70 nm remained stable in 60 d. In addition, efficient cellular uptake of MRO-SeNPs in macrophages was detected by contribution of MRO targeted to the receptors of macrophages. As expected, MRO-SeNPs exhibited improvement of macrophage reprogramming in vitro, thus leading to a typical M2 polarization profile of cytokine expression. Furthermore, in DSS-induced mouse colitis model, MRO-SeNPs effectively mitigated colitis by suppressing the pro-inflammatory such as IL-1β, IL-6, TNF-α, IL-12 and MCP-1 cytokines in situ, and exerted salient antioxidant capacity in colon tissues. As a result, MRO-SeNPs exhibited therapeutic potential by promoting macrophages reprogramming leading to inflammation resolution. Taken together, this study not only provides strategy for functionalization of nanomedicines using structure-optimized MRO, but also reveals the action mechanism on successful treatment of IBD and other intestinal diseases.

Hearing impairment as an early sign of alpha-mannosidosis in children with a mild phenotype: Report of seven new cases.

Alpha-mannosidosis (AM) is a very rare (prevalence: 1/500000 births) autosomal recessive lysosomal storage disorder. It is characterized by multi-systemic involvement associated with progressive intellectual disability, hearing loss, skeletal anomalies, and coarse facial features. The spectrum is wide, from very severe and lethal to a milder phenotype that usually progresses slowly. AM is caused by a deficiency of lysosomal alpha-mannosidase. A diagnosis can be established by measuring the activity of lysosomal alpha-mannosidase in leucocytes and screening for abnormal urinary excretion of mannose-rich oligosaccharides. Genetic confirmation is obtained with the identification of MAN2B1 mutations. Enzyme replacement therapy (LAMZEDER ) was approved for use in Europe in August 2018. Here, we describe seven individuals from four families, diagnosed at 3-23 years of age, and who were referred to a clinical geneticist for etiologic exploration of syndromic hearing loss, associated with moderate learning disabilities. Exome sequencing had been used to establish the molecular diagnosis in five cases, including a two-sibling pair. In the remaining two patients, the diagnosis was obtained with screening of urinary oligosaccharides excretion and the association of deafness and hypotonia. These observations emphasize that the clinical diagnosis of AM can be challenging, and that it is likely an underdiagnosed rare cause of syndromic hearing loss. Exome sequencing can contribute significantly to the early diagnosis of these nonspecific mild phenotypes, with advantages for treatment and management.

Multiple bovine abortions with evidence of fetal locoism from pre-clinically intoxicated dams

Chronic consumption of specific plant species from the genera Oxytropis, Astragalus, lpomeoa, and Swainsonia, commonly known as locoweeds, is well known to induce locoism in grazing animals. Locoism in cattle is characterized by sensory deficits, ataxia, behavior changes, loss of condition, and failure to grow. The toxic principle swainsonine is rapidly absorbed and distributed through the bloodstream to multiple organs where it inhibits cellular lysosomal alphamannosidase and Golgi mannosidase II, leading to lysosomal dysfunction and disruption of glycoprotein processing. Excessive mannose-rich oligosaccharide accumulations cause cytoplasmic vacuolation and cellular dysfunction. In addition to neurologic disease, chronic consumption of locoweeds is known to induce abortion and embryonic death in neurologically affected dams. We describe multiple cases of bovine abortion associated with chronic locoweed ingestion in non-clinical dams from a herd in southeastern Colorado. Neurons from the cerebral cortex, brainstem, cerebellum, and renal tubular epithelial cells displayed the characteristic cytoplasmic vacuolation observed in adult cases of locoism in the aborted animals. Although chronic ingestion of locoweed is widely known to cause early gestation embryonic death, late-term hydrops amnii, and fetal fluid accumulation, locoweed-induced abortion should be considered as a differential diagnosis for cases ofnon-infectious abortion in grazing cattle, even in the absence of maternal clinical signs. These findings indicate that the bovine fetus may be more sensitive to the abortive effects of swainsonine than previously thought.

