title: Probing protease sensitivity of recombinant human erythropoietin reveals α3-α4 inter-helical loop as a stability determinant.
abstract: 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.
authors.affiliation: CSIR- Institute of Microbial Technology, Sector-39A, Chandigarh, 160036, India.
authors.forename: Jesse
authors.initials: J
authors.lastname: Sebastian Samuel
authors.affiliation: CSIR- Institute of Microbial Technology, Sector-39A, Chandigarh, 160036, India.
authors.forename: Deepak
authors.initials: D
authors.lastname: Kumar
authors.affiliation: CSIR- Institute of Microbial Technology, Sector-39A, Chandigarh, 160036, India.
authors.forename: Sathi Babu
authors.initials: SB
authors.lastname: Chodisetti
authors.affiliation: CSIR- Institute of Microbial Technology, Sector-39A, Chandigarh, 160036, India.
authors.forename: Javed N
authors.initials: JN
authors.lastname: Agrewala
authors.affiliation: CSIR- Institute of Microbial Technology, Sector-39A, Chandigarh, 160036, India.
authors.forename: Balvinder
authors.initials: B
authors.lastname: Singh
authors.affiliation: Department of Biological Sciences, Indian Institute of Science Education & Research (IISER) Mohali, Punjab, 140306, India.
authors.forename: Purnananda
authors.initials: P
authors.lastname: Guptasarma
authors.affiliation: CSIR- Institute of Microbial Technology, Sector-39A, Chandigarh, 160036, India.
authors.forename: Dibyendu
authors.initials: D
authors.lastname: Sarkar
mesh_terms: D000595:Amino Acid Sequence; D056668:Cathepsin L / Q000378:metabolism; D045744:Cell Line, Tumor; D004921:Erythropoietin / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D055550:Protein Stability; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D004921:Erythropoietin; D056668:Cathepsin L
keywords: cathepsin L; circulating half-life; in vivo clearance; inter-helical loop region; proteolysis; recombinant HuEpo