Mass spectrometry analysis of intact protein N-glycosylation signatures of cells and sera in pancreatic adenocarcinomas / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Pancreatic cancer is among the most malignant cancers, and thus early intervention is the key to better survival outcomes. However, no methods have been derived that can reliably identify early precursors of development into malignancy. Therefore, it is urgent to discover early molecular changes during pancreatic tumorigenesis. As aberrant glycosylation is closely associated with cancer progression, numerous efforts have been made to mine glycosylation changes as biomarkers for diagnosis; however, detailed glycoproteomic information, especially site-specific N-glycosylation changes in pancreatic cancer with and without drug treatment, needs to be further explored. Herein, we used comprehensive solid-phase chemoenzymatic glycoproteomics to analyze glycans, glycosites, and intact glycopeptides in pancreatic cancer cells and patient sera. The profiling of N-glycans in cancer cells revealed an increase in the secreted glycoproteins from the primary tumor of MIA PaCa-2 cells, whereas human sera, which contain many secreted glycoproteins, had significant changes of glycans at their specific glycosites. These results indicated the potential role for tumor-specific glycosylation as disease biomarkers. We also found that AMG-510, a small molecule inhibitor against Kirsten rat sarcoma viral oncogene homolog (KRAS) G12C mutation, profoundly reduced the glycosylation level in MIA PaCa-2 cells, suggesting that KRAS plays a role in the cellular glycosylation process, and thus glycosylation inhibition contributes to the anti-tumor effect of AMG-510.

Congenital glycosylation disorder type Ⅱm caused by SLC35A2 mutations: a report of 3 cases and literature review / 中华神经科杂志

Objective:To summarize the clinical manifestations, gene variations, diagnosis and treatment of 3 cases with SLC35A2 variations characterized by congenital glycosylation disorder Ⅱm (CDG Ⅱm). Methods:A total of 3 patients admitted to the Department of Pediatrics of Xiangya Hospital of Central South University in China from 2018 to 2020 were examined in detail. The studies till January 2022 were searched with key words of "congenital disorders of glycosylation Ⅱm", " SLC35A2" and "CDG Ⅱm" in both English and Chinese in the databases of China National Knowledge Infrast Ructure (CNKI), Wanfang, Online Mendelian Inheritance in Man and PubMed, and the clinical manifestations, genetic variation, treatments and prognosis of patients with SLC35A2 mutation were summarized. Results:The patients all presented with intractable infantile spasm and global developmental delay, onset in infancy. A variety of antiepileptic treatments had temporary and partial efficacy. Otherwise, proband 2 and 3 presented with abnormal glutamic-pyruvic transaminase and increased platelets. Funduscopy showed dysplasia of the retinal pigment epithelium in both eyes, and they both received D-galactose treatment. A total of 22 relevant case reports, including 99 patients, were collected. The 99 patients all were heterozygous mutations, and a total of 75 different variation sites were reported. The clinical manifestations were characterized by global developmental delay or mental retardation ( n=89), epileptic seizure ( n=75), hypotonia ( n=57), facial deformity ( n=57), skeletal abnormality ( n=50), visual impairment ( n=42), elevated glutamic-pyruvic transaminase ( n=31), gastrointestinal symptoms ( n=28), skin deformity ( n=26), microcephaly ( n=23) and congenital heart disease ( n=12). Craniocerebral magnetic resonance imaging may be normal in the early stage. With age, magnetic resonance imaging may show abnormal white matter signals, brain atrophy, dysplasia of corpus callosum, delayed myelination, enlargement of lateral ventricle, brain stem atrophy and so on. Studies have shown that galactose treatment may be effective. Conclusions:SLC35A2 variants lead to CDG Ⅱm, whose clinical manifestations mainly include epileptic encephalopathy and global developmental delay. Multiple antiepileptic therapies can temporarily or partially control seizures, while oral galactose may improve the clinical symptoms, showing its prospect as a dietary therapy.

