Identification of O-linked oligosaccharide chains in the activation peptides of blood coagulation factor X

The role of the carbohydrate moieties in the activation of factor X

Authors


Correspondence to T. Morita, Department of Biochemistry, Meiji College of Pharmacy, Tanashi, Tokyo, Japan 188

Abstract

Conversion of factor X to factor Xa results in release of a heavily glycosylated activation peptide. Analysis of protease-digested glycopeptides derived from the activation peptides of bovine and human blood coagulation factor X allowed the identification of sites of the O-linked oligosaccharide chains in these peptides. Glycopeptides were prepared from the activation peptides by digestion with chymotrypsin or Staphylococcus aureus V8 protease. By combined analysis of amino acid sequence and sialic acid content, we found that bovine factor X had an O-linked oligosaccharide chain linked to Thr26, and human factor X had four carbohydrate-attachment sites, namely, O-glycosidic linkages to Thr17 and Thr29, respectively, and N-glycosidic linkages to Asn39 and Asn49, respectively, in their activation peptides. The O-linked carbohydrate-attachment sites were identified since the yields of phenylthiohydantoin derivatives of amino acids that corresponded to their residues were increased during amino acid sequencing after deglycosylation of the glycopeptides with sialidase and O-glycanase. The effect of deglycosylation of bovine factor X1 was investigated with factor-X-activating enzyme from Russell's viper venom or extrinsic Xase (factor VIIa/tissue factor/phosholipid) by examining the activation rates of derivatives of factor X prepared using O-glycanase, sialidase, and/or N-glycanase. The removal of O-linked carbohydrate resulted in a decrease in the rate of activation. It appears that carbohydrate residues in factor X play an important role in the activation of the zymogen.

Abbreviations
AP

activation peptide

AP1 and AP2

the activation peptides (residues 1–51 of the heavy chain) of bovine factor X1 and X2, respectively

Pth

phenylthiohydantoin

RVV-XCP

the factor-X-activating enzyme from Russell's viper venom

Boc

tert-butoxycarbonyl

NH-Np

p-nitroanilide

Enzymes
 

Coagulation factor Xa (EC 3.4.21.6)

 

peptide N-glycosidase F (EC 3.5.1.52)

 

endo-α-N-acetylgalactosaminidase (EC 3.2.1.97)

 

sialidase (EC 3.2.1.18)

 

chymotrypsin (EC 3.4.21.1)

 

Staphylococcus aureus V8 protease (EC 3.4.21.19)

 

pepsin (EC 3.4.23.1)

Ancillary