Mass spectrometric mapping of disulfide bonds in recombinant human interleukin-13.
Interleukin 13 (IL-13), a member of the a-helical family of cytokines, has approximately 30% primary sequence homology with IL-4 and shares a common receptor component. The biologically active rhIL-13 is monomeric and non-glycosylated, and contains two disulfide bonds as determined by comparative electrospray mass spectrometric (MS) analysis of the protein before and after reduction with dithiothreitol-dithioerythritol. A trypsin-resistant core peptide of rhIL-13 was isolated and analyzed by plasma desorption (PD) MS, identifying a disulfide-linked core peptide. Subsequent digestion of this core peptide by pepsin, followed by PDMS analysis of the resulting cystine-containing peptic fragments, provided rapid determination of the existing disulfide bonds between cysteine residues 28-56 and 44-70. This disulfide arrangement is similar to that observed for the analogous four internal cysteine residues in hIL-4. The conservation of disulfide bond arrangements between hIL-13 and hIL-4, coupled with their alpha-helical structure and sequence homologies, confirms that IL-13 and IL-4 are structural homologues. It is also consistent with their reported similarities in biological function and receptor binding kinetics.
Tsarbopoulos
A
A
Department of Bioisolation Process Development, Schering-Plough Research Institute, Union, New Jersey 07083, USA. atsarbop@goulandr.ath.forthnet.gr
Varnerin
J
J
Cannon-Carlson
S
S
Wylie
D
D
Pramanik
B
B
Tang
J
J
Nagabhushan
T L
TL
D000595:Amino Acid Sequence; D004220:Disulfides / Q000737:chemistry; D006801:Humans; D018793:Interleukin-13 / Q000737:chemistry* / Q000235:genetics; D015847:Interleukin-4 / Q000737:chemistry / Q000235:genetics; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
D004220:Disulfides; D018793:Interleukin-13; D010446:Peptide Fragments; D011994:Recombinant Proteins; D015847:Interleukin-4