Bridging the gap in cervical cancer screening for underserved communities: MCED and the promise of future technologies.

Cervical cancer screening is a critical public health measure, especially vital for underserved communities where disparities in access and outcomes are pronounced. Despite the life-saving potential of regular screening, numerous barriers-including geographical isolation, cultural and linguistic challenges, and socioeconomic factors-severely hinder accessibility for these populations. Multicancer early detection (MCED) tests emerge as a potentially effective intervention, offering a less invasive, more accessible approach that could transform how screenings are conducted. This paper explores the existing challenges in traditional cervical cancer screening methods, the potential of MCED tests to address these barriers, and the implications of these technologies for global health equity. Through a comprehensive review, we highlight the need for culturally sensitive, tailored interventions and the importance of effectively overcoming logistical and financial difficulties to implement MCED tests. Despite the promise shown by MCED tests, the paper acknowledges significant implementation challenges, including cost, logistical obstacles, and the need for cultural acceptance and validation studies. This study emphasizes the necessity for equitable MCED test implementation strategies, highlighting the potential of these innovative technologies to advance global health equity in cervical cancer prevention.

A non-invasive screening method using Caenorhabditis elegans for early detection of multiple cancer types: A prospective clinical study.

Cancer is the second leading cause of death worldwide, according to the World Health Organization, surpassed only by cardiovascular diseases. Early identification and intervention can significantly improve outcomes. However, finding a universal, non-invasive, economical, and precise method for early cancer detection remains a significant challenge. This study explores the efficacy of an innovative cancer detection test, N-NOSE, leveraging a Caenorhabditis elegans olfactory assay on urine samples across a diverse patient group exceeding 1600 individuals diagnosed with various cancers, with samples from the Shikoku Cancer Center (Ehime, Japan) under approved ethical standards. Current cancer screening techniques often require invasive procedures, can be painful or complex, with poor performance, and might be prohibitively costly, limiting accessibility for many. N-NOSE addresses these challenges head-on by offering a test based on urine analysis, eliminating the need for invasive methods, and being more affordable with higher performance at early stages than extensive blood tests or comprehensive body scans for cancer detection. In this study, N-NOSE demonstrated a capability to accurately identify upwards of 20 cancer types, achieving detection sensitivities between 60 and 90 %, including initial-stage cancers. The findings robustly advocate for N-NOSE's potential as a revolutionary, cost-effective, and minimally invasive strategy for broad-spectrum early cancer detection. It is also particularly significant in low- and middle-income countries with limited access to advanced cancer diagnostic methods, which may contribute to the improved outcome of affected individuals.

Structural insights into the C-terminus of the histone-lysine N-methyltransferase NSD3 by small-angle X-ray scattering.

NSD3 is a member of six H3K36-specific histone lysine methyltransferases in metazoans. Its overexpression or mutation is implicated in developmental defects and oncogenesis. Aside from the well-characterized catalytic SET domain, NSD3 has multiple clinically relevant potential chromatin-binding motifs, such as the proline-tryptophan-tryptophan-proline (PWWP), the plant homeodomain (PHD), and the adjacent Cys-His-rich domain located at the C-terminus. The crystal structure of the individual domains is available, and this structural knowledge has allowed the designing of potential inhibitors, but the intrinsic flexibility of larger constructs has hindered the characterization of mutual domain conformations. Here, we report the first structural characterization of the NSD3 C-terminal region comprising the PWWP2, SET, and PHD4 domains, which has been achieved at a low resolution in solution by small-angle X-ray scattering (SAXS) data on two multiple-domain NSD3 constructs complemented with size-exclusion chromatography and advanced computational modeling. Structural models predicted by machine learning have been validated in direct space, by comparison with the SAXS-derived molecular envelope, and in reciprocal space, by reproducing the experimental SAXS profile. Selected models have been refined by SAXS-restrained molecular dynamics. This study shows how SAXS data can be used with advanced computational modeling techniques to achieve a detailed structural characterization and sheds light on how NSD3 domains are interconnected in the C-terminus.

Clinical Possibility of Caenorhabditis elegans as a Novel Evaluation Tool for Esophageal Cancer Patients Receiving Chemotherapy: A Prospective Study.

