O-Sialoglycoprotein Endopeptidase Deficiency Impairs Proteostasis and Induces Autophagy in Human Embryonic Stem Cells

The OSGEP gene encodes O-sialoglycoprotein endopeptidase, a catalytic unit of the highly conserved KEOPS complex (Kinase, Endopeptidase, and Other Proteins of small Size) that regulates the second biosynthetic step in the formation of N-6-threonylcarbamoyladenosine (t6A). Mutations in KEOPS cause Galloway–Mowat syndrome (GAMOS), whose cellular function in mammals and underlying molecular mechanisms are not well understood. In this study, we utilized lentivirus-mediated OSGEP knockdown to generate OSGEP-deficient human embryonic stem cells (hESCs). OSGEP-knockdown hESCs exhibited reduced stemness factor expression and G2/M phase arrest, indicating a potential role of OSGEP in the regulation of hESC fate. Additionally, OSGEP silencing led to enhanced protein synthesis and increased aggregation of proteins, which further induced inappropriate autophagy, as evidenced by the altered expression of P62 and the conversion of LC3-I to LC3-II. The above findings shed light on the potential involvement of OSGEP in regulating pluripotency and differentiation in hESCs while simultaneously highlighting its crucial role in maintaining proteostasis and autophagy, which may have implications for human disease.

OSGEP (O-sialoglycoprotein endopeptidase) was first reported to be associated with GAMOS in 2017 [9].To date, OSGEP variants in 45 patients from 38 families have been reported and are the most prevalent certain stemness markers of the genetic etiology of GAMOS [2, [9][10][11][12][13][14][15][16].Most affected individuals die in early childhood, and the primary mutation type of OSGEP is a missense mutation.In a previous study, the knockout of OSGEP in zebrafish and mouse models resulted in early lethality [9].Such results suggest that the complete absence of OSGEP leads to cell death and that OSGEP is essential for normal growth and development in a variety of model organisms.However, research on its impact on humans is limited.Further investigation into the role of OSGEP in human cells is conducive to advancing our understanding of GAMOS.
OSGEP as a catalytic unit of the KEOPS complex participates in the biosynthesis of N6-threonylcarbamoyl adenosine (t6A), a universal transfer RNA (tRNA) modification at position 37 that decodes ANN codons in eukaryotes and archaea [17].tRNA modification is crucial for protein synthesis and is extensively characterized in Saccharomyces cerevisiae.The authors of several studies have linked tRNA hypomodification to elevated missense error rates and disrupted codon decoding efficiency, resulting in protein aggregation and harmful effects on the cell [18][19][20].A number of studies have demonstrated the role of proteostasis in mouse neural stem cells (NSCs) and their ability to exit quiescence, selfrenew, and differentiate [21,22].To remove abnormal protein aggregates and maintain protein homeostasis, autophagy is employed.Autophagy plays a crucial role in maintaining neuronal homeostasis and synaptic function.In numerous neurodegenerative diseases marked by the accumulation of protein aggregates-such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis (ALS)-this process is often dysregulated [23][24][25][26].ELP3 (Elongator Acetyltransferase Complex Subunit 2) is a susceptibility gene for ALS and affects the lifespan of some ALS patients [27,28].ELP3 is involved in the modification of the wobble uridine (U34) of tRNA, and its mutants can lead to inappropriate autophagy [29].The above studies suggest that tRNA modifications are closely related to protein homeostasis and autophagy.However, no study has elucidated the impact of the KEOPS complex on proteostasis and autophagy, especially in human cells.
In the present study, we developed an OSGEP-knockdown hESC model by using lentivirus shRNA infection.Based on the hECS model, we detected the proliferation, apoptosis, and translation of stem cell markers to investigate the impact of OSGEP on hESC fate.Moreover, we examined protein synthesis rates, protein aggregation, and alterations in autophagy levels to explore the global protein changes during OSGEP deficiency.