Enzyme replacement therapy with velmanase alfa (human recombinant alpha-mannosidase): Novel global treatment response model and outcomes in patients with alpha-mannosidosis

Alpha-mannosidosis is an ultra-rare monogenic disorder resulting from a deficiency in the lysosomal enzyme alpha-mannosidase, with a prevalence estimated to be as low as 1:1,000,000 live births. The resulting accumulation of mannose-rich oligosaccharides in all tissues leads to a very heterogeneous disorder with a continuum of clinical manifestations with no distinctive phenotypes. Long-term enzyme replacement therapy (ERT) with velmanase alfa is approved in Europe for the treatment of non-neurological manifestations in patients with mild to moderate alpha-mannosidosis. The clinical heterogeneity and rarity of the disease limit the sensitivity of single parameters to detect clinically relevant treatment effects. Thus, we propose a novel multiple variable responder analysis to evaluate the efficacy of ERT for alpha-mannosidosis and present efficacy analyses for velmanase alfa using this method.Global treatment response to velmanase alfa (defined by response to ≥2 domains comprising pharmacodynamic, functional, and quality of life outcomes) was applied post hoc to data from the pivotal placebo-controlled rhLAMAN-05 study and to the longer-term integrated data from all patients in the clinical development program (rhLAMAN-10). After 12 months of treatment, a global treatment response was achieved by 87% of patients receiving velmanase alfa (n = 15) compared with 30% of patients receiving placebo (n = 10). Longer-term data from all patients in the clinical program (n = 33) showed 88% of patients were global responders, including all (100%) pediatric patients (n = 19) and the majority (71%) of adult patients (n = 14). The responder analysis model demonstrates a clinically meaningful treatment effect with velmanase alfa and supports the early initiation and continued benefit of longer-term treatment of all patients with alpha-mannosidosis with this ERT.

PPS beyond MPS: Efficacy in Fabry and Gaucher in vitro studies

Alpha-mannosidosis is an ultra-rare monogenic disorder resulting from a deficiency in the lysosomal enzyme alpha-mannosidase, with a prevalence estimated to be as low as 1:1,000,000 live births. The resulting accumulation of mannose-rich oligosaccharides in all tissues leads to a very heterogeneous disorder with a continuum of clinical manifestations with no distinctive phenotypes. Long-term enzyme replacement therapy (ERT) with velmanase alfa is approved in Europe for the treatment of non-neurological manifestations in patients with mild to moderate alpha-mannosidosis. The clinical heterogeneity and rarity of the disease limit the sensitivity of single parameters to detect clinically relevant treatment effects. Thus, we propose a novel multiple variable responder analysis to evaluate the efficacy of ERT for alpha-mannosidosis and present efficacy analyses for velmanase alfa using this method.Global treatment response to velmanase alfa (defined by response to ≥2 domains comprising pharmacodynamic, functional, and quality of life outcomes) was applied post hoc to data from the pivotal placebo-controlled rhLAMAN-05 study and to the longer-term integrated data from all patients in the clinical development program (rhLAMAN-10). After 12 months of treatment, a global treatment response was achieved by 87% of patients receiving velmanase alfa (n = 15) compared with 30% of patients receiving placebo (n = 10). Longer-term data from all patients in the clinical program (n = 33) showed 88% of patients were global responders, including all (100%) pediatric patients (n = 19) and the majority (71%) of adult patients (n = 14). The responder analysis model demonstrates a clinically meaningful treatment effect with velmanase alfa and supports the early initiation and continued benefit of longer-term treatment of all patients with alpha-mannosidosis with this ERT.

Simple fluorimetric test for lysosomal swelling as a means to identify, monitor and develop therapies for all lysosomal diseases

Alpha-mannosidosis is an ultra-rare monogenic disorder resulting from a deficiency in the lysosomal enzyme alpha-mannosidase, with a prevalence estimated to be as low as 1:1,000,000 live births. The resulting accumulation of mannose-rich oligosaccharides in all tissues leads to a very heterogeneous disorder with a continuum of clinical manifestations with no distinctive phenotypes. Long-term enzyme replacement therapy (ERT) with velmanase alfa is approved in Europe for the treatment of non-neurological manifestations in patients with mild to moderate alpha-mannosidosis. The clinical heterogeneity and rarity of the disease limit the sensitivity of single parameters to detect clinically relevant treatment effects. Thus, we propose a novel multiple variable responder analysis to evaluate the efficacy of ERT for alpha-mannosidosis and present efficacy analyses for velmanase alfa using this method.Global treatment response to velmanase alfa (defined by response to ≥2 domains comprising pharmacodynamic, functional, and quality of life outcomes) was applied post hoc to data from the pivotal placebo-controlled rhLAMAN-05 study and to the longer-term integrated data from all patients in the clinical development program (rhLAMAN-10). After 12 months of treatment, a global treatment response was achieved by 87% of patients receiving velmanase alfa (n = 15) compared with 30% of patients receiving placebo (n = 10). Longer-term data from all patients in the clinical program (n = 33) showed 88% of patients were global responders, including all (100%) pediatric patients (n = 19) and the majority (71%) of adult patients (n = 14). The responder analysis model demonstrates a clinically meaningful treatment effect with velmanase alfa and supports the early initiation and continued benefit of longer-term treatment of all patients with alpha-mannosidosis with this ERT.

