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.

N-glycosylation modification of heat shock protein gp96 affects its immunological function / 生物工程学报

N-glycosylation modification, one of the most common protein post-translational modifications, occurs in heat shock protein gp96. The purpose of this study is to investigate the effect of N-glycosylation modification on immunologic function of the recombinant gp96 using the mutant gp96 in N-glycosylation sites. Firstly, wild-type and mutant gp96 proteins were expressed by insect expression system and their glycosylation levels were detected. To determine the effect of N-glycosylation on gp96 antigen presentation function, the IFN-γ+ CD8+ T cells in gp96-immunized mice and secretion level of IFN-γ were examined by flow cytometry and ELISA. The ATPase activity of gp96 was further detected by the ATPase kit. Finally, the effect of N-glycosylation on adjuvant function of gp96 for influenza vaccine was investigated in immunized mice. It was found that total sugar content of mutant recombinant gp96 was reduced by 27.8%. Compared to the wild type recombinant gp96, mutations in N-glycosylation sites resulted in decreased antigen presentation ability and ATPase activity of gp96. Furthermore, influenza vaccine-specific T cell levels induced by mutant gp96 as adjuvant were dramatically reduced compared to those by wild type recombinant gp96. These results demonstrate that N-glycosylation modification is involved in regulation of ATPase activity and antigen presentation function of gp96, thereby affecting its adjuvant function. The results provide the technical bases for development of gp96- adjuvanted vaccines.

Role of glycosylation in TGF-β signaling and epithelial-to-mesenchymal transition in cancer

Glycosylation is a common posttranslational modification on membrane-associated and secreted proteins that is of pivotal importance for regulating cell functions. Aberrant glycosylation can lead to uncontrolled cell proliferation, cell-matrix interactions, migration and differentiation, and has been shown to be involved in cancer and other diseases. The epithelial-to-mesenchymal transition is a key step in the metastatic process by which cancer cells gain the ability to invade tissues and extravasate into the bloodstream. This cellular transformation process, which is associated by morphological change, loss of epithelial traits and gain of mesenchymal markers, is triggered by the secreted cytokine transforming growth factor-β (TGF-β). TGF-β bioactivity is carefully regulated, and its effects on cells are mediated by its receptors on the cell surface. In this review, we first provide a brief overview of major types of glycans, namely, N-glycans, O-glycans, glycosphingolipids and glycosaminoglycans that are involved in cancer progression. Thereafter, we summarize studies on how the glycosylation of TGF-β signaling components regulates TGF-β secretion, bioavailability and TGF-β receptor function. Then, we review glycosylation changes associated with TGF-β-induced epithelial-to-mesenchymal transition in cancer. Identifying and understanding the mechanisms by which glycosylation affects TGF-β signaling and downstream biological responses will facilitate the identification of glycans as biomarkers and enable novel therapeutic approaches.

Comprehensive Characterization of Anti-CTLA4 Monoclonal Antibody by Liquid Chromatography High Resolution Mass Spectrometry / 中国药学杂志

OBJECTIVE: To establish a mass spectrometry method for the qualitative analysis of anti-CTLA4 monoclonal antibody and its N-linked glycosylation. METHODS The anti-CTLA4 monoclonal antibody was characterized by liquid-mass technique from the aspects of intact molecular weight, subunit molecular weight, amino acid sequence coverage, disulfide bond, N-linked glycosylation site and glycoform. RESULTS: The molecular weight of anti-CTLA4 mAb (A2G0F/A2G0F) is 147 992; the deglycosylation molecular weight of anti-CTLA4 mAb is 145 103; the molecular weight of light chain is 23 450; the molecular weight of heavy chain (A2G0F) is 50 548; the heavy chain Fd segment has a molecular weight of 25 355 and the heavy chain sFc segment (A2G0F,1xK_Loss) has a molecular weight of 25 189. Peptide mapping was performed with Trypsin & Chymotrypsin, and the coverage of the amino acid sequence was 100%. The peptide containing the N-linked glycosylations site is EEQYNSTYR, and the N-glycosylation site is located at Asn298 of the heavy chains. Thirteen glycoform were characterized including A2G0F(59.8%), A2G1Fa(18.66%), A2G1Fb(7.28%), A2G2F(3.2%), M5(1.66%), A2S1G0F(1.17%), A1G0F (1.16%), A3G1F(0.95%), A2G0(0.94%), A2S2F(0.78%), A2S1G1F(0.67%), A3G0F(0.53%)and A1G1F(0.51%). CONCLUSION: A method for qualitative and quantitative analysis of monoclonal antibody is established.

