PMID: 25485983

 

    Legend: Gene, Sites

Title : Increased N-glycosylation efficiency by generation of an aromatic sequon on N135 of antithrombin

Abstract :
  1. The inefficient glycosylation of consensus sequence on N135 in antithrombin explains the two glycoforms of this key anticoagulant serpin found in plasma: α and β, with four and three N-glycans, respectively
  2. The lack of this N-glycan increases the heparin affinity of the β-glycoform
  3. Recent studies have demonstrated that an aromatic sequon ( Phe-Y-Asn-X-Thr) in reverse β-turns enhances N-glycosylation efficiency and stability of different proteins
  4. We evaluated the effect of the aromatic sequon in this defective glycosylation site of antithrombin , despite of being located in a loop between the helix D and the strand 2A
  5. We analyzed the biochemical and functional features of variants generated in a recombinant cell system ( HEK-EBNA)
  6. Cells transfected with wild-type plasmid (K133-Y-N135-X-S137) generated 50% of α and β- antithrombin
  7. The S137T, as previously reported, K133F, and the double mutant (K133F/S137T) had improved glycosylation efficiency, leading to the secretion of α- antithrombin , as shown by electrophoretic and mass analysis
  8. The presence of the aromatic sequon did not significantly affect the stability of this conformationally sensitive serpin , as revealed by thermal denaturation assay
  9. Moreover, the aromatic sequon hindered the activation induced by heparin, in which is involved the helix D. Accordingly, K133F and particularly K133F/S137T mutants had a reduced anticoagulant activity
  10. Our data support that aromatic sequons in a different structural context from reverse turns might also improve the efficiency of N-glycosylation
Output (sent_index, trigger, protein, sugar, site):
  • 1. glycoforms, , serpin, -, -
  • 1. glycosylation, , -, -, N135
  • 1. glycosylation, , antithrombin, -, N135
  • 1. glycosylation, , antithrombin, -, sequence
  • 4. glycosylation, , antithrombin, -, site
Output(Part-Of) (sent_index, protein, site):
  • 0. -, N135
  • 0. antithrombin, N135
  • 4. antithrombin, site
*Output_Site_Fusion* (sent_index, protein, sugar, site):
  • 1. antithrombin, -, N135

 

 

Protein NCBI ID SENTENCE INDEX
antithrombin 462 1,4,6,7