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Gordan Lauc - One of the best experts on this subject based on the ideXlab platform.
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Change of transferrin Sialylation differs between mild sepsis and severe sepsis and septic shock.
Internal medicine (Tokyo Japan), 2011Co-Authors: Olga Gornik, Ivan Gornik, Ivana Zagorec Kolednjak, Gordan LaucAbstract:Objective and Design To investigate the association between the severity of sepsis and changes in Sialylation of serum proteins we have conducted a single center pilot study. Subjects and Methods Sialylation of transferrin (with enzyme-linked lectin assay-ELLA) and total serum proteins (with colorimetric assay) as well as serum iron and transferrin levels were measured in 27 patients with sepsis through the first eight days of the disease. Results Total serum Sialylation increased in the first two days, transferrin Sialylation decreased, while serum iron and transferrin fell. Patients who developed severe sepsis had either a small or marked change in transferrin Sialylation while in patients with mild sepsis Sialylation decreased moderately. Conclusion We hypothesize that the change in transferrin Sialylation could be a reflection of the intensity of inflammatory response which is insufficient if under-expressed and detrimental if over-expressed. This new feature is a potential marker of sepsis severity early in the disease.
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Change in transferrin Sialylation is a potential prognostic marker for severity of acute pancreatitis.
Clinical biochemistry, 2008Co-Authors: Olga Gornik, Ivan Gornik, Vladimir Gasparović, Gordan LaucAbstract:Early prediction of severe acute pancreatitis is one of the problems in clinical practice. Since many diseases are associated with alteration in glycosylation, in this work we studied Sialylation of transferrin and serum proteins in acute pancreatitis. Sialylation was analyzed during first eight days of hospitalization of 30 patients and compared to 28 healthy controls. Transferrin Sialylation was measured using enzyme linked lectin assay, while sialic acid on proteins was measured using resorcinol method. Both analyzed parameters changed during studied period. The change in transferrin Sialylation between Day1 and Day2 of hospitalization was shown to be an early prognostic marker of acute pancreatitis, with better sensitivity (88.9%) and specificity (90.5%) than other markers tested. Sialylation of transferrin and total serum proteins reflects the intensity of inflammatory response during acute pancreatitis and could be used as prognostic parameter for disease severity.
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Change in transferrin Sialylation is a potential prognostic marker for severity of acute pancreatitis.
Clinical Biochemistry, 2008Co-Authors: Olga Gornik, Ivan Gornik, Vladimir Gasparović, Gordan LaucAbstract:Abstract Objectives Early prediction of severe acute pancreatitis is one of the problems in clinical practice. Since many diseases are associated with alteration in glycosylation, in this work we studied Sialylation of transferrin and serum proteins in acute pancreatitis. Design and methods Sialylation was analyzed during first eight days of hospitalization of 30 patients and compared to 28 healthy controls. Transferrin Sialylation was measured using enzyme linked lectin assay, while sialic acid on proteins was measured using resorcinol method. Results Both analyzed parameters changed during studied period. The change in transferrin Sialylation between Day1 and Day2 of hospitalization was shown to be an early prognostic marker of acute pancreatitis, with better sensitivity (88.9%) and specificity (90.5%) than other markers tested. Conclusions Sialylation of transferrin and total serum proteins reflects the intensity of inflammatory response during acute pancreatitis and could be used as prognostic parameter for disease severity.
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Enzyme linked lectin assay (ELLA) for direct analysis of transferrin Sialylation in serum samples.
Clinical biochemistry, 2007Co-Authors: Olga Gornik, Gordan LaucAbstract:Glycosylation analysis provides many opportunities for diagnostics, but its complexity hampers its routine application. Aiming to alleviate this problem, we developed a simple assay that can measure Sialylation of transferrin directly from serum. Transferrin samples with different levels of Sialylation were prepared by deSialylation. Enzyme-linked-lectin assay (ELLA) and high-performance anion-exchange chromatography (HPAEC) have been used to analyze transferrin Sialylation. Periodate oxidation was used to oxidize carbohydrates on antibodies. ELLA was developed for the analysis of serum transferrin Sialylation. Antibodies oxidized in situ with periodate have been used to capture transferrin from serum samples. Sialic acid on transferrin has been detected with Sambucus nigra agglutinin (SNA) lectin. Transferrin samples with different Sialylation levels prepared by differential deSialylation have been used as standards. Accuracy of the method has been confirmed by comparison to HPAEC analysis. A rapid and simple ELLA that can be routinely used for the analysis of serum transferrin Sialylation has been developed.
