Serum Glycoproteins Research Articles

Unveiling Silent Atherosclerosis in Type 1 Diabetes: The Role of Glycoprotein and Lipoprotein Lipidomics, and Cardiac Autonomic Neuropathy.

Introduction: This study aimed to evaluate whether glycoprotein and lipoprotein lipidomics profiles could enhance a clinical predictive model for carotid subclinical atherosclerosis in patients with type 1 diabetes (T1D). Additionally, we assessed the influence of cardiac autonomic neuropathy (CAN) on these predictive models. Methods: We conducted a cross-sectional study including 256 patients with T1D. Serum glycoprotein and lipoprotein lipidomics profiles were determined using 1H-NMR spectroscopy. Subclinical atherosclerosis was defined as carotid intima-media thickness (cIMT) ≥ 1.5 mm. CAN was identified using the Clarke score. Predictive models were built and their performance evaluated using receiver operating characteristic curves and cross-validation. Results: Subclinical atherosclerosis was detected in 32% of participants. Patients with both CAN and atherosclerosis were older, had a longer duration of diabetes, and were more likely to present with bilateral carotid disease. Clinical predictors such as age, duration of diabetes, and smoking status remained the strongest determinants of subclinical atherosclerosis [AUC = 0.88 (95%CI: 0.84-0.93)]. While glycoprotein and lipoprotein lipidomics profiles were associated with atherosclerosis, their inclusion in the clinical model did not significantly improve its diagnostic performance. Stratification by the presence of CAN revealed no impact on the model's ability to predict subclinical atherosclerosis, underscoring its robustness across different risk subgroups. Conclusions: In a cohort of patients with T1D, subclinical atherosclerosis was strongly associated with traditional clinical risk factors. Advanced glycoprotein and lipoprotein lipidomics profiling, although associated with atherosclerosis, did not enhance the diagnostic accuracy of predictive models beyond clinical variables. The predictive model remained effective even in the presence of CAN, highlighting its reliability as a screening tool for identifying patients at risk of subclinical atherosclerosis.

Altered Pattern of Serum N-Glycome in Subarachnoid Hemorrhage and Cerebral Vasospasm.

Background: Subarachnoid hemorrhage is a serious condition caused by ruptured intracranial aneurysms, resulting in severe disability mainly in young adults. Cerebral vasospasm is one of the most common complication of subarachnoid hemorrhage; thus, active prevention is key to improve the prognosis. The glycosylation of proteins is a critical quality attribute which is reportedly altered in patients diagnosed with acute ischemic stroke. In this study, we examined the N-glycosylation profile of serum glycoproteins in patients with subarachnoid hemorrhage without vasospasm compared to patients with vasospasm. Methods: The serum N-glycans were released by PNGase F (Peptide: N-glycosidase F) digestion and subsequently labeled by procainamide via reductive amination. The samples were analyzed by hydrophilic-interaction liquid chromatography after solid-phase extraction-based sample purification. Results: Besides the glycosylation pattern, we also investigated the biomarkers following subarachnoid hemorrhage. Multiple statistical analyses were performed in order to find significant differences and identify potential prediction factors of cerebral vasospasm. Significant differences were identified such as higher sialylation on bi-, tri-, and tetra-antennary structures in patients with subarachnoid hemorrhage and cerebral vasospasm. Conclusions: Our results suggest that glycosylation analysis can improve the identification of patients with cerebral vasospasm in combination with laboratory parameters.

Intestinal epithelial cell-specific restoration of Nrf2 gene in whole-body-knockout mice ameliorates acute colitis.