Analysis of WCID-cIEF Spectrogram of Recombinant Interleukin-15 Fusion Protein / 中国药学杂志

OBJECTIVE: To analyze the glycosylated chains of recombinant interleukin-15 fusion protein using capillary isoelectric focusing-whole column imaging detection (WCID-cIEF) spectrograms. METHODS: Using established corresponding mathematical models and the least square method, the WCID-cIEF spectrograms of whole protein, de-salicylic-acid protein and de-N-glycosylation-chain protein were analyzed. Among the mathematical models, the interval-1-peak model was selected. And according to the model, the relationship between isoform peak-areas and isoelectric points was listed. RESULTS: The rationality of the interval-1-peak model was confirmed and a series of basic data was obtained according to the model as follows:the apparent m value of the protein was 25.53 reference(R), the apparent n value of the protein was 28.83R, the apparent m value of sialic acid was 0.86 (0.855) R, the apparent n value was 0.12 (0.119) R, the apparent n value of N-acetylglucosamine (undifferentiated from N-acetylgalactosamine) was 0.06(0.061) R, and the apparent m value of formed carboxyl after N-chain removal was 0.19 (0.186) R. Some information of protein sugar composition was also obtained: the sialylation degree was about 1.83 mol•mol-1, the percentage of prototype protein was about 8.3%, the percentage of single N-glycosated modification protein was about 19.8%, the percentage of double N-glycosated modification protein was about 28.4%, the percentage of triple N-glycosated modification protein was about 23.7%, and the percentage of O-glycosated modification (with sialic acid) protein was about 19.8%. The main sugar types should be G0 (F), G1 (F), G2 (F), G1A1 (F), and G2A1 (F). CONCLUSION: The structure of sugar chain is complex, but it also has some repeatability and regularity. We hope that through this study, the glycoprotein sugar chain can be quickly outlined, the understanding of glycoprotein and the study of protein interaction can be improved.

Expression, purification and characterization of N-glycosylation mutant human IFN-λ1 in Pichia pastoris / 生物工程学报

IFN-λ1 is a member of a new family of interferons called type Ⅲ IFNs with similar functions to type ⅠIFNs. Previously we obtained recombinant soluble human rhIFN-λ1 from Pichia pastoris. However, the hyper-glycosylation from P. pastoris brings immunogenicity and low purification recovery rate. To overcome this disadvantage, in this study, we constructed an rhIFN-λ1 mutant (rhIFN-λ1-Nm) devoid of the potential N-glycosylation sites by site-directed mutagenesis. rhIFN-λ1-Nm was successfully expressed and secreted extracellularly in P. pastoris (GS115) using methanol inducible AOX1 promoter with α-mating factor signal sequence. rhIFN-λ1-Nm was purified and characterized. There was no significant difference between rhIFN-λ1-Nm and rhIFN-λ1 in structure and bioactivity. The molecular weight was low after N-glycosylation mutation whereas the glycosylation was much lower. The mutational rhIFN-λ1 (rhIFN-λ1-Nm) could legitimately be developed as substitutes for rhIFN-λ1, and thus it may be developed into a more promising therapeutic reagent in the future.

