Production of Recombinant Human Ceruloplasmin: Improvements and Perspectives.

Maria Carmela Bonaccorsi Di Patti,Zuzana Pakanová,Peter Baráth,Marek Nemčovič,Antimo Cutone,Giovanni Musci

doi:10.3390/ijms22158228

Abstract

The ferroxidase ceruloplasmin (CP) plays a crucial role in iron homeostasis in vertebrates together with the iron exporter ferroportin. Mutations in the CP gene give rise to aceruloplasminemia, a rare neurodegenerative disease for which no cure is available. Many aspects of the (patho)physiology of CP are still unclear and would benefit from the availability of recombinant protein for structural and functional studies. Furthermore, recombinant CP could be evaluated for enzyme replacement therapy for the treatment of aceruloplasminemia. We report the production and preliminary characterization of high-quality recombinant human CP in glycoengineered Pichia pastoris SuperMan5. A modified yeast strain lacking the endogenous ferroxidase has been generated and employed as host for heterologous expression of the secreted isoform of human CP. Highly pure biologically active protein has been obtained by an improved two-step purification procedure. Glycan analysis indicates that predominant glycoforms HexNAc2Hex8 and HexNAc2Hex11 are found at Asn119, Asn378, and Asn743, three of the canonical four N-glycosylation sites of human CP. The availability of high-quality recombinant human CP represents a significant advancement in the field of CP biology. However, productivity needs to be increased and further careful glycoengineering of the SM5 strain is mandatory in order to evaluate the possible therapeutic use of the recombinant protein for enzyme replacement therapy of aceruloplasminemia patients.

Highlights

The ferroxidase ceruloplasmin (CP) belongs to the family of the blue multicopper oxidases, characterized by the presence of multiple copper ions, which safely couple the oxidation of substrates to the controlled reduction of oxygen to water without release of potentially toxic intermediates [1,2]
We have previously reported the expression of secreted human CP in the yeast P. pastoris
If compared to the yields that can be obtained by the isolation of CP from human plasma where the concentration of the protein is about 300 mg/L, it is evident that there is no advantage in heterologous expression, where production levels are much lower

Summary

Introduction

The ferroxidase ceruloplasmin (CP) belongs to the family of the blue multicopper oxidases, characterized by the presence of multiple copper ions, which safely couple the oxidation of substrates to the controlled reduction of oxygen to water without release of potentially toxic intermediates [1,2]. Domains 2, 4, and 6 each harbor a type 1 copper site, where electrons are accepted from the substrate. Depending on the cell type, the ferroxidase activity is provided either by CP (soluble or GPI-linked) or by hephaestin, a multicopper oxidase involved in intestinal iron absorption [8] and iron efflux in neurons [9]. The connection between ferroxidases and ferroportin is further strengthened by the finding that the ferroxidase activity of CP or of hephaestin is required for the stability of cell surface ferroportin and the transporter is rapidly internalized and degraded in the absence of the ferroxidase [10,11]

Methods

Results

Conclusion