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.

The endo-beta mannase MAN7 contributes to cadmium tolerance by modulating root cell wall binding capacity in Arabidopsis thaliana.

The heavy metal cadmium (Cd) is detrimental to crop growth and threatens human health through the food chain. To cope with Cd toxicity, plants employ multiple strategies to decrease Cd uptake and its root-to-shoot translocation. However, genes that participate in the Cd-induced transcriptional regulatory network, including those encoding transcription factors, remain largely unidentified. In this study, we demonstrate that ENDO-BETA-MANNASE 7 (MAN7) is necessary for the response of Arabidopsis thaliana to toxic Cd levels. We show that MAN7 is responsible for mannase activity and modulates mannose content in the cell wall, which plays a role in Cd compartmentalization in the cell wall under Cd toxicity conditions. Additionally, the repression of root growth by Cd was partially reversed via exogenous application of mannose, suggesting that MAN7-mediated cell wall Cd redistribution depends on the mannose pathway. Notably, we identified a basic leucine zipper (bZIP) transcription factor, bZIP44, that acts upstream of MAN7 in response to Cd toxicity. Transient dual-luciferase assays indicated that bZIP44 directly binds to the MAN7 promoter region and activates its transcription. Loss of bZIP44 function was associated with greater sensitivity to Cd treatment and higher accumulation of the heavy metal in roots and shoots. Moreover, MAN7 overexpression relieved the inhibition of root elongation seen in the bzip44 mutant under Cd toxicity conditions. This study thus reveals a pathway showing that MAN7-associated Cd tolerance in Arabidopsis is controlled by bZIP44 upon Cd exposure.

circRNA mannosidase alpha class 1A member 2 contributes to the proliferation and motility of papillary thyroid cancer cells through upregulating metadherin via absorbing microRNA-449a.

Papillary thyroid carcinoma (PTC) is a common malignancy in endocrine system globally. Accumulating articles have found that circular RNAs (circRNAs) were dysregulated, and they were involved in PTC development. The aim of this project was to explore the function and associated mechanism of circRNA mannosidase alpha class 1A member 2 (circMAN1A2) in PTC progression. The expression of RNA was determined by real-time quantitative PCR. Cell proliferation ability was analyzed by colony formation assay and 5-ethynyl-2'-deoxyuridine assay. Cell migration and invasion were assessed by wound healing assay and transwell invasion assay, respectively. Protein levels were determined by Western blot assay. Dual-luciferase reporter assay and RNA immunoprecipitation assay were applied to confirm the interaction between microRNA-449a (miR-449a) and circMAN1A2 or metadherin (MTDH). Xenograft tumor model was utilized to explore the effect of circMAN1A2 silencing on tumor growth in vivo . CircMAN1A2 expression was elevated in PTC specimens and three PTC cell lines relative to adjacent normal specimens and Nthy-ori 3-1 cell line. CircMAN1A2 silencing inhibited the proliferation and motility of PTC cells. CircMAN1A2 acted as a molecular sponge of miR-449a, and circMAN1A2 knockdown suppressed PTC development partly through upregulating miR-449a. MiR-449a bound to the 3' untranslated region of MTDH, and miR-449a restrained PTC progression partly through down-regulating MTDH. CircMAN1A2 interference suppressed PTC progression in vivo . CircMAN1A2 contributed to the proliferation ability and motility of PTC cells through enhancing MTDH expression via sponging miR-449a.

RNAi of Mannosidase-Ia in the Colorado potato beetle and changes in the midgut and peritrophic membrane.