Association between N-glycans of immunoglobulin G and central obesity / 中华健康管理学杂志

Objective To explore the association between immunoglobulin G (IgG) glycosylation and central obesity in a Chinese population. Methods We recruited 517 Chinese individuals from a community-based cohort in the Xicheng District, Beijing, China. The height, weight, waist circumference, and hip circumference were measured, and blood samples were collected. Plasma IgG N-glycome was determined using hydrophilic interaction liquid chromatography (HILIC). Central obesity was determined using the waist-to-hip ratio (WHR). Men and women with a WHR>0.90 and >0.80, respectively were assigned to the central obese group,and those with a WHR≤0.90(men)and≤0.80(women)were assigned to the normal group.The correlation between WHR and glycosylation was determined using the standardized regression coefficient,and P<0.05 was considered statistically significant.Results The study included 517 participants,with an average age of(47.81±5.58)years(range:27-68 years),and 164(31.70%)of them were men. The central obese and normal groups included 205 (39.65%) and 312 (60.35%) participants, respectively. The average age for the central obese group (49.21 ± 5.70) years was higher than that for the normal group (46.89 ± 5.30) years, and the difference was statistically significant (t=-4.73, P=0.001). The two groups showed no significant differences in the sex of the participants(χ2=0.34,P=0.558).Twenty-four N-glycan traits(GP1-GP24)were directly measured,and because of the high variability among the repeated measurements and the unknown structures, GP3 and GP20 were excluded from the analysis. The peak contents for GP4,GP8,GP14,and GP18 were higher than the others and accounted for about 50% of all the peak contents. IgG GP10 (b'=0.076, P=0.024) positively correlated with WHR, whereas IgG GP12, GP17, GP22 negatively correlated with WHR(b'=-0.076,-0.081,-0.080,all P<0.05).Additionally,the IgG GP12, GP17,and GP22(P=0.001,0.004 and 0.033,respectively)were significantly lower in the participants of the central obese group,compared to those of the normal group. Conclusion The loss of galactose and sialic acid,as well as the addition of fucose to IgG N-glycan,correlate with increased WHR.

Risk association of hepatocellular carcinoma with S-gene additional N-glycosylation mutation of hepatitis B virus in HBsAg and anti-HBs coexistent patients / 解放军医学杂志

Objective To investigate the association of additional N-glycosylation mutation in major hydrophilic region (MHR) of hepatitis B virus (HBV) S gene with the risk of hepatocellular carcinoma (HCC) in HBsAg and anti-HBs coexistent patients. Methods A total of 284 patients with coexistence of HBsAg and anti-HBs were enrolled in this study, who were admitted in 302 Hospital of PLA from July 2009 to June 2016. HBV DNA was extracted from serum samples and subjected to nested PCR for full-length S-gene sequencing. The association of MHR additional N-glycosylation mutation and clinical parameters with HCC occurrence risk was analyzed. Specifically, the additional N-glycosylation mutation was dynamically analyzed pre-and post-HCC occurrence for 18 patients. Results Multivariate analysis showed that age >40 years, HBsAg >median, HBeAg negativity, and additional N-glycosylation mutation in MHR were associated with HCC occurrence for the HBsAg and anti-HBs coexistent patients (OR=4.281, 95%CI 1.843-9.941, P=0.001; OR=3.146, 95%CI 1.633-6.060, P=0.001; OR=2.097, 95%CI 1.010-4.357, P=0.047; and OR=4.381, 95%CI 1.842-10.417, P=0.001). In contrast, ALT, anti-HBs, anti-HBe, and HBV DNA levels had no significant association with HCC occurrence. Dynamical analysis showed that the additional N-glycosylation mutation had already developed 1-4 years prior to HCC occurrence in the 8 of 18 observed patients. Conclusion Additional N-glycosylation mutation in MHR of HBV S gene had close association with HCC occurrence in HBsAg and anti-HBs coexistent patients, suggesting that HBsAg and anti-HBs coexistence and additional N-glycosylation mutation together could serve as a predictive indicator for HCC occurrence in chronic HBV-infected patients.