BACKGROUND: The nematode Caenorhabditis elegans (C. elegans) possesses a sophisticated sense of smell and is used for a novel cancer screening test that utilizes the chemotaxis index. We designed a single-institution, prospective study to confirm the ability of Nematode Nose (N-NOSE) to determine preoperative chemotherapy's efficacy for esophageal cancer patients. PATIENTS AND METHODS: We investigated the predictability of N-NOSE screening for the clinical effects of preoperative chemotherapy for esophageal cancer patients receiving radical surgery. The index reduction score (IRS) was calculated via the chemotaxis of C. elegans at three points: before treatment, before surgery, and after surgery, and its clinical relevance was examined. RESULT: Thirty-nine patients with esophageal cancer were enrolled from August 2020 to December 2021, and 30 patients receiving radical surgery were examined. Complete response or partial response was achieved in 23 cases (76.7%). When the target of the treatment effect was complete response only, the prediction accuracies of the IRS calculated by area under the curve was 0.85 (95% Confidence interval: 0.62-1) in clinically achieving complete response group, and the sensitivity and specificity were 1 and 0.63, respectively. CONCLUSION: Index reduction score using N-NOSE screening may reflect the efficacy of chemotherapy for esophageal cancer patients. A large-scale prospective study at multiple centers is desired in the future.

C. elegans as a Powerful Tool for Cancer Screening.

Regular cancer screening is critical for early cancer detection. Cancer screening tends to be burdensome, invasive, and expensive, especially for a comprehensive multi-organ check. Improving the rate and effectiveness of routine cancer screenings remain a challenge in health care. Multi-cancer early detection (MCED) is an exciting concept and a potentially effective solution for addressing current issues with routine cancer screening. In recent years, several technologies have matured for MCED, such as identifying cell-free tumor DNA in blood or using organisms such as Caenorhabditis elegans as a tool for early cancer detection. In Japan, N-NOSE is a commercially available multi-cancer detection test based on the chemotaxis of C. elegans using a urine sample showing 87.5% sensitivity and 90.2% specificity. In this review, we focus on using C. elegans as a powerful biosensor for universal cancer screening. We review N-NOSE clinical research results, spotlighting it as an effective primary cancer screening test.

Accuracy evaluation of the C. elegans cancer test (N-NOSE) using a new combined method.

Early cancer detection is critical for effective treatment. N-NOSE (Nematode-NOSE) is a simple, inexpensive, and highly sensitive cancer screening method based on the chemotaxis of the nematode Caenorhabditis elegans, which shows evasive action from the urine of healthy individuals while being attracted to the urine of cancer patients. Initially, N-NOSE relied on chemotaxis indexes obtained with 10-fold dilutions of urine samples. However, cancer tissue size and concentrations of cancer odors differ among cancer patients. In this study, we examined the accuracy improvement of N-NOSE method by using two types of dilutions, 10-fold and 100-fold. We have conducted N-NOSE tests with urine samples from 32 cancer patients (esophageal, gastric, colorectal, gallbladder, cholangiocarcinoma, breast, malignant lymphoma, and acute myeloid leukemia) along with 143 healthy subjects. Our data showed a significant difference in the N-NOSE at 10-fold dilution between the two groups (p < 0.0001), with an area under the ROC curve (AUC) of 0.9188 based on receiver operating characteristic (ROC) analysis. N-NOSE index at 100-fold dilutions was also significantly different between the two groups (p < 0.0001), with an AUC of 0.9032 based on ROC analysis. In this clinical study, we further improve N-NOSE with a combined method of two dilutions (10-fold and 100-fold) of urine samples, which results in a markedly improvement in cancer detection sensitivity of 87.5%. N-NOSE sensitivity improvement was significantly high even for early-stage cancer detection, which is in stark contrast with the sensitivity of detection using blood tumor markers (CEA, CA19-9 and CA15-3). These results strongly suggest that the N-NOSE test by this new combined method strikes a good balance between sensitivity and specificity.

High yield recombinant expression and purification of oncogenic NSD1, NSD2, and NSD3 with human influenza hemagglutinin tag.

The nuclear receptor-binding SET Domain (NSD) family consists of NSD1, NSD2/MMSET/WHSC1, and NSD3/WHSC1L1 histone methyltransferases that are crucial for chromatin remodeling. NSDs are implicated in developmental disorders such as Wolf-Hirschhorn and Sotos syndromes as well as various cancers including t(4; 14)(p16; q32) myeloma, an incurable cancer in plasma cells. NSDs have been the target of intensive study to understand their biological functions more fully and inform anti-cancer drug design. Recombinant protein expression and purification of human NSDs using an E. coli expression system are notoriously challenging, but the production of pure, stable, and active NSDs is essential for further studies. To overcome production challenges, we propose a cost-efficient approach optimized to produce a high yield of NSDs using a modified E. coli expression system. We found that tagging the NSDs with a human influenza hemagglutinin (HA) tag greatly improved the quality of the recombinant NSDs, resulting in more than 95% pure, stable, and active NSD-HAs, with an increase in production yield up to 22.4-fold and up to 6.25 mg/L from LB E. coli culture, and without further purification such as ion-exchange or size-exclusion chromatography.