Optimization of glycosylation type of recombinant human growth hormone-Fc fusion protein expressed in CHO cells / 中国生物制品学杂志

@#Objective To optimize the expression of recombinant human growth hormone-Fc(rhGH-Fc)fusion protein in CHO cells in order to obtain better glycosylation ratio and lower content of highmannose.Methods CHO cells expressing rhGH-Fc were cultured in a 7 L bioreactor.The glycosylation modifications of rhGH-Fc were adjusted by improving the composition of feeding media(using three commercial media:Gly-1:EX-CELL Glycosylation Adjust,Gly-2:SHEFF-CHO PLUS PG ACF and Gly-3:EfficientFeed C + AGT Supplement & GlycanTune C + Total Feed),and the glycosylation type and proportion of the target proteins were analyzed by mass spectrometry.Results The G0F(main glycosylation types:G0,G1 and G2;F:fucose)of Gly-1,Gly-2 and Gly-3 were 32.89%,58.66% and 33.28%,the G1F were 31.39%,18.03%and 34.90%,and the G2F were 31.39%,18.03% and 34.90%,respectively.Gly-1 and Gly-3 made the target protein contain less G0F while more G2F;Gly-3 feeding scheme-showed less high mannose modification than the other two schemes.Conclusion Gly-1 medium changed the glycosylation modification from G0F to G1F and G2F,while Gly-2 medium changed that from G2F and G1F to G0F.However,Gly-3 medium changed the glycosylation modification from G0F to G1F and G2F,and the contentof high mannose was less than 5%,which may have a better effect on modifying glycosylation type and proportion of the target protein.

Recent progress of protein glycosylation characterization utilizing native conformer-resolved mass spectrometry / 中国药科大学学报

@#Glycosylation of proteins, one of the most prevalent and complex post-translational modifications occurring in nature, plays a crucial role in regulating protein net charge, conformation, binding properties and, ultimately, biological function.Traditional structural techniques are not amenable for glycoproteins due to the inherent heterogeneity of oligosaccharides.With the advances in analytical technique, mass spectrometry displays an increasingly crucial role in elucidating the structure of glycoproteins.Mass spectrometry-based proteomic technique can dissect the chemical composition and site information of low-abundance glycosylation at the peptide level.Instead, native mass spectrometry (nMS) can analyze intact glycoproteins while maintaining the information for glycan heterogeneity, and the insights into the regulatory effects of glycosylation on protein higher order structures and interactions with other proteins or ligands.As a representative structural mass spectrometry tool, ion mobility-based nMS strategy is powered by its conformer-resolving capability and by the feasibility of conformer manipulation through collision-induced unfolding.Consequently, native IM-MS analysis can provide rich information of dynamic protein conformations, allowing for the rapid identification and differentiation of protein isoforms in an unprecedented manner.In this review, we briefly introduced two emerging native IM-MS analytical modes, dynamic conformer-resolving mode and glycoform-resolving mode.Besides, we also discussed the recent progress of conformational and topological characterization of intact glycoproteins with three typical model systems based on two above-mentioned emerging modes of native IM-MS.

Progresses of mass spectrometry-based analysis of N-glycoproteins / 中国药科大学学报

@#N-linked glycosylation is a common post-translational modification on proteins, which exhibits the same macro-heterogeneity of modification site as other small molecule modifications (such as methylation, acetylation, phosphorylation), i.e., the amino acid sequence of a protein has multiple putative modification sites. However, compared to small molecule modifications with single structures, N-glycosylation modification have tens of thousands of structures from multiple structural dimensions such as different monosaccharide compositions, sequence structures, linking structures, isomerism, and three-dimensional conformation.This results in additional micro-heterogeneity of modification site of N-glycosylation, i.e., the same N-glycosylation site can be modified with different glycans with a certain stoichiometric ratio.N-glycosylation modification regulates the structure and function of N-glycoproteins in a site- and structure-specific manner, and differential expression of N-glycosylation under disease conditions needs to be characterized through site- and structure-specific quantitative analysis.This article mainly introduces the latest development of mass spectrometry-based site- and structure-specific quantitative N-glycoproteomics and its applications in biomedical fields.