Implications of newly-added N-glycosylation mutation of hepatitis B virus s-gene in patients with coexistence of HBsAg and antiHBs / 解放军医学杂志

Objective To analyze the characteristics of newly added N-glycosylation mutation in major hydrophilic region (MHR) of HBV S gene in patients with coexistence of HBsAg and antiHBs, and reveal the generation mechanism and clinical implications of the coexistence. Methods HBV S genes from 284 patients with HBsAg+antiHBs and 314 patients with single HBsAg were amplified respectively for sequence analysis. A chronic hepatitis B (CHB) patient with HBsAg+antiHBs in MHR was found to harbor a novel double N-glycosylation mutation and selected for further study. Recombinant vectors harboring the novel mutant or control PreS/S genes were constructed and transfected in HepG2 cells respectively for phenotypic analysis, and the effects of the mutations on HBV duplication and antigenicity were investigated. Results The detection rate of MHR N-glycosylation mutation was significantly higher in HBsAg+antiHBs group than in single HBsAg group (11.3% vs. 2.9%, P<0.01, respectively). In HBsAg+antiHBs cohort, the proportion of hepatocellular carcinoma (HCC) patients accounted for 46.9%(15/32) in patients with N-glycosylation mutation at the time of testing; by contrast, the number was 22.6%(57/252) in patients with non-N-glycosylation mutation (P<0.01). N-glycosylation mutational pattern of the novel strain was s116-118TST → NST+s131-133TSM → NST concomitant with sP120 deletion+G145D mutation. The novel mutants accounted for 98.0%, 2.0% and 2.5%, respectively, of viral clones in three sequential serum samples. Mutants with single N-glycosylation mutation s130-132GTS→NSS without sP120 deletion+G145D were detected in sample 2, accounting for 17.6% of viral clones. Compared to the wild-type, the novel mutant had an increase of 31% in replication capacity, but a decrease of 99% in HBsAg level. Immunofluorescence showed that elimination of the two additional N-glycosylation mutations only partly restored HBsAg detection by antiHBs, suggesting that sP120 deletion+G145D mutation also attenuated HBsAg antigenicity. Conclusions Additional N-glycosylation mutation in MHR of HBV S gene is associated with coexisting HBsAg+antiHBs, and the two parameters together might be a better risk factor for HCC occurrence. Combination of two additional N-glycosylation mutation, sP120 deletion and sG145D mutation may co-play a role in silence of HBsAg antigenicity.

Effects of N-acyl glucosamines on GABA uptake activity of GABA transporter 1 / 中国药科大学学报

@#In order to screen the inhibitors of GAT1 protein, the effects of synthetic N-acylglucosamines on the GABA uptake activity of GAT1 were examined in the HEK-293 cell model stably expressing GAT1 by performing GABA uptake assay. And since the N-linked oligosaccharides, especially their terminal sialic acid of GAT1 are necessary for the function of GAT1, their effects on the modification of N-glycans of GAT1 were also determined by the quantitative analysis of the sialic acid of GAT1. The results showed that 10 mmol /L 3-O-methyl-N-acetylglucosamine(3-O-Met-GlcNAc)reduced the GABA uptake activity of GAT1 to 53%(P< 0. 01)through inhibiting the sialylation of GAT1(66. 8%). And the GABA uptake activities were decreased to 54%, 63%, 63% and 67%(P< 0. 01), respectively, by the treatments of 10 mmol /L N-propionylglucosamine(GlcNProp), N-hexanoylglucosamine(GlcNHex), N-cycloproylgormylglucosamine(GlcNCyclo)and N-acetamidoacetylglucosamine(GlcNAc-acetamido)through inhibiting N-glycan trimming. These results indicate that the analogues of glucosamines have great potential in the development of inhibitors of GAT1 activity or sialic acid biosynthesis.