OSGEP Knockdown Results in Reduced Proliferation and Increased Apoptosis
To investigate the functional role of OSGEP in stem cells, we constructed OSGEPknockdown (shOSGEP) hESCs, which demonstrated reductions of 40% and 63% in OSGEP mRNA and protein levels, respectively (Figure S1).Our morphological analysis results revealed that OSGEP silencing maintained the typical dome-shaped colonies but with a reduced surface area and increased intercellular spacing (Figure 1a,b).The shOSGEP hESCs showed a significantly reduced EdU staining area, indicating decreased cell proliferation (Figure 1c,d).Furthermore, OSGEP knockdown led to an enlargement of the nuclei compared with the control (Figure S2).Our cell cycle analysis results indicated a significant increase in the proportion of cells in the G2/M phase and a decrease in the proportion of cells in the G0/G1 phase in shOSGEP hESCs (Figure 1e,f), suggesting OSGEP knockdowninduced cell cycle arrest in the G2/M phase.Additionally, our apoptosis analysis results revealed a higher cell apoptosis index in shOSGEP hESCs (Figure 1g,h).The above findings collectively highlight the relevance of OSGEP in hESC proliferation and apoptosis.

OSGEP Knockdown Affects the Expression of Stemness Markers
We performed alkaline phosphatase (AP) staining assays, in which high AP levels serve as a marker for undifferentiated states.Remarkably, we observed a significant decrease in the AP-positive population in shOSGEP hESCs (Figure 2a,b).Additionally, we investigated several genes essential for maintaining the pluripotency of hESCs, including NANOG, OCT4, TBX3, and ESRRB.Although NANOG and OCT4 showed no significant changes in protein and mRNA levels, a significant reduction in TBX3 expression was noted (Figure 2c-e).Subsequently, we explored how the reduction in OSGEP affects the ability of hESCs to differentiate into various cell lineages.The increased expression of TBX6 in shOSGEP hESCs suggests that the cells might differentiate toward a mesodermal lineage.Elevated levels of PECAM1 suggest that the shOSGEP hESCs might differentiate into endothelial cells.The reduction in both OTX2 and SOX1 suggests a decrease in neural differentiation potential (Figure 2f).The above results indicate that OSGEP knockdown affects the expression of pluripotency markers of hESCs, making them more prone to differentiation and causing a shift away from the neural lineage.

OSGEP Knockdown Affects the Expression of Stemness Markers
We performed alkaline phosphatase (AP) staining assays, in which high AP levels serve as a marker for undifferentiated states.Remarkably, we observed a significant decrease in the AP-positive population in shOSGEP hESCs (Figure 2a,b).Additionally, we investigated several genes essential for maintaining the pluripotency of hESCs, including NANOG, OCT4, TBX3, and ESRRB.Although NANOG and OCT4 showed no significant changes in protein and mRNA levels, a significant reduction in TBX3 expression was noted (Figure 2c-e).Subsequently, we explored how the reduction in OSGEP affects the ability of hESCs to differentiate into various cell lineages.The increased expression of TBX6 in shOSGEP hESCs suggests that the cells might differentiate toward a mesodermal lineage.Elevated levels of PECAM1 suggest that the shOSGEP hESCs might differentiate into endothelial cells.The reduction in both OTX2 and SOX1 suggests a decrease in neural differentiation potential (Figure 2f).The above results indicate that OSGEP knockdown

OSGEP Deficiency Perturbs Proteostasis
Given the recognized influence of tRNA modifications on proteostasis, we examined the impact of OSGEP on global protein synthesis in hESCs.The results of a puromycin incorporation assay showed that de novo protein synthesis rates were remarkably elevated in shOSGEP hESCs (Figure 3a,b).These findings were further validated in the HEK293T cell line (Figure S3).In shOSGEP hESCs, numerous red punctate signals were observed in the cytoplasm (Figure 4a), and the fluorescence intensity was significantly higher than in the control cells (Figure 4b).We used MG-132 as a positive control, a relatively non-specific proteasome inhibitor known to accelerate the formation of perinuclear aggresomes and inclusion bodies.The MG-132 treatment group exhibited more numerous and stronger red fluorescent signals and fewer cells, indicating that the accumulation of proteins leads to cell death (Figure S4).The above results indicate that OSGEP depletion leads to elevated protein synthesis and the accumulation of aggregated proteins, highlighting the potential role of OSGEP in maintaining protein homeostasis in hESCs.
affects the expression of pluripotency markers of hESCs, making them more prone to dif ferentiation and causing a shift away from the neural lineage.