Progress of Basic and Clinical Research on Mucin-type O-Glycosylation and Colorectal Cancer / 胃肠病学

O⁃glycosylation is a common post⁃translational modification of mucins, widely present in both normal and tumor cells. In colorectal cancer (CRC) cells, there is a varying degree of dysregulation in O ⁃ glycosylation ⁃ related glycosyltransferases, molecular chaperones, and surface Tn antigen, sTn antigen, and T antigen. These dysregulations play a distinctive role in the occurrence and development of CRC, including invasion and metastasis, abnormal apoptosis and proliferation, immune escape, etc. They are extensively studied as novel tumor biomarkers and potential therapeutic targets. This article provides a comprehensive review of progress of research on mucin⁃type O⁃glycosylation and its relevance to the occurrence and development of CRC and its clinical application.

The Effect of EOGT-Mediated O-Glcnac Modification on Notch Signaling Pathway and Its Role in Diseases / 中国生物化学与分子生物学报

O-linked-N-acetylglucosamine (O-GlcNAc) modification is a unique post-translational modification that plays a regulatory role in many cellular processes, such as transcription, intracellular signaling, endocytosis, and protein stability. Epidermal growth factor (EGF) domain-specific O-GlcNAc transferase (EOGT) is an endoplasmic reticulum (ER) resident protein which can glycosylate the residues of Ser or Thr of secreted or membrane (transmembrane) glycoproteins containing EGF domain. Notch signaling pathway is involved in cell-to-cell communication which regulates cell biological processes through interactions between adjacent cells. To date, EOGT-mediated O-GlcNAc modification has been found to be involved in many human diseases, and shown significant relation with Notch signaling pathway. However, the specific molecular mechanisms have not been fully elucidated. In this review, we briefly introduce recent studies regarding to the roles of EOGT-mediated O-GlcNAc modification and its correlation with Notch signaling pathway in human diseases.

The Role of Post-Translational Modifications of Cav3. 2 in Neuropathic Pain / 中国生物化学与分子生物学报

Neuropathic pain is a common chronic pain that affects human health worldwide. As an important mediator of excitatory conduction in neurons, ion channels are important targets for mechanism research and drug research in this field. T-type calcium channel(Cav3) can be activated transiently when neurons are close to the resting potential of -70 mV, resulting in a transient Ca

Regulation mechanism of post-translational modification of farnesoid X receptor / 中国临床药理学与治疗学

Farnesoid X receptor (FXR) is a nuclear receptor activated by bile acid that is involved in regulating gene expression related to bile acid, fat, glucose, and amino acid metabolism. The activity of FXR is regulated by a variety of post-translational modifications. Common post-translational modifications of FXR include O-GlcNAcylation, phosphorylation, acetylation, sumoylation, methylation, etc. These post-translational modifications may affect FXR binding of DNA and ligand, heterodimerization, and subcellular localization, and may specifically regulate downstream gene transcription and expression. Different post-translational modifications can lead to changes in FXR stability and biological function, which are closely related to the occurrence of diseases. This paper aims to review the post-translational modification of FXR in the past five years and the mechanisms involved in disease regulation, to explore the effects of post-translational modification on the physiological function of FXR and to provide a theoretical basis for mechanism research targeting FXR.

Research on hypoglycemic activity of Osmanthus fragrans var. thunbergii extract / 药学学报

This study aimed to assess the hypoglycemic activity, and in vitro inhibition of α-glucosidase, inhibition of the advanced glycation end products (AGEs), and total antioxidant capacity were used to clarify its bioactivity. Furthermore, the potential hypoglycemic active chemical constituents in the aqueous extract of Osmanthus fragrans var. thunbergii flower were characterized using high performance liquid chromatography-electrospray ionization-quadruple time-of-flight mass spectrometry (HPLC-ESI-QTOF-MS) method. The result showed that in vitro inhibition of α-glucosidase of the extract (IC50 = 2.11 ± 0.26 mg·mL-1) were similar to acarbose (IC50 = 2.88 ± 0.32 mg·mL-1), and it inhibited the AGEs formation and the total antioxidant capacity in a certain extent. Based on the MS fragmentation pathway analysis of reference chemical acteoside contained in this extract, and related references, 73 constituents were tentatively identified from the aqueous extract of Osmanthus fragrans var. thunbergii flower, including 58 phenylethanoids, 8 caffeoylquinic acids, 1 flavonoid vicenin-2, and 6 common organic chemicals in plant. Furthermore, 8 unknown alkaloids were characterized in this work. Among of these chemicals, 61 phenylethanoids were supposed to be detected for the first time. In conclusion, this work disclosed the potential hypoglycemic active constituents of Osmanthus fragrans var. thunbergii flower.