BACKGROUND: In addition to its role in the digestive system, the peritrophic membrane (PM) provides a physical barrier protecting the intestine from abrasion and against pathogens. Because of its sensitivity to RNA interference (RNAi), the notorious pest insect, the Colorado potato beetle (CPB, Leptinotarsa decemlineata), has become a model insect for functional studies. Previously, RNAi-mediated silencing of Mannosidase-Ia (ManIa), a key enzyme in the transition from high-mannose glycan moieties to paucimannose N-glycans, was shown to disrupt the transition from larva to pupa and the metamorphosis into adult beetles. While these effects at the organismal level were interesting in a pest control context, the effects at the organ or tissue level and also immune effects have not been investigated yet. To fill this knowledge gap, we performed an analysis of the midgut and PM in ManIa-silenced insects. RESULTS: As marked phenotype, the ManIaRNAi insects, the PM pore size was found to be decreased when compared to the control GFPRNAi insects. These smaller pores are related to the observation of thinner microvilli (Mv) on the epithelial cells of the midgut of ManIaRNAi insects. A midgut and PM proteome study and reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis with a selection of marker genes was performed to characterize the midgut cells and understand their response to the silencing of ManIa. In agreement with the loss of ManIa activity, an accumulation of high-mannose N-glycans was observed in the ManIa-silenced insects. As a pathogen-associated molecular pattern (PAMP), the presence of these glycan structures could trigger the activation of the immune pathways. CONCLUSION: The observed decrease in PM pore size could be a response to prevent potential pathogens to access the midgut epithelium. This hypothesis is supported by the strong increase in transcription levels of the anti-fungal peptide drosomycin-like in ManIaRNAi insects, although further research is required to elucidate this possibility. The potential immune response in the midgut and the smaller pore size in the PM shed a light on the function of the PM as a physical barrier and provide evidence for the relation between the Mv and PM. © 2022 Society of Chemical Industry.

Identification of a Novel Theranostic Signature of Metabolic and Immune-Inflammatory Dysregulation in Myocardial Infarction, and the Potential Therapeutic Properties of Ovatodiolide, a Diterpenoid Derivative.

Myocardial infarction (MI) is a multifactorial global disease, recognized as one of the leading causes of cardiovascular morbidity and mortality. Timely and correct diagnoses and effective treatments could significantly reduce incidence of complications and improve patient prognoses. In this study, seven unconventional differentially expressed genes (DEGs) (MAN2A2, TNFRSF12A, SPP1, CSNK1D, PLAUR, PFKFB3, and CXCL16, collectively termed the MTSCPPC signature) were identified through integrating DEGs from six MI microarray datasets. The pathological and theranostic roles of the MTSCPPC signature in MI were subsequently analyzed. We evaluated interactions of the MTSCPPC signature with ovatodiolide, a bioactive compound isolated from Anisomeles indica (L.) Kuntze, using in silico molecular docking tools and compared it to specific inhibitors of the members of the MTSCPPC signature. Single-cell transcriptomic analysis of the public databases revealed high expression levels of the MTSCPPC signature in immune cells of adult human hearts during an MI event. The MTSCPPC signature was significantly associated with the cytokine-cytokine receptor interactions, chemokine signaling, immune and inflammatory responses, and metabolic dysregulation in MI. Analysis of a micro (mi)RNA regulatory network of the MTSCPPC signature suggested post-transcriptional activation and the roles of miRNAs in the pathology of MI. Our molecular docking analysis suggested a higher potential for ovatodiolide to target MAN2A2, CSNK1D, and TNFRSF12A. Collectively, the results derived from the present study further advance our understanding of the complex regulatory mechanisms of MI and provide a potential MI theranostic signature with ovatodiolide as a therapeutic candidate.

Analysis of fungal high-mannose structures using CAZymes.

Glycoengineering ultimately allows control over glycosylation patterns to generate new glycoprotein variants with desired properties. A common challenge is glycan heterogeneity, which may affect protein function and limit the use of key techniques such as mass spectrometry. Moreover, heterologous protein expression can introduce nonnative glycan chains that may not fulfill the requirement for therapeutic proteins. One strategy to address these challenges is partial trimming or complete removal of glycan chains, which can be obtained through selective application of exoglycosidases. Here, we demonstrate an enzymatic O-deglycosylation toolbox of a GH92 α-1,2-mannosidase from Neobacillus novalis, a GH2 ß-galactofuranosidase from Amesia atrobrunnea and the jack bean α-mannosidase. The extent of enzymatic O-deglycosylation was mapped against a full glycosyl linkage analysis of the O-glycosylated linker of cellobiohydrolase I from Trichoderma reesei (TrCel7A). Furthermore, the influence of deglycosylation on TrCel7A functionality was evaluated by kinetic characterization of native and O-deglycosylated forms of TrCel7A. This study expands structural knowledge on fungal O-glycosylation and presents a ready-to-use enzymatic approach for controlled O-glycan engineering in glycoproteins expressed in filamentous fungi.

