Assessing the Quick Inventory of Depressive Symptomatology Self-Report scores to predict continuous employment in mood disorder patients.

Objective: Depression significantly impacts the job performance and attendance of workers, leading to increased absenteeism. Predicting occupational engagement for individuals with depression is of paramount importance. This study aims to determine the cut-off score which predicts continuous employment for patients with mood disorders using the Quick Inventory of Depressive Symptomatology, Self-Report (QIDS-SR). Methods: In a prospective observational trial conducted in Tokyo, 111 outpatients diagnosed with either major depressive disorder or bipolar depression were enrolled. Their employment statuses of these participants were tracked over a six-month period after their QIDS-SR scores were recorded. Based on their employment trajectories, participants were categorized into either continuous or non-continuous employment groups. Binary logistic regression was applied to examine the relationship between the QIDS-SR scores and employment outcomes, with adjustments for age, gender, and psychiatric diagnoses. Receiver operating characteristic curves were utilized to identify the optimal QIDS-SR cut-off values for predicting continuous employment. Findings: Binary logistic regression demonstrated that a lower score on the QIDS-SR was linked to an elevated likelihood of continuous employment (adjusted odds ratio 1.15, 95% CI: 1.06-1.26, p=0.001). The optimal cut-off point, determined by the Youden Index, was 10/11, showcasing a 63% sensitivity and 71% specificity. Conclusion: The results emphasize the potential of the QIDS-SR as a prognostic instrument for predicting employment outcomes among individuals with depressive disorders. These findings further underscore the importance of managing depressive symptoms to mild or lower intensities to ensure ongoing employment.

Large-Scale and Site-Specific Mapping of the Murine Brain O-Glycoproteome with IMPa.

Altered protein glycosylation is typically associated with cognitive defects and other phenotypes, but there is a lack of knowledge about the brain glycoproteome. Here, we used the newly available O-glycoprotease IMPa from Pseudomonas aeruginosa for comprehensive O-glycoproteomic analyses of the mouse brain. In this approach, total tryptic glycopeptides were prepared, extracted, purified, and conjugated to a solid support before an enzymatic cleavage by IMPa. O-glycopeptides were analyzed by electron-transfer/higher-energy collision dissociation (EThcD), which permits site-specific and global analysis of all types of O-glycans. We developed two complementary approaches for the analysis of the total O-glycoproteome using HEK293 cells and derivatives. The results demonstrated that IMPa and EThcD facilitate the confident localization of O-glycans on glycopeptides. We then applied these approaches to characterize the O-glycoproteome of the mouse brain, which revealed the high frequency of various sialylated O-glycans along with the unusual presence of the Tn antigen. Unexpectedly, the results demonstrated that glycoproteins in the brain O-glycoproteome only partly overlap with those reported for the brain N-glycoproteome. These approaches will aid in identifying the novel O-glycoproteomes of different cells and tissues and foster clinical and translational insights into the functions of protein O-glycosylation in the brain and other organs.

Aberrant Glycosylation as Immune Therapeutic Targets for Solid Tumors.

Glycosylation occurs at all major types of biomolecules, including proteins, lipids, and RNAs to form glycoproteins, glycolipids, and glycoRNAs in mammalian cells, respectively. The carbohydrate moiety, known as glycans on glycoproteins and glycolipids, is diverse in their compositions and structures. Normal cells have their unique array of glycans or glycome which play pivotal roles in many biological processes. The glycan structures in cancer cells, however, are often altered, some having unique structures which are termed as tumor-associated carbohydrate antigens (TACAs). TACAs as tumor biomarkers are glycan epitopes themselves, or glycoconjugates. Some of those TACAs serve as tumor glyco-biomarkers in clinical practice, while others are the immune therapeutic targets for treatment of cancers. A monoclonal antibody (mAb) to GD2, an intermediate of sialic-acid containing glycosphingolipids, is an example of FDA-approved immune therapy for neuroblastoma indication in young adults and many others. Strategies for targeting the aberrant glycans are currently under development, and some have proceeded to clinical trials. In this review, we summarize the currently established and most promising aberrant glycosylation as therapeutic targets for solid tumors.

