Impaired catabolism of free oligosaccharides due to MAN2C1 variants causes a neurodevelopmental disorder.

Free oligosaccharides (fOSs) are soluble oligosaccharide species generated during N-glycosylation of proteins. Although little is known about fOS metabolism, the recent identification of NGLY1 deficiency, a congenital disorder of deglycosylation (CDDG) caused by loss of function of an enzyme involved in fOS metabolism, has elicited increased interest in fOS processing. The catabolism of fOSs has been linked to the activity of a specific cytosolic mannosidase, MAN2C1, which cleaves α1,2-, α1,3-, and α1,6-mannose residues. In this study, we report the clinical, biochemical, and molecular features of six individuals, including two fetuses, with bi-allelic pathogenic variants in MAN2C1; the individuals are from four different families. These individuals exhibit dysmorphic facial features, congenital anomalies such as tongue hamartoma, variable degrees of intellectual disability, and brain anomalies including polymicrogyria, interhemispheric cysts, hypothalamic hamartoma, callosal anomalies, and hypoplasia of brainstem and cerebellar vermis. Complementation experiments with isogenic MAN2C1-KO HAP1 cells confirm the pathogenicity of three of the identified MAN2C1 variants. We further demonstrate that MAN2C1 variants lead to accumulation and delay in the processing of fOSs in proband-derived cells. These results emphasize the involvement of MAN2C1 in human neurodevelopmental disease and the importance of fOS catabolism.

Bi-allelic variants in the ER quality-control mannosidase gene EDEM3 cause a congenital disorder of glycosylation.

EDEM3 encodes a protein that converts Man8GlcNAc2 isomer B to Man7-5GlcNAc2. It is involved in the endoplasmic reticulum-associated degradation pathway, responsible for the recognition of misfolded proteins that will be targeted and translocated to the cytosol and degraded by the proteasome. In this study, through a combination of exome sequencing and gene matching, we have identified seven independent families with 11 individuals with bi-allelic protein-truncating variants and one individual with a compound heterozygous missense variant in EDEM3. The affected individuals present with an inherited congenital disorder of glycosylation (CDG) consisting of neurodevelopmental delay and variable facial dysmorphisms. Experiments in human fibroblast cell lines, human plasma, and mouse plasma and brain tissue demonstrated decreased trimming of Man8GlcNAc2 isomer B to Man7GlcNAc2, consistent with loss of EDEM3 enzymatic activity. In human cells, Man5GlcNAc2 to Man4GlcNAc2 conversion is also diminished with an increase of Glc1Man5GlcNAc2. Furthermore, analysis of the unfolded protein response showed a reduced increase in EIF2AK3 (PERK) expression upon stimulation with tunicamycin as compared to controls, suggesting an impaired unfolded protein response. The aberrant plasma N-glycan profile provides a quick, clinically available test for validating variants of uncertain significance that may be identified by molecular genetic testing. We propose to call this deficiency EDEM3-CDG.

Diagnosis of alpha-Mannosidosis: Practical approaches to reducing diagnostic delays in this ultra-rare disease.

Alpha-mannosidosis is an ultra-rare lysosomal disease that is caused by variants of the MAN2B1 gene on chromosome 19p13. These variants result in faulty or absent alpha-mannosidase in lysosomes, which leads to intracellular accumulation of mannose-containing oligosaccharides. Diagnosis of alpha-mannosidosis is often delayed, in part because of the rarity of the disease, its gradual onset and heterogeneity of presentation, but also because of the similarity of many signs and symptoms of the disease to those of other lysosomal diseases. Treatment of alpha-mannosidosis was previously limited to hematopoietic stem cell transplantation, but outcomes are variable and not all patients are eligible or have a suitable donor. Recently, an enzyme replacement therapy, recombinant human alpha-mannosidase (velmanase alfa), was approved for the treatment of non-neurological manifestations in adult and pediatric patients with alpha-mannosidosis. Treatment with velmanase alfa reduces serum levels of oligosaccharides, increases levels of immunoglobulin G, and improves patients' functional capacity and quality of life, although it is not effective for the neurologic phenotype because it does not cross the blood-brain barrier. Since the effects of velmanase alfa are more marked in children than adults, early diagnosis to allow early initiation of treatment has become more important. To support this, patient, parent/caregiver, and clinician awareness and education is imperative. A number of approaches can be taken to meet this goal, such as the development of disease registries, validated diagnostic algorithms, and screening tools, improved under-/post-graduate clinician education, easily accessible and reliable information for patients/families (such as that made available on the internet), and the formation of patient advocacy groups. Such approaches may raise awareness of alpha-mannosidosis, reduce the diagnostic delay and thus improve the lives of those affected.