Unbalanced redox homeostasis leads to the production of reactive oxygen species and exacerbates inflammatory bowel disease. To investigate the role of the transcription factor Nrf2, a major antioxidative stress sensor, in intestinal epithelial cells (IECs), we generated IEC-specific Nrf2 gene knock-in mice (Nrf2-vRes), which express Nrf2 only in IECs, using the cre/loxp system. Colitis was induced in wild-type (WT) mice, whole-body Nrf2-knockout (Nrf2-KO) mice, and Nrf2-vRes mice by administering dextran sulfate sodium (DSS) for 1 week (acute model) or intermittently for 5 weeks (chronic model). The mRNA and protein levels of NAD(P)H:quinone oxidoreductase 1 (NQO1), which is involved in the oxidative stress response in a manner regulated by Nrf2, were reduced in Nrf2-KO compared with those in WT, while these decreases were reversed in Nrf2-vRes at all timepoints. Nrf2-KO mice administered DSS developed more severe colitis with higher disease activity index, higher leucine-rich α2 glycoprotein in serum, shorter colon length, and more severe epithelial damage and infiltration of inflammatory cells histopathologically than did WT mice in the acute model; moreover, these exacerbations of colitis were ameliorated in Nrf2-vRes mice. However, these differences were not observed among the three sets of mice in the chronic model. IEC-specific expression of Nrf2 ameliorated DSS-induced acute colitis. These results suggest that Nrf2 expression in IECs plays a protective role against early-stage colitis and undertakes important regulatory functions during intestinal inflammation.

Occurrence of free glycans in salmonid serum

Free N-glycans (FNGs) are oligosaccharides that are structurally related to N-linked glycans, and are widely found in nature. The mechanisms responsible for the formation and degradation of intracellular FNGs are well characterized in mammalian cells. More recent analysis in mammalian sera shows that there are various types of extracellular free glycans, including FNGs. However, it is unknown whether these free glycans are widely distributed in vertebrates. In this study, we investigated the occurrence of free glycans in salmonid serum. We found that it contained sialyl or neutral FNGs and sialyl lactose/N-acetyllactosamine (LacNAc)-type glycans, which was consistent with that found in mammalian sera. Many of the structures of FNGs matched those of N-glycans from serum glycoproteins. This study revealed that various types of free glycans are present in fish serum, demonstrating their wide occurrence among vertebrates.

Identification of serum N-glycans signatures in three major gastrointestinal cancers by high-throughput N-glycome profiling

BackgroundAlternative N-glycosylation of serum proteins has been observed in colorectal cancer (CRC), esophageal squamous cell carcinoma (ESCC) and gastric cancer (GC), while comparative study among those three cancers has not been reported before. We aimed to identify serum N-glycans signatures and introduce a discriminative model across the gastrointestinal cancers.MethodsThe study population was initially screened according to the exclusion criteria process. Serum N-glycans profiling was characterized by a high-throughput assay based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Diagnostic model was built by random forest, and unsupervised machine learning was performed to illustrate the differentiation between the three major gastrointestinal (GI) cancers.ResultsWe have found that three major gastrointestinal cancers strongly associated with significantly decreased mannosylation and mono-galactosylation, as well as increased sialylation of serum glycoproteins. A highly accurate discriminative power (> 0.90) for those gastrointestinal cancers was obtained with serum N-glycome based predictive model. Additionally, serum N-glycome profile exhibited distinct distributions across GI cancers, and several altered N-glycans were hyper-regulated in each specific disease.ConclusionsSerum N-glycome profile was differentially expressed in three major gastrointestinal cancers, providing a new clinical tool for cancer diagnosis and throwing a light upon the disease-specific molecular signatures.

Prevalence of autoantibodies neuromyelitis optica (NMO igg) and myelin oligodendrocyte glycoprotein (MOG) in NMOSD and the relationship between them in the general population

