Mass spectrometric mapping of disulfide bonds in recombinant human interleukin-13

Abstract

Interleukin 13 (IL-13), a member of the a-helical family of cytokines, has ∼30% primary sequence homology with IL-4 and shares a common receptor component. The biologically active rhIL-13 is monomeric and nonglycosylated, 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 disulfidelinked 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 a-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.

Interleukin-13 as an important cytokine: A review on its roles in some human diseases

“…The 25-amino-acid hydrophobic structural core is completely conserved between IL-13 and IL-4, with only some conservative hydrophobic substitutions, however IL-13 only shares 25% homology with IL-4 at the amino acid level [18]. IL-4 contains 3 disulfi de bridges and in contrast mass spectrophotometry revealed that L-13 contains only two bridges [19].…”