Germline C1GALT1C1 mutation causes a multisystem chaperonopathy.

Mutations in genes encoding molecular chaperones can lead to chaperonopathies, but none have so far been identified causing congenital disorders of glycosylation. Here we identified two maternal half-brothers with a novel chaperonopathy, causing impaired protein O-glycosylation. The patients have a decreased activity of T-synthase (C1GALT1), an enzyme that exclusively synthesizes the T-antigen, a ubiquitous O-glycan core structure and precursor for all extended O-glycans. The T-synthase function is dependent on its specific molecular chaperone Cosmc, which is encoded by X-chromosomal C1GALT1C1. Both patients carry the hemizygous variant c.59C>A (p.Ala20Asp; A20D-Cosmc) in C1GALT1C1. They exhibit developmental delay, immunodeficiency, short stature, thrombocytopenia, and acute kidney injury (AKI) resembling atypical hemolytic uremic syndrome. Their heterozygous mother and maternal grandmother show an attenuated phenotype with skewed X-inactivation in blood. AKI in the male patients proved fully responsive to treatment with the complement inhibitor Eculizumab. This germline variant occurs within the transmembrane domain of Cosmc, resulting in dramatically reduced expression of the Cosmc protein. Although A20D-Cosmc is functional, its decreased expression, though in a cell or tissue-specific manner, causes a large reduction of T-synthase protein and activity, which accordingly leads to expression of varied amounts of pathological Tn-antigen (GalNAcα1-O-Ser/Thr/Tyr) on multiple glycoproteins. Transient transfection of patient lymphoblastoid cells with wild-type C1GALT1C1 partially rescued the T-synthase and glycosylation defect. Interestingly, all four affected individuals have high levels of galactose-deficient IgA1 in sera. These results demonstrate that the A20D-Cosmc mutation defines a novel O-glycan chaperonopathy and causes the altered O-glycosylation status in these patients.

Targeting altered glycosylation in secreted tumor glycoproteins for broad cancer detection.

There is an urgent need to develop new tumor biomarkers for early cancer detection, but the variability of tumor-derived antigens has been a limitation. Here we demonstrate a novel anti-Tn antibody microarray platform to detect Tn+ glycoproteins, a near universal antigen in carcinoma-derived glycoproteins, for broad detection of cancer. The platform uses a specific recombinant IgG1 to the Tn antigen (CD175) as a capture reagent and a recombinant IgM to the Tn antigen as a detecting reagent. These reagents were validated by immunohistochemistry in recognizing the Tn antigen using hundreds of human tumor specimens. Using this approach, we could detect Tn+ glycoproteins at subnanogram levels using cell lines and culture media, serum, and stool samples from mice engineered to express the Tn antigen in intestinal epithelial cells. The development of a general cancer detection platform using recombinant antibodies for detection of altered tumor glycoproteins expressing a unique antigen could have a significant impact on cancer detection and monitoring.

Targeting Tn-positive tumors with an afucosylated recombinant anti-Tn IgG.

The aberrant expression of the Tn antigen (CD175) on surface glycoproteins of human carcinomas is associated with tumorigenesis, metastasis, and poor survival. To target this antigen, we developed Remab6, a recombinant, human chimeric anti-Tn-specific monoclonal IgG. However, this antibody lacks antibody-dependent cell cytotoxicity (ADCC) effector activity, due to core fucosylation of its N-glycans. Here we describe the generation of an afucosylated Remab6 (Remab6-AF) in HEK293 cells in which the FX gene is deleted (FXKO). These cells cannot synthesize GDP-fucose through the de novo pathway, and lack fucosylated glycans, although they can incorporate extracellularly-supplied fucose through their intact salvage pathway. Remab6-AF has strong ADCC activity against Tn+ colorectal and breast cancer cell lines in vitro, and is effective in reducing tumor size in an in vivo xenotransplant mouse model. Thus, Remab6-AF should be considered as a potential therapeutic anti-tumor antibody against Tn+ tumors.

Immunohistochemical analysis of Tn antigen expression in colorectal adenocarcinoma and precursor lesions.