Alpha-mannosidase-2 modulates arbovirus infection in a pathogen- and Wolbachia-specific manner in Aedes aegypti mosquitoes.

Multiple Wolbachia strains can block pathogen infection, replication and/or transmission in Aedes aegypti mosquitoes under both laboratory and field conditions. However, Wolbachia effects on pathogens can be highly variable across systems and the factors governing this variability are not well understood. It is increasingly clear that the mosquito host is not a passive player in which Wolbachia governs pathogen transmission phenotypes; rather, the genetics of the host can significantly modulate Wolbachia-mediated pathogen blocking. Specifically, previous work linked variation in Wolbachia pathogen blocking to polymorphisms in the mosquito alpha-mannosidase-2 (αMan2) gene. Here we use CRISPR-Cas9 mutagenesis to functionally test this association. We developed αMan2 knockouts and examined effects on both Wolbachia and virus levels, using dengue virus (DENV; Flaviviridae) and Mayaro virus (MAYV; Togaviridae). Wolbachia titres were significantly elevated in αMan2 knockout (KO) mosquitoes, but there were complex interactions with virus infection and replication. In Wolbachia-uninfected mosquitoes, the αMan2 KO mutation was associated with decreased DENV titres, but in a Wolbachia-infected background, the αMan2 KO mutation significantly increased virus titres. In contrast, the αMan2 KO mutation significantly increased MAYV replication in Wolbachia-uninfected mosquitoes and did not affect Wolbachia-mediated virus blocking. These results demonstrate that αMan2 modulates arbovirus infection in A. aegypti mosquitoes in a pathogen- and Wolbachia-specific manner, and that Wolbachia-mediated pathogen blocking is a complex phenotype dependent on the mosquito host genotype and the pathogen. These results have a significant impact for the design and use of Wolbachia-based strategies to control vector-borne pathogens.

An unusual diagnosis of alpha-mannosidosis with ocular anomalies: Behind the scenes of a hidden copy number variation.

Alpha-mannosidosis is a rare autosomal recessive lysosomal storage disorder caused by biallelic mutations in the MAN2B1 gene and characterized by a wide clinical heterogeneity. Diagnosis for this multisystemic disorder is confirmed by the presence of either a deficiency in the lysosomal enzyme acid alpha-mannosidase or biallelic mutations in the MAN2B1 gene. This diagnosis confirmation is crucial for both clinical management and genetic counseling purposes. Here we describe a late diagnosis of alpha-mannosidosis in a patient presenting with syndromic intellectual disability, and a rare retinopathy, where reverse phenotyping played a pivotal role in interpreting the exome sequencing result. While a first missense variant was classified as a variant of uncertain significance, the phenotype-guided analysis helped us detect and interpret an in-trans apparent alu-element insertion, which appeared to be a copy number variant (CNV) not identified by the CNV caller. A biochemical analysis showing abnormal excretion of urinary mannosyloligosaccharide and an enzyme assay permitted the re-classification of the missense variant to likely pathogenic, establishing the diagnosis of alpha-mannosidosis. This work emphasizes the importance of reverse phenotyping in the context of exome sequencing.

Involvement of the GH38 Family Exoglycosidase α-Mannosidase in Strawberry Fruit Ripening.