Set7 Is a H3K37 Methyltransferase in Schizosaccharomyces pombe and Is Required for Proper Gametogenesis.

Histone methylation by histone methyltransferases (HMTases) has a key role in transcriptional regulation. Discrepancies between the known HMTases and the histone lysine methylome suggest that HMTases remain to be identified. Here we report the discovery, characterization, and crystal structure of Schizosaccharomyces pombe Set7, an HMTase methylating the uncharted histone H3 lysine 37 (H3K37) mark. Set7 forms a dimer with its substrate-binding site structurally specific to K37, not the neighboring well-studied K36 mark. We also discovered that H3K37 methylation levels dramatically increase during gametogenesis. Set7 deletion mutant cells show defects in gametogenesis and produce the abnormal number of spores with aberrant morphology. S. pombe gametogenesis shares similarities with mammalian spermatogenesis. These findings extend our understanding of epigenetic regulation during gametogenesis and support a link between Set7, the epigenetic H3K37 methyl mark, and proper gametogenesis.

Identification of LEM-14 inhibitor of the oncoprotein NSD2.

The NSD family (NSD1, NSD2/MMSET/WHSC1, and NSD3/WHSC1L1) are histone lysine methyltransferases (HMTases) essential for chromatin regulation. The NSDs are oncoproteins, drivers of a number of tumors and are considered important drug-targets but the lack of potent and selective inhibitors hampers further therapeutic development and limits exploration of their biology. In particular, MMSET/NSD2 selective inhibition is being pursued for therapeutic interventions against multiple myeloma (MM) cases, especially in multiple myeloma t(4;14)(p16.3;q32) translocation that is associated with a significantly worse prognosis than other MM subgroups. Multiple myeloma is the second most common hematological malignancy, after non-Hodgkin lymphoma and remains an incurable malignancy. Here we report the discovery of LEM-14, an NSD2 specific inhibitor with an in vitro IC50 of 132 µM and that is inactive against the closely related NSD1 and NSD3. LEM-14-1189, a LEM-14 derivative, differentially inhibits the NSDs with in vitro IC50 of 418 µM (NSD1), IC50 of 111 µM (NSD2) and IC50 of 60 µM (NSD3). We propose LEM-14 and derivative LEM-14-1189 as tools for studying the biology of the NSDs and constitute meaningful steps toward potent NSDs therapeutic inhibitors.

Cloning, expression, purification and crystallization of Schizosaccharomyces pombe Set7, a putative histone methyltransferase.

Dysfunction of histone-modifying enzymes affects chromatin regulation and is involved in carcinogenesis, tumour progression and other diseases. Histone methyltransferases are a family of key histone-modifying enzymes, but their structures, functions and mechanisms are incompletely understood, thus constraining drug-design efforts. Here, preliminary steps towards structure-function studies of Schizosaccharomyces pombe Set7, a putative histone methyltransferase and the first yeast full-length SET-domain-containing protein to be studied using X-ray crystallography, are reported. The methods from cloning to X-ray diffraction and phasing are discussed and the results will aid in prospective studies of histone-modifying enzymes.

In vitro histone lysine methylation by NSD1, NSD2/MMSET/WHSC1 and NSD3/WHSC1L.

BACKGROUND: Histone lysine methylation has a pivotal role in regulating the chromatin. Histone modifiers, including histone methyl transferases (HMTases), have clear roles in human carcinogenesis but the extent of their functions and regulation are not well understood. The NSD family of HMTases comprised of three members (NSD1, NSD2/MMSET/WHSC1, and NSD3/WHSC1L) are oncogenes aberrantly expressed in several cancers, suggesting their potential to serve as novel therapeutic targets. However, the substrate specificity of the NSDs and the molecular mechanism of histones H3 and H4 recognition and methylation have not yet been established. RESULTS: Herein, we investigated the in vitro mechanisms of histones H3 and H4 recognition and modifications by the catalytic domain of NSD family members. In this study, we quantified in vitro mono-, di- and tri- methylations on H3K4, H3K9, H3K27, H3K36, H3K79, and H4K20 by the carboxyl terminal domain (CTD) of NSD1, NSD2 and NSD3, using histone as substrate. Next, we used a molecular modelling approach and docked 6-mer peptides H3K4 a.a. 1-7; H3K9 a.a. 5-11; H3K27 a.a. 23-29; H3K36 a.a. 32-38; H3K79 a.a. 75-81; H4K20 a.a. 16-22 with the catalytic domain of the NSDs to provide insight into lysine-marks recognition and methylation on histones H3 and H4. CONCLUSIONS: Our data highlight the versatility of NSD1, NSD2, and NSD3 for recognizing and methylating several histone lysine marks on histones H3 and H4. Our work provides a basis to design selective and specific NSDs inhibitors. We discuss the relevance of our findings for the development of NSD inhibitors amenable for novel chemotherapies.

