ARHGEF5 binds Drebrin and affects α-tubulin acetylation to direct neuronal morphogenesis and migration during mouse brain development.

Rho guanine nucleotide exchange factors (Rho GEFs) activate Rho GTPases, which act as molecular switches regulating various essential cellular functions. This study investigated the role of ARHGEF5, a Rho GEF known for its involvement in cell migration and invasion processes, in the context of brain development. We found that ARHGEF5 is essential for dendrite development during the early stages of neuronal growth. We also discovered that ARHGEF5 binds to Drebrin E, which is vital for coordinating actin and microtubule dynamics, and facilitates the interaction between Drebrin E and Cyclin-dependent kinase 5, which phosphorylates Drebrin E. Notably, ARHGEF5 deficiency resulted in a decrease in acetylated α-tubulin levels, and the expression of an α-tubulin acetylation mimetic mutant (K40Q) rescued the defects in dendrite development and neuronal migration, suggesting ARHGEF5's role in modulating microtubule stability. Additionally, ARHGEF5 was shown to influence Golgi positioning in the leading processes of migrating cortical neurons during brain development. Our study suggests that ARHGEF5 plays a crucial role in integrating cytoskeletal dynamics with neuronal morphogenesis and migration processes during brain development.

The intricate role of Sir2 in oxidative stress response during the post-diauxic phase in Saccharomyces cerevisiae.

Silent information regulator 2 (Sir2) is a conserved NAD+-dependent histone deacetylase crucial for regulating cellular stress response and the aging process in Saccharomyces cerevisiae. In this study, we investigated the molecular mechanism underlying how the absence of Sir2 can lead to altered stress susceptibilities in S. cerevisiae under different environmental and physiological conditions. In a glucose-complex medium, the sir2Δ strain showed increased sensitivity to H2O2 compared to the wild-type strain during the post-diauxic phase. In contrast, it displayed increased resistance during the exponential growth phase. Transcriptome analysis of yeast cells in the post-diauxic phase indicated that the sir2Δ mutant expressed several oxidative defense genes at lower levels than the wild-type, potentially accounting for its increased susceptibility to H2O2. Interestingly, however, the sir2Δras2Δ double mutant exhibited greater resistance to H2O2 than the ras2Δ single mutant counterpart. We found that the expression regulation of the cytoplasmic catalase encoded by CTT1 was critical for the increased resistance to H2O2 in the sir2Δras2Δ strain. The expression of the CTT1 gene was influenced by the combined effect of RAS2 deletion and the transcription factor Azf1, whose level was modulated by Sir2. These findings provide insights into the importance of understanding the intricate interactions among various factors contributing to cellular stress response.

Secreted Akkermansia muciniphila threonyl-tRNA synthetase functions to monitor and modulate immune homeostasis.

Commensal bacteria are critically involved in the establishment of tolerance against inflammatory challenges, the molecular mechanisms of which are just being uncovered. All kingdoms of life produce aminoacyl-tRNA synthetases (ARSs). Thus far, the non-translational roles of ARSs have largely been reported in eukaryotes. Here, we report that the threonyl-tRNA synthetase (AmTARS) of the gut-associated bacterium Akkermansia muciniphila is secreted and functions to monitor and modulate immune homeostasis. Secreted AmTARS triggers M2 macrophage polarization and orchestrates the production of anti-inflammatory IL-10 via its unique, evolutionary-acquired regions, which mediates specific interactions with TLR2. This interaction activates the MAPK and PI3K/AKT signaling pathways, which converge on CREB, leading to an efficient production of IL-10 and suppression of the central inflammatory mediator NF-κB. AmTARS restores IL-10-positive macrophages, increases IL-10 levels in the serum, and attenuates the pathological effects in colitis mice. Thus, commensal tRNA synthetases can act as intrinsic mediators that maintain homeostasis.

Corrigendum: The intricate role of Sir2 in oxidative stress response during the post-diauxic phase in Saccharomyces cerevisiae.

[This corrects the article DOI: 10.3389/fmicb.2023.1285559.].

Effectiveness and safety of motion style acupuncture treatment of the pelvic joint for herniated lumbar disc with radiating pain: A prospective, observational pilot study.