Michael J. Betenbaugh - One of the best experts on this subject based on the ideXlab platform.
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The impact of Sialylation linkage-type on the pharmacokinetics of recombinant butyrylcholinesterases
Biotechnology and bioengineering, 2019Co-Authors: Cheng-yu Chung, Qiong Wang, Shuang Yang, Sandra Chough, Younji Seo, John F. Cipollo, Joseph P. Balthasar, Michael J. BetenbaughAbstract:Chinese hamster ovary (CHO) cells typically produce glycoproteins with N-glycans terminating in α-2,3 Sialylation. Human cells produce glycoproteins that include α-2,3 and α-2,6 sialic acids. To examine the impact of altering protein Sialylation on pharmacokinetic properties, recombinant human butyrylcholinesterase (BChE) was produced in CHO cells by knocking out the α-2,3 sialyltransferase genes followed by overexpression of the α-2,6 sialyltransferase (26BChE) enzyme. The N-glycan composition of 26BChE was compared to BChE with α-2,3 Sialylation (23BChE) derived from wild-type CHO cells. Both 23BChE and 26BChE exhibited comparable antennarity distributions with bi-antennary di-sialylated glycans representing the most abundant glycoform. CD-1 mice were intravenously injected with the 23BChE or 26BChE, and residual BChE activities from blood collected at various time points for pharmacokinetic analyses. Although 23BChE contained a slightly lower initial Sialylation level compared to 26BChE, the molecule exhibited higher residual activity between 5 and 24 hr postinjection. Pharmacokinetic analyses indicated that 23BChE exhibited an increase in area under the curve and a lower volume of distribution at steady state than that of 26BChE. These findings suggest that the type of Sialylation linkage may play a significant role in the pharmacokinetic behavior of a biotherapeutic when tested in in vivo animal models.
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integrated genome and protein editing swaps α 2 6 Sialylation for α 2 3 sialic acid on recombinant antibodies from cho
Biotechnology Journal, 2017Co-Authors: Cheng-yu Chung, Bojiao Yin, Qiong Wang, Shuang Yang, Hui Zhang, Michael J. BetenbaughAbstract:Immunoglobin G with α-2,6 Sialylation has been reported to have an impact on antibody-dependent cellular cytotoxicity and anti-inflammatory efficacy. However, production of antibodies with α-2,6 Sialylation from Chinese hamster ovary cells is challenging due to the inaccessibility of sialyltransferases for the heavy chain N-glycan site and the presence of exclusively α-2,3 sialyltransferases. In this study, combining mutations on the Fc regions to allow sialyltransferase accessibility with overexpression of α-2,6 sialyltransferase produced IgG with significant levels of both α-2,6 and α-2,3 Sialylation. Therefore, ST3GAL4 and ST3GAL6 genes were disrupted by CRISPR/Cas9 to minimize the α-2,3 Sialylation. Sialidase treatment and SNA lectin blot indicated greatly increased α-2,6 Sialylation level relative to α-2,3 Sialylation for the α-2,3 sialyltransferase knockouts when combined with α-2,6 sialyltransferase overexpression. Indeed, α-2,3 linked sialic acids were not detected on IgG produced from the α-2,3 sialyltransferase knockout-α-2,6 sialyltransferase overexpression pools. Finally, glycoprofiling of IgG with four amino acid substitutions expressed from an α-2,3 sialyltransferase knockout-α-2,6 sialyltransferase stable clone resulted in more than 77% sialylated glycans and more than 62% biantennary disialylated glycans as indicated by both MALDI-TOF and LC-ESI-MS. Engineered antibodies from these modified Chinese hamster ovary cell lines will provide biotechnologists with IgGs containing N-glycans with different structural variations for examining the role of glycosylation on protein performance.