Siblings with MAN1B1-CDG Showing Novel Biochemical Profiles.

Congenital disorders of glycosylation (CDG), inherited metabolic diseases caused by defects in glycosylation, are characterized by a high frequency of intellectual disability (ID) and various clinical manifestations. Two siblings with ID, dysmorphic features, and epilepsy were examined using mass spectrometry of serum transferrin, which revealed a CDG type 2 pattern. Whole-exome sequencing showed that both patients were homozygous for a novel pathogenic variant of MAN1B1 (NM_016219.4:c.1837del) inherited from their healthy parents. We conducted a HPLC analysis of sialylated N-linked glycans released from total plasma proteins and characterized the α1,2-mannosidase I activity of the lymphocyte microsome fraction. The accumulation of monosialoglycans was observed in MAN1B1-deficient patients, indicating N-glycan-processing defects. The enzymatic activity of MAN1B1 was compromised in patient-derived lymphocytes. The present patients exhibited unique manifestations including early-onset epileptic encephalopathy and cerebral infarction. They also showed coagulation abnormalities and hypertransaminasemia. Neither sibling had truncal obesity, which is one of the characteristic features of MAN1B1-CDG.

BdPUL12 depolymerizes ß-mannan-like glycans into mannooligosaccharides and mannose, which serve as carbon sources for Bacteroides dorei and gut probiotics.

Symbiotic bacteria, including members of the Bacteroides genus, are known to digest dietary fibers in the gastrointestinal tract. The metabolism of complex carbohydrates is restricted to a specified subset of species and is likely orchestrated by polysaccharide utilization loci (PULs) in these microorganisms. ß-Mannans are plant cell wall polysaccharides that are commonly found in human nutrients. Here, we report the structural basis of a PUL cluster, BdPUL12, which controls ß-mannan-like glycan catabolism in Bacteroides dorei. Detailed biochemical characterization and targeted gene disruption studies demonstrated that a key glycoside hydrolase, BdP12GH26, performs the initial attack on galactomannan or glucomannan likely via an endo-acting mode, generating mannooligosaccharides and mannose. Importantly, coculture assays showed that the B. dorei promoted the proliferation of Lactobacillus helveticus and Bifidobacterium adolescentis, likely by sharing mannooligosaccharides and mannose with these gut probiotics. Our findings provide new insights into carbohydrate metabolism in gut-inhabiting bacteria and lay a foundation for novel probiotic development.

MAN1B1-CDG: novel patients and novel variant.

OBJECTIVES: Congenital disorders of glycosylation (CDGs) are a group of genetic disorders due to hypoglycosylation of proteins and lipids. A type I pattern is associated with defects in glycan assembly and transfer (CDG-I; cytosol; and endoplasmic reticulum defects), a type II pattern is seen in processing defects of the Golgi apparatus. MAN1B1-CDG is an autosomal recessive CDG-II due to mutations in the α 1,2-mannosidase gene (MAN1B1), mainly characterized by psychomotor disability, facial dysmorphism, truncal obesity, and hypotonia. CASE PRESENTATION: Three patients (two males and one female), with MAN1B1-CDG who had elevated transaminase levels are presented. All patients had presented due to dysmorphic and neurological findings and hypertransaminasemia was remarkable. A type 2 pattern was found on serum transferrin isoelectrofocusing analysis of the presented cases. MAN1B1-CDG was confirmed by genetic analysis. CONCLUSIONS: Although the cause of the increased serum transaminase levels in the present patients is not clear, no evidence for an infection or underlying liver pathology could be identified. In order to know if this is a consistent feature, we suggest measuring serum transaminase levels regularly in MAN1B1-CDG patients.