Exoglycosidase enzymes hydrolyze the N-glycosylations of cell wall enzymes, releasing N-glycans that act as signal molecules and promote fruit ripening. Vesicular exoglycosidase α-mannosidase enzymes of the GH38 family (EC 3.2.1.24; α-man) hydrolyze N-glycans in non-reduced termini. Strawberry fruit (Fragaria × ananassa) is characterized by rapid softening as a result of cell wall modifications during the fruit ripening process. Enzymes acting on cell wall polysaccharides explain the changes in fruit firmness, but α-man has not yet been described in F. × ananassa, meaning that the indirect effects of N-glycan removal on its fruit ripening process are unknown. The present study identified 10 GH38 α-man sequences in the F. × ananassa genome with characteristic conserved domains and key residues. A phylogenetic tree built with the neighbor-joining method and three groups of α-man established, of which group I was classified into three subgroups and group III contained only Poaceae spp. sequences. The real-time qPCR results demonstrated that FaMAN genes decreased during fruit ripening, a trend mirrored by the total enzyme activity from the white to ripe stages. The analysis of the promoter regions of these FaMAN genes was enriched with ripening and phytohormone response elements, and contained cis-regulatory elements related to stress responses to low temperature, drought, defense, and salt stress. This study discusses the relevance of α-man in fruit ripening and how it can be a useful target to prolong fruit shelf life.

Evolution and phylogenetic distribution of endo-α-mannosidase.

While glycans underlie many biological processes, such as protein folding, cell adhesion, and cell-cell recognition, deep evolution of glycosylation machinery remains an understudied topic. N-linked glycosylation is a conserved process in which mannosidases are key trimming enzymes. One of them is the glycoprotein endo-α-1,2-mannosidase which participates in the initial trimming of mannose moieties from an N-linked glycan inside the cis-Golgi. It is unique as the only endo-acting mannosidase found in this organelle. Relatively little is known about its origins and evolutionary history; so far it was reported to occur only in vertebrates. In this work, a taxon-rich bioinformatic survey to unravel the evolutionary history of this enzyme, including all major eukaryotic clades and a wide representation of animals, is presented. The endomannosidase was found to be more widely distributed in animals and other eukaryotes. The protein motif changes in context of the canonical animal enzyme were tracked. Additionally, the data show the two canonical vertebrate endomannosidase genes, MANEA and MANEAL, arose at the second round of the two vertebrate genome duplications and one more vertebrate paralog, CMANEAL, is uncovered. Finally, a framework where N-glycosylation co-evolved with complex multicellularity is described. A better understanding of the evolution of core glycosylation pathways is pivotal to understanding biology of eukaryotes in general, and the Golgi apparatus in particular. This systematic analysis of the endomannosidase evolution is one step toward this goal.

Affinity Proteomics and Deglycoproteomics Uncover Novel EDEM2 Endogenous Substrates and an Integrative ERAD Network.

Various pathologies result from disruptions to or stress of endoplasmic reticulum (ER) homeostasis, such as Parkinson's disease and most neurodegenerative illnesses, diabetes, pulmonary fibrosis, viral infections, and cancers. A critical process in maintaining ER homeostasis is the selection of misfolded proteins by the ER quality-control system for destruction via ER-associated degradation (ERAD). One key protein proposed to act during the first steps of misfolded glycoprotein degradation is the ER degradation-enhancing α-mannosidase-like protein 2 (EDEM2). Therefore, characterization of the EDEM2-associated proteome is of great interest. We took advantage of using melanoma cells overexpressing EDEM2 as a cancer model system, to start documenting at the deglycoproteome level (N-glycosites identification) the emerging link between ER homeostasis and cancer progression. The dataset created for identifying the EDEM2 glyco clients carrying high mannose/hybrid N-glycans provides a comprehensive N-glycosite analysis mapping over 1000 N-glycosites on more than 600 melanoma glycoproteins. To identify EDEM2-associated proteins, we used affinity proteomics and proteome-wide analysis of sucrose density fractionation in an integrative workflow. Using intensity and spectral count-based quantification, we identify seven new EDEM2 partners, all of which are involved in ER quality-control system and ERAD. Moreover, we defined novel endogenous candidates for EDEM2-dependent ERAD by combining deglycoproteomics, stable isotope labeling with amino acids in cell culture-based proteomics, and biochemical methods. These included tumor antigens and several ER-transiting endogenous melanoma proteins, including integrin alpha-1 and protocadherin 2, the expression of which was negatively correlated with that of EDEM2. Tumor antigens are key in the antigen presentation process, whereas integrin alpha-1 and protocadherin 2 are involved in melanoma metastasis and invasion. EDEM2 could therefore have a regulatory role in melanoma through the modulation of degradation and trafficking in these glycoproteins. The data presented herein suggest that EDEM2 is involved in ER homeostasis to a greater extent than previously suggested.