Structural insights into the regulation and the recognition of histone marks by the SET domain of NSD1.

The development of epigenetic therapies fuels cancer hope. DNA-methylation inhibitors, histone-deacetylase and histone-methyltransferase (HMTase) inhibitors are being developed as the utilization of epigenetic targets is emerging as an effective and valuable approach to chemotherapy as well as chemoprevention of cancer. The nuclear receptor binding SET domain (NSD) protein is a family of three HMTases, NSD1, NSD2/MMSET/WHSC1, and NSD3/WHSC1L1 that are critical in maintaining the chromatin integrity. A growing number of studies have reported alterations or amplifications of NSD1, NSD2, or NSD3 in numerous carcinogenic events. Reducing NSDs activity through specific lysine-HMTase inhibitors appears promising to help suppressing cancer growth. However, little is known about the NSD pathways and our understanding of the histone lysine-HMTase mechanism is partial. To shed some light on both the recognition and the regulation of epigenetic marks by the SET domain of the NSD family, we investigate the structural mechanisms of the docking of the histone-H4 tail on the SET domain of NSD1. Our finding exposes a key regulatory and recognition mechanism driven by the flexibility of a loop at the interface of the SET and postSET region. Finally, we prospect the special value of this regulatory region for developing specific and selective NSD inhibitors for the epigenetic therapy of cancers.

Cancers and the NSD family of histone lysine methyltransferases.

Both genetic and epigenetic alterations are responsible for the stepwise initiation and progression of cancers. Only epigenetic aberrations can be reversible, allowing the malignant cell population to revert to a more benign phenotype. The epigenetic therapy of cancers is emerging as an effective and valuable approach to both the chemotherapy and the chemoprevention of cancer. The utilization of epigenetic targets that include histone methyltransferase (HMTase), Histone deacetylatase, and DNA methyltransferase, are emerging as key therapeutic targets. The nuclear receptor binding SET domain (NSD) protein is a family of three HMTases, NSD1, NSD2/MMSET/WHSC1, and NSD3/WHSC1L1, and plays a critical part in chromatin integrity as evidenced by a growing number of conditions linked to the alterations and/or amplification of NSD1, NSD2, and/or NSD3. NSD1, NSD2 and NSD3 are associated with multiple cancers. The amplification of either NSD1 or NSD2 triggers the cellular transformation and thus is key in the early carcinogenesis events. In most cases, reducing the levels of NSD proteins would suppress cancer growth. NSD1 and NSD2 were isolated as genes linked to developmental diseases, such as Sotos syndrome and Wolf-Hirschhorn syndrome, respectively, implying versatile aspects of the NSD proteins. The NSD pathways, however, are not well understood. It is noteworthy that the NSD family is phylogenetically distinct compared to other known lysine-HMTases, Here, we review the current knowledge on NSD1/NSD2/NSD3 in tumorigenesis and prospect their special value for developing novel anticancer drugs.

Phosphate-induced apoptosis in human peritoneal mesothelial cells in vitro.