Mannose- and Mannobiose-Specific Responses of the Insect-Associated Cellulolytic Bacterium Streptomyces sp. Strain SirexAA-E.

The cellulolytic insect symbiont bacterium Streptomyces sp. strain SirexAA-E secretes a suite of carbohydrate-active enzymes (CAZymes), which are involved in the degradation of various polysaccharides in the plant cell wall, in response to the available carbon sources. Here, we examined a poorly understood response of this bacterium to mannan, one of the major plant cell wall components. SirexAA-E grew well on mannose, carboxymethyl cellulose (CMC), and locust bean gum (LBG) as sole carbon sources in the culture medium. The secreted proteins from each culture supernatant were tested for their polysaccharide-degrading ability, and the composition of secreted CAZymes in each sample was determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results indicated that mannose, LBG, and CMC induced the secretion of mannan and cellulose-degrading enzymes. Interestingly, two α-1,2-mannosidases were abundantly secreted during growth on mannose and LBG. Using genomic analysis, we found a unique 12-bp palindromic sequence motif at 4 locations in the SirexAA-E genome, two of which were found upstream of the above-mentioned α-1,2-mannosidase genes, along with a newly identified mannose and mannobiose-responsive transcriptional regulator, SsManR. Furthermore, the previously reported cellobiose-responsive repressor, SsCebR, was determined to also use mannobiose as an effector ligand. To test whether mannobiose induces the sets of genes under the control of the two regulators, SirexAA-E was grown on mannobiose, and the secretome composition was analyzed. As hypothesized, the composition of the mannobiose secretome combined sets of CAZymes found in both LBG and CMC secretomes, and thus they are likely under the regulation of both SsManR and SsCebR. IMPORTANCEStreptomyces sp. SirexAA-E, a microbial symbiont of biomass-harvesting insects, secretes a suite of polysaccharide-degrading enzymes dependent on the available carbon sources. However, the response of this bacterium to mannan has not been documented. In this study, we investigated the response of this bacterium to mannose, mannobiose, and galactomannan (LBG). By combining biochemical, proteomic, and genomic approaches, we discovered a novel mannose and mannobiose responsive transcriptional regulator, SsManR, which selectively regulates three α-1,2-mannosidase-coding genes. We also demonstrated that the previously described cellobiose responsive regulator, SsCebR, could use mannobiose as an effector ligand. Overall, our findings suggest that the Streptomyces sp. SirexAA-E responds to mannose and mannooligosaccharides through two different transcriptional repressors that regulate the secretion of the plant cell wall-degrading enzymes to extract carbon sources in the host environment.

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.

Down-regulation of MANNANASE7 gene in Brassica napus L. enhances silique dehiscence-resistance.

KEY MESSAGE: MANNANASE7 gene in Brassica napus L. encodes a hemicellulose which located at cell wall or extracellular space and dehiscence-resistance can be manipulated by altering the expression of MANNANASE7. Silique dehiscence is an important physiological process in plant reproductive development, but causes heavy yield loss in crops. The lack of dehiscence-resistant germplasm limits the application of mechanized harvesting and greatly restricts the rapeseed (Brassica napus L.) production. Hemicellulases, together with cellulases and pectinases, play important roles in fruit development and maturation. The hemicellulase gene MANNANASE7 (MAN7) was previously shown to be involved in the development and dehiscence of Arabidopsis (Arabidopsis thaliana) siliques. Here, we cloned BnaA07g12590D (BnMAN7A07), an AtMAN7 homolog from rapeseed, and demonstrate its function in the dehiscence of rapeseed siliques. We found that BnMAN7A07 was expressed in both vegetative and reproductive organs and significantly highly expressed in leaves, flowers and siliques where the abscission or dehiscence process occurs. Subcellular localization experiment showed that BnMAN7A07 was localized in the cell wall. The biological activity of the BnMAN7A07 protein isolated and purified through prokaryotic expression system was verified to catalyse the decomposition of xylan into xylose. Phenotypic studies of RNA interference (RNAi) lines revealed that down-regulation of BnMAN7A07 in rapeseed could significantly enhance silique dehiscence-resistance. In addition, the expression of upstream silique development regulators is altered in BnMAN7A07-RNAi plants, suggesting that a possible feedback regulation mechanism exists in the regulation network of silique dehiscence. Our results demonstrate that dehiscence-resistance can be manipulated by altering the expression of hemicellulase gene BnMAN7A07, which could provide an available genetic resource for breeding practice in rapeseed which is beneficial to mechanized harvest.