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Assessment of the coordinated role of ST3GAL3, ST3GAL4 and ST3GAL6 on the α2,3 Sialylation linkage of mammalian glycoproteins.
Biochemical and biophysical research communications, 2015Co-Authors: Cheng-yu Chung, Bojiao Yin, Qiong Wang, Kai-yun Chuang, Jeffrey H. Chu, Michael J. BetenbaughAbstract:In this research, we examined which genes are involved in N-linked Sialylation in Chinese Hamster Ovary (CHO) cells using siRNA knockdown approaches. Three genes from the sialyltransferase family (ST3GAL3, ST3GAL4 and ST3GAL6) were chosen as knockdown targets with siRNA applied to reduce their expression. Single, double and triple gene knockdowns were investigated, and the reduction levels of Sialylation on the total cell lysate were monitored by enzyme-linked lectin absorption assays (ELLA) and sialic acid quantification with high performance liquid chromatography (HPLC). All transfection groups showed effective reduction in 2,3-linked Sialylation whereas the trend of reduction levels of triple siRNA transfection outweighed both the dual siRNA groups and single siRNA transfection groups. Next, this transfection approach was applied to CHO cells producing erythropoietin (EPO). Quantification of EPO Sialylation showed similar result to total cell lysate except that the ST3GAL4 siRNA transfection exhibited the largest reduction according to the HPLC analysis as compared with other single siRNA transfections. Finally, the N-glycan released from the EPO transfected with ST3GAL4 siRNA showed a prominent reduction in sialyation level among the single siRNA transfections. From these experiments, we concluded that each of these three genes were involved in N-linked Sialylation and ST3GAL4 may play the critical role in glycoprotein Sialylation of recombinant proteins such as EPO.
Olga Gornik - One of the best experts on this subject based on the ideXlab platform.
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Change of transferrin Sialylation differs between mild sepsis and severe sepsis and septic shock.
Internal medicine (Tokyo Japan), 2011Co-Authors: Olga Gornik, Ivan Gornik, Ivana Zagorec Kolednjak, Gordan LaucAbstract:Objective and Design To investigate the association between the severity of sepsis and changes in Sialylation of serum proteins we have conducted a single center pilot study. Subjects and Methods Sialylation of transferrin (with enzyme-linked lectin assay-ELLA) and total serum proteins (with colorimetric assay) as well as serum iron and transferrin levels were measured in 27 patients with sepsis through the first eight days of the disease. Results Total serum Sialylation increased in the first two days, transferrin Sialylation decreased, while serum iron and transferrin fell. Patients who developed severe sepsis had either a small or marked change in transferrin Sialylation while in patients with mild sepsis Sialylation decreased moderately. Conclusion We hypothesize that the change in transferrin Sialylation could be a reflection of the intensity of inflammatory response which is insufficient if under-expressed and detrimental if over-expressed. This new feature is a potential marker of sepsis severity early in the disease.
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Change in transferrin Sialylation is a potential prognostic marker for severity of acute pancreatitis.
Clinical biochemistry, 2008Co-Authors: Olga Gornik, Ivan Gornik, Vladimir Gasparović, Gordan LaucAbstract:Early prediction of severe acute pancreatitis is one of the problems in clinical practice. Since many diseases are associated with alteration in glycosylation, in this work we studied Sialylation of transferrin and serum proteins in acute pancreatitis. Sialylation was analyzed during first eight days of hospitalization of 30 patients and compared to 28 healthy controls. Transferrin Sialylation was measured using enzyme linked lectin assay, while sialic acid on proteins was measured using resorcinol method. Both analyzed parameters changed during studied period. The change in transferrin Sialylation between Day1 and Day2 of hospitalization was shown to be an early prognostic marker of acute pancreatitis, with better sensitivity (88.9%) and specificity (90.5%) than other markers tested. Sialylation of transferrin and total serum proteins reflects the intensity of inflammatory response during acute pancreatitis and could be used as prognostic parameter for disease severity.
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Change in transferrin Sialylation is a potential prognostic marker for severity of acute pancreatitis.
