Intracellular trafficking and glycosylation of hydroxyproline-O-glycosylation module in tobacco BY-2 cells is dependent on medium composition and transcriptome analysis

Document Type

Article

Publication Title

Scientific reports

PubMed ID

37598266

MeSH Headings (Medical Subject Headings)

Nicotiana (genetics); Glycosylation; Hydroxyproline; Plant Cells; Gene Expression Profiling

Abstract

Expression of recombinant proteins in plant cells with a "designer" hydroxyproline (Hyp)-O-glycosylated peptide (HypGP), such as tandem repeats of a "Ser-Pro" motif, has been shown to boost the secreted protein yields. However, dramatic secretion and Hyp-O-glycosylation of HypGP-tagged proteins can only be achieved when the plant cells were grown in nitrogen-deficient SH medium. Only trace amounts of secreted fusion protein were detected in MS medium. This study aims to gain a deeper understanding of the possible mechanism underlying these results by examining the intracellular trafficking and Hyp-O-glycosylation of enhanced green fluorescent protein (EGFP) fused with a (SP) tag, consisting of 32 repeats of a "Ser-Pro" motif, in tobacco BY-2 cells. When cells were grown in MS medium, the (SP)-EGFP formed protein body-like aggregate and was retained in the ER, without undergoing Hyp-O-glycosylation. In contrast, the fusion protein becomes fully Hyp-O-glycosylated, and then secreted in SH medium. Transcriptome analysis of the BY-2 cells grown in SH medium vs. MS medium revealed over 16,000 DEGs, with many upregulated DEGs associated with the microtubule-based movement, movement of subcellular component, and microtubule binding. These DEGs are presumably responsible for the enhanced ER-Golgi transport of HypGP-tagged proteins, enabling their glycosylation and secretion in SH medium.

First Page

13506

DOI

10.1038/s41598-023-40723-3

Publication Date

8-19-2023

E-ISSN

2045-2322

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