A Polyglutamic Acid Motif Confers IL-27 Hydroxyapatite and Bone-Binding Properties

A common design principle of heteromeric signaling proteins is the use of shared subunits. This allows encoding of complex messages while maintaining evolutionary flexibility. How cells regulate and control assembly of such composite signaling proteins remains an important open question. An example of particular complexity and biological relevance is the interleukin 12 (IL-12) family. Four functionally distinct αβ heterodimers are assembled from only five subunits to regulate immune cell function and development. In addition, some subunits act as independent signaling molecules. Here we unveil key molecular mechanisms governing IL-27 biogenesis, an IL-12 family member that limits infections and autoimmunity. In mice, the IL-27α subunit is secreted as a cytokine, whereas in humans only heterodimeric IL-27 is present. Surprisingly, we find that differences in a single amino acid determine if IL-27α can be secreted autonomously, acting as a signaling molecule, or if it depends on heterodimerization for secretion. By combining computer simulations with biochemical experiments, we dissect the underlying structural determinants: a protein folding switch coupled to disulfide bond formation regulates chaperone-mediated retention versus secretion. Using these insights, we rationally change folding and assembly control for this protein. This provides the basis for a more human-like IL-27 system in mice and establishes a secretion-competent human IL-27α that signals on its own and can regulate immune cell function. Taken together, our data reveal a close link between protein folding and immunoregulation. Insights into the underlying mechanisms can be used to engineer immune modulators.

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“…3A). Next, we focused on the characteristic poly-Glu stretch of IL-27α (31). It was predicted to be unstructured ( Fig.…”

Section: Resultsmentioning

confidence: 99%

A folding switch regulates interleukin 27 biogenesis and secretion of its α-subunit as a cytokine

Müller

Friedl

Aschenbrenner

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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“…B9-IL-27R␣ and Ba/F3-gp130 cells were generated as described previously (20,47). The cells were grown in RPMI 1640 (Wisent Bio Products, St-Bruno, Canada) supplemented with 10% FBS, 10 mM HEPES, 2 mM L-glutamine, 1% nonessential amino acids, 100 units/ml penicillin, and 100 g/ml streptomycin (all from Wisent) in the presence of IL-6 (10 ng/ml; R&D Systems, Cedarlane) or IL-3 (10 ng/ml; PeproTech Inc., Cedarlane) and hygromycin (1 mg/ml) for B9-IL-27R␣ and Ba/F3-gp130, respectively.…”

Section: Cells and Reagentsmentioning

confidence: 99%

Epstein-Barr virus-induced gene 3 (EBI3) can mediate IL-6 trans-signaling

Chehboun

Labrecque-Carbonneau

Pasquin

et al. 2017

Journal of Biological Chemistry

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Epstein-Barr virus-induced gene 3 (EBI3) is a subunit of the composite cytokines IL-27 and IL-35. Both have beneficial functions or effects in models of infectious and autoimmune diseases. This suggests that administration of EBI3 could be therapeutically useful by binding free p28 and p35 to generate IL-27 and IL-35. IL-27- and IL-35-independent functions of EBI3 could compromise its therapeutic uses. We therefore assessed the effects of EBI3 on cytokine receptor-expressing cells. We observed that EBI3 activates STAT3 and induces the proliferation of the IL-6-dependent B9 mouse plasmacytoma cell line. Analyses using blocking mAbs and Ba/F3 transfectants expressing gp130 indicate that EBI3 activity was linked to its capacity to mediate IL-6 -signaling, albeit less efficiently than soluble IL-6Rα. In line with this interpretation, co-immunoprecipitation and SPR experiments indicated that EBI3 binds IL-6. An important pro-inflammatory function of IL-6 -signaling is to activate blood vessel endothelial cells. We observed that EBI3 in combination with IL-6 could induce the expression of chemokines by human venal endothelial cells. Our results indicate that EBI3 can promote pro-inflammatory IL-6 functions by mediating-signaling. These unexpected observations suggest that use of EBI3 as a therapeutic biologic for autoimmune diseases will likely require co-administration of soluble gp130 to prevent the side effects associated with IL-6 -signaling. Together with previous studies that demonstrated activation of IL-6R by p28 (IL-30), new findings further suggest a complex interrelation between IL-27 and IL-6.

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“…The p28 is structurally similar to an IL-6/IL-12 family, composed of a long chain of four α helix bundle named A-D from the N terminus to the C terminus 49 . The polypeptide loop connecting the p28 C and D α helices contains a stretch of polyglutamic acids (poly-E) unique among helical cytokines and is highly conserved 50 . The p28 alone can suppress IL-27 mediated Th1 responses 51 and IL-6 mediated signaling 52 .…”

Section: Molecular Characteristics Of Il-27mentioning

confidence: 99%

Biology of IL-27 and its Role in the Host Immunity against Mycobacterium Tuberculosis

Abdalla¹,

Li²,

Xie³

2015

Int. J. Biol. Sci.

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IL-27, a heterodimeric cytokine of IL-12 family, regulates both innate and adaptive immunity largely via Jak-Stat signaling. IL-27 can induce IFN-γ and inflammatory mediators from T lymphocytes and innate immune cells. IL-27 has unique anti-inflammatory properties via both Tr1 cells dependent and independent mechanisms. Here the role and biology of IL-27 in innate and adaptive immunity are summarized, with special interest with immunity against Mycobacterium tuberculosis.

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A Polyglutamic Acid Motif Confers IL-27 Hydroxyapatite and Bone-Binding Properties

Cited by 18 publications

References 65 publications

A folding switch regulates interleukin 27 biogenesis and secretion of its α-subunit as a cytokine

A folding switch regulates interleukin 27 biogenesis and secretion of its α-subunit as a cytokine

Epstein-Barr virus-induced gene 3 (EBI3) can mediate IL-6 trans-signaling

Biology of IL-27 and its Role in the Host Immunity against Mycobacterium Tuberculosis

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