Oxidation of Methionine Residues in Coagulation Factor VIIa

Kornfelt, Troels; Persson, Egon; Palm, Lisbeth

doi:10.1006/abbi.1998.1071

Cited by 40 publications

(26 citation statements)

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“…Complementary experiments confirmed this assumption and showed that these two peaks correspond to heavy chains bearing an oxidized methionine residue at position 298 and 306, respectively. This is in agreement with previously published data [28]. The other peaks originate from hydrolysis of HC either after Arg 290 or Arg 315 as deduced from accurate mass measurement (Table 1).…”

Section: Maldi-tofms Of Intact and N-deglycosylated Fviiasupporting

confidence: 92%

Mass spectrometric characterization of N- and O-glycans of plasma-derived coagulation factor VII

et al. 2008

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Factor VII (FVII) is a vitamin K-dependent glycoprotein which, in its activated form (FVIIa), participates in the coagulation process by activating factor X and factor IX. FVII is secreted as single peptide chain of 406 residues. Plasma-derived FVII undergoes many post-translational modifications such as gamma-carboxylation, N- and O-glycosylation, beta-hydroxylation. Despite glycosylation of recombinant FVIIa has been fully characterized, nothing is reported on the N- and O-glycans of plasma-derived FVII (pd-FVII) and on their structural heterogeneity at each glycosylation site. N- and O-glycosylation sites and site specific heterogeneity of pd-FVII were studied by various complementary qualitative and quantitative techniques. A MALDI-MS analysis of the native protein indicated that FVII is a 50.1 kDa glycoprotein modified on two sites by diantennary, disialylated non-fucosylated (A2S2) glycans. LC-ESIMS/MS analysis revealed that both light chain and heavy chain were N-glycosylated mainly by A2S2 but also by triantennary sialylated glycans. Nevertheless, lower amounts of triantennary structures were found on Asn(322) compared to Asn(145). Moreover, the triantennary glycans were shown to be fucosylated. In parallel, quantitative analysis of the isolated glycans by capillary electrophoresis indicated that the diantennary structures represented about 50% of the total glycan content. Glycan sequencing using different glycanases led to the identification of triantennary difucosylated structures. Last, MS and MS/MS analysis revealed that FVII is O-glycosylated on the light chain at position Ser(60) and Ser(52) which are modified by oligosaccharide structures such as fucose and Glc(Xyl)(0-1-2), respectively. These latter three O-glycans coexist in equal amounts in plasma-derived FVII.

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Section: Maldi-tofms Of Intact and N-deglycosylated Fviiasupporting

confidence: 92%

Mass spectrometric characterization of N- and O-glycans of plasma-derived coagulation factor VII

et al. 2008

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“…While the other two Met residues in hGH as well as the other five Met residues in hCS had dramatically different oxidation rates depending on their microenvironments. Different oxidation rates of Met residues in the same protein were also obtained for human parathyroid hormone [6], recombinant human granulocyte colony stimulating factor [7], recombinant interferon ␥ and recombinant tissue-type plasminogen activator [8], human cystatin C expressed in E. Coli [9], recombinant human leptin [10], recombinant coagulation factor exposed to H 2 O 2 [11] and wheat germ calmodulin [12]. Met oxidation is a concern for protein therapeutics due to the possible adverse effects such as in the case of recombinant human leptin [10], where decreased thermostability and a significant loss of in vitro bioactivity was observed when Met1 and Met69 were both oxidized.…”

Section: Introductionmentioning

confidence: 99%

Comparison of methionine oxidation in thermal stability and chemically stressed samples of a fully human monoclonal antibody

Chumsae

Gaza-Bulseco

Sun

et al. 2007

Journal of Chromatography B

174

158

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“…[7][8][9][10] Met oxidation can have adverse effects on proteins, including decreased stability, 5,[11][12][13][14][15][16] and decreased biological activity. 5,8,12,[17][18][19][20] The immunoglobulin gamma (IgG) monoclonal antibody (mAb) has emerged as one of the most promising drug class in the biopharmaceutical industry. More than 20 antibody drug products have been approved by the FDA for human therapeutic use across many clinical settings.…”

Section: Introductionmentioning

confidence: 99%

Methionine oxidation in human IgG2 Fc decreases binding affinities to protein A and FcRn

et al. 2009

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Susceptibility of methionine residues to oxidation is a significant issue of protein therapeutics. Methionine oxidation may limit the product's clinical efficacy or stability. We have studied kinetics of methionine oxidation in the Fc portion of the human IgG2 and its impact on the interaction with FcRn and Protein A. Our results confirm previously published observations for IgG1 that two analogous solvent-exposed methionine residues in IgG2, Met 252 and Met 428, oxidize more readily than the other methionine residue, Met 358, which is buried inside the Fc. Met 397, which is not present in IgG1 but in IgG2, oxidizes at similar rate as Met 358. Oxidation of two labile methionines, Met 252 and Met 428, weakens the binding of the intact antibody with Protein A and FcRn, two natural protein binding partners. Both of these binding partners share the same binding site on the Fc. Additionally, our results shows that Protein A may serve as a convenient and inexpensive surrogate for FcRn binding measurements.

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Oxidation of Methionine Residues in Coagulation Factor VIIa

Cited by 40 publications

References 0 publications

Mass spectrometric characterization of N- and O-glycans of plasma-derived coagulation factor VII

Mass spectrometric characterization of N- and O-glycans of plasma-derived coagulation factor VII

Comparison of methionine oxidation in thermal stability and chemically stressed samples of a fully human monoclonal antibody

Methionine oxidation in human IgG2 Fc decreases binding affinities to protein A and FcRn

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