Construction of a glycoengineering yeast overexpressing N-glycosyltransferase / 军事医学

Objective To obtain a strain of glycoengineering yeast with higher N-glycosylation efficiency by overexpressing N-glycosyltransferase.Methods Through the selecting marker URA3 gene, a new glycoengineering yeast strain named 4-32-STT3D was constructed, which could overexpress the Leishmania major N-glycosyltransferase staurosporine and temperature sensitivity3 D subunit(STT3D) under the control of an inducible alcohol oxidase 1(AOX1) promoter.We analyzed the N-glycosylation status of anti-human epidermal growth factor receptor 2 ( HER2 ) antibody and granulocyte macrophage colony stimulating factor (GM-CSF) expressed in 4-32-STT3D using SDS-PAGE,Western blotting and peptide-N-asparigineamidase F(PNGase F).Finally the effect of STT3D on the growth rate of glycoengineering yeast was detected.Results SDS-PAGE showed that anti-HER2 antibody expressed in 4-32-HL had two components:the first one with a relative molecular mass 55 ×103 was glycosylated,while the second one with 50 ×103 was non-glycosylated,but anti-HER2 antibody expressed in 4-32-HL-STT3D had the component of 55 ×103 only without any non-glycosylated 50 ×103 .The above components became 50 ×103 with the digestion of PNGaseF.All of them proved to be antibodies by Western blotting.As a report protein,GM-CSF expressed in 4-32-GM-CSF had two components: 22 ×103 and 20 ×103, while in 4-32-GM-CSF-STT3D there was only one with 22 ×103 .All these components became 18 ×103 with the digestion of PNGase F.Statistical analysis showed that without induction,STT3D had no effect on the growth rate of glycoengineering yeast, while great effect was observed when STT3D was induced.Conclusion Glycoengineering yeast with the overexpression of N-glycosyltransferase has higher N-glycosylation efficiency.

Expression of endo-beta-N-acetylglucosaminidase H in Pichia pastoris and its applica-tion to N-glycosylation analysis / 军事医学

Objective To prepare endo-beta-N-acetylglucosaminidase H (Endo-H) expressed in Pichia pastoris, and apply it to N-glycosylation analysis .Methods One complete gene was synthesized on the basis of the cDNA sequence encoding Streptomyces plicatus reported in GenBank .The gene was cloned into the expression vector pPIC 9.The expression vector pPIC9-Endo-H was transformed into P.pastoris(JC308).The expression products were induced by methanol , puri-fied by two-step chromatography , used to analyze the glycan structures of RNaseB by the DNA sequencer assisted fluoro-phore-assisted carbohydrate electrophoresis (DSA-FACE)methods, and finally compared with peptide-N-asparagine amidase F(PNGase F).Results This enzyme expressed in P.pastoris(JC308) had the ability to hydrolyze natural or denatured high-mannose type of oligosaccharide linked by β-1,4-glycosidic bonds , but not complex-type oligosaccharide .The result of DSA-FACE showed that carbohydrate chains of Man 5 GlcNAc-Man9 GlcNAc could be obtained when RNaseB was hydrolyzed by Endo-H, and that Man5 GlcNAc2-Man9 GlcNAc2 chains became available when RNaseB was hydrolyzed by PNGase F . Conclusion Endo-H expressed in P.pastoris has bioactivity which can be used to analyze N-glycosylation with the method of DSA-FACE.

Comparative analysis of Gp120 sequences of enterotropic and neurotropic SIV strains / 中国比较医学杂志