BACKGROUND: The Tn antigen (CD175) is an O-glycan expressed in various types of human adenocarcinomas, including colorectal cancer (CRC), though prior studies have relied heavily upon poorly characterized in-house generated antibodies and lectins. In this study, we explored Tn expression in CRC using ReBaGs6, a well-characterized recombinant murine antibody with high specificity for clustered Tn antigen. METHODS: Using well-defined monoclonal antibodies, expression patterns of Tn and sialylated Tn (STn) antigens were characterized by immunostaining in CRC, in matched peritumoral [transitional margin (TM)] mucosa, and in normal colonic mucosa distant from the tumor, as well as in adenomas. Vicia villosa agglutinin lectin was used to detect terminal GalNAc expression. Histo-scoring (H scoring) of staining was carried out, and pairwise comparisons of staining levels between tissue types were performed using paired samples Wilcoxon rank sum tests, with statistical significance set at 0.05. RESULTS: While minimal intracellular Tn staining was seen in normal mucosa, significantly higher expression was observed in both TM mucosa (p < 0.001) and adenocarcinoma (p < 0.001). This pattern was reflected to a lesser degree by STn expression in these tissue types. Interestingly, TM mucosa demonstrates a Tn expression level even higher than that of the adenocarcinoma itself (p = 0.019). Colorectal adenomas demonstrated greater Tn and STn expression relative to normal mucosa (p < 0.001 and p = 0.012, respectively). CONCLUSIONS: In summary, CRC is characterized by alterations in Tn/STn antigen expression in neoplastic epithelium as well as peritumoral benign mucosa. Tn/STn antigens are seldom expressed in normal mucosa. This suggests that TM mucosa, in addition to CRC itself, represents a source of glycoproteins rich in Tn that may offer future biomarker targets.

Identification and characterization of circulating immune complexes in IgA nephropathy.

The underlying pathology of immunoglobulin A (IgA) nephropathy (IgAN), the most common glomerulonephritis worldwide, is driven by the deposition of immune complexes containing galactose-deficient IgA1 [Tn(+)IgA1] in the glomerular mesangium. Here, we report that novel anti-Tn circulating immune complexes (anti-Tn CICs) contain predominantly IgM, representing large macromolecular complexes of ~1.2 megadaltons to several megadalton sizes together with Tn(+)IgA1 and some IgG. These complexes are significantly elevated in sera of patients with IgAN, which contains higher levels of complement C3, compared to healthy individuals. Anti-Tn CICs are bioactive and induce specific proliferation of human renal mesangial cells. We found that these anti-Tn CICs can be dissociated with small glycomimetic compounds, which mimic the Tn antigen of Tn(+)IgA1, releasing IgA1 from anti-Tn CICs. This glycomimetic compound can also significantly inhibit the proliferative activity of anti-Tn CICs of patients with IgAN. These findings could enhance both the diagnosis of IgAN and its treatment, as specific drug treatments are now unavailable.

Sialylated and sulfated N-Glycans in MDCK and engineered MDCK cells for influenza virus studies.

The Madin-Darby canine kidney (MDCK) cell line is an in vitro model for influenza A virus (IAV) infection and propagation. MDCK-SIAT1 (SIAT1) and humanized MDCK (hCK) cell lines are engineered MDCK cells that express N-glycans with elevated levels of sialic acid (Sia) in α2,6-linkage (α2,6-Sia) that are recognized by many human IAVs. To characterize the N-glycan structures in these cells and the potential changes compared to the parental MDCK cell line resulting from engineering, we analyzed the N-glycans from these cells at different passages, using both mass spectrometry and specific lectin and antibody binding. We observed significant differences between the three cell lines in overall complex N-glycans and terminal galactose modifications. MDCK cells express core fucosylated, bisected complex-type N-glycans at all passage stages, in addition to expressing α2,6-Sia on short N-glycans and α2,3-Sia on larger N-glycans. By contrast, SIAT1 cells predominantly express α2,6-Sia glycans and greatly reduced level of α2,3-Sia glycans. Additionally, they express bisected, sialylated N-glycans that are scant in MDCK cells. The hCK cells exclusively express α2,6-Sia glycans. Unexpectedly, hCK glycoproteins bound robustly to the plant lectin MAL-1, indicating α2,3-Sia glycans, but such binding was not Sia-dependent and closely mirrored that of an antibody that recognizes glycans with terminal 3-O-sulfate galactose (3-O-SGal). The 3-O-SGal epitope is highly expressed in N-glycans on multiple hCK glycoproteins. These results indicate vastly different N-glycomes between MDCK cells and the engineered clones that could relate to IAV infectivity.