Biochemical characterization and analysis of gene expression of an α-mannosidase secreted by Paracoccidioides brasiliensis.

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by fungi of the Paracoccidioides genus, being endemic in Latin America and with the highest number of cases in Brazil. Paracoccidioides spp. release a wide range of molecules, such as enzymes, which may be important for PCM establishment. Here, we identified the 85- and 90-kDa proteins from the supernatants of P. brasiliensis cultures as being an α-mannosidase. Because the expected mass of this α-mannosidase is 124.2-kDa, we suggest that the proteins were cleavage products. Indeed, we found an α-mannosidase activity in the culture supernatants among the excreted/secreted antigens (ESAg). Moreover, we determined that the enzyme activity was optimal in buffer at pH 5.6, at the temperature of 45ºC, and with a concentration of 3 mM of the substrate p-NP-α-D-Man. Remarkably, we showed that the gene expression of this α-mannosidase was higher in yeasts than hyphae in three P. brasiliensis isolates with different virulence degrees that were grown in Ham's F12 synthetic medium for 15 days. But in complex media YPD and Fava Netto, the significantly higher gene expression in yeasts than in hyphae was seen only for the virulent isolate Pb18, but not for intermediate virulence Pb339 and low virulence Pb265 isolates. These results about the high expression of the α-mannosidase gene in the pathogenic yeast form of P. brasiliensis open perspectives for studying this α-mannosidase concerning the virulence of P. brasiliensis isolates. LAY SUMMARY: Paracoccidioides brasiliensis causes deep mycosis, paracoccidioidomycosis. We determined for the first time the biochemical properties of an α-mannosidase released by this fungus. We suggest that the enzyme gene expression in the fungus is associated with fungal morphology, stress, and virulence.

The Competitive Endogenous RNA (ceRNA) Regulation in Porcine Alveolar Macrophages (3D4/21) Infected by Swine Influenza Virus (H1N1 and H3N2).

H1N1 and H3N2 are the two most common subtypes of swine influenza virus (SIV). They not only endanger the pig industry, but are also a huge risk of zoonotic diseases. However, the molecular mechanism and regulatory network of pigs (hosts) against influenza virus infection are still unclear. In this study, porcine alveolar macrophage cell (3D4/21) models infected by swine influenza virus (H1N1 and H3N2) were constructed. The expression profiles of miRNAs, mRNAs, lncRNAs and circRNAs after H1N1 and H3N2 infected 3D4/21 cells were revealed in this study. Then, two ceRNAs (TCONS_00166432-miR10391-MAN2A1 and novel_circ_0004733-miR10391-MAN2A1) that regulated H1N1 and H3N2 infection in 3D4/21 cells were verified by the methods of bioinformatics analysis, gene overexpression, gene interference, real-time quantitative PCR (qPCR), dual luciferase activity assay and RNA immunoprecipitation (RIP). In addition, the important candidate molecules (miR-10391, TCONS_00166432, and novel_circ_0004733) were identified by qPCR and enzyme linked immunosorbent assay (ELISA). Finally, the regulatory effect and possible molecular mechanism of the target gene MAN2A1 were identified by the methods of gene interference, qPCR, Western blot and ELISA. The results of this study suggested that TCONS_00166432 and novel_circ_0004733 could competitively bind miR-10391 to target the MAN2A1 gene to regulate swine influenza virus infecting 3D4/21 cells. This study reported for the first time the ceRNA networks involved in the regulation of the swine influenza virus infecting 3D4/21 cells, which provided a new insight into the molecular mechanism of 3D4/21 cells against swine influenza virus infection.

EDEM3 Domains Cooperate to Perform Its Overall Cell Functioning.

EDEM3 recognizes and directs misfolded proteins to the ER-associated protein degradation (ERAD) process. EDEM3 was predicted to act as lectin or as a mannosidase because of its homology with the GH47 catalytic domain of the Man1B1, but the contribution of the other regions remained unresolved. Here, we dissect the molecular determinants governing EDEM3 function and its cellular interactions. LC/MS analysis indicates very few stable ER interactors, suggesting EDEM3 availability for transient substrate interactions. Sequence analysis reveals that EDEM3 consists of four consecutive modules defined as GH47, intermediate (IMD), protease-associated (PA), and intrinsically disordered (IDD) domain. Using an EDEM3 knock-out cell line, we expressed EDEM3 and domain deletion mutants to address EDEM3 function. We find that the mannosidase domain provides substrate binding even in the absence of mannose trimming and requires the IMD domain for folding. The PA and IDD domains deletions do not impair the trimming, but specifically modulate the turnover of two misfolded proteins, NHK and the soluble tyrosinase mutant. Hence, we demonstrate that EDEM3 provides a unique ERAD timing to misfolded glycoproteins, not only by its mannose trimming activity, but also by the positive and negative feedback modulated by the protease-associated and intrinsically disordered domain, respectively.