The cyanobacterial lectin scytovirin displays potent in vitro and in vivo activity against Zaire Ebola virus

The cyanobacterial lectin scytovirin (SVN) binds with high affinity to mannose-rich oligosaccharides on the envelope glycoprotein (GP) of a number of viruses, blocking entry into target cells. In this study, we assessed the ability of SVN to bind to the envelope GP of Zaire Ebola virus (ZEBOV) and inhibit its replication. SVN interacted specifically with the protein’s mucin-rich domain. In cell culture, it inhibited ZEBOV replication with a 50% virus-inhibitory concentration (EC50) of 50nM, and was also active against the Angola strain of the related Marburg virus (MARV), with a similar EC50. Injected subcutaneously in mice, SVN reached a peak plasma level of 100nm in 45min, but was cleared within 4h. When ZEBOV-infected mice were given 30mg/kg/day of SVN by subcutaneous injection every 6h, beginning the day before virus challenge, 9 of 10 animals survived the infection, while all infected, untreated mice died. When treatment was begun one hour or one day after challenge, 70–90% of mice survived. Quantitation of infectious virus and viral RNA in samples of serum, liver and spleen collected on days 2 and 5 postinfection showed a trend toward lower titers in treated than control mice, with a significant decrease in liver titers on day 2. Our findings provide further evidence of the potential of natural lectins as therapeutic agents for viral infections.

Successful unrelated bone marrow transplantation in two siblings with alpha‐mannosidosis

Alpha-mannosidosis is a rare lysosomal storage disorder with an autosomal recessive inheritance. Deficient alpha-mannosidase activity leads to lysosomal accumulation of mannose-rich oligosaccharides. The disease characterized by mental retardation, skeletal changes, hearing impairment, and recurrent infections. Stem cell transplantation has been shown to be an effective treatment. It works by providing increased levels of α-mannosidase in the localized extracellular milieu to provide improvements in skeletal malformations, neurocognitive, and sensorineural function. In this case report, we describe a pair of siblings with α-mannosidosis who successfully underwent HSCT from matched unrelated donors. In both siblings, enzyme levels reached to normal limits and improvements in clinical symptoms were recognized early after HSCT. We conclude that HSCT should be considered as a therapeutic approach in patients with alpha-mannosidosis before disease-related complications have developed.

Cell Walls of Saccharomyces cerevisiae Differentially Modulated Innate Immunity and Glucose Metabolism during Late Systemic Inflammation

Background Salmonella causes acute systemic inflammation by using its virulence factors to invade the intestinal epithelium. But, prolonged inflammation may provoke severe body catabolism and immunological diseases. Salmonella has become more life-threatening due to emergence of multiple-antibiotic resistant strains. Mannose-rich oligosaccharides (MOS) from cells walls of Saccharomyces cerevisiae have shown to bind mannose-specific lectin of Gram-negative bacteria including Salmonella, and prevent their adherence to intestinal epithelial cells. However, whether MOS may potentially mitigate systemic inflammation is not investigated yet. Moreover, molecular events underlying innate immune responses and metabolic activities during late inflammation, in presence or absence of MOS, are unknown.Methods and Principal FindingsUsing a Salmonella LPS-induced systemic inflammation chicken model and microarray analysis, we investigated the effects of MOS and virginiamycin (VIRG, a sub-therapeutic antibiotic) on innate immunity and glucose metabolism during late inflammation. Here, we demonstrate that MOS and VIRG modulated innate immunity and metabolic genes differently. Innate immune responses were principally mediated by intestinal IL-3, but not TNF-α, IL-1 or IL-6, whereas glucose mobilization occurred through intestinal gluconeogenesis only. MOS inherently induced IL-3 expression in control hosts. Consequent to LPS challenge, IL-3 induction in VIRG hosts but not differentially expressed in MOS hosts revealed that MOS counteracted LPS's detrimental inflammatory effects. Metabolic pathways are built to elucidate the mechanisms by which VIRG host's higher energy requirements were met: including gene up-regulations for intestinal gluconeogenesis (PEPCK) and liver glycolysis (ENO2), and intriguingly liver fatty acid synthesis through ATP citrate synthase (CS) down-regulation and ATP citrate lyase (ACLY) and malic enzyme (ME) up-regulations. However, MOS host's lower energy demands were sufficiently met through TCA citrate-derived energy, as indicated by CS up-regulation.ConclusionsMOS terminated inflammation earlier than VIRG and reduced glucose mobilization, thus representing a novel biological strategy to alleviate Salmonella-induced systemic inflammation in human and animal hosts.