Objective To study the mutations of Env sequence of SIVmac239 after infection of Chinese rhesus monkeys, and compare the differences and characteristics of Gp120 sequences of enterotropic and neurotropic SIV strains. Methods Six strains of simian immunodeficiency virus were analyzed in this study: four separated from peripheral blood mononuclear cells of SIVmac239-infected monkeys and two neurotropic SIVmac251 strains.Isolated and cultured monoclonal virus was obtained by limiting dilution assay.Gp120 sequences were amplified after the RNA extraction and phylogenetic analysis was processed thereafter.So did the Gp120 amino acid sequence and N-glycosylation sites analysis of the enterotropic and neurotropic strains.Results SIVmac239 had different mutations in four rhesus monkeys.The diversity in amino acid sequences of the enterotropic and neurotropic strains concentrated in the V1 and V4 regions of Gp120.The enterotropic strains had an addition of glycosylation site in V4 but the glycosylation site changes of neurotropic strains were located in the conservative regions of C1, C2 and C3.Conclusions The addition of one glycosylation site in V4 region of GP120 and loss of one glycosylation site in C1 region are associated with enhanced enterotropism and neurotropism.The differences between the enterotropic and neurotropic strains are not dipicted in Gp120 V3 region which is closely related with the tropism of strains.

Papel del receptor 1 de transferrina en la captación del hierro y su relación con la deficiencia gestacional de hierro y la preeclampsia / Function of Receptor 1 in uptaking transferrin and its relation to iron deficiency and iron gestational preeclampsia

La anemia ferropénica gestacional afecta al 48 porciento de las mujeres y se asocia con efectos deletéreos para la madre y el feto. Para la captación del hierro de la gestante es necesaria la expresión en el sincitiotrofoblasto de la glicoproteína receptor 1 de transferrina (TfR1). En ensayos celulares, en modelos animales y en humanos la deprivación de hierro se ha asociado a un aumento en la transcripción y expresión del TfR1, que se ha explicado como un mecanismo compensatorio para la captación del hierro a favor del feto. De otra parte, en alteraciones de la gestación como la preeclampsia se espera un aumento en la expresión del TfR1 placentario, sin embargo se ha evidenciado una reducción de este. Este evento se ha explicado como una regulación de tipo transcripcional relacionada con el factor de transcripción inducible por la hipoxia. El objetivo fue revisar evidencia que soporte que en la reducción de la expresión del TfR1 en preeclampsia, estén implicados cambios en la glicosilación como una modificación postraduccional relacionada con el adecuado plegamiento, maduración y exportación del receptor a la membrana celular. La base de datos Pubmed fue consultada para identificar los artículos más relevantes. Los descriptores usados fueron metabolismo de hierro, anemia, placenta, receptor de transferrrina, preeclampsia, glicosilación. Se propone una regulación postranscripcional relacionada con la glicosilación que explica como, pese al aumento en la expresión del RNA mensajero del TfR1 inducido por la hipoxia en la placenta preeclámptica, se genera una reducción en su expresión

Iron deficiency gestational anemia affects 48 per cent of women and it is associated with deleterious effects for the mother and her fetus. For mother iron uptake, the syncytiotrophoblast expression of glycoprotein transferrin receptor 1 (TfR1) is required. In cellular trails, in animal models and in humans, iron deprivation has been associated with an increase in TfR1 transcription and expression, which has been explained as a compensatory mechanism for the fetus iron uptake. On the other hand, pregnancy alterations such as preeclampsia are expected to increase in the expression of placental TfR1; however, a reduction of it has shown. This event has been explained as a type transcriptional regulatory factor related to the hypoxia-inducible transcription. Our objective was to review evidence to support that in the reduction of TfR1 preeclampsia expression, changes in glycosylation are involved as a posttranslational modification regarding the appropriate folding, maturation and receptor export to the cell membrane. PubMed database was consulted to identify the most relevant articles. The descriptors used were iron metabolism, anemia, placenta, transferrin receiver, preeclampsia, glycosylation. A posttranscriptional regulation related to glycosylation is proposed explaining how a reduction in expression is generated, despite of the increase in RNA expression of TfR1messenger induced by hypoxia in the preeclamptic placenta

Purification and biochemica characterisation of endoplasmic reticulum á 1-2-mannosidase from Sporothrix schenckiil