OSGEP Deficiency Perturbs Proteostasis
Given the recognized influence of tRNA modifications on proteostasis, we examine the impact of OSGEP on global protein synthesis in hESCs.The results of a puromyci incorporation assay showed that de novo protein synthesis rates were remarkably ele vated in shOSGEP hESCs (Figure 3a,b).These findings were further validated in th HEK293T cell line (Figure S3).In shOSGEP hESCs, numerous red punctate signals wer observed in the cytoplasm (Figure 4a), and the fluorescence intensity was significantl higher than in the control cells (Figure 4b).We used MG-132 as a positive control, a rela tively non-specific proteasome inhibitor known to accelerate the formation of perinuclea aggresomes and inclusion bodies.The MG-132 treatment group exhibited more numerou and stronger red fluorescent signals and fewer cells, indicating that the accumulation o proteins leads to cell death (Figure S4).The above results indicate that OSGEP depletio leads to elevated protein synthesis and the accumulation of aggregated proteins, high lighting the potential role of OSGEP in maintaining protein homeostasis in hESCs.

OSGEP Deficiency Induces Inappropriate Autophagy
To investigate the involvement of autophagy in aggregated proteins, we examined alterations in autophagy-associated protein levels.P62, a cargo protein specific to autophagosomes, forms aggregates that can be selectively degraded through autophagy [30].The expression levels of P62 decreased in shOSGEP hESCs (Figure 5a,b).The conversion of LC3-I to LC3-II serves as a key marker of autophagosome formation.Upon the silencing of OSGEP, we observed a notable decrease in LC3-I expression levels and a significant elevation in the LC3-II/LC3-I ratio (Figure 5c,d).The above findings collectively indicate that OSGEP depletion triggers autophagy as a cellular response to counteract the accumulation of aggregated proteins.

OSGEP Deficiency Induces Inappropriate Autophagy
To investigate the involvement of autophagy in aggregated proteins, we examined alterations in autophagy-associated protein levels.P62, a cargo protein specific to autophagosomes, forms aggregates that can be selectively degraded through autophagy [30].The expression levels of P62 decreased in shOSGEP hESCs (Figure 5a,b).The conversion of LC3-I to LC3-II serves as a key marker of autophagosome formation.Upon the silencing of OSGEP, we observed a notable decrease in LC3-I expression levels and a significant elevation in the LC3-II/LC3-I ratio (Figure 5c,d).The above findings collectively indicate that OSGEP depletion triggers autophagy as a cellular response to counteract the accumulation of aggregated proteins.

Discussion
In the study presented herein, we observed that OSGEP knockdown in hESCs led to decreased cell proliferation and increased apoptosis.These findings are consistent with observations in OSGEP-knockdown human podocyte cell lines [9], indicating that impaired cell proliferation and increased apoptosis are common pathogenic features of GAMOS.
Cell cycle regulation is important for the self-renewal and pluripotency of hESCs [31].Our findings suggest that OSGEP silencing results in cell cycle arrest in hESCs, which may impact their self-renewal capacity.This finding is consistent with previous studies demonstrating that strongly mitotic tissues are highly sensitive to the loss of kae1 (the Drosophila ortholog of OSGEP), whereas non-proliferating tissues are less affected [32].Additionally,