Mutagenesis of Surfactant Protein D Informed by Evolution and X-ray Crystallography Enhances Defenses against Influenza A Virus in Vivo

The recognition of influenza A virus (IAV) by surfactant protein D (SP-D) is mediated by interactions between the SP-D carbohydrate recognition domains (CRD) and glycans displayed on envelope glycoproteins. Although native human SP-D shows potent antiviral and aggregating activity, trimeric recombinant neck+CRDs (NCRDs) show little or no capacity to influence IAV infection. A mutant trimeric NCRD, D325A/R343V, showed marked hemagglutination inhibition and viral neutralization, with viral aggregation and aggregation-dependent viral uptake by neutrophils. D325A/R343V exhibited glucose-sensitive binding to Phil82 hemagglutinin trimer (HA) by surface plasmon resonance. By contrast, there was very low binding to the HA trimer from another virus (PR8) that lacks glycans on the HA head. Mass spectrometry demonstrated the presence of high mannose glycans on the Phil82 HA at positions known to contribute to IAV binding. Molecular modeling predicted an enhanced capacity for bridging interactions between HA glycans and D325A/R343V. Finally, the trimeric D325A/R343V NCRD decreased morbidity and increased viral clearance in a murine model of IAV infection using a reassortant A/WSN/33 virus with a more heavily glycosylated HA. The combined data support a model in which altered binding by a truncated mutant SP-D to IAV HA glycans facilitates viral aggregation, leading to significant viral neutralization in vitro and in vivo. These studies demonstrate the potential utility of homology modeling and protein structure analysis for engineering effective collectin antivirals as in vivo therapeutics.

Intracellular trafficking of lysosomal proteins and lysosomes

In the synthesis and trafficking of precursors of most lysosomal matrix proteins, the stages necessary for lysosomal delivery include the addition of phosphorylated mannose-rich oligosaccharides, binding of the modified proteins to receptors, their segregation from the secretory pathways and delivery to the endosomal pathway. Targeting of both internally synthesized and externally provided enzymes (as in enzyme replacement therapy) to endosomes is executed by a complex machinery of membrane and cytosolic proteins. Recently, the homotypic fusion and vacuolar protein sorting (HOPS) complex has been identified in lysosomes from human cells. This complex is likely to play an important role in the exchange of enzymes between endosomal and lysosomal compartments. The present review describes the interactions and functions of proteins that participate in delivering lysosomal proteins to different lysosomal compartments. In summary, lysosomal trafficking depends on the recognition of many structural signals. It delivers soluble and membrane proteins, and can be exploited for therapeutic substitution of missing enzymes.

Alpha-mannosidase activity in goats fed with Sida carpinifolia

Human α-mannosidosis results from α-mannosidase deficiency and progressive accumulation of mannose-rich oligosaccharides in lysosomes. Two days before Saanen goats were fed with Sida carpinifolia, α-mannosidase activity in leukocytes was 128±28 nmoles 4-MU/h/mg protein (first trial) and 104±6 nmoles 4-MU/h/mg protein (second trial). At day 5, after the introduction of S. carpinifolia diet, the α-mannosidase activity in leukocytes was significantly increased, both in the first (288±13 nmoles 4-MU/h/mg protein) and in the second trial (303±45 nmoles 4-MU/h/mg protein), and it returned to normal levels 2 days after the withdrawal of the plant from the diet (114±7 nmoles 4-MU/h/mg protein in first trial, and 108±25 nmoles 4-MU/h/mg protein in the second one). Plasma α-mannosidase activity decreased significantly 4 days after animal exposure to the S. carpinifolia diet (769±167 nmoles 4-MU/h/ml) and returned to normal values 10 days after the withdrawal of the plant from the diet (1289±163 nmoles 4-MU/h/ml). Thin-layer chromatography showed an abnormal excretion of oligosaccharides in urine as of day 2 after diet exposure, which persisted until one day after the withdrawal of the plant. Animals presented neurological clinical signs beginning at day 37 (in the first trial) and at day 25 (in the second trial) after being fed with the plant. The results obtained herein suggest that oligosaccharides observed in urine are a result of a decrease in α-mannosidase activity in plasma. S. carpinifolia seems to have other compounds that act on α-mannosidase enzyme in leukocytes in a competitive manner with swainsonine. The increase in α-mannosidase enzyme in leukocytes could be attributed to one of these compounds present in S. carpinifolia.