Identification of a Novel MAN1A1-ROS1 Fusion Gene Through mRNA-based Screening for Tyrosine Kinase Gene Aberrations in a Patient with Leiomyosarcoma.

BACKGROUND: Soft tissue sarcomas are a heterogeneous group of rare malignant tumors. Advanced soft tissue sarcomas have a poor prognosis, and effective systemic therapies have not been established. Tyrosine kinases are increasingly being used as therapeutic targets for a variety of cancers and soft tissue sarcomas. Although complex karyotype sarcomas typically tend to carry more potentially actionable genetic alterations than do translocation-associated sarcomas (fusion gene sarcomas), based on our database review, we found that leiomyosarcoma and malignant peripheral nerve sheath tumors have lower frequencies of potential targets than other nontranslocation soft tissue sarcomas. We theorized that both leiomyosarcoma and malignant peripheral nerve sheath tumors might be included in any unique translocations. Furthermore, if tyrosine kinase imbalances, especially fusion genes, occur in patients with leiomyosarcomas and malignant peripheral nerve sheath tumors, tyrosine kinase inhibitors might be a drug development target for this sarcoma. In this study, we used a tyrosine kinase screening system that could detect an imbalance in mRNA between 5'- and 3'-sides in tyrosine kinase genes to identify potential novel therapeutic tyrosine kinase targets for soft tissue sarcomas. QUESTIONS/PURPOSES: (1) Are there novel therapeutic tyrosine kinase targets in tumors from patients with soft tissue sarcomas that are detectable using mRNA screening focusing on imbalance expressions between the 5' and 3' end of the kinase domain? (2) Can potential targets be verified by RNA sequencing and reverse transcription PCR (RT-PCR)? (3) Will potential fusion gene(s) transform cells in in vitro assays? (4) Will tumors in mice that have an identified fusion gene respond to treatment with a therapeutic drug directed at that target? METHODS: We used mRNA screening to look for novel tyrosine kinase targets that might be of therapeutic potential. Using functional assays, we verified whether the identified fusion genes would be good therapeutic candidates for soft tissue sarcomas. Additionally, using in vivo assays, we assessed whether suppressing the fusion's kinase activity has therapeutic potential. Study eligibility was based on a patient having high-grade spindle cell and nontranslocation sarcomas, including leiomyosarcoma, malignant peripheral nerve sheath tumor, and high-grade myxofibrosarcoma. Between 2015 and 2019, of the 172 patients with soft tissue sarcomas treated with surgical resection at Juntendo University Hospital, 72 patients had high-grade nontranslocation sarcomas. The analysis was primarily for leiomyosarcoma and malignant peripheral nerve sheath tumors, and there was a limitation of analysis size (reagent limitations) totaling 24 samples at the start of the study. We collected additional samples from a sample bank at the Tokyo Medical and Dental University to increase the number of sarcomas to study. Therefore, in this study, a total of 15 leiomyosarcoma samples, five malignant peripheral nerve sheath tumors samples, and four high-grade myxofibrosarcoma samples were collected to achieve the sample size of 24 patients. To identify tyrosine kinase fusion genes, we designed a NanoString-based assay (NanoString Technologies Inc, Seattle, WA, USA) to query the expression balances regarding transcripts of 90 tyrosine kinases at two points: the 5' end of the kinase domain and within the kinase domain or 3' end of the kinase domain. The tumor's RNA was hybridized to the NanoString probes and analyzed for the expression ratios of outliers from the 3' to 5' end of the kinase domain. Presumed novel fusion events in these positive tumors that were defined by NanoString-based assays were confirmed tyrosine kinase fusion genes by RNA sequencing and confirmatory RT-PCR. Functional analyses consisting of in vitro and in vivo assays were also performed to elucidate whether the identified tyrosine kinase gene fusions were associated with oncogenic abilities and drug responses. RESULTS: We identified aberrant expression ratios regarding the 3' to 5' end of the kinase domain ratios in ROS1 transcripts in a leiomyosarcoma in a 90-year-old woman. A novel MAN1A1-ROS1 fusion gene was identified from her thigh tumor through RNA sequencing, which was confirmed with real-time PCR. In functional assays, MAN1A1-ROS1 rearrangement revealed strong transforming potential in 3T3 cells. Moreover, in an in vivo assay, crizotinib, a ROS1 inhibitor, markedly inhibited the growth of MAN1A1-ROS1 rearrangement-induced transformed cells in a dose-dependent manner. CONCLUSION: We conducted tyrosine kinase screening to identify new therapeutic targets in soft tissue sarcomas. We found a novel MAN1A1-ROS1 fusion gene that may be a therapeutic target in patients with leiomyosarcoma. This study demonstrates that the mRNA screening system may aid in the development of useful therapeutic options for soft tissue sarcomas. CLINICAL RELEVANCE: If novel tyrosine fusions such as MAN1A1-ROS1 fusion can be found in sarcomas from other patients, they could offer avenues for new molecular target therapies for sarcomas that currently do not have effective chemotherapeutic options. Therefore, the establishment of a screening system that includes both genomic and transcript analyses in the clinical setting is needed to verify our discoveries and take the developmental process of treatment to the next step.