BACKGROUND: It has been demonstrated that phosphate uptake through the type III sodium-dependent phosphate co-transporter, Pit-1, induced apoptosis of aortic vascular smooth muscle cells and endothelial cells in vitro. However, the apoptotic effects of high phosphate (HP) level in human peritoneal mesothelial cells (HPMCs) are not known. METHODS: To examine whether Pit-1 is expressed in HPMCs, we checked the Western blot assay of immunoreactive Pit-1 and the transcription of Pit-1 by reverse transcriptase PCR. We treated several different phosphate concentrations (1-4 mM) and calcium concentrations (1.8 and 2.8 mM) on HPMCs to assess the effects of concentration. MTT, TUNEL assays, and flow cytometry analysis using Annexin V and propidium iodide were performed to identify cell death and apoptosis. Bax and Bcl-2 by Western blot and caspase-3 activity were evaluated by colorimetric assay. In addition, phosphonoformic acid (PFA) and pan-caspase inhibitor, Z-VAD-FMK, were given to prevent phosphate-induced apoptosis. RESULTS: Pit-1 expression on HPMCs was demonstrated. Apoptosis in HPMCs significantly increased with a high concentration of phosphate in a dose- and time-dependent manner, and was enhanced in the presence of 2.8 mM calcium. HP concentrations significantly decreased the anti-apoptotic Bcl-2/Bax ratio and increased caspase-3 activity. The treatment with PFA and Z-VAD-FMK prevented cell death by HP. CONCLUSION: Phosphate uptake through Pit-1 induces apoptosis in HPMCs by a caspase-related mechanism.

Sorting signals within the Saccharomyces cerevisiae sporulation-specific dityrosine transporter, Dtr1p, C terminus promote Golgi-to-prospore membrane transport.

During sporulation in Saccharomyces cerevisiae, the dityrosine transporter Dtr1p, which is required for formation of the outermost layer of the spore wall, is specifically expressed and transported to the prospore membrane, a novel double-lipid-bilayer membrane. Dtr1p consists of 572 amino acids with predicted N- and C-terminal cytoplasmic extensions and 12 transmembrane domains. Dtr1p missing the largest internal cytoplasmic loop was trapped in the endoplasmic reticulum in both mitotically dividing cells and cells induced to sporulate. Deletion of the carboxyl 15 amino acids, but not the N-terminal extension of Dtr1p, resulted in a protein that failed to localize to the prospore membrane and was instead observed in cytoplasmic puncta. The puncta colocalized with a cis-Golgi marker, suggesting that Dtr1p missing the last 15 amino acids was trapped in an early Golgi compartment. Deletion of the C-terminal 10 amino acids resulted in a protein that localized to the prospore membrane with a delay and accumulated in cytoplasmic puncta that partially colocalized with a trans-Golgi marker. Both full-length Dtr1p and Dtr1p missing the last 10 amino acids expressed in vegetative cells localized to the plasma membrane and vacuoles, while Dtr1p deleted for the carboxyl-terminal 15 amino acids was observed only at vacuoles, suggesting that transport to the prospore membrane is mediated by distinct signals from those that specify plasma membrane localization. Transfer-of-function experiments revealed that both the carboxyl transmembrane domain and the C-terminal tail are important for Golgi complex-to-prospore membrane transport.

Snc1p v-SNARE transport to the prospore membrane during yeast sporulation is dependent on endosomal retrieval pathways.

Vesicular traffic is essential for sporulation in Saccharomyces cerevisiae. The Golgi-associated retrograde protein (GARP) tethering complex is required for retrograde traffic from both the early and late endosomes to the Golgi. Analyses of GARP complex mutants in sporulation reveal defects in meiotic progression and spore formation. In contrast, inactivation of the retromer complex, which mediates vesicle budding and cargo selection from the late endosome, or Snx4p, which is involved in retrieval of proteins from the early endosome, has little effect on sporulation. A retromer GARP double mutant is defective in the formation of the prospore membrane (PSM) that surrounds the haploid nuclei. In the retromer GARP double mutant, PSM precursor vesicles carrying the cargo, Dtr1p, are transported to the spindle pole body (SPB), where PSM formation is initiated. However, the v-SNARE Snc1p is not transported to the SPB in the double mutant, suggesting that the defect in PSM formation is because of the failure to retrieve Snc1p, and perhaps other proteins, from the endosomal pathway. Taken together, these results indicate that retrograde trafficking from the endosome is essential for sporulation by retrieving molecules important for PSM and spore wall formation.

Phospholipase D and the SNARE Sso1p are necessary for vesicle fusion during sporulation in yeast.

Spore formation in Saccharomyces cerevisiae requires the de novo formation of prospore membranes. The coalescence of secretory vesicles into a membrane sheet occurs on the cytoplasmic surface of the spindle pole body. Spo14p, the major yeast phospholipase D, is necessary for prospore membrane formation; however, the specific function of Spo14p in this process has not been elucidated. We report that loss of Spo14p blocks vesicle fusion, leading to the accumulation of prospore membrane precursor vesicles docked on the spindle pole body. A similar phenotype was seen when the t-SNARE Sso1p, or the partially redundant t-SNAREs Sec9p and Spo20p were mutated. Although phosphatidic acid, the product of phospholipase D action, was necessary to recruit Spo20p to the precursor vesicles, independent targeting of Spo20p to the membrane was not sufficient to promote fusion in the absence of SPO14. These results demonstrate a role for phospholipase D in vesicle fusion and suggest that phospholipase D-generated phosphatidic acid plays multiple roles in the fusion process.