Alpha-mannosidosis

Alpha-mannosidosis is an inherited lysosomal storage disorder characterized by immune deficiency, facial and skeletal abnormalities, hearing impairment, and intellectual disability. It occurs in approximately 1 of 500,000 live births. The children are often born apparently normal, and their condition worsens progressively. Some children are born with ankle equinus or develop hydrocephalus in the first year of life. Main features are immune deficiency (manifested by recurrent infections, especially in the first decade of life), skeletal abnormalities (mild-to-moderate dysostosis multiplex, scoliosis and deformation of the sternum), hearing impairment (moderate-to-severe sensorineural hearing loss), gradual impairment of mental functions and speech, and often, periods of psychosis. Associated motor function disturbances include muscular weakness, joint abnormalities and ataxia. The facial trait include large head with prominent forehead, rounded eyebrows, flattened nasal bridge, macroglossia, widely spaced teeth, and prognathism. Slight strabismus is common. The clinical variability is significant, representing a continuum in severity. The disorder is caused by lysosomal alpha-mannosidase deficiency. Alpha-mannosidosis is inherited in an autosomal recessive fashion and is caused by mutations in the MAN2B1 gene located on chromosome 19 (19 p13.2-q12). Diagnosis is made by measuring acid alpha-mannosidase activity in leukocytes or other nucleated cells and can be confirmed by genetic testing. Elevated urinary secretion of mannose-rich oligosaccharides is suggestive, but not diagnostic. Differential diagnoses are mainly the other lysosomal storage diseases like the mucopolysaccharidoses. Genetic counseling should be given to explain the nature of the disease and to detect carriers. Antenatal diagnosis is possible, based on both biochemical and genetic methods. The management should be pro-active, preventing complications and treating manifestations. Infections must be treated frequently. Otolaryngological treatment of fluid in the middle ear is often required and use of hearing aids is invariably required. Early educational intervention for development of social skills is needed and physiotherapy is important to improve bodily function. Orthopedic surgery may be necessary. The long-term prognosis is poor. There is an insidiously slow progression of neuromuscular and skeletal deterioration over several decades, making most patients wheel-chair dependent. No patients manage to be completely socially independent. Many patients are over 50 years of age.

The Comparative Pathology of the Glycosidase Inhibitors Swainsonine, Castanospermine, and Calystegines A3, B2, and C1 in Mice

To study various polyhydroxy-alkaloid glycosidase inhibitors, 16 groups of 3 mice were dosed using osmotic minipumps with swainsonine (0, 0.1, 1, and 10 mg/kg/day), castanospermine, and calystegines A(3), B(2), and C(1) (1, 10, and 100 mg/kg/day). After 28 days, the mice were euthanized, necropsied, and examined using light and electron microscopy. The high-dose swainsonine-treated mice developed neurologic disease with neuro-visceral vacuolation typical of locoweed poisoning. Castanospermine- and calystegines-treated mice were clinically normal; however, high-dose castanospermine-treated mice had thyroid, renal, hepatic, and skeletal myocyte vacuolation. Histochemically, swainsonine- and castanospermine-induced vacuoles contained mannose-rich oligosaccharides. High-dose calystegine A(3)-treated mice had increased numbers of granulated cells in the hepatic sinusoids. Electron microscopy, lectin histochemistry, and immunohistochemistry suggest these are pit cells (specialized NK cells). Histochemically, the granules contain glycoproteins or oligosaccharides with abundant terminal N-acetylglucosamine residues. Other calystegine-treated mice were histologically normal. These findings indicate that swainsonine produced lesions similar to locoweed, castanospermine caused vacuolar changes with minor changes in glycogen metabolism, and only calystegine A(3) produced minimal hepatic changes. These also suggest that in mice calystegines and castanospermine are less toxic than swainsonine, and as rodents are relatively resistant to disease, they are poor models to study such induced storage diseases.

Conformational gating of dimannose binding to the antiviral protein cyanovirin revealed from the crystal structure at 1.35 A resolution.

Cyanovirin (CV-N) is a small lectin with potent HIV neutralization activity, which could be exploited for a mucosal defense against HIV infection. The wild-type (wt) protein binds with high affinity to mannose-rich oligosaccharides on the surface of gp120 through two quasi-symmetric sites, located in domains A and B. We recently reported on a mutant of CV-N that contained a single functional mannose-binding site, domain B, showing that multivalent binding to oligomannosides is necessary for antiviral activity. The structure of the complex with dimannose determined at 1.8 A resolution revealed a different conformation of the binding site than previously observed in the NMR structure of wt CV-N. Here, we present the 1.35 A resolution structure of the complex, which traps three different binding conformations of the site and provides experimental support for a locking and gating mechanism in the nanoscale time regime observed by molecular dynamics simulations.

Developmental Analysis of CNS Pathology in the Lysosomal Storage Disease α-Mannosidosis

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.