Clinical Biochemistry, 2008Co-Authors: Olga Gornik, Ivan Gornik, Vladimir Gasparović, Gordan LaucAbstract:Abstract Objectives Early prediction of severe acute pancreatitis is one of the problems in clinical practice. Since many diseases are associated with alteration in glycosylation, in this work we studied Sialylation of transferrin and serum proteins in acute pancreatitis. Design and methods Sialylation was analyzed during first eight days of hospitalization of 30 patients and compared to 28 healthy controls. Transferrin Sialylation was measured using enzyme linked lectin assay, while sialic acid on proteins was measured using resorcinol method. Results Both analyzed parameters changed during studied period. The change in transferrin Sialylation between Day1 and Day2 of hospitalization was shown to be an early prognostic marker of acute pancreatitis, with better sensitivity (88.9%) and specificity (90.5%) than other markers tested. Conclusions Sialylation of transferrin and total serum proteins reflects the intensity of inflammatory response during acute pancreatitis and could be used as prognostic parameter for disease severity.
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Enzyme linked lectin assay (ELLA) for direct analysis of transferrin Sialylation in serum samples.
Clinical biochemistry, 2007Co-Authors: Olga Gornik, Gordan LaucAbstract:Glycosylation analysis provides many opportunities for diagnostics, but its complexity hampers its routine application. Aiming to alleviate this problem, we developed a simple assay that can measure Sialylation of transferrin directly from serum. Transferrin samples with different levels of Sialylation were prepared by deSialylation. Enzyme-linked-lectin assay (ELLA) and high-performance anion-exchange chromatography (HPAEC) have been used to analyze transferrin Sialylation. Periodate oxidation was used to oxidize carbohydrates on antibodies. ELLA was developed for the analysis of serum transferrin Sialylation. Antibodies oxidized in situ with periodate have been used to capture transferrin from serum samples. Sialic acid on transferrin has been detected with Sambucus nigra agglutinin (SNA) lectin. Transferrin samples with different Sialylation levels prepared by differential deSialylation have been used as standards. Accuracy of the method has been confirmed by comparison to HPAEC analysis. A rapid and simple ELLA that can be routinely used for the analysis of serum transferrin Sialylation has been developed.
Cheng-yu Chung - One of the best experts on this subject based on the ideXlab platform.
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The impact of Sialylation linkage-type on the pharmacokinetics of recombinant butyrylcholinesterases
Biotechnology and bioengineering, 2019Co-Authors: Cheng-yu Chung, Qiong Wang, Shuang Yang, Sandra Chough, Younji Seo, John F. Cipollo, Joseph P. Balthasar, Michael J. BetenbaughAbstract:Chinese hamster ovary (CHO) cells typically produce glycoproteins with N-glycans terminating in α-2,3 Sialylation. Human cells produce glycoproteins that include α-2,3 and α-2,6 sialic acids. To examine the impact of altering protein Sialylation on pharmacokinetic properties, recombinant human butyrylcholinesterase (BChE) was produced in CHO cells by knocking out the α-2,3 sialyltransferase genes followed by overexpression of the α-2,6 sialyltransferase (26BChE) enzyme. The N-glycan composition of 26BChE was compared to BChE with α-2,3 Sialylation (23BChE) derived from wild-type CHO cells. Both 23BChE and 26BChE exhibited comparable antennarity distributions with bi-antennary di-sialylated glycans representing the most abundant glycoform. CD-1 mice were intravenously injected with the 23BChE or 26BChE, and residual BChE activities from blood collected at various time points for pharmacokinetic analyses. Although 23BChE contained a slightly lower initial Sialylation level compared to 26BChE, the molecule exhibited higher residual activity between 5 and 24 hr postinjection. Pharmacokinetic analyses indicated that 23BChE exhibited an increase in area under the curve and a lower volume of distribution at steady state than that of 26BChE. These findings suggest that the type of Sialylation linkage may play a significant role in the pharmacokinetic behavior of a biotherapeutic when tested in in vivo animal models.