Neuromyelitis optica spectrum disorder (NMOSD) and anti-myelin oligodendrocyte glycoprotein (anti-MOG) syndromes are inflammatory conditions of the central nervous system that often involve the optic nerves and spinal cord. They can be mistaken for MS due to their similar symptoms. Therefore, we investigated the relationship between NMO antibody (NMO-Ab) and anti-MOG antibody (MOG-Ab) and the positivity of these antibodies in the general Indian population.: This retrospective study analyzed 40186 patients for Neuromyelitis Optica Antibodies serum and Myelin Oligodendrocyte Glycoprotein antibodies serum. Additionally, 5762 patients were analyzed specifically for these antibodies in their cerebrospinal fluid. The study included patients of all ages, unaccounting for their clinical history, and was conducted between January 2019 and July 2023 at the Global Reference Lab.: Overall, it was observed that MOG serum antibodies were more prevalent (18.59%) than NMO serum antibodies (8.12%). Females had a higher prevalence of NMO serum antibodies (13.23%) than males (2.16%), whereas the prevalence of MOG serum antibodies was similar in females (14.27%) and males (14.59%). The highest percentage of MOG serum positivity (31.40%) was observed in 1-12 years age group, and for NMO, it was 9.44% in the 19-30 years age group. The overlap in the positivity between NMO serum and CSF was 6.09% while for MOG serum and CSF, it was 3.86%. A concordance of 92.04% was observed for samples tested negative for NMO in serum as well as CSF. Only 2 cases of 259 cases tested for MOG antibodies showed negativity in serum but positivity in CSF.The study highlights the prevalence of MOG and NMO antibodies in serum and CSF among patients with suspected or known autoimmune neurological disorders, with notable difference in positivity rates between genders for NMO antibodies. These findings underscore the importance of comprehensive antibody testing in the diagnosis and management of demyelinating diseases such as MOG-Ab NMOSD and AQP4-Ab NMOSD. Further research is warranted to explore the clinical implications of these antibodies and their role in guiding therapeutic interventions for affected individuals.

Distinct O-Acetylation Patterns of Serum Glycoproteins among Humans, Mice, and Rats.

O-Acetylation is a significant chemical modification of sialic acids on glycoproteins with diverse biological functions. As two important animal models, mice and rats have been widely used for various biomedical studies. In this study, we show that the sialic acid types and their O-acetylation patterns have large differences among serum glycoproteins of humans, rats, and mice. Based on intact N-glycopeptide analyses, all sialoglycopeptides in human sera were modified by Neu5Ac without any O-acetylation; 90% of sialoglycopeptides in rat sera were also modified by Neu5Ac, with more than 60% that were further O-acetylated. In contrast, 99% of sialoglycopeptides in mouse sera contained Neu5Gc including 12% in O-acetylated forms. Among all O-acetylated N-glycans, rat sera had hybrid glycans fivefold those of mouse sera, while mouse sera contained 5.5-fold core-fucosylated glycans and 4.6-31.5-fold mono-/penta-/hexa-antenna glycans compared to mice. The overall O-acetylation proportions of serum glycoproteins in rats were much higher than those in mice, and diverse O-acetylation proportions also commonly existed at different glycosites of the same glycoproteins. This study enhances our understanding of O-acetylated sialoglycan diversities and underscores the necessity of considering glycosylation profiles when selecting suitable animal models for various biomedical studies.

8381 The Pro-atrophic Effect of Vitamin D Binding Protein in Skeletal Muscle and Its Involvement in Cancer-Induced Muscle Wasting

