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.

A Homozygous MAN2B1 Missense Mutation in a Doberman Pinscher Dog with Neurodegeneration, Cytoplasmic Vacuoles, Autofluorescent Storage Granules, and an α-Mannosidase Deficiency.

A 7-month-old Doberman Pinscher dog presented with progressive neurological signs and brain atrophy suggestive of a hereditary neurodegenerative disorder. The dog was euthanized due to the progression of disease signs. Microscopic examination of tissues collected at the time of euthanasia revealed massive accumulations of vacuolar inclusions in cells throughout the central nervous system, suggestive of a lysosomal storage disorder. A whole genome sequence generated with DNA from the affected dog contained a likely causal, homozygous missense variant in MAN2B1 that predicted an Asp104Gly amino acid substitution that was unique among whole genome sequences from over 4000 dogs. A lack of detectable α-mannosidase enzyme activity confirmed a diagnosis of a-mannosidosis. In addition to the vacuolar inclusions characteristic of α-mannosidosis, the dog exhibited accumulations of autofluorescent intracellular inclusions in some of the same tissues. The autofluorescence was similar to that which occurs in a group of lysosomal storage disorders called neuronal ceroid lipofuscinoses (NCLs). As in many of the NCLs, some of the storage bodies immunostained strongly for mitochondrial ATP synthase subunit c protein. This protein is not a substrate for α-mannosidase, so its accumulation and the development of storage body autofluorescence were likely due to a generalized impairment of lysosomal function secondary to the accumulation of α-mannosidase substrates. Thus, it appears that storage body autofluorescence and subunit c accumulation are not unique to the NCLs. Consistent with generalized lysosomal impairment, the affected dog exhibited accumulations of intracellular inclusions with varied and complex ultrastructural features characteristic of autophagolysosomes. Impaired autophagic flux may be a general feature of this class of disorders that contributes to disease pathology and could be a target for therapeutic intervention. In addition to storage body accumulation, glial activation indicative of neuroinflammation was observed in the brain and spinal cord of the proband.

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.

Audiological and radiological study of eight polish patients with alpha-mannosidosis.

Alpha-mannosidase catalyze lysosomal cleaving of mannose residues from glycoproteins. The enzyme is encoded by the MAN2B1 gene. Biallelic pathogenic variants cause enzymatic deficiency, which clinically results in alpha-mannosidosis (AM), an autosomal recessively inherited condition. Typical features observed in AM patients include intellectual disability, loss of speech, dysmorphic features, progressive motor problems, ataxia, hearing impairment and recurrent otitis. The cause of the latter is mainly attributed to immunodeficiency. The aim of our study was to demonstrate the otolaryngologic and hearing outcomes in patients with AM. The study group consisted of 8 AM patients: 6 males and 2 females, aged 2.5-37 yrs. The clinical course, dysmorphic ENT features, hearing status and the HRCT scans of the temporal bones were analyzed. MS Excel for Windows and Statistica software package were used for the comparison of interaural audiometric loss, mean hearing loss and mean hearing threshold for each patient's audiometric frequency tested. We identified ENT dysmorphic features in all of our AM patients, while the hearing loss was detected in 6 out of our 8 patients. For those cases, the onset of deafness was noted in the first decade of life, this impairment was sensorineural, of cochlear origin, bilateral, of a moderate degree (mean loss 62.76 dB; median 60 dB, standard deviation 12.5 dB), symmetrical and stable. The shape of the audiometric curves of our patients can be described as slightly sloping towards the higher tested frequencies, with a marked improvement at 4 kHz. The radiological examination revealed normal structures of the ears, with the exception of one case where a persistent otitis generated a cochlear gap. We therefore concluded that the hearing loss in our AM patients derived from cochlear impairment unrelated with recurrent otitis.

Biochemical characteristics of point mutated Capra hircus lysosome α-mannosidase.

Locoweeds, a type of poisonous weedare, are widely distributed throughout the world and have a significant impact on the development of herbivore animal husbandry. Swainsonine (SW), the main toxin in locoweeds, can competitively inhibit lysosomes α-mannosidase (LAM) in animal cells, resulting in α-mannosidosis. However, the specifics of the interaction between SW and LAM are still unclear. Here, we used molecular docking to predicte the interaction points between SW and LAM, built mutated lysosomes α-mannosidase (LAMM), and analyzed its biochemical properties changes in presumption points. The Trp at the 28th position and the Tyr at the 599th position of the LAM were interaction point candidates, and the above two amino acids in Capra hircus LAM (chLAM), were successfully mutated to glycine by constructing recombinant yeast GS115/PIC9K- LAMM. The results showed that the sensitivity of Capra hircus LAMM (chLAMM), to SW decreased significantly compared with wild-type LAM, the enzyme activity of LAM decreased approximately threefold, the optimum temperature of LAMM decreased from 55°C to 50°C, the optimum pH value increased from 4.5 to 5.0, and the effects of Mn2+, Fe3+, Al3+, Co2+, Cr3+, and ethylenediaminetetraacetic acid (EDTA) on LAM enzyme activity before and after point mutation changed significantly. These findings help us better understanding the molecular mechanism of the interaction mechanism between SW and chLAM, and provide new reference for solving locoweeds poisoning.

