Disulfide bond assignments and secondary structure analysis of human and murine interleukin 10

Abstract

Interleukin 10 (IL-10), which was first discovered by its ability to inhibit the synthesis of various cytokines, most notably gamma interferon, from Th1 helper cells, displays pleiotropic immunoregulatory properties. Human and murine IL-10 have a high amino acid sequence identity (ca. 73%) which includes the conservation of all four cysteine residues in human IL-10 and the first four out of five cysteine residues for murine IL-10. Chemical analysis was used to determine that both recombinant human and recombinant murine IL-10 contain two disulfide bonds. The disulfide pairs for each were determined by mass spectrometric and reversed-phase HPLC analysis of trypsin-derived polypeptide fragments. The disulfide bond assignments for both species were similar in that the first cysteine residue in the sequence paired with the third and the second paired with the fourth. The fifth cysteine in murine IL-10 was determined by chemical modification to be unpaired. Far-UV circular dichroism analysis indicated that the secondary structure of recombinant human and murine IL-10 are composed of ca. 60% alpha-helix. Reduction of the disulfide bonds structurally destabilized the protein and led to a structure containing only 53% alpha-helix. The reduced protein displayed no in vitro biological activity in a mast cell proliferation assay. These studies indicate that IL-10 is a highly alpha-helical protein containing two disulfide bonds, either one or both of which are critical for its structure and function. In addition, these properties suggest that this interesting cytokine may belong to the alpha helical cytokine class of hematopoietic ligands.

Purification of receptor complexes of interleukin‐10

“…Several of these crystals were washed with the mother liquor, dissolved in water and analyzed by SDS/PAGE in order to assess their composition. We found that both hIL-10 and the three forms of shIL-10R1 (41, 36, and 33 kDa) were present in the crystals, indicating that 3 . Small crystals were captured from hanging drops and washed in MembFac solution #23, using a small nylon loop.…”

“…These crystals could be enlarged (0.35 mm £ 0.35 mm £ 0.5 mm) by serial seeding, but they did not diffract beyond 0.7 nm resolution. After further deglycosylation using endoF 3 , crystals were obtained in the same fashion using MembFac crystal screen solution #18 (12% polyethylene glycol 6000, 0.1 m sodium citrate pH 5.6, 0.1 m Li 2 SO 4 ). A crystal of size 0.1 mm £ 0.1 mm £ 0.05 mm diffracted to <0.45 nm resolution.…”

“…As shIL-10R1 remained in solution after deglycosylation with endoF 3 , an attempt was made to further purify the receptor by isoelectric focusing chromatography. After elution from a preparative monoP column (Pharmacia Biotech), three major isoforms of shIL-10R1 were observed, having isoelectric points of 7.0±7.2, 6.7 and 6.5, respectively (Fig.…”

“…This is probably due to the vIL-10 ligand becoming detached from the complex, as it is bound 1000-fold weaker than hIL-10. It is also possible that specific sugar residues, removed by endoF 3 , strengthen the binding of ligand within the complex.…”

“…The dimer corresponds to the biologically active form of the protein [3,4]. hIL-10 mediates its biological activities by interacting with specific cell surface receptors.…”