Establishment and evaluation of in vitro galectin-3 inhibitor screening model / 药学学报

Galectin-3 (Gal-3) belongs to the galectin family and is specific in binding β-galactoside. Through its C-terminal domain, Gal-3 binds to the galactoside group of the glycosylated insulin receptor (IR) and inhibits IR signaling pathway, which leads to the insulin resistance. Thus, Gal-3 is a potential therapeutic target for the treatment of insulin resistance and type 2 diabetes. Here we report a simple Gal-3 screening model based on the property that Gal-3 binds to the galactoside. We expressed and purified human Gal-3 in Escherichia coli (E.coli), and labeled it with fluorescein isothiocyanate (FITC) in vitro. After incubating FITC labeled Gal-3 (Gal-3-FITC) with PANC-1 cells, which express glycosylated membrane protein, PANC-1 cells started to show green fluorescent signal due to the Gal-3-FITC binding to the glycosylated membrane protein. Gal-3 inhibitor disrupts the binding of Gal-3-FITC and PANC1 cells, subsequently leads to the decrease of the fluorescent signal in PANC-1 cells. We can evaluate the inhibitory efficiency of Gal-3 inhibitors through measurement of the fluorescent signal. Further studies show this model is simple, stable, and repeatable with a Z' factor between 0.7 and 0.85. In sum, we have successfully established an in vitro high-throughput screening model for Gal-3 inhibitors.

Chemical approaches for the stereocontrolled synthesis of 1,2-cis-β-D-rhamnosides / 中国天然药物

In carbohydrate chemistry, the stereoselective synthesis of 1,2-cis-glycosides remains a formidable challenge. This complexity is comparable to the synthesis of 1,2-cis-β-D-mannosides, primarily due to the adverse anomeric and Δ-2 effects. Over the past decades, to attain β-stereoselectivity in D-rhamnosylation, researchers have devised numerous direct and indirect methodologies, including the hydrogen-bond-mediated aglycone delivery (HAD) method, the synthesis of β-D-mannoside paired with C6 deoxygenation, and the combined approach of 1,2-trans-glycosylation and C2 epimerization. This review elaborates on the advancements in β-D-rhamnosylation and its implications for the total synthesis of tiacumicin B and other physiologically relevant glycans.

Advances in the diagnosis and treatment of phosphomannomutase 2 deficiency / 中国当代儿科杂志

Phosphomannomutase 2 deficiency is the most common form of N-glycosylation disorders and is also known as phosphomannomutase 2-congenital disorder of glycosylation (PMM2-CDG). It is an autosomal recessive disease with multi-system involvements and is caused by mutations in the PMM2 gene (OMIM: 601785), with varying severities in individuals. At present, there is still no specific therapy for PMM2-CDG, and early identification, early diagnosis, and early treatment can effectively prolong the life span of pediatric patients. This article reviews the advances in the diagnosis and treatment of PMM2-CDG.

Characterization of the SRD5A3-CDG Clinical Spectrum

Abstract We aimed to characterize the clinical spectrum of patients diagnosed with SRD5A3-CDG, a subtype of congenital disorders of glycosylation (CDG) due to variants in the steroid 5a-reductase type 3 (SRD5A3) gene. It presents with multi-systemic involvement including neurological disability, dermatologic abnormalities, and ophthalmological defects. We conducted a cross-sectional study of children (n=6, ages 4-16 years) with a confirmed diagnosis of SRD5A3-CDG (c.57G>A, p.W19X). Families completed a detailed medical history questionnaire, two quality of life measures, and an adaptive behavior scale. Prevalent clinical features in our cohort included visual impairment (6/6), developmental delay (6/6), nystagmus (5/6), retinal dystrophy (4/6), and hypotonia (3/6). The Vineland Adaptive Behavior Scales demonstrated deficits across all functional domains (Composite Mean 36.17 ± 26.88), although one child did not show significant deficits. The QI-Disability Form demonstrated a mean total score of 64.8 (±12.7), and the PedsQL-Family Impact Module demonstrated a mean total score of 56.5 (±31.5). Vineland composite scores did not correlate with levels of disability captured by the QI-Disability Form (Pearson Correlation range -0.63 to +0.69, p>0.05 on all subscales). Ultimately, despite genotypic homogeneity, there is notable variability in adaptive functioning and quality of life among affected children that does not correlate with age.

