A cytosolic reductase pathway is required for efficient N-glycosylation of an STT3B-dependent acceptor site.

Marcel Van Lith,Neil J Bulleid,Jisu Im,Bethany Fleming,Giorgia Gaeta,Marie Anne Pringle,Reid Gilmore

doi:10.1242/jcs.259340

Abstract

ABSTRACTN-linked glycosylation of proteins entering the secretory pathway is an essential modification required for protein stability and function. Previously, it has been shown that there is a temporal relationship between protein folding and glycosylation, which influences the occupancy of specific glycosylation sites. Here, we used an in vitro translation system that reproduces the initial stages of secretory protein translocation, folding and glycosylation under defined redox conditions. We found that the efficiency of glycosylation of hemopexin was dependent upon a robust NADPH-dependent cytosolic reductive pathway, which could be mimicked by the addition of a membrane-impermeable reducing agent. We identified a hypoglycosylated acceptor site that is adjacent to a cysteine involved in a short-range disulfide. We show that efficient glycosylation at this site is influenced by the cytosolic reductive pathway acting on both STT3A- and STT3B-dependent glycosylation. Our results provide further insight into the important role of the endoplasmic reticulum redox conditions in glycosylation site occupancy and demonstrate a link between redox conditions in the cytosol and glycosylation efficiency.

Highlights

Proteins entering the secretory pathway are subject to a variety of modifications, the most prevalent of which include N-linked glycosylation and disulfide formation (Bulleid, 2012; Cherepanova et al, 2016)
A cytosolic reductive pathway determines the extent of sequon usage in an STT3B-dependent glycoprotein Our initial experiments aimed to determine whether the redox conditions within the endoplasmic reticulum (ER) had any effect on the fidelity of sequon usage within a model protein, hemopexin, which has previously been shown to undergo hypoglycosylation when expressed in cells – a phenomenon that is exacerbated in the absence of STT3B (Shrimal and Gilmore, 2013)
As glucose 6-phosphate (G6-P) most likely alters the redox conditions by recycling NADP to NADPH in the cytosol, and because TCEP is membrane impermeable, these results suggest that the redox conditions on the cytosolic side of the ER membrane affect the glycosylation efficiency of ER-translocated hemopexin

Summary

Introduction

Proteins entering the secretory pathway are subject to a variety of modifications, the most prevalent of which include N-linked glycosylation and disulfide formation (Bulleid, 2012; Cherepanova et al, 2016). Handling Editor: Jennifer Lippincott-Schwartz Received 31 August 2021; Accepted 27 October 2021 complexes characterised by the catalytic subunits STT3A or STT3B They have common subunits as well as complex-specific subunits, including DC2 ( known as OSTC) and KCP2 ( known as KRTCAP2) for the STT3A complex and the thioredoxindomain-containing proteins MagT1 or TUSC3 for the STT3B complex (Blomen et al, 2015; Roboti and High, 2012; Shibatani et al, 2005). It has been demonstrated previously that the STT3A complex associates with the endoplasmic reticulum (ER) translocon (Braunger et al, 2018; Shibatani et al, 2005) and catalyses the co-translational glycosylation of proteins, whereas the STT3B complex glycosylates sites skipped by STT3A, acting predominantly post-translationally (Cherepanova et al, 2014; Ruiz-Canada et al, 2009). Deficiency of the STT3B complex cannot be compensated by the STT3A complex, resulting in hypoglycosylation of substrates, affecting their function and leading to disease pathologies linked to immunodeficiency (Blommaert et al, 2019; Matsuda-Lennikov et al, 2019)

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