N-Glycans of Phaeodactylum tricornutum Diatom and Functional Characterization of Its N-Acetylglucosaminyltransferase I Enzyme

Bérengère Baïet,Carole Burel,Bruno Saint-Jean,Romain Louvet,Laurence Menu-Bouaouiche,Marie-Christine Kiefer-Meyer,Elodie Mathieu-Rivet,Thomas Lefebvre,Hélène Castel,Aude Carlier,Jean-Paul Cadoret,Patrice Lerouge,Muriel Bardor

doi:10.1074/jbc.m110.175711

Abstract

N-glycosylation, a major co- and post-translational event in the synthesis of proteins in eukaryotes, is unknown in aquatic photosynthetic microalgae. In this paper, we describe the N-glycosylation pathway in the diatom Phaeodactylum tricornutum. Bio-informatic analysis of its genome revealed the presence of a complete set of sequences potentially encoding for proteins involved in the synthesis of the lipid-linked Glc(3)Man(9)GlcNAc(2)-PP-dolichol N-glycan, some subunits of the oligosaccharyltransferase complex, as well as endoplasmic reticulum glucosidases and chaperones required for protein quality control and, finally, the α-mannosidase I involved in the trimming of the N-glycan precursor into Man-5 N-glycan. Moreover, one N-acetylglucosaminyltransferase I, a Golgi glycosyltransferase that initiates the synthesis of complex type N-glycans, was predicted in the P. tricornutum genome. We demonstrated that this gene encodes for an active N-acetylglucosaminyltransferase I, which is able to restore complex type N-glycans maturation in the Chinese hamster ovary Lec1 mutant, defective in its endogeneous N-acetylglucosaminyltransferase I. Consistent with these data, the structural analyses of N-linked glycans demonstrated that P. tricornutum proteins carry mainly high mannose type N-glycans ranging from Man-5 to Man-9. Although representing a minor glycan population, paucimannose N-glycans were also detected, suggesting the occurrence of an N-acetylglucosaminyltransferase I-dependent maturation of N-glycans in this diatom.

Highlights

In Silico Analysis of the P. tricornutum Genome Revealed a Set of Genes Encoding for Proteins Involved in the N-Glycosylation Pathway—In eukaryotes, the N-glycan biosynthetic pathway can be divided into three steps: (i) the synthesis of the Glc3Man9GlcNAc2-PP-dolichol precursor and its transfer by the OST onto asparagine residues of nascent polypeptides entering the lumen of the rough endoplasmic reticulum (ER); (ii) deglucosylation/reglucosylation of the precursor N-glycan in the ER, allowing the interaction with chaperones responsible for proper folding and oligomerization; and (iii) maturation in the Golgi apparatus of the high mannose type N-linked oligosaccharides into complex type N-glycans
We identified in the genome of P. tricornutum a set of putative sequences that are likely involved in the different steps of the N-glycan biosynthesis and maturation (Fig. 1 and Table 1)
All of the genes encoding for enzymes involved in the biosynthesis of dolichol pyrophosphate-linked oligosaccharide on the cytosolic face and in the lumen of the ER were identified in the genome of P. tricornutum (Fig. 1 and Table 1)

Summary

Introduction

N-Glycan processing occurs in the secretory pathway and is essential for glycoproteins destined to be secreted or integrated in the membranes In this process, a Man5GlcNAc2-PP-dolichol oligosaccharide intermediate is assembled by the stepwise addition of monosaccharides to dolichol pyrophosphate on the cytosolic face of the endoplasmic reticulum (ER).. Arabidopsis cgl mutant, defective in GnT I activity, was demonstrated to grow normally in standard culture conditions [12] These plants exhibited a strong phenotype in salt-induced stress conditions, for example, suggesting a role for mature plant N-glycans in specific physiological processes [13,14,15]. Major data regarding protein N-glycosylation have been established in yeast and higher eukaryotes, nothing is known on N-glycan biosynthesis and structures in microalgae. To the best of our knowledge, this is the first functional characterization of a N-glycan glycosyltransferase from microalgae

Methods

Results

Conclusion