Alpha 1,2-mannosidases from glycosyl hydrolase family 47 participate in N-glycan biosynthesis. In filamentous fungi and mammalian cells, á1,2-mannosidases are present in the endoplasmic reticulum (ER) and Golgi complex and are required to generate complex N-glycans. However, lower eukaryotes such Saccharomyces cerevisiae contain only one á1,2-mannosidase in the lumen of the ER and synthesise high-mannose N-glycans. Little is known about the N-glycan structure and the enzyme machinery involved in the synthesis of these oligosaccharides in the dimorphic fungus Sporothrix schenckii. Here, a membrane-bound á-mannosidase from S. schenckii was solubilised using a high-temperature procedure and purified by conventional methods of protein isolation. Analytical zymograms revealed a polypeptide of 75 kDa to be responsible for enzyme activity and this purified protein was recognised by anti-á1,2-mannosidase antibodies. The enzyme hydrolysed Man9GlcNAc2 into Man8GlcNAc2 isomer B and was inhibited preferentially by 1-deoxymannojirimycin. This á1,2-mannosidase was localised in the ER, with the catalytic domain within the lumen of this compartment. These properties are consistent with an ER-localised á1,2-mannosidase of glycosyl hydrolase family 47. Our results also suggested that in contrast to other filamentous fungi, S. schenckii lacks Golgi á1,2-mannosidases and therefore, the processing of N-glycans by á1,2-mannosidases is similar to that present in lower eukaryotes.

The Binding Properties of Glycosylated and Non-Glycosylated Tim-3 Molecules on CD4+CD25+ T Cells

BACKGROUND: T cell immunoglobulin and mucin domain containing 3 protein (Tim-3) expressed on terminally differentiated Th1 cells plays a suppressive role in Th1-mediated immune responses. Recently, it has been shown that N-glycosylation affects the binding activity of the Tim-3-Ig fusion protein to its ligand, galectin-9, but the binding properties of non-glycosylated Tim-3 on CD4+CD25+ T cells has not been fully examined. In this study, we produced recombinant Tim-3-Ig fusion proteins in different cellular sources and its N-glycosylation mutant forms to evaluate their binding activities to CD4+CD25+ T cells. METHODS: We isolated and cloned Tim-3 cDNA from BALB/C mouse splenocytes. Then, we constructed a mammalian expression vector and a prokaryotic expression vector for the Tim-3-Ig fusion protein. Using a site directed mutagenesis method, plasmid vectors for Tim-3-Ig N-glycosylation mutant expression were produced. The recombinant protein was purified by protein A sepharose column chromatography. The binding activity of Tim-3-Ig fusion protein to CD4+CD25+ T cells was analyzed using flow cytometry. RESULTS: We found that the nonglycosylated Tim-3-Ig fusion proteins expressed in bacteria bound to CD4+CD25+ T cells similarly to the glycosylated Tim-3-Ig protein produced in CHO cells. Further, three N-glycosylation mutant forms (N53Q, N100Q, N53/100Q) of Tim-3-Ig showed similar binding activities to those of wild type glycosylated Tim-3-Ig. CONCLUSION: Our results suggest that N-glycosylation of Tim-3 may not affect its binding activity to ligands expressed on CD4+CD25+ T cells.

Heterologous expression and biochemical characterization of an alpha 1, 2-mannosidase encoded by the Candida albicans MNS1 gene

Protein glycosylation pathways, commonly found in fungal pathogens, offer an attractive new area of study for the discovery of antifungal targets. In particular, these post-translational modifications are required for virulence and proper cell wall assembly in Candida albicans, an opportunistic human pathogen. The C. albicans MNS1 gene is predicted to encode a member of the glycosyl hydrolase family 47, with 1,2-mannosidase activity. In order to characterise its activity, we first cloned the C. albicans MNS1 gene into Escherichia coli, then expressed and purified the enzyme. The recombinant Mns1 was capable of converting a Man9GlcNAc2 N-glycan core into Man8GlcNAc2 isomer B, but failed to process a Man5GlcNAc2-Asn N-oligosaccharide. These properties are similar to those displayed by Mns1 purified from C. albicansmembranes and strongly suggest that the enzyme is an ±1,2-mannosidase that is localised to the endoplasmic reticulum and involved in the processing of N-linked mannans. Polyclonal antibodies specifically raised against recombinant Mns1 also immunoreacted with the soluble ±1,2-mannosidases E-I and E-II, indicating that Mns1 could share structural similarities with both soluble enzymes. Due to the high degree of similarity between the members of family 47, it is conceivable that these antibodies may recognise ±1,2-mannosidases in other biological systems as well.