Abstract Disclosure: N. Filigheddu: None. T. Raiteri: None. S. Reano: None. A. Scircoli: None. A. Antonioli: None. F. Prodam: None. Vitamin D binding protein (VDBP), encoded by the Gc gene, is a multifunctional serum glycoprotein synthesized by hepatocytes, whose primary function is the transport of vitamin D metabolites in the bloodstream. In addition, VDBP enhances the chemotactic activity of neutrophil chemoattractants and takes part in the actin-scavenger system by acting as a monomeric G-actin-binding protein. Several studies have reported a correlation between increased levels of VDBP and various pathologies often associated with muscle wasting, including different types of tumors. Given these findings, we hypothesized that VDBP may play a role in skeletal muscle homeostasis.In vitro, treatment of C2C12 myotubes with 100 mM VDBP for 24 h induced the perturbation of intracellular actin dynamics due to VDBP's ability to bind G-actin that, in turn, led to mitochondrial dysfunction (i.e., mitochondrial membrane potential dissipation, respiratory impairment, increased ROS production, induction of the fission machinery), exacerbation of autophagy, and, eventually, atrophy, seen as the reduction of myotube diameter. Remarkably, pharmacological intervention on myotubes with jasplakinolide (250 mM, 30 min pre-treatment) to counteract VDBP effects on intracellular actin dynamics was sufficient to prevent VDBP-induced atrophy. To assess if VDBP had a causative role in muscle atrophy in vivo, we injected VDBP (1.5 mg/Kg) every 48 h for one week in the tibialis anterior muscles of Gc knock-out mice (VDBP KO). At the end of the experimental period, we observed the induction of the mitophagic gene Bnip3, impairment of muscle performances (26% reduction of grip strength at the endpoint), and a 16% reduction of muscle mass compared to the saline-injected contralateral muscles, confirming the atrophic effect of VDBP in vivo.Coherently with the upregulation of VDBP observed in cancer patients, VDBP levels also increase in murine models of cancer cachexia. To test the hypothesis that VDBP could play a role in cancer-associated muscle wasting, we induced cancer cachexia in VDBP KO mice by inoculating 106 Lewis Lung Carcinoma (LLC) cells resuspended in 100 μl of saline on the back of mice. Tumor-bearing VDBP KO mice preserved their body weight and performances, and the muscle loss was lessened by more than 50% compared to cachectic WT mice. Notably, between the two groups, there were no differences in tumor growth or food intake, ruling out the possibility that the reduction in muscle wasting could depend on smaller tumors or differential development of anorexia in the two genotypes. In conclusion, we demonstrated that VDBP acts as a hormone per se, having a direct pro-atrophic activity on skeletal muscle. Our data suggest that VDBP could represent a potential therapeutic target to treat cancer cachexia and other pathologies in which the rise of VDBP could impinge muscle mass and functionality. Presentation: 6/1/2024

9268 The Pro-atrophic Effect of Vitamin D Binding Protein in Skeletal Muscle and Its Involvement in Cancer-Induced Muscle Wasting

Abstract Disclosure: N. Filigheddu: None. T. Raiteri: None. S. Reano: None. A. Scircoli: None. A. Antonioli: None. F. Prodam: None. Vitamin D binding protein (VDBP), encoded by the Gc gene, is a multifunctional serum glycoprotein synthesized by hepatocytes, whose primary function is the transport of vitamin D metabolites in the bloodstream. In addition, VDBP enhances the chemotactic activity of neutrophil chemoattractants and takes part in the actin-scavenger system by acting as a monomeric G-actin-binding protein. Several studies have reported a correlation between increased levels of VDBP and various pathologies often associated with muscle wasting, including different types of tumors. Given these findings, we hypothesized that VDBP may play a role in skeletal muscle homeostasis.In vitro, treatment of C2C12 myotubes with 100 mM VDBP for 24 h induced the perturbation of intracellular actin dynamics due to VDBP's ability to bind G-actin that, in turn, led to mitochondrial dysfunction (i.e., mitochondrial membrane potential dissipation, respiratory impairment, increased ROS production, induction of the fission machinery), exacerbation of autophagy, and, eventually, atrophy, seen as the reduction of myotube diameter. Remarkably, pharmacological intervention on myotubes with jasplakinolide (250 mM, 30 min pre-treatment) to counteract VDBP effects on intracellular actin dynamics was sufficient to prevent VDBP-induced atrophy. To assess if VDBP had a causative role in muscle atrophy in vivo, we injected VDBP (1.5 mg/Kg) every 48 h for one week in the tibialis anterior muscles of Gc knock-out mice (VDBP KO). At the end of the experimental period, we observed the induction of the mitophagic gene Bnip3, impairment of muscle performances (26% reduction of grip strength at the endpoint), and a 16% reduction of muscle mass compared to the saline-injected contralateral muscles, confirming the atrophic effect of VDBP in vivo.Coherently with the upregulation of VDBP observed in cancer patients, VDBP levels also increase in murine models of cancer cachexia. To test the hypothesis that VDBP could play a role in cancer-associated muscle wasting, we induced cancer cachexia in VDBP KO mice by inoculating 106 Lewis Lung Carcinoma (LLC) cells resuspended in 100 μl of saline on the back of mice. Tumor-bearing VDBP KO mice preserved their body weight and performances, and the muscle loss was lessened by more than 50% compared to cachectic WT mice. Notably, between the two groups, there were no differences in tumor growth or food intake, ruling out the possibility that the reduction in muscle wasting could depend on smaller tumors or differential development of anorexia in the two genotypes. In conclusion, we demonstrated that VDBP acts as a hormone per se, having a direct pro-atrophic activity on skeletal muscle. Our data suggest that VDBP could represent a potential therapeutic target to treat cancer cachexia and other pathologies in which the rise of VDBP could impinge muscle mass and functionality. Presentation: 6/1/2024