Discussion
In the study presented herein, we observed that OSGEP knockdown in hESCs led to decreased cell proliferation and increased apoptosis.These findings are consistent with observations in OSGEP-knockdown human podocyte cell lines [9], indicating that impaired cell proliferation and increased apoptosis are common pathogenic features of GAMOS.
Cell cycle regulation is important for the self-renewal and pluripotency of hESCs [31].Our findings suggest that OSGEP silencing results in cell cycle arrest in hESCs, which may impact their self-renewal capacity.This finding is consistent with previous studies demonstrating that strongly mitotic tissues are highly sensitive to the loss of kae1 (the Drosophila ortholog of OSGEP), whereas non-proliferating tissues are less affected [32].Additionally, the results of a previous study showed that the KEOPS complex may inhibit Mis17-Mis6 complex-mediated centromere formation in Schizosaccharomyces pombe [33], which could be associated with G2/M phase arrest in hESCs.
The results of our puromycin incorporation assay demonstrate that OSGEP knockdown leads to increased protein synthesis in both hESCs and HEK293T cells.The observed increase in protein synthesis may be related to an increase in the frequency of leaky scanning through start codons, leading to enhanced protein translation.This phenomenon has also been observed in yeast lacking the function of SUA5/YDRC, which is involved in t6A modifications [34].Additionally, in yeast with kae1 mutations, the upregulation of genes related to amino acid synthesis can be observed [35].In our study, an abnormal increase in protein aggregates in OSGEP-knockdown hESCs was identified.This observation supports the result of increased protein synthesis, potentially due to erroneous translation products caused by defective t6A modifications.The results of previous studies have shown that abnormalities in t6A modifications can lead to increased +1 frameshift frequencies and readthrough of stop codons [34].Whether the increase in abnormal protein synthesis observed in OSGEP-knockdown human cells is due to the same underlying mechanism requires further investigation.
In our study, we observed that OSGEP knockdown in hESCs leads to increased autophagic activity.This enhanced autophagy likely represents a cellular attempt to clear the increased protein aggregates resulting from OSGEP deficiency.Maintaining proper proteostasis is a fundamental requirement for cellular functionality and viability.Our results indicate that OSGEP knockdown affects protein synthesis, which in turn impacts the proliferation and apoptosis of hESCs, highlighting the importance of t6A modification in maintaining protein homeostasis.
In conclusion, the results of our study elucidate the impact of OSGEP on stem cell development, demonstrating that OSGEP is crucial for maintaining the fate of hESCs.Moreover, our study results reveal that OSGEP deficiency leads to proteostasis dysregulation and autophagy activation.The above findings thus provide new insights into the pathogenic mechanisms of GAMOS syndrome.Further research is warranted to unravel the intricate mechanisms underlying the interplay between OSGEP, proteostasis, and autophagy in the context of neurodevelopmental disorders.

Cell Culture
H9 hESCs were regularly cultured in mTeSR Plus medium (STEMCELL Technologies, Vancouver, BC, Canada) on plates coated with Matrigel.HEK293T cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS).Cell passaging was performed at 70-80% confluence.All cell cultures were maintained in a humidified incubator set at 37 • C with 5% CO 2 .

Lentivirus Production and Transduction of hESCs
To generate OSGEP-knockdown hESCs, hESCs were infected with pLKO.1 lentiviruses containing OSGEP shRNA1 (sh1), OSGEP shRNA2 (sh2), and a non-target shRNA (negative control, NC).The sequences of shRNAs used are listed in Table S1.Briefly, HEK293T cells were transfected with the specified lentiviral vector utilizing Lipofectamine 3000 reagent (Invitrogen, Waltham, MA, USA) following the manufacturer's guidelines.The target and packaging plasmids psPAX2 and pMD2.G (VSV-G envelope protein) were used in a 3:2:1 ratio.After 48 h, the culture medium was collected and filtered with a 0.45 µm filter (Millipore, Burlington, MA, USA).Subsequently, hESCs were incubated with the obtained lentivirus-containing medium at 37 • C for 12 h.Following an additional 24 h, cells were selected by using 1 µg/mL puromycin for 1 week.

HESC Self-Renewal and Proliferation Analysis
For the colony formation assay, 1000 cells were plated in 6-well plates.Colony size was assessed on day 7. Alkaline phosphatase (AP) staining was carried out using the Alkaline Phosphatase Staining Kit for Stem Cell (Maokang Biotechnology Co., Ltd., Shanghai, China).Briefly, 2-3 days after seeding, cells were washed with PBS, fixed for 2-5 min, and then incubated with AP solution for 5-10 min at 25 • C, shielded from light.Stained colonies were examined and documented using a microscope.At least 100 colonies per sample were counted for data analysis.

Flow Cytometry Analysis
For cell cycle analysis, cells were collected using Accutase (STEMCELL Technologies) and fixed overnight in 70% ice-cold ethanol.After fixation, cells were stained for 30 min with a solution containing 100 µg/mL RNase A and 50 µg/mL propidium iodide (PI) (Coolaber, Beijing, China).Flow cytometry was performed with a DxP Athena™ flow cytometer, and the data were analyzed using ModFit software version 4.1.To measure apoptotic cells, the Annexin V-FITC/PI Apoptosis Detection Kit (Vazyme, Nanjing, China) was used according to the manufacturer's instructions.A flow cytometer and FlowJo version 10.8.1 were used for the analysis of the flow cytometric results.For the assessment of protein aggregation, cells were stained by using the Proteostat Protein Aggregation Assay Kit (Enzo Life Science, Farmingdale, NY, USA) following the manufacturer's guidelines.