End3p-mediated endocytosis is required for spore wall formation in Saccharomyces cerevisiae.

During sporulation in Saccharomyces cerevisiae, vesicles transported to the vicinity of spindle pole bodies are fused to each other to generate bilayered prospore membranes (PSMs). PSMs encapsulate the haploid nuclei that arise from the meiotic divisions and serve as platforms for spore wall deposition. Membrane trafficking plays an important role in supplying vesicles for these processes. The endocytosis-deficient mutant, end3Delta, sporulated poorly and the spores produced lost resistance to ether vapor, suggesting that END3-mediated endocytosis is important for sporulation. End3p-GFP localized to cell and spore peripheries in vegetative and sporulating cells and colocalized with actin structures. Correspondingly, the actin cytoskeleton appeared aberrant during sporulation in end3Delta. Analysis of meiosis in end3Delta mutants revealed that the meiotic divisions occurred with wild-type kinetics. Furthermore, PSMs were assembled normally. However, the levels of proteins required for spore wall synthesis and components of the spore wall layers at spores were reduced, indicating that end3Delta mutants are defective in spore wall synthesis. Thus, END3-mediated endocytosis is important for spore wall formation. Additionally, cytological analyses suggest that trafficking between the plasma membrane and PSMs is important earlier during sporulation.

Response of fission yeast to toxic cations involves cooperative action of the stress-activated protein kinase Spc1/Sty1 and the Hal4 protein kinase.

Stress-activated protein kinases (SAPKs), members of a mitogen-activated protein kinase (MAPK) subfamily, are highly conserved among eukaryotes. Studies of yeasts demonstrated that SAPKs play pivotal roles in survival responses to high osmolarity, oxidative stress, and heat shock. Here we report a novel physiological role of the fission yeast Spc1 SAPK in cellular resistance to certain cations, such as Na(+), Li(+), and Ca(2+). Strains lacking Spc1 or its activator, Wis1 MAPK kinase, are hypersensitive to these cations. Spc1 positively regulates expression of sod2(+) encoding a Na(+)/H(+) antiporter through Atf1 and other transcription factors. In addition, we have identified a novel Spc1-interacting protein, Hal4, which is highly homologous to the budding yeast Sat4/Hal4 protein kinase. Like its budding yeast counterpart, the fission yeast Hal4 kinase is essential for cellular resistance to Na(+), Li(+), and Ca(2+). The hal4-null phenotype is complemented by overexpression of the Trk1 potassium transporter or increased K(+) in the growth medium, suggesting that Hal4 promotes K(+) uptake, which consequently increases cellular resistance to other cations. Interestingly, the Spc1-Hal4 interaction appears to be required for cellular resistance to Ca(2+) but not Na(+) and Li(+). We propose that Spc1 SAPK and Hal4 kinase cooperatively function to protect cells from the toxic cations.

Characterization of O-mannosyltransferase family in Schizosaccharomyces pombe.

Protein O-glycosylation is an essential protein modification in eukaryotic cells. In Saccharomyces cerevisiae, O-mannosylation is initiated in the lumen of the endoplasmic reticulum by O-mannosyltransferase gene products (Pmt1p-7p). A search of the Schizosaccharomyces pombe genome database revealed a total of three O-glycoside mannosyltransferase homologs (ogm1+, ogm2+, and ogm4+), closely related to Saccharomyces cerevisiae PMT1, PMT2, and PMT4. Although individual ogm genes were not found to be essential, ogm1Delta and ogm4Delta mutants exhibited aberrant morphology and failed to agglutinate during mating. The phenotypes of the ogm4Delta mutant were not complemented by overexpression of ogm1+ or ogm2+, suggesting that each of the Ogm proteins does not have overlapping functions. Heterologous expression of a chitinase from S. cerevisiae in the ogm mutants revealed that O-glycosylation of chitinase had decreased in ogm1Delta cells. A GFP-tagged Fus1p from S. cerevisiae was specifically not glycosylated and accumulated in the Golgi in ogm4Delta cells. These results indicate that O-glycosylation initiated by Ogm proteins plays crucial physiological roles and can serve as a sorting determinant for protein transport of membrane glycoproteins in S. pombe.