A novel gene in early childhood diabetes: EDEM2 silencing decreases SLC2A2 and PXD1 expression, leading to impaired insulin secretion.

Monogenic diabetes is a rare type of diabetes resulting from mutations in a single gene. To date, most cases remain genetically unexplained, posing a challenge for accurate diabetes treatment, which leads to on a molecular diagnosis. Therefore, a trio exome scan was performed in a lean, nonsyndromic Caucasian girl with diabetes onset at 2½ years who was negative for autoantibodies. The lean father had diabetes from age 11 years. A novel heterozygous mutation in EDEM2, c.1271G > A; p.Arg424His, was found in the proband and father. Downregulation of Edem2 in rat RIN-m ß-cells resulted in a decrease in insulin genes Ins1 to 67.9% (p = 0.006) and Ins2 to 16.8% (p < 0.001) and reduced insulin secretion by 60.4% (p = 0.0003). Real-time PCR revealed a major disruption of endocrine pancreas-specific genes, including Glut2 and Pxd1, with mRNA suppression to 54% (p < 0.001) and 85.7% (p = 0.01), respectively. No other expression changes related to stress or apoptotic genes were observed. Extended clinical follow-up involving ten family members showed that two healthy individuals carried the same mutation with no sign of diabetes in the clinical screen except for a slight increase in IA-2 antibody in one of them, suggesting incomplete penetrance. In conclusion, we describe EDEM2 as a likely/potential novel diabetes gene, in which inhibition in vitro reduces the expression of ß-cell genes involved in the glucose-stimulated insulin secretion (GSIS) pathway, leading to an overall suppression of insulin secretion but not apoptosis.

α-Mannosidosis - An underdiagnosed lysosomal storage disease in individuals with an 'MPS-like' phenotype.

Individuals affected by alpha-Mannosidosis suffer from similar clinical symptoms such as respiratory infections, skeletal changes as patients with mucopolysaccharidoses (MPS). α-Mannosidosis is considered as an ultra-rare disorders and also diagnostic testing is often limited. With the availability of novel therapies and easy-to-access diagnostic tests (e.g. Tandem mass spectrometry) using dried blood spots for both enzymatic and genetic testing, the chance for the development of a better understanding of disease and awareness may be triggered. In a pilot study, we have investigated 1010 residual dried blood spot samples from individuals suspicious to MPS. In these study cohort, 158/1010 individuals were genetically confirmed for MPS. Additional biochemical and genetic confirmatory testing for α-mannosidases revealed four individuals with a final diagnosis of α-mannosidosis. This unexpected high number of individuals with α-mannosidosis demonstrated the urgent need of taking this rare disorder in clinical and diagnostic consideration particularly in patients suspicious to MPS.

Overexpression of wheat α-mannosidase gene TaMP impairs salt tolerance in transgenic Brachypodium distachyon.

KEY MESSAGE: The TaMP gene from wheat encodes an α-mannosidase induced by salt stress that functions as negative regulator of salt tolerance in plants. Salt stress significantly affects growth and yield of crop plants. The α-mannosidases function in protein folding, trafficking, and endoplasmic reticulum-associated degradation in eukaryotic cells, and they are involved in abiotic stress tolerance in plants. Previously, we identified the α-mannosidase gene TaMP in wheat (Triticum aestivum). In this study, we investigated the function of TaMP in salt stress tolerance. TaMP expression was induced in wheat leaves by salt, drought, abscisic acid, and H2O2 treatments. Overexpressing TaMP in Brachypodium distachyon was associated with a salt-sensitive phenotype. Under salt stress, the overexpressing plants had reduced height, delayed growth status, low photosynthetic rate, decreased survival rate, and diminished yield. Moreover, the overexpression of TaMP aggravated the tendency for ions to become toxic under salt stress by significantly affecting the Na+ and K+ contents in cells. In addition, TaMP could negatively regulate salt tolerance by affecting the antioxidant enzyme system capacity and increasing the reactive oxygen species accumulation. Our study was helpful to understand the underlying physiological and molecular mechanisms of salt stress tolerance in plants.