Enzymatic N-glycan analysis of 31 kDa molecule in plerocercoid of Spirometra mansoni (sparganum) and its antigenicity after chemical oxidation

A highly specific antigenic protein of 31 kDa from plerocercoid of Spirometra mansoni (sparganum) was obtained by gelatin affinity and Mono Q anion-exchange column chromatography. The purified 31 kDa protein was subjected to N-glycan enzymatic digestion for structural analysis. The relative electrophoretic mobility was analyzed by SDS-PAGE, before and after digestion. On SDS-PAGE after enzymatic digestion, the 31 kDa protein showed a molecular shift of approximately 2 kDa, which indicated the possession of complex N-linked oligosaccharides (N-glycosidase F sensitive) but not of high-mannose oligosaccharides (endo-beta-N-acetylglucosaminidase H, non-sensitive). Chemically periodated 31 kDa protein showed statistically non-significant changes with human sparganosis sera by enzyme linked immunosorbent assay (ELISA). Therefore, the dominant epitopes of the 31 kDa molecule in human sparganosis were found to be mainly polypeptide, while N-glycans of the antigenic molecule in sparganum was minimal in anti-carbohydrate antibody production.

Expression of recombinant N-glycosylation interleukin 24 and study of inducing tumor cells apoptosis in vitro / 中国药理学通报

Aim To obtain recombinant human IL-24 secretorily expressed in Pichia pastoris,and study the activity of inducing tumor cells apoptosis of this N-glycosylation protein. Methods By the recombinant plasmid ?/pUC18,the confirmed IL-24 gene was inserted between the sites of BamH Ⅰ and EcoR Ⅰ of expression plasmid pPIC9K. The recombinant plasmid IL-24/pPIC9K was transformed into P. pastoris strain GS115. Yeast transformants were induced for expression of recombinant human IL-24 with methanol. The desired protein was identified with Tricine-SDS-PAGE and Western blot.Amount of IL-24 was assayed with ELISA and the glycosylation was analyzed by PNGase F.The activity of inducing tumor cells apoptosis was confirmed by MTT assay and Hoechst assay in vitro.Results Recombinant expression plasmid IL-24/pPIC9K was successfully constructed. 5 transformants were screened with G418 and PCR. Induced with methanol,the expression level of IL-24 was (81.31?14.46) mg?L-1 at flask fermentation,and 70 % IL-24 generated N-glycosylation.Recombinant IL-24 induced apoptosis in MCF-7 cells,but not in NHLF.Conclusion The secretorily expression of the N-glycosylation IL-24 protein in P. pastoris and the study of inducing tumor cells apoptosis lay the foundation for the further study of molecular mechanism of IL-24 on anti-tumors and the potential application.

Amphomycin: A tool to study protein N-glycosylation.

Radio-labelled amphomycin (3H-amphomycin) forms a complex with dolichylmonophosphate in presence of Ca2+ . Complex formation has also been documented with retinylmonophosphate and perhydromonoeneretinylmonophosphate. Analysis of the space-filling model suggested both fatty acylated aspartic acid residue at the N-terminus of the lipopeptide and phosphate head group of dolichylmonophosphate are necessary for the complex formation. The binding ability of amphomycin is then utilized to localize dolichylmonophosphate in the microsomal membrane. Studies with microsomal membranes from hen oviduct suggested that dolichylmonophosphate is located in the cytoplasmic side of the membrane.