Expression and Characterization of a GH38 α-Mannosidase from the Hyperthermophile Pseudothermotoga thermarum.

This study focused on the bio-characterization of a GH38 α-mannosidase from the hyperthermophile Pseudothermotoga thermarum DSM 5069. We aimed to successfully express and characterize this thermophilic α-mannosidase and to assess its functional properties. Subsequently, recombinant α-mannosidase PtαMan was expressed in Escherichia coli BL21(DE3) and purified via affinity chromatography, and native protein was verified as a tetramer by size exclusion chromatography. In addition, the activity of α-mannosidase PtαMan was relatively stable at pH 5.0-6.5 and temperatures up to 75 ℃. α-Mannosidase PtαMan was active toward Co2+ and had a good catalytic efficiency deduced from the kinetic parameters. However, its activity was strongly inhibited by Cu2+, Zn2+, SDS, and swainsonine. In summary, this cobalt-required α-mannosidase is putatively involved in the direct modification of glycoproteins.

Protein disulfide isomerases (PDIs) negatively regulate ebolavirus structural glycoprotein expression in the endoplasmic reticulum (ER) via the autophagy-lysosomal pathway.

Zaire ebolavirus (EBOV) causes a severe hemorrhagic fever in humans and non-human primates with high morbidity and mortality. EBOV infection is dependent on its structural glycoprotein (GP), but high levels of GP expression also trigger cell rounding, detachment, and downregulation of many surface molecules that is thought to contribute to its high pathogenicity. Thus, EBOV has evolved an RNA editing mechanism to reduce its GP expression and increase its fitness. We now report that the GP expression is also suppressed at the protein level in cells by protein disulfide isomerases (PDIs). Although PDIs promote oxidative protein folding by catalyzing correct disulfide formation in the endoplasmic reticulum (ER), PDIA3/ERp57 adversely triggered the GP misfolding by targeting GP cysteine residues and activated the unfolded protein response (UPR). Abnormally folded GP was targeted by ER-associated protein degradation (ERAD) machinery and, unexpectedly, was degraded via the macroautophagy/autophagy-lysosomal pathway, but not the proteasomal pathway. PDIA3 also decreased the GP expression from other ebolavirus species but increased the GP expression from Marburg virus (MARV), which is consistent with the observation that MARV-GP does not cause cell rounding and detachment, and MARV does not regulate its GP expression via RNA editing during infection. Furthermore, five other PDIs also had a similar inhibitory activity to EBOV-GP. Thus, PDIs negatively regulate ebolavirus glycoprotein expression, which balances the viral life cycle by maximizing their infection but minimizing their cellular effect. We suggest that ebolaviruses hijack the host protein folding and ERAD machinery to increase their fitness via reticulophagy during infection.Abbreviations: 3-MA: 3-methyladenine; 4-PBA: 4-phenylbutyrate; ACTB: ß-actin; ATF: activating transcription factor; ATG: autophagy-related; BafA1: bafilomycin A1; BDBV: Bundibugyo ebolavirus; CALR: calreticulin; CANX: calnexin; CHX: cycloheximide; CMA: chaperone-mediated autophagy; ConA: concanamycin A; CRISPR: clusters of regularly interspaced short palindromic repeats; Cas9: CRISPR-associated protein 9; dsRNA: double-stranded RNA; EBOV: Zaire ebolavirus; EDEM: ER degradation enhancing alpha-mannosidase like protein; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; Env: envelope glycoprotein; ER: endoplasmic reticulum; ERAD: ER-associated protein degradation; ERN1/IRE1: endoplasmic reticulum to nucleus signaling 1; GP: glycoprotein; HA: hemagglutinin; HDAC6: histone deacetylase 6; HMM: high-molecular-mass; HIV-1: human immunodeficiency virus type 1; HSPA5/BiP: heat shock protein family A (Hsp70) member 5; IAV: influenza A virus; IP: immunoprecipitation; KIF: kifenesine; Lac: lactacystin; LAMP: lysosomal associated membrane protein; MAN1B1/ERManI: mannosidase alpha class 1B member 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MARV: Marburg virus; MLD: mucin-like domain; NHK/SERPINA1: alpha1-antitrypsin variant null (Hong Kong); NTZ: nitazoxanide; PDI: protein disulfide isomerase; RAVV: Ravn virus; RESTV: Reston ebolavirus; SARS-CoV: severe acute respiratory syndrome coronavirus; SBOV: Sudan ebolavirus; sGP: soluble GP; SQSTM1/p62: sequestosome 1; ssGP: small soluble GP; TAFV: Taï Forest ebolavirus; TIZ: tizoxanide; TGN: thapsigargin; TLD: TXN (thioredoxin)-like domain; Ub: ubiquitin; UPR: unfolded protein response; VLP: virus-like particle; VSV: vesicular stomatitis virus; WB: Western blotting; WT: wild-type; XBP1: X-box binding protein 1.

