Elsevier

Experimental Cell Research

Volume 317, Issue 7, 15 April 2011, Pages 976-993
Experimental Cell Research

Research Article
Diverse functions of reactive cysteines facilitate unique biosynthetic processes of aggregate-prone interleukin-31

https://doi.org/10.1016/j.yexcr.2010.12.012Get rights and content

Abstract

Interleukin-31 (IL-31) is a member of the four helical-bundle gp130/IL-6 cytokine family. Despite its implicated roles in inflammatory diseases, the biosynthetic processes of IL-31 have been poorly investigated. A detailed understanding of IL-31 biosynthesis and the nature of ligand–receptor interactions can provide insights into effective strategies for the design of therapeutic approaches. By using various heterologous protein expression systems, we demonstrated that murine IL-31 was secreted as inter-molecularly disulfide-bonded covalent aggregates. Covalently aggregated IL-31 appeared while trafficking in the secretory pathway, but was not actively retained in the ER. The aggregate formation was not caused by a dysfunctional ER quality control mechanism or an intrinsic limitation in protein folding capacity. Furthermore, secreted IL-31 aggregates were part of a large complex composed of various pleiotropic secretory factors and immune-stimulators. The extent and the heterogeneous nature of aggregates may imply that IL-31 was erroneously folded, but it was capable of signaling through cognate receptors. Mutagenesis revealed the promiscuity of all five cysteines in inter-molecular disulfide formation with components of the hetero-aggregates, but no cysteine was required for IL-31 secretion itself. Our present study not only illustrated various functions that cysteines perform during IL-31 biosynthesis and secretion, but also highlighted their potential roles in cytokine effector functions.

Introduction

Nascent polypeptide chains that co-translationally enter the ER's oxidizing environment are subjected to stringent quality control which determines the subsequent intracellular fate of polypeptides. For instance, when protein folding is productive, secretory cargoes are allowed to exit the ER and are trafficked to the later secretory compartments for further modifications [1], [2]. If the folding attempts are unproductive, proteins may be allowed to go through additional folding cycles or they can be singled out for retrotranslocation to the cytosol where they are degraded by proteasomes [3], [4], [5]. Protein quality control persists even after proteins exit the ER in order to ensure that only the properly-folded proteins are exocytosed from the cells [6], [7].

Whether individual secretory glycoproteins can reach a thermodynamically stable conformation in the ER largely depends on the concerted action of molecular chaperones, lectin-like proteins, oxidoreductases, and their associated co-factors which constitute the protein folding and quality control mechanisms [8], [9]. A group of lectin-chaperones monitors the trimming status of individual N-linked glycans which is a key biochemical attribute that reflects folding status and the export readiness of client proteins [10]. Molecular chaperones monitor exposed hydrophobic residues which are typically buried to form a hydrophobic core that determines the packaging strategy and the stability of the fold [11]. Their solvent exposure often suggests that the folding clients have yet to reach their thermodynamically favorable state. The presence of reactive free thiol groups offers another protein quality attribute which is monitored by a family of oxidoreductases. Because the ER's oxidizing environment favors spontaneous disulfide formation, any erroneously bridged cysteine pairs must be isomerized until the correct pairings are achieved [12]. Oxidation of some cysteines may not take place until their cognate cysteine partners become available, whereas other cysteines are prevented from engaging in any disulfide bonding. Regardless of the protein folding/assembly difficulties, interactions with ER-resident proteins result in the retention of folding client proteins in the ER until they reach an export-competent conformation [13], [14].

Interleukin-31 (IL-31) is a recently-listed member of the four helical-bundle gp130/IL-6 cytokine family. Its mRNA is highly expressed in the activated Th2 CD4+ T cells, and the secreted cytokine is reported to signal through a heterodimeric receptor consisting of IL-31 receptor A (IL-31RA) and oncostatin M receptor beta (OSMRb) [15]. The receptor activation induces the expression of other pro-inflammatory cytokines and chemokines to amplify inflammatory responses [16]. IL-31 has been implicated in playing roles in various immunological disorders including atopic dermatitis and asthma through its pleiotropic effector functions [17], [18]. Neutralization of this pro-inflammatory cytokine or the blockade of the receptor signaling can thus be a promising path for treating pertinent inflammatory diseases. In order to develop effective therapeutic approaches, a detailed understanding of IL-31 biosynthesis, secretion, and the nature of ligand–receptor interactions is indispensable. Despite its importance in inflammatory disease settings, the biosynthetic processes of IL-31 have been poorly investigated partly because its mRNA is induced only under certain physiological conditions in limited cell populations. Detailed characterization of native IL-31 protein expressed in Th2 cells would be most physiologically relevant, but to circumvent technical challenges in inducing native IL-31 protein to a level amenable to cell biological and biochemical studies, we employed robust heterologous expression systems to characterize biosynthetic steps of recombinant IL-31 as well as the biochemical attributes of secreted recombinant IL-31.

