Disulfide bond assignments and secondary structure analysis of human and murine interleukin 10.
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.
Windsor
W T
WT
Schering-Plough Research Institute, Kenilworth, New Jersey 07033.
Syto
R
R
Tsarbopoulos
A
A
Zhang
R
R
Durkin
J
J
Baldwin
S
S
Paliwal
S
S
Mui
P W
PW
Pramanik
B
B
Trotta
P P
PP
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D006224:Cricetinae; D004220:Disulfides / Q000032:analysis*; D006801:Humans; D016753:Interleukin-10 / Q000737:chemistry*; D051379:Mice; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D013438:Sulfhydryl Compounds / Q000032:analysis
D004220:Disulfides; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D016753:Interleukin-10