The Production of Human β-Glucocerebrosidase in Nicotiana benthamiana Root Culture.

Uthailak Naphatsamon,Takao Ohashi,Ryo Misaki,Kazuhito Fujiyama

doi:10.3390/ijms19071972

Abstract

Gaucher disease is caused by a deficiency of the enzyme glucocerebrosidase (GCase). Currently, enzyme-replacement therapy using recombinant GCase produced in mammalian cells is considered the most effective treatment. Plants are an attractive alternative host for recombinant protein production due to the low cost of large-scale production and lack of risk of contamination by human pathogens. Compared to whole plants, root cultures can grow faster. Therefore, this study aimed to produce recombinant GCase in a Nicotiana benthamiana root culture. Root culture of a GCase-producing transgenic plant was induced by indole-3-acetic acid at the concentration of 1 mg/L. Recombinant GCase was successfully produced in roots as a functional protein with an enzyme activity equal to 81.40 ± 17.99 units/mg total protein. Crude proteins were extracted from the roots. Recombinant GCase could be purified by concanavalin A and phenyl 650C chromatography. The productivity of GCase was approximately 1 µg/g of the root. A N-glycan analysis of purified GCase was performed using nano LC/MS. The Man3XylFucGlcNAc2 structure was predominant in purified GCase with two plant-specific glycan residues. This study presents evidence for a new, safe and efficient system of recombinant GCase production that might be applied to other recombinant proteins.

Highlights

Gaucher disease is an inherited lysosomal storage disorder caused by mutation of the GBA1 gene, which is located on chromosome 1
There are several commercial recombinant GCases for Enzyme replacement therapy (ERT), including imiglucerase (Cerezyme®; Genzyme Corporation, Cambridge, MA, USA), velaglucerase alpha (VPRIV®; Shire Plc, Dublin, Ireland) and taliglucerase alpha (Elelyso®; Pfizer, New York, NY, USA), which are produced in Chinese hamster ovary (CHO) cells, human fibroblasts and carrot cells, respectively
Crude protein extracted from N. benthamiana WT showed a small amount of β-glucosidase enzymatic activity (9.60 ± 2.11 units/mg total protein), which was considered to originate from internal β-glucosidase [28,29], since the β-glucosidase found in plants plays important roles in lignification, chemical defense and phytohormone regulation

Summary

Introduction

Gaucher disease is an inherited lysosomal storage disorder caused by mutation of the GBA1 (acid-β-glucosidase) gene, which is located on chromosome 1. The mutation leads to deficiency of a lysosomal enzyme called glucocerebrosidase (acid β-glucosidase or GCase; EC: 4.2.1.25), which catalyzes the hydrolysis of glucosylceramide to glucose and ceramide [1,2]. A lack of GCase leads to accumulation of glucosylceramide in the lysosomes of macrophage cells, resulting in the symptoms of Gaucher disease’s, including bone fractures and pathological enlargement of the spleen and liver [3]. Enzyme replacement therapy (ERT) has been used as one of the most efficient treatments for Gaucher disease type I. Treating patients with the recombinant enzyme can reduce both spleen and liver sizes as well as improve bone health [3,5]

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