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.

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.

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.

Purified EDEM3 or EDEM1 alone produces determinant oligosaccharide structures from M8B in mammalian glycoprotein ERAD.

Sequential mannose trimming of N-glycan, from M9 to M8B and then to oligosaccharides exposing the α1,6-linked mannosyl residue (M7A, M6, and M5), facilitates endoplasmic reticulum-associated degradation of misfolded glycoproteins (gpERAD). We previously showed that EDEM2 stably disulfide-bonded to the thioredoxin domain-containing protein TXNDC11 is responsible for the first step (George et al., 2020). Here, we show that EDEM3 and EDEM1 are responsible for the second step. Incubation of pyridylamine-labeled M8B with purified EDEM3 alone produced M7 (M7A and M7C), M6, and M5. EDEM1 showed a similar tendency, although much lower amounts of M6 and M5 were produced. Thus, EDEM3 is a major α1,2-mannosidase for the second step from M8B. Both EDEM3 and EDEM1 trimmed M8B from a glycoprotein efficiently. Our confirmation of the Golgi localization of MAN1B indicates that no other α1,2-mannosidase is required for gpERAD. Accordingly, we have established the entire route of oligosaccharide processing and the enzymes responsible.

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.

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.

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.

Inhibition of MAN2A1 Enhances the Immune Response to Anti-PD-L1 in Human Tumors.

PURPOSE: Immune checkpoint blockade has shown remarkable efficacy, but in only a minority of patients with cancer, suggesting the need to develop additional treatment strategies. Aberrant glycosylation in tumors, resulting from the dysregulated expression of key enzymes in glycan biosynthesis, modulates the immune response. However, the role of glycan biosynthesis enzymes in antitumor immunity is poorly understood. We aimed to study the immunomodulatory effects of these enzymes. EXPERIMENTAL DESIGN: We integrated transcriptional profiles of treatment-naïve human tumors and functional CRISPR screens to identify glycometabolism genes with immunomodulatory effects. We further validated our findings using in vitro coculture and in vivo syngeneic tumor growth assays. RESULTS: We identified MAN2A1, encoding an enzyme in N-glycan maturation, as a key immunomodulatory gene. Analyses of public immune checkpoint blockade trial data also suggested a synergy between MAN2A1 inhibition and anti-PD-L1 treatment. Loss of Man2a1 in cancer cells increased their sensitivity to T-cell-mediated killing. Man2a1 knockout enhanced response to anti-PD-L1 treatment and facilitated higher cytotoxic T-cell infiltration in tumors under anti-PD-L1 treatment. Furthermore, a pharmacologic inhibitor of MAN2A1, swainsonine, synergized with anti-PD-L1 in syngeneic melanoma and lung cancer models, whereas each treatment alone had little effect. CONCLUSIONS: Man2a1 loss renders cancer cells more susceptible to T-cell-mediated killing. Swainsonine synergizes with anti-PD-L1 in suppressing tumor growth. In light of the limited efficacy of anti-PD-L1 and failed phase II clinical trial on swainsonine, our study reveals a potential therapy combining the two to overcome tumor immune evasion.See related commentary by Bhat and Kabelitz, p. 5778.

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.

Mitotic cyclin Clb4 is required for the intracellular adaptation to glucose starvation in Saccharomyces cerevisiae.

Cellular homeostasis in response to glucose availability is maintained through the tight coordination of various physiological processes, including cell proliferation, transcription, and metabolism. In this study, we use the budding yeast Saccharomyces cerevisiae to identify proteins implicated in carbon source-dependent modulation of physiological processes. We find that the mitotic cyclin Clb4 is required for optimal regulation of glucose-starvation-responsive pathways through the target of rapamycin complex 1. Cells lacking Clb4 are characterized by dysregulation of autophagy and impaired modulation of cell size. Notably, cell viability after prolonged glucose starvation is severely reduced by disruption of Clb4. We conclude that Clb4, in addition to its function in the cell cycle, plays a role in the intracellular adaptation to glucose starvation.