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integrated genome and protein editing swaps α 2 6 Sialylation for α 2 3 sialic acid on recombinant antibodies from cho
Biotechnology Journal, 2017Co-Authors: Cheng-yu Chung, Bojiao Yin, Qiong Wang, Shuang Yang, Hui Zhang, Michael J. BetenbaughAbstract:Immunoglobin G with α-2,6 Sialylation has been reported to have an impact on antibody-dependent cellular cytotoxicity and anti-inflammatory efficacy. However, production of antibodies with α-2,6 Sialylation from Chinese hamster ovary cells is challenging due to the inaccessibility of sialyltransferases for the heavy chain N-glycan site and the presence of exclusively α-2,3 sialyltransferases. In this study, combining mutations on the Fc regions to allow sialyltransferase accessibility with overexpression of α-2,6 sialyltransferase produced IgG with significant levels of both α-2,6 and α-2,3 Sialylation. Therefore, ST3GAL4 and ST3GAL6 genes were disrupted by CRISPR/Cas9 to minimize the α-2,3 Sialylation. Sialidase treatment and SNA lectin blot indicated greatly increased α-2,6 Sialylation level relative to α-2,3 Sialylation for the α-2,3 sialyltransferase knockouts when combined with α-2,6 sialyltransferase overexpression. Indeed, α-2,3 linked sialic acids were not detected on IgG produced from the α-2,3 sialyltransferase knockout-α-2,6 sialyltransferase overexpression pools. Finally, glycoprofiling of IgG with four amino acid substitutions expressed from an α-2,3 sialyltransferase knockout-α-2,6 sialyltransferase stable clone resulted in more than 77% sialylated glycans and more than 62% biantennary disialylated glycans as indicated by both MALDI-TOF and LC-ESI-MS. Engineered antibodies from these modified Chinese hamster ovary cell lines will provide biotechnologists with IgGs containing N-glycans with different structural variations for examining the role of glycosylation on protein performance.
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Assessment of the coordinated role of ST3GAL3, ST3GAL4 and ST3GAL6 on the α2,3 Sialylation linkage of mammalian glycoproteins.
Biochemical and biophysical research communications, 2015Co-Authors: Cheng-yu Chung, Bojiao Yin, Qiong Wang, Kai-yun Chuang, Jeffrey H. Chu, Michael J. BetenbaughAbstract:In this research, we examined which genes are involved in N-linked Sialylation in Chinese Hamster Ovary (CHO) cells using siRNA knockdown approaches. Three genes from the sialyltransferase family (ST3GAL3, ST3GAL4 and ST3GAL6) were chosen as knockdown targets with siRNA applied to reduce their expression. Single, double and triple gene knockdowns were investigated, and the reduction levels of Sialylation on the total cell lysate were monitored by enzyme-linked lectin absorption assays (ELLA) and sialic acid quantification with high performance liquid chromatography (HPLC). All transfection groups showed effective reduction in 2,3-linked Sialylation whereas the trend of reduction levels of triple siRNA transfection outweighed both the dual siRNA groups and single siRNA transfection groups. Next, this transfection approach was applied to CHO cells producing erythropoietin (EPO). Quantification of EPO Sialylation showed similar result to total cell lysate except that the ST3GAL4 siRNA transfection exhibited the largest reduction according to the HPLC analysis as compared with other single siRNA transfections. Finally, the N-glycan released from the EPO transfected with ST3GAL4 siRNA showed a prominent reduction in sialyation level among the single siRNA transfections. From these experiments, we concluded that each of these three genes were involved in N-linked Sialylation and ST3GAL4 may play the critical role in glycoprotein Sialylation of recombinant proteins such as EPO.
Loïc Guillevin - One of the best experts on this subject based on the ideXlab platform.
