Fragmentation stability and retention time-shift obtained by LC-MS/MS to distinguish sialylated N-glycan linkage isomers in therapeutic glycoproteins / 药物分析学报

Sialylated N-glycan isomers with α2-3 or 42-6 linkage(s)have distinctive roles in glycoproteins,but are difficult to distinguish.Wild-type(WT)and glycoengineered(mutant)therapeutic glycoproteins,cyto-toxic T lymphocyte-associated antigen-4-immunoglobulin(CTLA4-Ig),were produced in Chinese ham-ster ovary cell lines:however,their linkage isomers have not been reported.In this study,N-glycans of CTLA4-Igs were released,labeled with procainamide,and analyzed by liquid chromatography-tandem mass spectrometry(MS/MS)to identify and quantify sialylated N-glycan linkage isomers.The linkage isomers were distinguished by comparison of 1)intensity of the N-acetylglucosamine ion to the sialic acid ion(Ln/Nn)using different fragmentation stability in MS/MS spectra and 2)retention time-shift for a selective m/z value in the extracted ion chromatogram.Each isomer was distinctively identified,and each quantity(＞0.1％)was obtained relative to the total N-glycans(100％)for all observed ionization states.Twenty sialylated N-glycan isomers with only α2-3 linkage(s)in WT were identified,and each isomer's sum of quantities was 50.4％.Furthermore,39 sialylated N-glycan isomers(58.8％)in mono-(3 N-glycans;0.9％),bi-(18;48.3％),tri-(14;8.9％),and tetra-(4;0.7％)antennary structures of mutant were obtained,which comprised mono-(15 N-glycans;25.4％),di-(15;28.4％),tri-(8;4.8％),and tetra-(1;0.2％)sialy-lation,respectively,with only α2-3(10 N-glycans;4.8％),both α2-3 and α2-6(14;18.4％),and only α2-6(15;35.6％)linkage(s).These results are consistent with those for α2-3 neuraminidase-treated N-glycans.This study generated a novel plot of Ln/Nn versus retention time to distinguish sialylated N-glycan linkage isomers in glycoprotein.

Sialylation is involved in cell fate decision during development, reprogramming and cancer progression

Sialylation, or the covalent addition of sialic acid to the terminal end of glycoproteins, is a biologically important modification that is involved in embryonic development, neurodevelopment, reprogramming, oncogenesis and immune responses. In this review, we have given a comprehensive overview of the current literature on the involvement of sialylation in cell fate decision during development, reprogramming and cancer progression. Sialylation is essential for early embryonic development and the deletion of UDP-GlcNAc 2-epimerase, a rate-limiting enzyme in sialic acid biosynthesis, is embryonically lethal. Furthermore, the sialyltransferase ST6GAL1 is required for somatic cell reprogramming, and its downregulation is associated with decreased reprogramming efficiency. In addition, sialylation levels and patterns are altered during cancer progression, indicating the potential of sialylated molecules as cancer biomarkers. Taken together, the current evidences demonstrate that sialylation is involved in crucial cell fate decision.

Clinical significances of sialylation level in fragment crystaline of serum immunoglobulin G / 中华风湿病学杂志

Objective To establish the method of testing immunoglobulin G (IgG) with Fc sialylation, and to investigate the clinical significance of IgG with Fc sialylation in systemic lupus erythematosus (SLE), especially in those with neuropsychiatric manifestations (NPSLE). Methods Seventy-five SLE including thirty patients with neuropsychiatric manifestations (NPSLE) and forty-five non-NPSLE patients, 30 rheuma-toid arthritis (RA) patients, 32 juvenile idiopathic arthritis (JIA) patients and 41 healthy controls were recruited in this study. Standard method of testing lgG with Fc sialylation was established by a lectin based sandwiched enzyme linked immunosorbent assay (ELISA). The levels of IgG with Fc sailylation were assayed, and its clinical significance was evaluated. Results There were no difference in the levels of serum IgG with Fc sailylation in RA group (0.82±1.81) mg/ml, JIA group (0.69±1.30) mg/ml, healthy control group (0.64± 1.09) mg/ml, and the levels of IgG with Fc sailylation in SLE group (0.12±0.17) mg/ml (P<0.01), especially in NPSLE group [(0.03±0.03) mg/ml, P<0.01] was significantly lower than that of control groups. The perc-entage of IgG with Fc sialylation in control group (4.64±5.90)% were significantly higher than that in non-NPSLE (1.88±2.16)% (P<0.01) and in NPSLE (0.29±0.47)% (P<0.01). The percentage of IgG with Fc sial-ylation was negatively associated with SLEDAI score (r=-0.43, P<0.01). Conclusion Significantly low level of serum IgG with Fc sialylation was associated with disease activity in SLE patients, especially in NPSLE patients, lgG with Fc sialylation may be a new target for therapeutic strategy.