Dystroglycanopathies: Genetic Bases of Muscular Dystrophies Due to Alteration in the O-Glycosylation of α-Dystroglycan

Abstract Congenital muscular dystrophies (CMDs) are inherited, progressive and heterogeneous muscle disorders. A group of CMDs are dystroglycanopathies, also called α-dystroglycanopathies, where there is an abnormal glycosylation of protein α-dystroglycan. Hypoglycosylation of α-DG results in different severities of congenital muscular dystrophies and they present with progressive muscle weakness and loss of motor functions. This article first focuses on the CMDs, their classification according to the observed symptoms or the protein involved in the resulting phenotype. We then focus on dystroglycanopathies, the importance of its correct O-glycosylation of the α-dystroglycan given its important structural function, considering the enzymes involved in said glycosylation and the phenotypes that can result, to finally address current therapeutics for these diseases with the aim of increasing current knowledge.

Clinical and genetic features of a patient with COG5-CDG caused by COG5 gene variation / 中华检验医学杂志

The patient in this study was a 3 years 8 month old boy. The patient presented with facial dysmorphism including wide eye distance and flat nose. The major clinical manifestations were poor response, backward language and motor development; and his fingers cannot be bent. Moreover, the patient′s hands were also uncoordinated. In addition the patient suffered from congenital myopia and nystagmus; and the teeth were fall off easily. The abnormal reproductive system was characterized by small penis and small testicle. No obvious abnormality was found in liver and kidney function and serum immunoglobulin level through laboratory biochemical test. The results of the spine X-Ray examination indicates scoliosis. Results from brain MRI showed cerebellar dysplasia. Compound heterozygous variants in COG5 gene (c.1039C>T and c.928+3A>G), each inheranted from his parents were found in this patient by high-throughput sequencing and Sanger sequencing. After a clear diagnosis, the patient received language rehabilitation training and motor rehabilitation training. In this study, we found two new variants in COG5 gene and increased the mutation spectrum of this gene.

Correlation between serum IgG N-glycan and Lauren classification of gastric cancer / 中华检验医学杂志

Objective:Analyze the correlation between serum immunoglobulin G (IgG) N-glycan and Lauren classification of gastric cancer.Methods:A retrospective study was performed on 17 patients with diffuse type gastric cancer and 21 patients with intestinal type who received treatment in Zhongshan Hospital from 2017 to 2018, and the general medical history data and disease characteristics were summarized. The serum IgG glycome profiles were analyzed by ultraperformance liquid chromatography, and the difference between intestinal type and diffuse type gastric cance was compared.Logistic regression was used to evaluate the correlation between serum IgG N-glycan and Lauren classification.Results:IgG N-glycome analysis included 27 directly detected glycans and 4 derived traits. H=Hexose, N=N-acetylglucosamine, F=Fucose, S=Sialic acid.There was no significant difference in IgG N-glycan among different chemotherapy protocol. Compared with intestinal type, H3N3F1 ( t=3.785, P=0.001), H3N4( t=3.919, P=0.002), H3N4F1( t=2.770, P=0.005), H3N5F1( t=2.888, P=0.010) were decreased in diffuse type; H4N4F1(6)( t=?3.488, P<0.001), H5N4F1( t=?3.401, P=0.003), H5N5F1( t=?2.303, P=0.023), H5N4F1S1 ( t=?3.068, P=0.008) were increased.H3N3F1( OR:1.20, P=0.008), H3N4( OR:1.32, P=0.005), H3N4F1 ( OR:1.13, P=0.017), H3N5F1 ( OR:1.78, P=0.015), H4N4F1(6)( OR:0.43, P=0.008), H5N4F1(6)( OR:0.74, P=0.008), H5N5F1 ( OR:0.32, P=0.036), H5N4F1S1( OR:0.48, P=0.009) were significantly correlated with Lauren classification. Sialylated ( t=?2.717, P=0.012) and galactosylated ( t=?3.400, P=0.001) IgG N-glycan were reduced in patients with intestinal type gastric cancer.Galactosylated ( OR:0.87, P=0.007) and sialylated ( OR:0.62, P=0.015) IgG N-glycan were significantly correlated with Lauren classification. Conclusion:Some IgG N-glycan are significantly correlated with Lauren classification, which can be used as potential biomarkers.