Immunofluorescence Staining
Cells were seeded onto Matrigel-covered glass coverslips in mTeSR Plus medium.The cells were fixed in 4% paraformaldehyde for 20 min and incubated at 4 • C in a blocking buffer (PBS containing 5% BSA).The cells were stained in accordance with the manufacturer's procedure and then washed three times in PBS.

Real-Time Quantitative PCR
Total RNA was extracted using TRIzol reagent (Invitrogen).For cDNA synthesis, 2 µg of RNA was utilized with the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA).Gene expression analysis was performed using the PowerUp SYBR Green Master Mix (Applied Biosystems, Waltham, MA, USA) on an ASA-9600 RT-qPCR System (BAIYUAN GENE-TECH, Lanzhou, China).Relative gene expression was determined using the comparative cycle threshold (Ct) method and analyzed using the 2 −∆∆Ct method.The RT-qPCR primer details are provided in Table S2.

Measurement of Protein Synthesis by Puromycin Incorporation
The cells were incubated for 30 min in complete culture media containing 10 µg/mL puromycin.After treatment, the cells were washed twice with PBS, collected, and lysed using loading buffer.We used an anti-puromycin antibody to detect the puromycin by Western blot.

Western Blot Assay
Cell pellets were collected and lysed with a loading buffer.The lysates were denatured by heating at 100 • C for 10 min.Proteins from the whole-cell lysates were separated using SDS-PAGE and transferred to PVDF membranes (Millipore).The membranes were then incubated with specific primary antibodies, as detailed in Table S3, and the antibodyprotein complexes were visualized using secondary antibodies conjugated to horseradish peroxidase (Sigma, Tokyo, Japan) and the SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Scientific).The images were captured by the Molecular Imager ChemiDoc XRS+ System.

Figure 1 .
Figure 1.OSGEP knockdown affects hESC proliferation and apoptosis.shNC represents the control group, and shOSGEP represents the OSGEP-knockdown group.(a) Images of the colony formation assay, scale bar: 50 μm; (b) quantification of the colony area in the colony formation assay; (c) images of EdU staining, scale bar: 50 µm; (d) quantification of the EdU-positive cell area; (e) the flow cytometry results using propidium iodide (PI) staining; (f) quantification of the cell cycle analysis; (g) the flow cytometry results of PI and Annexin-V; (h) quantification of the cell apoptosis index.n.s ≥ 0.05; * p < 0.05; ** p < 0.01.

Figure 1 .
Figure 1.OSGEP knockdown affects hESC proliferation and apoptosis.shNC represents the control group, and shOSGEP represents the OSGEP-knockdown group.(a) Images of the colony formation assay, scale bar: 50 µm; (b) quantification of the colony area in the colony formation assay; (c) images of EdU staining, scale bar: 50 µm; (d) quantification of the EdU-positive cell area; (e) the flow cytometry results using propidium iodide (PI) staining; (f) quantification of the cell cycle analysis; (g) the flow cytometry results of PI and Annexin-V; (h) quantification of the cell apoptosis index.n.s ≥ 0.05; * p < 0.05; ** p < 0.01.

Figure 3 .
Figure 3. OSGEP knockdown affects protein synthesis rate.(a) An image of the puromycin incorporation assay and (b) the quantification of puromycin.* p < 0.05.

Figure 3 .
Figure 3. OSGEP knockdown affects protein synthesis rate.(a) An image of the puromycin incorporation assay and (b) the quantification of puromycin.* p < 0.05.

Figure 3 .
Figure 3. OSGEP knockdown affects protein synthesis rate.(a) An image of the puromycin incorporation assay and (b) the quantification of puromycin.* p < 0.05.

Figure 5 .
Figure 5. OSGEP deficiency induces inappropriate autophagy in hESCs.(a) An image of the Western blot of P62 and LC3; (b) quantification of the P62 protein expression level; (c) quantification of the LC3-І protein expression level; (d) quantification of the LC3-II/LC3-І protein expression level.Calibrated with GPAPH and normalized by the control group.* p < 0.05; ** p < 0.01.

Figure 5 .
Figure 5. OSGEP deficiency induces inappropriate autophagy in hESCs.(a) An image of the Western blot of P62 and LC3; (b) quantification of the P62 protein expression level; (c) quantification of the LC3-I protein expression level; (d) quantification of the LC3-II/LC3-I protein expression level.Calibrated with GPAPH and normalized by the control group.* p < 0.05; ** p < 0.01.