Prognostic relevance of the Golgi mannosidase MAN1A1 in ovarian cancer: impact of N-glycosylation on tumour cell aggregation.

BACKGROUND: Maturation of complex N-glycans involves the action of Golgi mannosidases and plays a major role in cancer progression. We recently showed a favourable prognostic role of α-mannosidase MAN1A1 in breast cancer mainly caused by alteration of certain adhesion molecules. METHODS: We analysed the protein expression of MAN1A1 in ovarian cancer (n = 204) using western blot and studied the impact of MAN1A1 itself and of MAN1A1-related glycosylation on the prognostic relevance of two adhesion molecules. Functional consequences of mannosidase inhibition using kifunensine and MAN1A1 knock out were investigated in ovarian cancer cells in vitro. RESULTS: Patients with high MAN1A1 expression in tumours showed significantly shorter RFS than those with low-MAN1A1 levels. Moreover, high MAN1A1 expression correlated significantly with advanced stage, lymph node involvement and distant metastasis. Further, the glycosylated adhesion molecule ALCAM reveals a significant adverse prognostic effect only in the presence of high MAN1A1 expression. In spheroid-formation assays, mannosidase inhibition and especially MAN1A1 knock out led to strong reduction of tumour cell aggregation. CONCLUSIONS: Our study demonstrates the unfavourable prognostic role of MAN1A1 in ovarian cancer, probably caused by an altered ability of spheroid formation, and the strong influence of this glycosylation enzyme on the prognostic impact of ALCAM.

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.

Distortion of mannoimidazole supports a B2,5 boat transition state for the family GH125 α-1,6-mannosidase from Clostridium perfringens.

Enzyme transition-state mimics can act as powerful inhibitors and allow structural studies that report on the conformation of the transition-state. Here, mannoimidazole, a mimic of the transition state of mannosidase catalyzed hydrolysis of mannosides, is shown to bind in a B2,5 conformation on the Clostridium perfringens GH125 α-1,6-mannosidase, providing additional evidence of a OS2-B2,5-1S5 conformational itinerary for enzymes of this family.

MAN2A1-FER Fusion Gene Is Expressed by Human Liver and Other Tumor Types and Has Oncogenic Activity in Mice.

BACKGROUND & AIMS: Human tumors and liver cancer cell lines express the product of a fusion between the first 13 exons in the mannosidase α class 2A member 1 gene (MAN2A1) and the last 6 exons in the FER tyrosine kinase gene (FER), called MAN2A1-FER. We investigated whether MAN2A1-FER is expressed by human liver tumors and its role in liver carcinogenesis. METHODS: We performed reverse transcription polymerase chain reaction analyses of 102 non-small cell lung tumors, 61 ovarian tumors, 70 liver tumors, 156 glioblastoma multiform samples, 27 esophageal adenocarcinomas, and 269 prostate cancer samples, as well as 10 nontumor liver tissues and 20 nontumor prostate tissues, collected at the University of Pittsburgh. We also measured expression by 15 human cancer cell lines. We expressed a tagged form of MAN2A1-FER in NIH3T3 and HEP3B (liver cancer) cells; Golgi were isolated for analysis. MAN2A1-FER was also overexpressed in PC3 or DU145 (prostate cancer), NIH3T3 (fibroblast), H23 (lung cancer), and A-172 (glioblastoma multiforme) cell lines and knocked out in HUH7 (liver cancer) cells. Cells were analyzed for proliferation and in invasion assays, and/or injected into flanks of severe combined immunodeficient mice; xenograft tumor growth and metastasis were assessed. Mice with hepatic deletion of PTEN were given tail-vein injections of MAN2A1-FER. RESULTS: We detected MAN2A1-FER messenger RNA and fusion protein (114 kD) in the hepatocellular carcinoma cell line HUH7, as well as in liver tumors, esophageal adenocarcinoma, glioblastoma multiforme, prostate tumors, non-small cell lung tumors, and ovarian tumors, but not nontumor prostate or liver tissues. MAN2A1-FER protein retained the signal peptide for Golgi localization from MAN2A1 and translocated from the cytoplasm to Golgi in cancer cell lines. MAN2A1-FER had tyrosine kinase activity almost 4-fold higher than that of wild-type FER, and phosphorylated the epidermal growth factor receptor at tyrosine 88 in its N-terminus. Expression of MAN2A1-FER in 4 cell lines led to epidermal growth factor receptor activation of BRAF, MEK, and AKT; HUH7 cells with MAN2A1-FER knockout had significant decreases in phosphorylation of these proteins. Cell lines that expressed MAN2A1-FER had increased proliferation, colony formation, and invasiveness and formed larger (>2-fold) xenograft tumors in mice, with more metastases, than cells not expressing the fusion protein. HUH7 cells with MAN2A1-FER knockout formed smaller xenograft tumors, with fewer metastases, than control HUH7 cells. HUH7, A-172, and PC3 cells that expressed MAN2A1-FER were about 2-fold more sensitive to the FER kinase inhibitor crizotinib and the epidermal growth factor receptor kinase inhibitor canertinib; these drugs slowed growth of xenograft tumors from MAN2A1-FER cells and prevented their metastasis in mice. Hydrodynamic tail-vein injection of MAN2A1-FER resulted in rapid development of liver cancer in mice with hepatic disruption of Pten. CONCLUSIONS: Many human tumor types and cancer cell lines express the MAN2A1-FER fusion, which increases proliferation and invasiveness of cancer cell lines and has liver oncogenic activity in mice.