Site-specific N-glycan alterations on haptoglobin as potential biomarkers for distinguishing intrahepatic cholangiocarcinoma from hepatocellular carcinoma

Intrahepatic cholangiocellular carcinoma (ICC) is a challenging malignancy marked by subtle early symptoms and a high mortality rate, making effective diagnostic markers crucial for early detection and improved patient outcomes. Currently, the conventional diagnosis of ICC is not easily distinguishable from Hepatocellular Carcinoma (HCC) and lacks highly specific and sensitive diagnostic markers. Protein glycosylation, pivotal in biological processes, shows promise for cancer biomarkers due to its association with disease progression. This study aims to develop a novel biomarker discovery framework for ICC utilizing site-specific quantitative N-glycoproteomics to overcome the limitations of existing diagnostic approaches. Employing a tandem mass tag (TMT)-based quantitative analysis, we profiled serum glycoproteins from ICC, HCC, and control cohorts at site-specific glycosylation level. The identified markers underwent further validation in an independent cohort using label-free quantitative methods. Ultimately, we identified five site-specific N-glycans on haptoglobin (HP) as potential biomarkers (AUC > 0.9) for distinguishing ICC from HCC. This finding represents a considerable advance over traditional biomarkers, highlighting the significance of protein glycosylation alterations in ICC pathogenesis. This research, therefore, sets a new precedent for biomarker discovery in ICC, with potential applications in other cancers characterized by glycosylation abnormalities.

High-abundance serum glycoproteins as valuable resources for glycopeptide standards

High-abundance serum proteins, mostly modified by N-glycans, are usually depleted from human sera to achieve in-depth analyses of serum proteome and sub-proteomes. In this study, we show that these high-abundance glycoproteins (HAGPs) can be used as valuable standard glycopeptide resources, as long as the structural features of their glycans have been well defined at the glycosite-specific level. By directly analyzing intact glycopeptides enriched from serum, we identified 1322 unique glycopeptides at 48 N-glycosites from the top 12 HAGPs (19 subclasses). These HAGPs could be further classified into four major groups based on the structural features of their attached N-glycans. Immunoglobins including IGHG1/2/3/4, IGHA1/2 and IGHM were mostly modified by core fucosylated and bisected N-glycans with rarely sialic acids. Alpha-1-acid glycoproteins (ORM1/2) and haptoglobins (HP) were mainly modified by tri-and tetra-antennary (40 %) N-glycans with antenna-fucoses and sialic acids. Complement components C3 and C4A/B were highly modified by oligo-mannose glycans. The other HAGPs including SERPINA1, A2M, TF, FGB/G and APOB mainly contain bi-antennary complex glycans with the common core structure and (sialyl-) LacNAc branch structures. These HAGPs are easily detected by LC-MS analysis and therefore could be used as standard glycopeptides for glycoproteomic methodology studies as well as possible clinical utilities.