CONTEXT: Conservative treatment is effective for treating and managing herniated lumbar disc with radiating leg pain. OBJECTIVES: To investigate the effects of motion style acupuncture treatment (MSAT) on the pelvic joint for this condition. DESIGN: This prospective observational study was a pilot study for a future randomized, controlled trial (RCT). SETTING: [masked for review]. PATIENTS/INTERVENTIONS: We enroled 40 patients and allocated them to two groups (both n = 20). Groups 1 and 2 received integrative Korean medicine treatment (KMT) and integrative KMT with MSAT for pelvic joint, respectively. Primary outcome was the Numeric Rating Scale (NRS) score for low back pain. Secondary outcomes were the Oswestry Disability Index (ODI), Visual analogue Scale (VAS), and EuroQol 5-Dimension-5-level (EQ-5D-5 L) scores. Efficacy was assessed by comparing the baseline and Day 4 results. Safety was assessed based on the frequency and severity of all adverse events. RESULTS: On Day 14, except for ODI in Group 1, the NRS, VAS, and EQ-5D-5 L scores showed significant improvements in both groups. On Day 90, both groups showed significant improvements in the NRS, ODI, and EQ-5D-5 L scores. There was a significant between-group difference in the NRS score on Day 7. On Day 14, Group 2 had a significantly lower VAS score for radiating leg pain than Group 1. Twelve patients reported adverse events associated with integrative KMT; however, there was no association with pelvic joint MSAT. CONCLUSION: Adding MSAT for pelvic joint to conventional integrative KMT may ameliorate radiating leg pain and improve the quality of life.

The transcription factor Cas5 suppresses hyphal morphogenesis during yeast-form growth in Candida albicans.

Candida albicans is an opportunistic human pathogen that exists as yeast, hyphal or pseudohyphal forms depending on pH, nutrients, and temperature. The morphological transition from yeast to hyphae, which is required for the complete virulence of C. albicans, is controlled by many transcription factors that activate or repress hypha-specific genes. The C. albicans transcriptional factor Cas5, a key regulator of genes involved in cell wall integrity, affects the susceptibility of C. albicans to fluconazole, an inhibitor of ergosterol synthesis. In this study, we found that deletion of CAS5 in C. albicans decreased the expression levels of a set of ergosterol biosynthesis genes, such as ERG2, ERG3, ERG5, ERG6, ERG11, and ERG24, resulting in the accumulation of lanosterol and zymosterol, which are intermediate metabolites in the ergosterol biosynthesis pathway. Interestingly, it was observed that the cas5Δ/Δ mutant could not maintain the yeast form under non-hypha-inducing conditions, while the CAS5-overexpressing cells could not form hyphae under hypha-inducing conditions. Consistent with these observations, the cas5Δ/Δ mutant highly expressed hypha-specific genes, ALS3, ECE1, and HWP1, under non-hypha-inducing conditions. In addition, CAS5 transcription was significantly downregulated immediately after hyphal initiation in the wild-type strain. Furthermore, the cas5Δ/Δ mutant reduced the transcription of NRG1, which encodes a major repressor of hyphal morphogenesis, while Cas5 overexpression increased the transcription of NRG1 under hypha-inducing conditions. Collectively, this study suggests the potential role of Cas5 as a repressor of hypha-specific genes during yeast-form growth of C. albicans.

Myonectin inhibits adipogenesis in 3T3-L1 preadipocytes by regulating p38 MAPK pathway.

In current times, obesity is a major health problem closely associated with metabolic disease such as diabetes, dyslipidemia, and cardiovascular disease. The direct cause of obesity is known as an abnormal increase in fat cell size and the adipocyte pool. Hyperplasia, the increase in number of adipocytes, results from adipogenesis in which preadipocytes differentiate into mature adipocytes. Adipogenesis is regulated by local and systemic cues that alter transduction pathways and subsequent control of adipogenic transcription factors. Therefore, the regulation of adipogenesis is an important target for preventing obesity. Myonectin, a member of the CTRP family, is a type of myokine released by skeletal muscle cells. Although several studies have shown that myonectin is associated with lipid metabolism, the role of myonectin during adipogenesis is not known. Here, we demonstrate the role of myonectin during adipocyte differentiation of 3T3-L1 cells. We found that myonectin inhibits the adipogenesis of 3T3-L1 preadipocytes with a reduction in the expression of adipogenic transcription factors such as C/EBPα, ß and PPARγ. Furthermore, we show that myonectin has an inhibitory effect on adipogenesis through the regulation of the p38 MAPK pathway and CHOP. These findings suggest that myonectin may be a novel therapeutic target for the prevention of obesity. [BMB Reports 2021; 54(2): 124-129].