High Mannose N-Glycans Promote Migration of Bone-Marrow-Derived Mesenchymal Stromal Cells.

For hundreds of indications, mesenchymal stromal cells (MSCs) have not achieved the expected therapeutic efficacy due to an inability of the cells to reach target tissues. We show that inducing high mannose N-glycans either chemically, using the mannosidase I inhibitor Kifunensine, or genetically, using an shRNA to silence the expression of mannosidase I A1 (MAN1A1), strongly increases the motility of MSCs. We show that treatment of MSCs with Kifunensine increases cell migration toward bone fracture sites after percutaneous injection, and toward lungs after intravenous injection. Mechanistically, high mannose N-glycans reduce the contact area of cells with its substrate. Silencing MAN1A1 also makes cells softer, suggesting that an increase of high mannose N-glycoforms may change the physical properties of the cell membrane. To determine if treatment with Kifunensine is feasible for future clinical studies, we used mass spectrometry to analyze the N-glycan profile of MSCs over time and demonstrate that the effect of Kifunensine is both transitory and at the expense of specific N-glycoforms, including fucosylations. Finally, we also investigated the effect of Kifunensine on cell proliferation, differentiation, and the secretion profile of MSCs. Our results support the notion of inducing high mannose N-glycans in MSCs in order to enhance their migration potential.

The cytoplasmic tail of human mannosidase Man1b1 contributes to catalysis-independent quality control of misfolded alpha1-antitrypsin.

The failure of polypeptides to achieve conformational maturation following biosynthesis can result in the formation of protein aggregates capable of disrupting essential cellular functions. In the secretory pathway, misfolded asparagine (N)-linked glycoproteins are selectively sorted for endoplasmic reticulum-associated degradation (ERAD) in response to the catalytic removal of terminal alpha-linked mannose units. Remarkably, ER mannosidase I/Man1b1, the first alpha-mannosidase implicated in this conventional N-glycan-mediated process, can also contribute to ERAD in an unconventional, catalysis-independent manner. To interrogate this functional dichotomy, the intracellular fates of two naturally occurring misfolded N-glycosylated variants of human alpha1-antitrypsin (AAT), Null Hong Kong (NHK), and Z (ATZ), in Man1b1 knockout HEK293T cells were monitored in response to mutated or truncated forms of transfected Man1b1. As expected, the conventional catalytic system requires an intact active site in the Man1b1 luminal domain. In contrast, the unconventional system is under the control of an evolutionarily extended N-terminal cytoplasmic tail. Also, N-glycans attached to misfolded AAT are not required for accelerated degradation mediated by the unconventional system, further demonstrating its catalysis-independent nature. We also established that both systems accelerate the proteasomal degradation of NHK in metabolic pulse-chase labeling studies. Taken together, these results have identified the previously unrecognized regulatory capacity of the Man1b1 cytoplasmic tail and provided insight into the functional dichotomy of Man1b1 as a component in the mammalian proteostasis network.

