The Expression and Characterization of Functionally Active Soluble CD83 by Pichia pastoris Using High-Density Fermentation

Yugang Guo,Jiajia Ma,Rui Li,Wenyao Kang,Yongjun Zhong,Dong Wang,Zhigang Tian,Hao Jia,Lidan Wu,Jie Sun,Weihua Xiao,Chenguang Wang,Xiaoping Song,Fang Fang,Muriel Moser

doi:10.1371/journal.pone.0089264

Abstract

CD83 is a highly glycosylated type I transmembrane glycoprotein that belongs to the immunoglobulin superfamily. CD83 is upregulated during dendritic cell (DC) maturation, which is critical for the initiation of adaptive immune responses. The soluble isoform of CD83 (sCD83) is encoded by alternative splicing from full-length CD83 mRNA and inhibits DC maturation, which suggests that sCD83 acts as a potential immune suppressor. In this study, we developed a sound strategy to express functional sCD83 from Pichia pastoris in extremely high-density fermentation. Purified sCD83 was expressed as a monomer at a yield of more than 200 mg/L and contained N-linked glycosylation sites that were characterized by PNGase F digestion. In vitro tests indicated that recombinant sCD83 bound to its putative counterpart on monocytes and specifically blocked the binding of anti-CD83 antibodies to cell surface CD83 on DCs. Moreover, sCD83 from yeast significantly suppressed ConA-stimulated PBMC proliferation. Therefore, sCD83 that was expressed from the P. pastoris was functionally active and may be used for in vivo and in vitro studies as well as future clinical applications.

Highlights

CD83 is a highly glycosylated type I transmembrane glycoprotein with 186 amino acids
Full-length CD83 is a 45-kDa highly glycosylated transmembrane protein that consists of 186 amino acids and extracellular, transmembrane, and intracellular domains
The higher level of expression of sCD83 was obtained when it was cultured at 25uC, while significant reductions of total expression levels and the proportion of glycosylated sCD83 were observed at 20uC cultures, suggesting that the culture temperature has a great impact on the intensity of peripheral blood mononuclear cell (PBMC) were incubated with soluble CD83 or bovine serum albumin (BSA), and coated with either PE-anti-CD83 (A) or anti-His followed by FITC-Anti-mouse IgG (B)

Summary

Introduction

CD83 is a highly glycosylated type I transmembrane glycoprotein with 186 amino acids. CD83 is upregulated during dendritic cell (DC) maturation and plays an essential role in the initiation of adaptive immune responses [1,2]. Recent studies have demonstrated that CD83 is expressed in most immune cells and plays an important role in regulating innate and adaptive immune responses, including mediating the activation of T cells by DCs [3,4,5,6,7,8], controlling the thymic maturation and activation of CD4+ single-positive lymphocytes, and maintaining the homeostasis of B lymphocytes [9,10]. In addition to the transmembrane form of CD83, an alternative isoform of soluble CD83 (sCD83), which results from the alternative splicing of full-length CD83, had been found in the serum of healthy adults and patients with leukemia, malignant tumors, and rheumatoid arthritis [8,11,12,13,14]. SCD83 represents a potential therapeutic tool and immune regulation target

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Conclusion