Cortactin deacetylation by HDAC6 and SIRT2 regulates neuronal migration and dendrite morphogenesis during cerebral cortex development.

Proper dendrite morphogenesis and neuronal migration are crucial for cerebral cortex development and neural circuit formation. In this study, we sought to determine if the histone deacetylase HDAC6 plays a role in dendrite development and neuronal migration of pyramidal neurons during cerebral cortex development. It was observed that knockdown of HDAC6 leads to defective dendrite morphogenesis and abnormal Golgi polarization in vitro, and the expression of wild type cortactin or deacetyl-mimetic cortactin 9KR rescued the defective phenotypes of the HDAC6 knockdown neurons. This suggests that HDAC6 promotes dendritic growth and Golgi polarization through cortactin deacetylation in vitro. We also demonstrated that ectopic expression of SIRT2, a cytoplasmic NAD+ - dependent deacetylase, suppresses the defects of HDAC6 knockdown neurons. These results indicate that HDAC6 and SIRT2 may be functionally redundant during dendrite development. Neurons transfected with both HDAC6 and SIRT2 shRNA or acetyl-mimetic cortactin 9KQ showed slow radial migration compared to the control cells during cerebral cortex development. Furthermore, a large portion of cortactin 9KQ-expressing pyramidal neurons at layer II/III in the cerebral cortex failed to form an apical dendrite toward the pial surface and had an increased number of primary dendrites, and the percentage of neurons with dendritic Golgi decreased in cortactin 9KQ-expressing cells, compared to control neurons. Taken together, this study suggests that HDAC6 and SIRT2 regulate neuronal migration and dendrite development through cortactin deacetylation in vivo.

Cohesion Between Research Literature and Health System Level Efforts to Address Global Neurosurgical Inequity: A Scoping Review.

BACKGROUND: Research output on global neurosurgery (GNS) has exponentially increased in recent years. As research efforts increase, we must first analyze how the current body of GNS literature fits into the macroscopic schema of systems-based policies. The aim of this study was to identify and categorize GNS research based on health system domains. METHODS: PubMed, CINAHL, and Embase were searched for GNS literature published from 1999 to 2019. Then, health system domains were defined and itemized based on publicly available documents from the Program in Global Surgery and Social Change. This items chart was subsequently used to categorize the GNS literature into health system domains. RESULTS: A total 63 articles were determined to focus on a health system domain. Of these articles, 6 focused on multiple domains, yielding an adjusted total of 70 articles. Overall, the most represented health system domain was service delivery (21 articles), followed by workforce (19), infrastructure (15), financing (12) and information management (3). A total of 30 low- and middle-income countries (LMICs) were represented across all articles. In addition, the first author was affiliated with an institution from a high-income country for 71.4% of the articles. CONCLUSIONS: This review highlighted the pressing need for more research into information management in the context of GNS. In addition, health system-focused GNS literature represented only 20% of all LMICs (30/143). The trends in authorship should be noted, because many ethical (and practical) issues may arise if there is a disconnect in the objectives of the authors and the neurosurgeons in LMICs.

Bacteria-derived metabolite, methylglyoxal, modulates the longevity of C. elegans through TORC2/SGK-1/DAF-16 signaling.

Gut microbes play diverse roles in modulating host fitness, including longevity; however, the molecular mechanisms underlying their mediation of longevity remain poorly understood. We performed genome-wide screens using 3,792 Escherichia coli mutants and identified 44 E. coli mutants that modulated Caenorhabditis elegans longevity. Three of these mutants modulated C. elegans longevity via the bacterial metabolite methylglyoxal (MG). Importantly, we found that low MG-producing E. coli mutants, Δhns E. coli, extended the lifespan of C. elegans through activation of the DAF-16/FOXO family transcription factor and the mitochondrial unfolded protein response (UPRmt). Interestingly, the lifespan modulation by Δhns did not require insulin/insulin-like growth factor 1 signaling (IIS) but did require TORC2/SGK-1 signaling. Transcriptome analysis revealed that Δhns E. coli activated novel class 3 DAF-16 target genes that were distinct from those regulated by IIS. Taken together, our data suggest that bacteria-derived MG modulates host longevity through regulation of the host signaling pathways rather than through nonspecific damage on biomolecules known as advanced glycation end products. Finally, we demonstrate that MG enhances the phosphorylation of hSGK1 and accelerates cellular senescence in human dermal fibroblasts, suggesting the conserved role of MG in controlling longevity across species. Together, our studies demonstrate that bacteria-derived MG is a novel therapeutic target for aging and aging-associated pathophysiology.

