Characterization of an antagonist interleukin-6 dimer by stable isotope labeling, cross-linking, and mass spectrometry

Taverner, T; Hall, NE; O&#39;Hair, RAJ; Simpson, RJ

Author(s): Taverner, T; Hall, NE; O'Hair, RAJ; Simpson, RJ;
Details: Publication Year 2002-11-29,Volume 277,Issue #48,Page 46487-46492
Journal Title: JOURNAL OF BIOLOGICAL CHEMISTRY
Publication Type: Journal Article
Abstract: The homodimeric form of a recombinant cytokine interleukin-6 (IL-6(D)) is known to antagonize IL-6 signaling. In this study, spatially proximal residues between IL-6 chains in IL-6, were identified using a method for specific recognition of intermolecular cross-linked peptides. Our strategy involved mixing 1:1 N-15-labeled and unlabeled (N-14) protein to form a mixture of isotopically labeled and unlabeled homodimers, which was chemically cross-linked. This cross-linked IL-6(D) was subjected to proteolysis by trypsin and the generated peptides were analyzed by electrospray ionization time-of-flight mass spectrometry (MS). Molecular ions from crosslinked peptides of intermolecular origin are labeled with [N-15/N-15] + [N-15/N-14] + [N-14/N-15] + [N-14/N-14] yielding readily identified triplet/quadruplet MS peaks. All other peptide species are labeled with [N-15] + [N-14] yielding doublet peaks. Intermolecular cross-linked peptides were identified by MS, and cross-linked residues were identified. This intermolecular cross-link detection method, which we have designated "mixed isotope cross-linking" MIX may have more general application to protein-protein interaction studies. The pattern of proximal residues found was consistent with IL-6(D) having a domain-swapped fold similar to IL-10 and interferon-gamma. This fold implies that IL-6(D)-mediated antagonism of IL-6 signaling is caused by obstruction of cooperative gp130 binding on IL-6(D), rather than direct blocking of gp-130-binding sites on IL-6(D).
Publisher: AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
Keywords: RECOMBINANT MURINE INTERLEUKIN-6; CRYSTAL-STRUCTURE; RECEPTOR COMPLEX; IL-6 RECEPTOR; BINDING; REAGENTS; DOMAIN; GP130; CELLS; IDENTIFICATION
Publisher's Version: https://doi.org/10.1074/jbc.M207370200
Terms of Use/Rights Notice: Refer to copyright notice on published article.

Creation Date: 2002-11-29 12:00:00