Establishment of lectin-ELISA for sialylated fetuin-A and its diagnostic value in primary hepatocellular carcinoma / 中华检验医学杂志

Objective:To establish a lectin enzyme-linked immunosorbent assay (lectin-ELISA) for the dection of sialylated fetuin-A and to explore the clinical diagnostic value of sialylated fetuin-A in hepatocellular carcinoma (HCC).Methods:From January 2017 to December 2020, 300 HCC patients and 160 disease controls, including 36 liver cirrhosis subgroups and 124 chronic hepatitis B subgroups, were collected from Shanghai Eastern Hepatobiliary Surgery Hospital. At the same time, 100 healthy subjects were collected as healthy controls. Lectin-ELISA method for detecting sialylated fetuin A was established based on the principle that Sambucus nigra lectin (SNA) can recognize the structure of α-2, 6-linked sialic acid residues. Differences between groups were compared using t-test or analysis of variance. Logistic regression method was used to establish the multi-index joint detection model, and receiver operating characteristic curve (ROC) was used to evaluate the efficacy of single index and joint detection model in the diagnosis of HCC.Results:A lectin-ELISA method for the detection of serum Sia-fetuin A was established. The linear regression coefficient of the system was 0.978 5, and the precision evaluation and interference experiments were in line with the clinical detection requirements. Using this method to detect serum Sia-fetuin A levels in each group, the levels of HCC group, disease control group and healthy control group were 1.362±0.310, 1.199±0.370, 1.086±0.420, respectively, and the three groups decreased in turn. The areas under the curve of Sia-fetuin A, α-fetoprotein, and their combined detection models for differential diagnosis of HCC were 0.790, 0.809, and 0.860, respectively. The diagnostic model had a sensitivity of 79.3% (238/300) and a specificity of 95.0% (247/260). Among the 300 patients in the HCC group, 138 (46%) patients were negative for serum AFP (<20 μg/L), and their serum Sia-fetuin A level was 1.364±0.305. Combining the disease control group and the healthy control group into the non-Cancer group, the serum Sia-fetuin A level was 1.146±0.381. The serum level of Sia-fetuin A in AFP-negative HCC patients was higher than that in non-HCC group ( t=6.134, P<0.001). The areas under the curve of Sia-fetuin A and the combined diagnostic model for the diagnosis of AFP-negative HCC were 0.776 and 0.919, respectively. The combined diagnostic model had a sensitivity of 93.4% (129/138) and a specificity of 77.3% (201/260). Conclusion:Serum Sia-fetuin A and combined determination model can provide a new auxiliary diagnostic index for AFP-negative HCC.

Application of IgG sialylation in the diagnosis and therapy of autoimmune diseases / 中华检验医学杂志

Glycosylation is a part of the structure and function of immunoglobulin G (IgG). Sialic acid is located at the end of IgG N-glycan and regulates IgG anti-inflammatory and pro-inflammatory activities by changing the binding of IgG with fragment crystallizable gamma receptors (FcγRs) and complements. Low IgG sialylation is closely related to the occurrence, development and prognosis of autoimmune diseases and shows great potential in the field of diagnosis, monitoring and treatment, and may function as a new biomarker and therapeutic target for autoimmune diseases.

A potential therapeutic target: platelet glycosylation / 中华检验医学杂志

Platelet surface is rich in glycocalyx. It has been found that platelet glycosylation plays an important role in the physiological hemostasis mechanism, regulating the interaction between platelets and receptor proteins, and dynamically reshaping the surface glycosylation through its own glucose metabolism system. Platelet glycosylation also participates in platelet aging and clearance, and regulates platelet counts. Meanwhile, abnormal platelet glycosylation is closely related to primary immune thrombocytopenia, coronary heart disease and other related diseases, being a potential therapeutic target.