Although numerous past studies using model cargoes such as VSV-G (ts045), immunoglobulins, α1-antitrypsin (null Hong Kong), etc. increased our conceptual and mechanistic understanding of the protein quality control system in the secretory pathway [19], we were puzzled to find that recombinant IL-31 formed inter-molecularly disulfide-bonded covalent aggregates while trafficking in the secretory pathway and was secreted as covalently aggregated complexes which widely ranged from 50 kDa to ~ 700 kDa in apparent size. In this report, we demonstrate that IL-31 covalent aggregate formation was not due to a lack of folding capacity or a deficiency in the ER quality control mechanism. We also show that the IL-31 aggregates were part of a large complex composed of various pleiotropic secretory factors and immune-stimulators. The extent and the heterogeneous nature of covalent aggregation might suggest that IL-31 was erroneously-folded and dysfunctional, but the ‘hetero-aggregates’ of IL-31 were capable of specifically binding to and activating through the cognate heterodimeric receptors. Mutagenesis revealed the promiscuity of all five cysteine residues within the mature domain of murine IL-31, but, surprisingly, no cysteine was required for protein secretion itself. Our study not only illustrated diverse functions of cysteine residues during the biosynthesis of IL-31, but also in the effector functions of IL-31 after secretion. The underlying mechanism of IL-31 aggregate formation and the biological relevance of macromolecular complex formation are discussed.

Section snippets

Covalent aggregate formation of murine interleukin-31 is not a post-secretion event

Production of bioactive glycoproteins is sometimes challenging because we often encounter secretory proteins whose behaviors are unconventional or at odds with the paradigm of ER quality control mechanisms. When full-length mu IL-31, with a C-terminal FLAG-6His (FH) tag, was expressed using a widely used cytomegalovirus immediate-early (CMV-IE) promoter/enhancer or an elongation factor 1α (EF1α) promoter, it was secreted from various heterologous mammalian hosts to a high level (~ 42 mg purified

Discussion

Technical challenges in the induction of native IL-31 protein to a detectable level hampered our efforts to perform similar studies using an endogenously expressed IL-31 secreted from activated mouse primary CD4+ T cells skewed to a Th2 phenotype. Due to the lack of practical and scalable methods to isolate native IL-31 protein, we used a surrogate system to understand the biosynthetic mechanisms of IL-31 by analyzing the characteristics of both intracellular and secreted recombinant IL-31

Antibodies

Mouse anti-FLAG (clone M2), rabbit anti-FLAG, and rabbit anti-calnexin were from Sigma-Aldrich. Rabbit anti-GPP130 was from Covance. Goat anti-mu IL-31, goat anti-galectin 3-binding protein, goat anti-SPARC, goat anti-LAP, and mouse anti-LTBP1 were from R&D Systems. Rabbit anti-thioredoxin, mouse anti-Hsp90, mouse anti-Hsp70, and mouse anti-LTBP1 were from Santa Cruz Biotechnology. Mouse anti-GAPDH was from Chemicon.

Chemicals and proteins

Unless specifically stated, all the chemicals were purchased from

Acknowledgment

The authors thank Mary Gerhart and Rick Sorenson for the technical assistance in N-terminal sequencing experiments and cell-based assay, respectively. We thank Jamie Harvey and Ian Dunleavy for a constant supply of HEK293(6E) suspension culture. We also thank Carla Forte, Elham Ettehadieh, Jue Zhang, and Shaunda Brouns for their help in making some of the expression constructs used in this study. We are indebted to Margaret Karow for her critical inputs on the manuscript.

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