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Probing protease sensitivity of recombinant human erythropoietin reveals α3-α4 inter-helical loop as a stability determinant.

DIR@IMTECH: CSIR-Institute of Microbial Technology

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Title Probing protease sensitivity of recombinant human erythropoietin reveals α3-α4 inter-helical loop as a stability determinant.
 
Creator Sebastian Samuel, Jesse
Kumar, Deepak
Babu Chodisetti, Sathi
Agrewala, Javed N
Singh, Balvinder
Guptasarma, Purnananda
Sarkar, Dibyendu
 
Subject QR Microbiology
 
Description Although unglycosylated HuEpo is fully functional, it has very short serum half-life. However, the mechanism of in vivo clearance of human Epo (HuEpo) remains largely unknown. In this study, the relative importance of protease-sensitive sites of recombinant HuEpo (rHuEpo) has been investigated by analysis of structural data coupled with in vivo half-life measurements. Our results identify α3-α4 inter-helical loop region as a target site of lysosomal protease Cathepsin L. Consistent with previously-reported lysosomal degradation of HuEpo, these results for the first time identify cleavage sites of rHuEpo by specific lysosomal proteases. Furthermore, in agreement with the lowered exposure of the peptide backbone around the cleavage site, remarkably substitutions of residues with bulkier amino acids result in significantly improved in vivo stability. Together, these results have implications for the mechanism of in vivo clearance of the protein in humans.
 
Publisher Wiley Online
 
Date 2015-10
 
Type Article
PeerReviewed
 
Relation http://dx.doi.org/10.1002/prot.24865
http://crdd.osdd.net/open/1758/
 
Identifier Sebastian Samuel, Jesse and Kumar, Deepak and Babu Chodisetti, Sathi and Agrewala, Javed N and Singh, Balvinder and Guptasarma, Purnananda and Sarkar, Dibyendu (2015) Probing protease sensitivity of recombinant human erythropoietin reveals α3-α4 inter-helical loop as a stability determinant. Proteins, 83 (10). pp. 1813-22. ISSN 1097-0134