Cosmc deficiency causes spontaneous autoimmunity by breaking B cell tolerance.

Factors regulating the induction and development of B cell­mediated autoimmunity are not well understood. Here, we report that targeted deletion in murine B cells of X-linked Cosmc, encoding the chaperone required for expression of core 1 O-glycans, causes the spontaneous development of autoimmune pathologies due to a breakdown of B cell tolerance. BC-CosmcKO mice display multiple phenotypic abnormalities, including severe weight loss, ocular manifestations, lymphadenopathy, and increased female-associated mortality. Disruption of B cell tolerance in BC-CosmcKO mice is manifested as elevated self-reactive IgM and IgG autoantibodies. Cosmc-deficient B cells exhibit enhanced basal activation and responsiveness to stimuli. There is also an elevated frequency of spontaneous germinal center B cells in BC-CosmcKO mice. Mechanistically, loss of Cosmc confers enhanced B cell receptor (BCR) signaling through diminished BCR internalization. The results demonstrate that Cosmc, through control of core 1 O-glycans, is a previously unidentified immune checkpoint gene in maintaining B cell tolerance.

Major differences in glycosylation and fucosyltransferase expression in low-grade versus high-grade bladder cancer cell lines.

Bladder cancer is the ninth most frequently diagnosed cancer worldwide, and there is a need to develop new biomarkers for staging and prognosis of this disease. Here we report that cell lines derived from low-grade and high-grade bladder cancers exhibit major differences in expression of glycans in surface glycoproteins. We analyzed protein glycosylation in three low-grade bladder cancer cell lines RT4 (grade-1-2), 5637 (grade-2), and SW780 (grade-1), and three high-grade bladder cancer cell lines J82COT (grade-3), T24 (grade-3) and TCCSUP (grade-4), with primary bladder epithelial cells, A/T/N, serving as a normal bladder cell control. Using a variety of approaches including flow cytometry, immunofluorescence, glycomics and gene expression analysis, we observed that the low-grade bladder cancer cell lines RT4, 5637 and SW780 express high levels of the fucosylated Lewis-X antigen (Lex, CD15) (Galß1-4(Fucα1-3)GlcNAcß1-R), while normal bladder epithelial A/T/N cells lack Lex expression. T24 and TCCSUP cells also lack Lex, whereas J82COT cells express low levels of Lex. Glycomics analyses revealed other major differences in fucosylation and sialylation of N-glycans between these cell types. O-glycans are highly differentiated, as RT4 cells synthesize core 2-based O-glycans that are lacking in the T24 cells. These differences in glycan expression correlated with differences in RNA expression levels of their cognate glycosyltransferases, including α1-3/4-fucosyltransferase genes. These major differences in glycan structures and gene expression profiles between low- and high-grade bladder cancer cells suggest that glycans and glycosyltransferases are candidate biomarkers for grading bladder cancers.

Differential recognition of oligomannose isomers by glycan-binding proteins involved in innate and adaptive immunity.

The recognition of oligomannose-type glycans in innate and adaptive immunity is elusive due to multiple closely related isomeric glycan structures. To explore the functions of oligomannoses, we developed a multifaceted approach combining mass spectrometry assignments of oligomannose substructures and the development of a comprehensive oligomannose microarray. This defined microarray encompasses both linear and branched glycans, varying in linkages, branching patterns, and phosphorylation status. With this resource, we identified unique recognition of oligomannose motifs by innate immune receptors, including DC-SIGN, L-SIGN, Dectin-2, and Langerin, broadly neutralizing antibodies against HIV gp120, N-acetylglucosamine-1-phosphotransferase, and the bacterial adhesin FimH. The results demonstrate that each protein exhibits a unique specificity to oligomannose motifs and suggest the potential to rationally design inhibitors to selectively block these protein-glycan interactions.