387. VALIDATION OF PROMARKERESO, A DIAGNOSTIC BLOOD TEST TO IDENTIFY ESOPHAGEAL ADENOCARCINOMA

Abstract Background Esophageal adenocarcinoma (EAC) has become the predominant type of esophageal cancer in Western countries. Early diagnosis and intervention of EAC significantly reduces the disease mortality. A previous study identified a panel of novel serum glycoprotein biomarkers that were associated with EAC, enabling the development of a prototype diagnostic blood test, PromarkerEso. The current study aimed to validate PromarkerEso in an independent cohort of patients with EAC. Methods Blood samples from an independent cohort of 165 participants (n=99 negative controls, n=66 diagnosed with EAC) were analyzed. Five candidate biomarkers were measured after lectin pulldown using a targeted mass spectrometry assay. Model performance was assessed by the area under the receiver operating characteristics curve (AUC-ROC) with sensitivity and specificity determined. Results After adjusting for clinical variables, diagnostic models showed good discrimination to distinguish participants with EAC from negative control participants. Conclusions This study validates a panel of serum glycoprotein biomarkers for the diagnosis of EAC. These biomarkers have potential utility as a simple blood test, PromarkerEso, that could be used for screening EAC in a large population.

Comprehensive serum glycopeptide spectra analysis to identify early-stage epithelial ovarian cancer

Epithelial ovarian cancer (EOC) is widely recognized as the most lethal gynecological malignancy; however, its early-stage detection remains a considerable clinical challenge. To address this, we have introduced a new method, named Comprehensive Serum Glycopeptide Spectral Analysis (CSGSA), which detects early-stage cancer by combining glycan alterations in serum glycoproteins with tumor markers. We detected 1712 glycopeptides using liquid chromatography–mass spectrometry from the sera obtained from 564 patients with EOC and 1149 controls across 13 institutions. Furthermore, we used a convolutional neural network to analyze the expression patterns of the glycopeptides and tumor markers. Using this approach, we successfully differentiated early-stage EOC (Stage I) from non-EOC, with an area under the curve (AUC) of 0.924 in receiver operating characteristic (ROC) analysis. This method markedly outperforms conventional tumor markers, including cancer antigen 125 (CA125, 0.842) and human epididymis protein 4 (HE4, 0.717). Notably, our method exhibited remarkable efficacy in differentiating early-stage ovarian clear cell carcinoma from endometrioma, achieving a ROC-AUC of 0.808, outperforming CA125 (0.538) and HE4 (0.557). Our study presents a promising breakthrough in the early detection of EOC through the innovative CSGSA method. The integration of glycan alterations with cancer-related tumor markers has demonstrated exceptional diagnostic potential.

Potential contribution of alpha-fetoprotein level to biomarker of pregnancy outcome in Asian elephants.

Alpha-fetoprotein (AFP) is a structural serum glycoprotein that plays vital roles in reproduction and mammalian development. Analysis of serum prolactin (PRL) is considered one of the useful methods for diagnosing pregnancy in Asian elephants. However, the expression profiles of AFP in pregnant and nonpregnant Asian elephants remain unclear, nor is the relationship with PRL. In this study, serum seven gonadal hormones and AFP in three pregnant and seven nonpregnant Asian elephants were analysed by via radioimmunoassay (RIA) and enzyme-linked immunosorbent (ELISA) assay. We found that the mean (±SD) concentration of prolactin (PRL) in pregnant (136.782 ± 30.987ng/mL) elephants was significantly higher than that in nonpregnant elephants (52.803 ± 21.070ng/mL; p ≤ 0.0005). The mean (±SD) concentration of AFP in pregnant elephants (11.598 ± 0.824ng/mL) was significantly higher than that in nonpregnant elephants (7.200 ± 2.283ng/mL; p ≤ 0.05). Furthermore, the AFP concentration was positively correlated with the PRL concentration in the 10 Asian elephants studied. In conclusion, our findings suggest that serum AFP concentration is a potential biomarker of pregnancy outcomes in Asian elephants.

Immunoglobulin G glycosylation and its alterations in aging-related diseases.