TORC1 signaling regulates cytoplasmic pH through Sir2 in yeast.

Glucose controls the phosphorylation of silent information regulator 2 (Sir2), a NAD+ -dependent protein deacetylase, which regulates the expression of the ATP-dependent proton pump Pma1 and replicative lifespan (RLS) in yeast. TORC1 signaling, which is a central regulator of cell growth and lifespan, is regulated by glucose as well as nitrogen sources. In this study, we demonstrate that TORC1 signaling controls Sir2 phosphorylation through casein kinase 2 (CK2) to regulate PMA1 expression and cytoplasmic pH (pHc) in yeast. Inhibition of TORC1 signaling by either TOR1 deletion or rapamycin treatment decreased PMA1 expression, pHc, and vacuolar pH, whereas activation of TORC1 signaling by expressing constitutively active GTR1 (GTR1Q65L) resulted in the opposite phenotypes. Deletion of SIR2 or expression of a phospho-mutant form of SIR2 increased PMA1 expression, pHc, and vacuolar pH in the tor1Δ mutant, suggesting a functional interaction between Sir2 and TORC1 signaling. Furthermore, deletion of TOR1 or KNS1 encoding a LAMMER kinase decreased the phosphorylation level of Sir2, suggesting that TORC1 signaling controls Sir2 phosphorylation. It was also found that Sit4, a protein phosphatase 2A (PP2A)-like phosphatase, and Kns1 are required for TORC1 signaling to regulate PMA1 expression and that TORC1 signaling and the cyclic AMP (cAMP)/protein kinase A (PKA) pathway converge on CK2 to regulate PMA1 expression through Sir2. Taken together, these findings suggest that TORC1 signaling regulates PMA1 expression and pHc through the CK2-Sir2 axis, which is also controlled by cAMP/PKA signaling in yeast.

Quantitative proteomic analyses reveal that GPX4 downregulation during myocardial infarction contributes to ferroptosis in cardiomyocytes.

Ischaemic heart disease (IHD) is the leading cause of death worldwide. Although myocardial cell death plays a significant role in myocardial infarction (MI), its underlying mechanism remains to be elucidated. To understand the progression of MI and identify potential therapeutic targets, we performed tandem mass tag (TMT)-based quantitative proteomic analysis using an MI mouse model. Gene ontology (GO) analysis and gene set enrichment analysis (GSEA) revealed that the glutathione metabolic pathway and reactive oxygen species (ROS) pathway were significantly downregulated during MI. In particular, glutathione peroxidase 4 (GPX4), which protects cells from ferroptosis (an iron-dependent programme of regulated necrosis), was downregulated in the early and middle stages of MI. RNA-seq and qRT-PCR analyses suggested that GPX4 downregulation occurred at the transcriptional level. Depletion or inhibition of GPX4 using specific siRNA or the chemical inhibitor RSL3, respectively, resulted in the accumulation of lipid peroxide, leading to cell death by ferroptosis in H9c2 cardiomyoblasts. Although neonatal rat ventricular myocytes (NRVMs) were less sensitive to GPX4 inhibition than H9c2 cells, NRVMs rapidly underwent ferroptosis in response to GPX4 inhibition under cysteine deprivation. Our study suggests that downregulation of GPX4 during MI contributes to ferroptotic cell death in cardiomyocytes upon metabolic stress such as cysteine deprivation.

An Antibody Designed to Improve Adoptive NK-Cell Therapy Inhibits Pancreatic Cancer Progression in a Murine Model.

Natural killer (NK) cells are primary immune cells that target cancer cells and can be used as a therapeutic agent against pancreatic cancer. Despite the usefulness of NK cells, NK-cell therapy is limited by tumor cell inhibition of NK-cell homing to tumor sites, thereby preventing a sustained antitumor immune response. One approach to successful cancer immunotherapy is to increase trafficking of NK cells to tumor tissues. Here, we developed an antibody-based NK-cell-homing protein, named NK-cell-recruiting protein-conjugated antibody (NRP-body). The effect of NRP-body on infiltration of NK cells into primary and metastatic pancreatic cancer was evaluated in vitro and in murine pancreatic ductal adenocarcinoma models. The NRP-body increased NK-cell infiltration of tumors along a CXCL16 gradient (CXCL16 is cleaved from the NRP-body by furin expressed on the surface of pancreatic cancer cells). CXCL16 induced NK-cell infiltration by activating RhoA via the ERK signaling cascade. Administration of the NRP-body to pancreatic cancer model mice increased tumor tissue infiltration of transferred NK cells and reduced the tumor burden compared with that in controls. Overall survival of NRP-body-treated mice (even the metastasis models) was higher than that of mice receiving NK cells alone. In conclusion, increasing NK-cell infiltration into tumor tissues improved response to this cancer immunotherapy. The combination of an NRP-body with NK-cell therapy might be useful for treating pancreatic cancer.