SARS-CoV-2 Spike Protein Interacts with Multiple Innate Immune Receptors.

The spike (S) glycoprotein in the envelope of SARS-CoV-2 is densely glycosylated but the functions of its glycosylation are unknown. Here we demonstrate that S is recognized in a glycan-dependent manner by multiple innate immune receptors including the mannose receptor MR/CD206, DC-SIGN/CD209, L-SIGN/CD209L, and MGL/CLEC10A/CD301. Single-cell RNA sequencing analyses indicate that such receptors are highly expressed in innate immune cells in tissues susceptible to SARS-CoV-2 infection. Binding of the above receptors to S is characterized by affinities in the picomolar range and consistent with S glycosylation analysis demonstrating a variety of N- and O-glycans as receptor ligands. These results indicate multiple routes for SARS-CoV-2 to interact with human cells and suggest alternative strategies for therapeutic intervention.

Parallel Glyco-SPOT Synthesis of Glycopeptide Libraries.

Glycan recognition is typically studied using free glycans, but glycopeptide presentations represent more physiological conditions for glycoproteins. To facilitate studies of glycopeptide recognition, we developed Glyco-SPOT synthesis, which enables the parallel production of diverse glycopeptide libraries at microgram scales. The method uses a closed system for prolonged reactions required for coupling Fmoc-protected glycoamino acids, including O-, N-, and S-linked glycosides, and release conditions to prevent side reactions. To optimize reaction conditions and sample reaction progress, we devised a biopsy testing method. We demonstrate the efficient utilization of such microscale glycopeptide libraries to determine the specificity of glycan-recognizing antibodies (e.g., CTD110.6) using microarrays, enzyme specificity on-array and in-solution (e.g., ST6GalNAc1, GCNT1, and T-synthase), and binding kinetics using fluorescence polarization. We demonstrated that the glycosylation on these peptides can be expanded using glycosyltransferases both in-solution and on-array. This technology will promote the discovery of biological functions of peptide modifications by glycans.

The Human Lung Glycome Reveals Novel Glycan Ligands for Influenza A Virus.

Glycans within human lungs are recognized by many pathogens such as influenza A virus (IAV), yet little is known about their structures. Here we present the first analysis of the N- and O- and glycosphingolipid-glycans from total human lungs, along with histological analyses of IAV binding. The N-glycome of human lung contains extremely large complex-type N-glycans with linear poly-N-acetyllactosamine (PL) [-3Galß1-4GlcNAcß1-]n extensions, which are predominantly terminated in α2,3-linked sialic acid. By contrast, smaller N-glycans lack PL and are enriched in α2,6-linked sialic acids. In addition, we observed large glycosphingolipid (GSL)-glycans, which also consists of linear PL, terminating in mainly α2,3-linked sialic acid. Histological staining revealed that IAV binds to sialylated and non-sialylated glycans and binding is not concordant with respect to binding by sialic acid-specific lectins. These results extend our understanding of the types of glycans that may serve as binding sites for human lung pathogens.

Cosmc-dependent mucin-type O-linked glycosylation is essential for podocyte function.

Mucin-type O-linked glycosylation, a posttranslational modification affecting the stability and biophysical characteristics of proteins, requires C1GalT1 (T synthase) and its obligate, X-linked chaperone Cosmc. Hypomorphic C1GalT1 mutations cause renal failure via not yet established mechanisms. We hypothesize that impaired Cosmc-dependent O-glycosylation in podocytes is sufficient to cause disease. Podocyte-specific Cosmc knockout mice were generated and phenotyped to test this hypothesis. Female heterozygous mice displaying mosaic inactivation of Cosmc in podocytes due to random X-linked inactivation were also examined. Mice with podocyte-specific Cosmc deletion develop profound albuminuria, foot process effacement, glomerular sclerosis, progressive renal failure, and impaired survival. Glomerular transcriptome analysis reveals early changes in cell adhesion, extracellular matrix organization, and chemokine-mediated signaling pathways, coupled with podocyte loss. Expression of the O-glycoprotein podoplanin was lost, while Tn antigen, representing immature O-glycans, was most abundantly found on podocalyxin. In contrast to hemizygous male and homozygous female animals, heterozygous female mosaic animals developed only mild albuminuria, focal foot process effacement, and nonprogressive kidney disease. Ultrastructurally, Cosmc-deficient podocytes formed Tn antigen-positive foot processes interdigitating with those of normal podocytes but not with other Cosmc-deficient cells. This suggests a cell nonautonomous mechanism for mucin-type O-glycoproteins in maintaining podocyte function. In summary, our findings demonstrated an essential and likely cell nonautonomous role for mucin-type O-glycosylation for podocyte function.