Immunoglobulin G (IgG) is an important serum glycoprotein and a major component of antibodies. Glycans on IgG affect the binding of IgG to the Fc receptor or complement C1q, which in turn affects the biological activity and biological function of IgG. Altered glycosylation patterns on IgG emerge as important biomarkers in the aging process and age-related diseases. Key aging-related alterations observed in IgG glycosylation include reductions in galactosylation and sialylation, alongside increases in agalactosylation, and bisecting GlcNAc. Understanding the role of IgG glycosylation in aging-related diseases offers insights into disease mechanisms and provides opportunities for the development of diagnostic and therapeutic strategies. This review summarizes five aspects of IgG: an overview of IgG, IgG glycosylation, IgG glycosylation with inflammation mediation, IgG glycan changes with normal aging, as well as the relevance of IgG glycan changes to aging-related diseases. This review provides a reference for further investigation of the regulatory mechanisms of IgG glycosylation in aging-related diseases, as well as for evaluating the potential of IgG glycosylation changes as markers of aging and aging-related diseases.

Syndecan-1 Levels in Females with Active Rheumatoid Arthritis.

Background: The relationship between serum glycoprotein syndecan-1 and disease activity in rheumatoid arthritis (RA) is still unknown. This study aimed to evaluate whether serum syndecan-1 concentrations are associated with moderate/severe disease activity. Methods: Study Design: This was a cross-sectional study. Seventy-five adult women with RA were classified into (a) moderate/severe RA based on the disease activity score, using the erythrocyte sedimentation rate (DAS28-ESR ≥ 3.2, n = 50), and (b) RA in remission (DAS28-ESR < 2.6, n = 25). Twenty-five healthy women were taken as the reference group. Syndecan-1 levels were determined using enzyme-linked immunosorbent assay (ELISA). High values of serum syndecan-1 levels (≥24 ng/mL) were used to identify the utility values of this biomarker. Results: The patients with RA had higher levels of syndecan-1 than the controls (p < 0.001). RA patients with active disease had higher syndecan-1 levels than RA patients in remission (57.6 vs. 23.5 ng/mL, respectively; p = 0.002). High syndecan-1 concentrations demonstrated the following utility values for identifying disease activity: sensitivity, 84% (95%CI: 71-93); specificity, 52% (95%CI: 31-72); positive predictive value, 78% (95%CI: 70-84); and negative predictive value, 62% (95%CI: 44-77). Conclusions: High syndecan-1 levels have good sensitivity and positive predictive value for identifying disease activity; however, their specificity is limited. Future prospective studies are needed to assess whether syndecan-1 levels can predict treatment failure in RA.

Glyco-signatures in patients with advanced lung cancer during anti-PD-1/PD-L1 immunotherapy.

Immune checkpoint inhibitors (ICIs) targeting programmed cell death 1/programmed cell death ligand-1 (PD-1/PD-L1) have significantly prolonged the survival of advanced/metastatic patients with lung cancer. However, only a small proportion of patients can benefit from ICIs, and clinical management of the treatment process remains challenging. Glycosylation has added a new dimension to advance our understanding of tumor immunity and immunotherapy. To systematically characterize anti-PD-1/PD-L1 immunotherapy-related changes in serum glycoproteins, a series of serum samples from 12 patients with metastatic lung squamous cell carcinoma (SCC) and lung adenocarcinoma (ADC), collected before and during ICIs treatment, are firstly analyzed with mass-spectrometry-based label-free quantification method. Second, a stratification analysis is performed among anti-PD-1/PD-L1 responders and non-responders, with serum levels of glycopeptides correlated with treatment response. In addition, in an independent validation cohort, a large-scale site-specific profiling strategy based on chemical labeling is employed to confirm the unusual characteristics of IgG N-glycosylation associated with anti-PD-1/PD-L1 treatment. Unbiased label-free quantitative glycoproteomics reveals serum levels' alterations related to anti-PD-1/PD-L1 treatment in 27 out of 337 quantified glycopeptides. The intact glycopeptide EEQFN 177STYR (H3N4) corresponding to IgG4 is significantly increased during anti-PD-1/PD-L1 treatment (FC=2.65, P=0.0083) and has the highest increase in anti-PD-1/PD-L1 responders (FC=5.84, P=0.0190). Quantitative glycoproteomics based on protein purification and chemical labeling confirms this observation. Furthermore, obvious associations between the two intact glycopeptides (EEQFN 177STYR (H3N4) of IgG4, EEQYN 227STFR (H3N4F1) of IgG3) and response to treatment are observed, which may play a guiding role in cancer immunotherapy. Our findings could benefit future clinical disease management.