Oxidoreductases in Glycoprotein Glycosylation, Folding, and ERAD.

N-linked glycosylation and sugar chain processing, as well as disulfide bond formation, are among the most common post-translational protein modifications taking place in the endoplasmic reticulum (ER). They are essential modifications that are required for membrane and secretory proteins to achieve their correct folding and native structure. Several oxidoreductases responsible for disulfide bond formation, isomerization, and reduction have been shown to form stable, functional complexes with enzymes and chaperones that are involved in the initial addition of an N-glycan and in folding and quality control of the glycoproteins. Some of these oxidoreductases are selenoproteins. Recent studies also implicate glycan machinery-oxidoreductase complexes in the recognition and processing of misfolded glycoproteins and their reduction and targeting to ER-associated degradation. This review focuses on the intriguing cooperation between the glycoprotein-specific cell machineries and ER oxidoreductases, and highlights open questions regarding the functions of many members of this large family.

Golgi Alpha-Mannosidase II as a Novel Biomarker Predicts Prognosis in Clear Cell Renal Cell Carcinoma.

OBJECTIVE: Golgi alpha-mannosidase II (GM II) is one of the crucial enzymes in the process of N-glycan processing. The aim of our study was to examine the clinical significance of GM II in patients with clear cell renal cell carcinoma (ccRCC). METHODS: Quantitative reverse transcription polymerase chain reaction analysis and immunohistochemical staining were used to analyze GM II expression in patients with ccRCC. The clinical data of 62 patients with ccRCC were collected to analyze the clinical significance of GM II. The clinical significance among GM II expression, clinicopathological staging, and histological grade of ccRCC was explored. Survival analyses were performed to identify the relevance between the expression of GM II and the overall survival of patients with ccRCC. A uni-/multivariate Cox regression model was used to detect risk factors affecting the prognosis of patients with ccRCC. Subsequently, the proliferation and migration of ccRCC cells were detected after transfecting with GM II-short hairpin RNA (shRNA). RESULTS: The results of these comparisons suggested that GM II expression of ccRCC tissues was dramatically higher than that of para-carcinoma tissues (p < 0.05). GM II expression in the high-differentiation group was lower than that in the median- and low-differentiation groups (p < 0.05). GM II expression in stage I and II tissues was lower than that in stage III and IV tissues (p < 0.05). The expression levels of GM II in the group without lymph node metastasis were lower than those in the group with lymph node metastasis (p < 0.05). Survival analysis indicated that patients with ccRCC with high GM II expression generally had decreased overall survival. Uni-/multivariate Cox model analyses further suggested an association between GM II expression and prognosis of patients with breast cancer. High GM II expression is a potential and independent prognostic biomarker in ccRCC. The inhibition of GM II by transfecting with GM II-shRNA could reduce the proliferation and migration of ccRCC. CONCLUSION: GM II expression in human ccRCC tissues was upregulated compared with that found in normal human renal tissue, and GM II may promote the progression and migration of ccRCC. Furthermore, the GM II gene may be used as a promising tumor marker for the diagnosis and prognosis of ccRCC.

Constructing a human complex type N-linked glycosylation pathway in Kluyveromyces marxianus.