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Sialylation levels of anti proteinase 3 antibodies are associated with the activity of granulomatosis with polyangiitis wegener s
Arthritis & Rheumatism, 2011Co-Authors: Cécile Espy, Willy Morelle, Niloufar Kavian, Philippe A. Grange, Claire Goulvestre, Vivian Viallon, Christiane Chéreau, Christian Pagnoux, Jean-claude Michalski, Loïc GuillevinAbstract:Objective. To investigate whether the glycosylation and Sialylation levels of anti–proteinase 3 (antiPR3) antibodies could affect their pathogenicity, and whether these levels could be correlated with the activity of granulomatosis with polyangiitis (Wegener’s) (GPA). Methods. Forty-two serum samples positive for anti-PR3 antibodies from 42 patients with active or weakly active/inactive GPA were included. Anti-PR3 antibodies were assayed by enzyme-linked immunosorbent assay, and their levels of glycosylation and Sialylation were assessed by enzyme-linked lectin assay. The glycosylation and Sialylation levels of IgG purified from the serum of healthy donors and patients with active, remitted, or weakly active disease were assessed by permethylation and mass spectrometry analysis of glycans, following neuraminidase digestion. The neutrophil oxidative burst induced by purified IgG was assayed by spectrofluorimetry. Results. The mean Sialylation ratio of anti-PR3 antibodies was significantly lower in patients with active disease than in patients with weakly active or inactive disease, and this was inversely correlated with the Birmingham Vasculitis Activity Score (BVAS) (P < 0.0001). Similar results were obtained using the BVAS/ GPA. The area under the receiver operating characteristic curve for the Sialylation ratio of anti-PR3 antibodies, as a test to determine the activity of GPA, was 0.82 (P 0.0006). The characterization of N-glycans showed a decrease in 2,6-linked sialylated N-glycans and an increase in dHex1Hex3HexNAc4 (mass/charge 1,836) agalactosylated structures in purified IgG from patients with active disease compared with controls. The antiPR3 antibody–induced oxidative burst of neutrophils was inversely correlated with the Sialylation levels of anti-PR3 IgG. Conclusion. The Sialylation level of anti-PR3 antibodies contributes to the clinical activity of GPA, by modulating the oxidative burst of neutrophils induced by these autoantibodies.
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Sialylation levels of anti–proteinase 3 antibodies are associated with the activity of granulomatosis with polyangiitis (Wegener's)
Arthritis and rheumatism, 2011Co-Authors: Cécile Espy, Willy Morelle, Niloufar Kavian, Philippe A. Grange, Claire Goulvestre, Vivian Viallon, Christiane Chéreau, Christian Pagnoux, Jean-claude Michalski, Loïc GuillevinAbstract:Objective. To investigate whether the glycosylation and Sialylation levels of anti–proteinase 3 (antiPR3) antibodies could affect their pathogenicity, and whether these levels could be correlated with the activity of granulomatosis with polyangiitis (Wegener’s) (GPA). Methods. Forty-two serum samples positive for anti-PR3 antibodies from 42 patients with active or weakly active/inactive GPA were included. Anti-PR3 antibodies were assayed by enzyme-linked immunosorbent assay, and their levels of glycosylation and Sialylation were assessed by enzyme-linked lectin assay. The glycosylation and Sialylation levels of IgG purified from the serum of healthy donors and patients with active, remitted, or weakly active disease were assessed by permethylation and mass spectrometry analysis of glycans, following neuraminidase digestion. The neutrophil oxidative burst induced by purified IgG was assayed by spectrofluorimetry. Results. The mean Sialylation ratio of anti-PR3 antibodies was significantly lower in patients with active disease than in patients with weakly active or inactive disease, and this was inversely correlated with the Birmingham Vasculitis Activity Score (BVAS) (P < 0.0001). Similar results were obtained using the BVAS/ GPA. The area under the receiver operating characteristic curve for the Sialylation ratio of anti-PR3 antibodies, as a test to determine the activity of GPA, was 0.82 (P 0.0006). The characterization of N-glycans showed a decrease in 2,6-linked sialylated N-glycans and an increase in dHex1Hex3HexNAc4 (mass/charge 1,836) agalactosylated structures in purified IgG from patients with active disease compared with controls. The antiPR3 antibody–induced oxidative burst of neutrophils was inversely correlated with the Sialylation levels of anti-PR3 IgG. Conclusion. The Sialylation level of anti-PR3 antibodies contributes to the clinical activity of GPA, by modulating the oxidative burst of neutrophils induced by these autoantibodies.