Factors Related to Increased CA19-9 andLewis Antigen in Pancreatic Cancer Cell Lines

PURPOSE: The 8 pancreatic cancer cell lines (BxPC-3, Capan-2, CFPAC-1, HPAC, Capan-1, AsPC-1, MIA PaCa-2, and PANC-1) were investigated to identify the factors which would increase CA19-9 related to the Lewis antigen. CA19-9 in serum is a well-known tumor marker, and is frequently used for the clinical diagnosis of pancreatic cancer. The oligosaccharide on the CA19-9 epitope is a sialylated Lewis A blood group antigen. METHODS: beta3Gal-T was detected by reverse transcriptase polymerase chain reaction (RT-PCR). The phenotypes and genotypes of Lewis antigen were determined by flow cytometry analysis and restriction fragment length polymorphism (RFLP), respectively. The phenotypes of sLe(a) were assessed by flow cytometry analysis and the sLe(a) on supernatants was detected by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE). CA19-9 and DUPAN-2 on supernatants were measured by enzyme immunoassay. RESULTS: CA19-9 productions were possible from all cell lines since they all had beta3Gal-T and there were no genotypical Lewis negative (le/le). The elevation of CA19-9 was noted on Capan-2 and CFPAC-1, which were phenotypically Lewis positive (Le(a+b+)), as expected. Interestingly, it was also elevated in BxPC-3 even though the line was known to be phenotypically Lewis negative (Le(a-b-)). Sialyl Le(a) appeared to play an important role in this phenomenon. Although CA 19-9 was not detected in the phenotypically Lewis negative pancreatic cell line without sialyl Le(a), the levels of DUPAN-2 were variable. CONCLUSION: It was revealed that an elevated CA19-9 was related with increased expression of Lewis gene, not merely the existence of the gene. Further investigations on the role of ST3Gal are warranted to explain the mechanisms of the variable levels of DUPAN-2 in Le(a-b-) cell lines.

Altered Expression of Lewis Antigen on Tissue and Erythrocytes in Gastric Cancer Patients

To elucidate the clinical significance of phenotypic alterations of Lewis antigen in gastric cancer patients, we investigated Lewis antigens by analyzing the genotypes of the Le and Se genes and by comparing the results obtained with the phenotypic expression of Lewis antigen in gastric cancer tissue and blood cells. One hundred and twenty gastric cancer patients were examined and compared with respect to Lewis blood phenotype and genotype. The expression of Lea, Leb, sialylated Lea, and sialylated Lex antigens was immunohistochemically examined in uninvolved gastric mucosa, intestinal metaplasia, and cancerous tissue. We also analyzed the significance of Lewis antigen expression by analyzing patient survival. The frequencies of the Lewis phenotypes of RBCs corresponding to Le(a+b-), Le(a-b+), and Le(a-b-) were 16%, 58%, and 26%, respectively. The Le and le allele gene frequencies calculated from genotyping in gastric cancer patients were 0.623 and 0.377, respectively. The frequency for Le(a-b-) of the RBC phenotype had a tendency to be higher in cancer patients than in normal healthy Koreans. However, no difference in the Lewis gene frequency was found between these gastric cancer patients and healthy persons. The phenotype of Le(a-b+) was most prevalent in uninvolved gastric mucosal tissue, whereas the most prevalent form in tumor tissue was Le(a-b-). Sialyl-Lea and sialyl-Lex antigens were hardly detectable in uninvolved gastric mucosa, whereas the two antigens were expressed highly in intestinal metaplastic mucosa and tumor cells. In conclusion, the loss of Lewis antigen expression in tissue and on RBCs in gastric cancer patients is not a result of genetic influences, but rather a result of sialylation in tissue. We also confirm that poor prognosis is associated with dimeric sialyl-Lex and vascular spread.