Structural characteristics of alpha-fetoprotein, including N-glycosylation, metal ion and fatty acid binding sites

Alpha-fetoprotein (AFP), a serum glycoprotein, is expressed during embryonic development and the pathogenesis of liver cancer. It serves as a clinical tumor marker, function as a carcinogen, immune suppressor, and transport vehicle; but the detailed AFP structural information has not yet been reported. In this study, we used single-particle cryo-electron microscopy(cryo-EM) to analyze the structure of the recombinant AFP obtained a 3.31 Å cryo-EM structure and built an atomic model of AFP. We observed and identified certain structural features of AFP, including N-glycosylation at Asn251, four natural fatty acids bound to distinct domains, and the coordination of metal ions by residues His22, His264, His268, and Asp280. Furthermore, we compared the structural similarities and differences between AFP and human serum albumin. The elucidation of AFP’s structural characteristics not only contributes to a deeper understanding of its functional mechanisms, but also provides a structural basis for developing AFP-based drug vehicles.

Differential expression of N-glycopeptides derived from serum glycoproteins in mild cognitive impairment (MCI) patients.

Mild cognitive impairment (MCI) is an early stage of memory loss that affects cognitive abilities with the aging of individuals, such as language or visual/spatial comprehension. MCI is considered a prodromal phase of more complicated neurodegenerative diseases such as Alzheimer's. Therefore, accurate diagnosis and better understanding of the disease prognosis will facilitate prevention of neurodegeneration. However, the existing diagnostic methods fail to provide precise and well-timed diagnoses, and the pathophysiology of MCI is not fully understood. Alterations of the serum N-glycoproteome expression could represent an essential contributor to the overall pathophysiology of neurodegenerative diseases and be used as a potential marker to assess MCI diagnosis using less invasive procedures. In this approach, we identified N-glycopeptides with different expressions between healthy and MCI patients from serum glycoproteins. Seven of the N-glycopeptides showed outstanding AUC values, among them the antithrombin-III Asn224 + 4-5-0-2 with an AUC value of 1.00 and a p value of 0.0004. According to proteomics and ingenuity pathway analysis (IPA), our data is in line with recent publications, and the glycoproteins carrying the identified N-sites play an important role in neurodegeneration.

Site-Specific Analysis of Core and Antenna Fucosylation on Serum Glycoproteins.

Fucosylation is an important structural feature of glycans and plays an essential role in the regulation of glycoprotein functions. Fucosylation can be classified into core- (CF) and antenna-fucosylation (AF, also known as (sialyl-) Lewis) based on the location on N-glycans, and they perform distinct biological functions. In this study, core- and antenna-fucosylated N-glycans on human serum glycoproteins that hold great clinical application values were systematically characterized at the site-specific level using StrucGP combined with the recently developed fucosylation assignment method. The results showed that fucosylation was widely distributed on serum glycoproteins, with 50% of fucosylated glycopeptides modified by AF N-glycans, 37% by CF N-glycans, and 13% by dual-fucosylated N-glycans. Interestingly, CF and AF N-glycans preferred to modify different groups of serum glycoproteins with different tissue origins and were involved in distinctive biological processes. Specifically, AF N-glycoproteins are mainly from the liver and participated in complement activation, blood coagulation, and endopeptidase activities, while CF N-glycoproteins originate from diverse tissues and are mainly involved in cell adhesion and signaling transduction. These data further enhanced our understanding of fucosylation on circulation glycoproteins.