Glycosylation can affect various protein properties such as stability, biological activity, and immunogenicity. To produce human therapeutic proteins, a host that can produce glycoproteins with correct glycan structures is required. Microbial expression systems offer economical, rapid and serum-free production and are more amenable to genetic manipulation. In this study, we developed a protocol for CRISPR/Cas9 multiple gene knockouts and knockins in Kluyveromyces marxianus, a probiotic yeast with a rapid growth rate. As hyper-mannosylation is a common problem in yeast, we first knocked out the α-1,3-mannosyltransferase (ALG3) and α-1,6-mannosyltransferase (OCH1) genes to reduce mannosylation. We also knocked out the subunit of the telomeric Ku domain (KU70) to increase the homologous recombination efficiency of K. marxianus. In addition, we knocked in the MdsI (α-1,2-mannosidase) gene to reduce mannosylation and the GnTI (ß-1,2-N-acetylglucosaminyltransferase I) and GnTII genes to produce human N-glycan structures. We finally obtained two strains that can produce low amounts of the core N-glycan Man3GlcNAc2 and the human complex N-glycan Man3GlcNAc4, where Man is mannose and GlcNAc is N-acetylglucosamine. This study lays a cornerstone of glycosylation engineering in K. marxianus toward producing human glycoproteins.

High-resolution structure of a modular hyperthermostable endo-ß-1,4-mannanase from Thermotoga petrophila: The ancillary immunoglobulin-like module is a thermostabilizing domain.

The endo-ß-1,4-mannanase from the hyperthermostable bacterium Thermotoga petrophila (TpMan) is an enzyme that catalyzes the hydrolysis of mannan and heteromannan polysaccharides. Of the three domains that comprise TpMan, the N-terminal GH5 catalytic domain and the C-terminal carbohydrate-binding domain are connected through a central ancillary domain of unknown structure and function. In this study, we report the partial crystal structure of the TpMan at 1.45 Å resolution, so far, the first modular hyperthermostable endo-ß-1,4-mannanase structure determined. The structure exhibits two domains, a (ß/α)8-barrel GH5 catalytic domain connected via a linker to the central domain with an immunoglobulin-like ß-sandwich fold formed of seven ß-strands. Functional analysis showed that whereas the immunoglobulin-like domain does not have the carbohydrate-binding function, it stacks on the GH5 catalytic domain acting as a thermostabilizing domain and allowing operation at hyperthermophilic conditions. The carbohydrate-binding domain is absent in the crystal structure most likely due to its high flexibility around the immunoglobulin-like domain which may act also as a pivot. These results represent new structural and functional information useful on biotechnological applications for biofuel and food industries.

Spatially remote motifs cooperatively affect substrate preference of a ruminal GH26-type endo-ß-1,4-mannanase.

ß-Mannanases from the glycoside hydrolase 26 (GH26) family are retaining hydrolases that are active on complex heteromannans and whose genes are abundant in rumen metagenomes and metatranscriptomes. These enzymes can exhibit distinct modes of substrate recognition and are often fused to carbohydrate-binding modules (CBMs), resulting in a molecular puzzle of mechanisms governing substrate preference and mode of action that has not yet been pieced together. In this study, we recovered a novel GH26 enzyme with a CBM35 module linked to its N terminus (CrMan26) from a cattle rumen metatranscriptome. CrMan26 exhibited a preference for galactomannan as substrate and the crystal structure of the full-length protein at 1.85 Å resolution revealed a unique orientation of the ancillary domain relative to the catalytic interface, strategically positioning a surface aromatic cluster of the ancillary domain as an extension of the substrate-binding cleft, contributing to galactomannan preference. Moreover, systematic investigation of nonconserved residues in the catalytic interface unveiled that residues Tyr195 (-3 subsite) and Trp234 (-5 subsite) from distal negative subsites have a key role in galactomannan preference. These results indicate a novel and complex mechanism for substrate recognition involving spatially remote motifs, distal negative subsites from the catalytic domain, and a surface-associated aromatic cluster from the ancillary domain. These findings expand our molecular understanding of the mechanisms of substrate binding and recognition in the GH26 family and shed light on how some CBMs and their respective orientation can contribute to substrate preference.