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Probing protease sensitivity of recombinant human erythropoietin reveals α3–α4 inter‐helical loop as a stability determinant
Author(s) -
Sebastian Samuel Jesse,
Kumar Deepak,
Chodisetti Sathi Babu,
Agrewala Javed N.,
Singh Balvinder,
Guptasarma Purnananda,
Sarkar Dibyendu
Publication year - 2015
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.24865
Subject(s) - recombinant dna , erythropoietin , protease , loop (graph theory) , sensitivity (control systems) , chemistry , biophysics , biology , biochemistry , genetics , enzyme , mathematics , engineering , gene , combinatorics , electronic engineering
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. Proteins 2015; 83:1813–1822. © 2015 Wiley Periodicals, Inc.