Identification of Tn antigen O-GalNAc-expressing glycoproteins in human carcinomas using novel anti-Tn recombinant antibodies.

The Tn antigen is a neoantigen abnormally expressed in many human carcinomas and expression correlates with metastasis and poor survival. To explore its biomarker potential, new antibodies are needed that specifically recognize this antigen in tumors. Here we generated two recombinant antibodies to the Tn antigen, Remab6 as a chimeric human IgG1 antibody and ReBaGs6 as a murine IgM antibody and characterized their specificities using multiple biochemical and biological approaches. Both Remab6 and ReBaGs6 recognize clustered Tn structures, but most importantly do not recognize glycoforms of human IgA1 that contain potential cross-reactive Tn antigen structures. In flow cytometry and immunofluorescence analyses, Remab6 recognizes human cancer cell lines expressing the Tn antigen, but not their Tn-negative counterparts. In immunohistochemistry (IHC), Remab6 stains many human cancers in tissue array format but rarely stains normal tissues and then mostly intracellularly. We used these antibodies to identify several unique Tn-containing glycoproteins in Tn-positive Colo205 cells, indicating their utility for glycoproteomics in future biomarker studies. Thus, recombinant Remab6 and ReBaGs6 are useful for biochemical characterization of cancer cells and IHC of tumors and represent promising tools for Tn biomarker discovery independently of recognition of IgA1.

[Corrigendum] Increased expression levels of ppGalNAc-T13 in lung cancers: Significance in the prognostic diagnosis.

Following the publication of this article, the authors noted that there was an error in Fig. 4. The lines representing the two groups were indicated incorrectly in all graphs in Fig. 4. A corrected version of Fig. 4 is presented below. Furthermore, we regret any inconvenience this error may have caused. [the original article was published in the International Journal of Oncology 49: 1369-1376, 2016; DOI: 10.3892/ijo.2016.3638].

Increased expression levels of ppGalNAc-T13 in lung cancers: Significance in the prognostic diagnosis.

ppGalNAc-T13 is upregulated along with reduced expression of GM1 in high metastatic sublines of the murine Lewis lung cancer cell line, but little is known about the implication of ppGalNAc-T13 expression in human cancers. Since lung cancer cell lines showed high expression levels of ppGalNAc-T13, we analyzed ppGalNAc-T13 expression in surgical lung cancer specimens to examine whether ppGalNAc-T13 can be used as a prognostic marker or a therapeutic target. We analyzed mRNA expression levels of GALNT13 and its variant exon usages in surgical specimens by real-time RT-PCR, and the results were evaluated by correlating with clinical data. Ninety-one surgical specimens were analyzed. Consequently, recurrence-free survival was significantly shorter (P=0.045) in high expression group of GALNT13 mRNA. In the analysis of tumor specific exon usage in GALNT13 RNA sequence, one variant exon was significantly associated with worse prognosis. By contrast, in another variant exon, positive variant expression group showed better prognosis than negative group. We also tried to detect GALNT13 mRNA in 63 serum samples from patients with lung cancers to examine whether GALNT13 mRNA can be measured in body fluids, detecting significant levels in 4 samples. Finally, expression of GM1, ppGalNAc-T13 and trimeric Tn antigen was examined by immunohistochemistry in order to evaluate them as a prognostic factor. It was demonstrated that ppGalNAc-T13 and trimeric Tn antigen had a relationship with worse prognosis in 35 investigated lung cancer patients. In conclusion, our results suggest that ppGalNAc-T13 might be a useful prognostic factor of lung cancers.