Comparative proteomic analysis: SclR is importantly involved in carbohydrate metabolism in Aspergillus oryzae.

The helix-loop-helix (HLH) family of transcriptional factors is a key player in a wide range of developmental processes in organisms from mammals to microbes. We previously identified the bHLH transcription factor SclR in Aspergillus oryzae and found that the loss of SclR function led to significant phenotypic changes, such as rapid protein degradation and cell lysis in dextrin-polypeptone-yeast extract liquid medium. The result implied that SclR is potentially important in both traditional fermentative manufacturing and commercial enzyme production in A. oryzae because of its effect on growth. Therefore, this study presents a comparative assessment at the proteome level of the intracellular differences between an sclR-disrupted strain and a control strain using isobaric tandem mass tag (TMT) labeling for quantification. A total of 5447 proteins were identified, and 568 were differentially expressed proteins (DEPs). Of the DEPs, 251 proteins were increased by 1.5-fold, and 317 proteins were decreased by 1.5-fold in an sclR-disrupted strain compared to the control. The comparison of the quantitative TMT results revealed that SclR was mainly involved in carbon metabolism, especially carbohydrate metabolism. In addition, an enzyme profile by a semi-quantitative method (API-ZYM) indicated that three enzymes (ß-galactosidase, α-glucosidase, and α-mannosidase) were significantly less active in the ∆sclR strain than in the control. Moreover, quantitative RT-PCR showed that the expression of certain genes was changed similarly to their corresponding proteins. These results suggested that a possible function of SclR during growth of A. oryzae is its important involvement in carbohydrate metabolism.

Alpha-Mannosidosis: Therapeutic Strategies.

Alpha-mannosidosis (α-mannosidosis) is a rare lysosomal storage disorder with an autosomal recessive inheritance caused by mutations in the gene encoding for the lysosomal α-d-mannosidase. So far, 155 variants from 191 patients have been identified and in part characterized at the biochemical level. Similarly to other lysosomal storage diseases, there is no relationship between genotype and phenotype in alpha-mannosidosis. Enzyme replacement therapy is at the moment the most effective therapy for lysosomal storage disease, including alpha-mannosidosis. In this review, the genetic of alpha-mannosidosis has been described together with the results so far obtained by two different therapeutic strategies: bone marrow transplantation and enzyme replacement therapy. The primary indication to offer hematopoietic stem cell transplantation in patients affected by alpha-mannosidosis is preservation of neurocognitive function and prevention of early death. The results obtained from a Phase I⁻II study and a Phase III study provide evidence of the positive clinical effect of the recombinant enzyme on patients with alpha-mannosidosis.