Identification of anti-SF3B1 autoantibody as a diagnostic marker in patients with hepatocellular carcinoma.

BACKGROUND: Tumor-associated (TA) autoantibodies, which are generated by the immune system upon the recognition of abnormal TA antigens, are promising biomarkers for the early detection of tumors. In order to detect autoantibody biomarkers effectively, antibody-specific epitopes in the diagnostic test should maintain the specific conformations that are as close as possible to those presenting in the body. However, when using patients' serum as a source of TA autoantibodies the characterization of the autoantibody-specific epitope is not easy due to the limited amount of patient-derived serum. METHODS: To overcome these limits, we constructed a B cell hybridoma pool derived from a hepatocellular carcinoma (HCC) model HBx-transgenic mouse and characterized autoantibodies derived from them as tumor biomarkers. Their target antigens were identified by mass spectrometry and the correlations with HCC were examined. With the assumption that TA autoantibodies generated in the tumor mouse model are induced in human cancer patients, the enzyme-linked immunosorbent assays (ELISA) based on the characteristics of mouse TA autoantibodies were developed for the detection of autoantibody biomarkers in human serum. To mimic natural antigenic structures, the specific epitopes against autoantibodies were screened from the phage display cyclic random heptapeptide library, and the streptavidin antigens fused with the specific epitopes were used as coating antigens. RESULTS: In this study, one of HCC-associated autoantibodies derived from HBx-transgenic mouse, XC24, was characterized. Its target antigen was identified as splicing factor 3b subunit 1 (SF3B1) and the high expression of SF3B1 was confirmed in HCC tissues. The specific peptide epitopes against XC24 were selected and, among them, XC24p11 cyclic peptide (-CDATPPRLC-) was used as an epitope of anti-SF3B1 autoantibody ELISA. With this epitope, we could effectively distinguish between serum samples from HCC patients (n = 102) and healthy subjects (n = 85) with 73.53% sensitivity and 91.76% specificity (AUC = 0.8731). Moreover, the simultaneous detection of anti-XC24p11 epitope autoantibody and AFP enhanced the efficiency of HCC diagnosis with 87.25% sensitivity and 90.59% specificity (AUC = 0.9081). CONCLUSIONS: ELISA using XC24p11 peptide epitope that reacts against anti-SF3B1 autoantibody can be used as a novel test to enhance the diagnostic efficiency of HCC.

BIG2-ARF1-RhoA-mDia1 Signaling Regulates Dendritic Golgi Polarization in Hippocampal Neurons.

Proper dendrite development is essential for establishing neural circuitry, and Rho GTPases play key regulatory roles in this process. From mouse brain lysates, we identified Brefeldin A-inhibited guanine exchange factor 2 (BIG2) as a novel Rho GTPase regulatory protein involved in dendrite growth and maintenance. BIG2 was highly expressed during early development, and knockdown of the ARFGEF2 gene encoding BIG2 significantly reduced total dendrite length and the number of branches. Expression of the constitutively active ADP-ribosylation factor 1 ARF1 Q71L rescued the defective dendrite morphogenesis of ARFGEF2-null neurons, indicating that BIG2 controls dendrite growth and maintenance by activating ARF1. Moreover, BIG2 co-localizes with the Golgi apparatus and is required for Golgi deployment into major dendrites in cultured hippocampal neurons. Simultaneous overexpression of BIG2 and ARF1 activated RhoA, and treatment with the RhoA activator lysophosphatidic acid in neurons lacking BIG2 or ARF1 increased the number of cells with dendritic Golgi, suggesting that BIG2 and ARF1 activate RhoA to promote dendritic Golgi polarization. mDia1 was identified as a downstream effector of BIG2-ARF1-RhoA axis, mediating Golgi polarization and dendritic morphogenesis. Furthermore, in utero electroporation of ARFGEF2 shRNA into the embryonic mouse brain confirmed an in vivo role of BIG2 for Golgi deployment into the apical dendrite. Taken together, our results suggest that BIG2-ARF1-RhoA-mDia1 signaling regulates dendritic Golgi polarization and dendrite growth and maintenance in hippocampal neurons.

