Human Indoleamine 2,3-dioxygenase 1 (IDO1) Expressed in Plant Cells Induces Kynurenine Production.

Michele Bellucci,Andrea Pompa,Elena Orecchini,Elisa Maricchiolo,Eleonora Panfili,Maria Teresa Pallotta,Daniele Fraternale,Ciriana Orabona,Carine De Marcos Lousa,Francesca De Marchis

doi:10.3390/ijms22105102

Abstract

Genetic engineering of plants has turned out to be an attractive approach to produce various secondary metabolites. Here, we attempted to produce kynurenine, a health-promoting metabolite, in plants of Nicotiana tabacum (tobacco) transformed by Agrobacterium tumefaciens with the gene, coding for human indoleamine 2,3-dioxygenase 1 (IDO1), an enzyme responsible for the kynurenine production because of tryptophan degradation. The presence of IDO1 gene in transgenic plants was confirmed by PCR, but the protein failed to be detected. To confer higher stability to the heterologous human IDO1 protein and to provide a more sensitive method to detect the protein of interest, we cloned a gene construct coding for IDO1-GFP. Analysis of transiently transfected tobacco protoplasts demonstrated that the IDO1-GFP gene led to the expression of a detectable protein and to the production of kynurenine in the protoplast medium. Interestingly, the intracellular localisation of human IDO1 in plant cells is similar to that found in mammal cells, mainly in cytosol, but in early endosomes as well. To the best of our knowledge, this is the first report on the expression of human IDO1 enzyme capable of secreting kynurenines in plant cells.

Highlights

The production of recombinant proteins in plants dates back to the end of 1980s [1], and since many recombinant proteins have been produced in plants for different uses, spanning from biomedical applications to industrial production [2,3]
The IDO1 ability to restrain inflammation depends on two distinct functions of the IDO1 protein: one is enzymatic while the other is independent from its enzymatic function
Several recombinant human proteins with therapeutic activity have been produced in plants, for example, the enzyme alpha-mannosidase is produced in stably transformed plants for the replacement therapy of the lysosomal storage disease alpha-mannosidosis

Summary

Introduction

The production of recombinant proteins in plants dates back to the end of 1980s [1], and since many recombinant proteins have been produced in plants for different uses, spanning from biomedical applications to industrial production [2,3]. IDO1 is a hemecontaining enzyme that catalyses the oxidative cleavage of the indole ring in L-tryptophan, regulating the catabolism of this essential amino acid at an initial, rate-limiting level in a specific pathway. This activity leads to the kynurenine production, which is the upstream metabolite along the so-called kynurenine pathway [14]. Apart from being a cytoplasmic enzyme, IDO1 acts as a signal-transducing molecule by binding tyrosine phosphatases in its immunoreceptor tyrosine-based inhibitory motif (ITIM) domains [23] and anchoring to the early endosomes This localisation is a result of the IDO1 interaction with activated class IA PI3K subunits [24]. The IDO1 ability to restrain inflammation depends on two distinct functions of the IDO1 protein: one is enzymatic (tryptophan degradation to kynurenine) while the other is independent from its enzymatic function

Methods

Results

Conclusion