Functional analysis of Mpk1-mediated cell wall integrity signaling pathway in the thermotolerant methylotrophic yeast Hansenula polymorpha.

Understanding the characteristics and regulation mechanisms of cell wall integrity (CWI) in yeast is important not only for basic research but also in biotechnological applications. We found significantly different CWIs in two representative strains of the thermotolerant methylotrophic yeast Hansenula polymorpha. Compared to the A16 strain (classified as Ogataea polymorpha), the DL1-L strain (classified as Ogataea parapolymorpha) has a thinner cell wall that was found to be more fragile following long-term cultivation and more sensitive to zymolyase. To gain a deeper insight into this difference, we compared the characteristics of the Mpk1pmediated CWI signaling pathway in the two strains. While a DL1-L mutant deficient in Mpk1p (mpk1Δ) showed severe growth retardation at both normal and high growth temperatures and in the presence of cell-wall disrupting agents, the A16 mpk1Δ mutant displayed only a mild defect in cell growth. Sorbitol effect on rescuing growth retardation was different in the two mpk1Δ strains, which could partly be ascribed to subtle differences in the activation of HOG pathway. Among the cell wall disruptors evaluated, only caffeine clearly increased phosphorylation of Mpk1p in DL1-L, but not in A16. A transcriptome analysis of the DL1-L strain revealed that caffeine significantly increased the expression of a subset of cell-wall related genes in an Mpk1p-dependent manner, but not the expected Rlm1-target genes. Taken together, our data support an essential role for Mpk1p in maintaining CWI in H. polymorpha, although the requirement for Mpk1p and its regulation under diverse stress conditions varies depending on the strain background.

HST1 increases replicative lifespan of a sir2Δ mutant in the absence of PDE2 in Saccharomyces cerevisiae.

Silent information regulator 2 (Sir2), which is the founding member of the sirtuin family of proteins, is a pro-longevity factor for replicative lifespan (RLS) in Saccharomyces cerevisiae. Sir2 is required for transcriptional silencing at mating type loci, telomeres, and rDNA loci. Sir2 also represses transcription of highly expressed growth-related genes, such as PMA1 and some ribosomal protein genes. Although the Sir2 paralogues Hst1, Hst2, Hst3, and Hst4 occur in S. cerevisiae, none of them could replace the transcriptional regulation of PMA1 by Sir2 in the wild type. In this study, we demonstrate that Hst1, the closest Sir2 paralogue, deacetylates the acetylated lysine 16 of histone H4 (H4K16Ac) and represses PMA1 transcription in the sir2Δ pde2Δ mutant. We further show that Hst1 plays a role in extending the RLS of the sir2Δ pde2Δ mutant. Collectively, our results suggest that Hst1 can substitute for Sir2 by deacetylating H4K16Ac only in the sir2Δ pde2Δ.

Abolishment of N-glycan mannosylphosphorylation in glyco-engineered Saccharomyces cerevisiae by double disruption of MNN4 and MNN14 genes.

Mannosylphosphorylated glycans are found only in fungi, including yeast, and the elimination of mannosylphosphates from glycans is a prerequisite for yeast glyco-engineering to produce human-compatible glycoproteins. In Saccharomyces cerevisiae, MNN4 and MNN6 genes are known to play roles in mannosylphosphorylation, but disruption of these genes does not completely remove the mannosylphosphates in N-glycans. This study was performed to find unknown key gene(s) involved in N-glycan mannosylphosphorylation in S. cerevisiae. For this purpose, each of one MNN4 and five MNN6 homologous genes were deleted from the och1Δmnn1Δmnn4Δmnn6Δ strain, which lacks yeast-specific hyper-mannosylation and the immunogenic α(1,3)-mannose structure. N-glycan profile analysis of cell wall mannoproteins and a secretory recombinant protein produced in mutants showed that the MNN14 gene, an MNN4 paralog with unknown function, is essential for N-glycan mannosylphosphorylation. Double disruption of MNN4 and MNN14 genes was enough to eliminate N-glycan mannosylphosphorylation. Our results suggest that the S. cerevisiae och1Δmnn1Δmnn4Δmnn14Δ strain, in which all yeast-specific N-glycan structures including mannosylphosphorylation are abolished, may have promise as a useful platform for glyco-engineering to produce therapeutic glycoproteins with human-compatible N-glycans.