Abstract

N-glycosylation is an important posttranslational modification in all eukaryotes, but little is known about the N-glycoproteins and N-glycans in microalgae. Here, N-glycoproteomic and N-glycomic approaches were used to unveil the N-glycoproteins and N-glycans in the model diatom Phaeodactylum tricornutum. In total, 863 different N-glycopeptides corresponding to 639 N-glycoproteins were identified from P. tricornutum. These N-glycoproteins participated in a variety of important metabolic pathways in P. tricornutum. Twelve proteins participating in the N-glycosylation pathway were identified as N-glycoproteins, indicating that the N-glycosylation of these proteins might be important for the protein N-glycosylation pathway. Subsequently, 69 N-glycans corresponding to 59 N-glycoproteins were identified and classified into high mannose and hybrid type N-glycans. High mannose type N-glycans contained four different classes, such as Man-5, Man-7, Man-9, and Man-10 with a terminal glucose residue. Hybrid type N-glycan harbored Man-4 with a terminal GlcNAc residue. The identification of N-glycosylation on nascent proteins expanded our understanding of this modification at a N-glycoproteomic scale, the analysis of N-glycan structures updated the N-glycan database in microalgae. The results obtained from this study facilitate the elucidation of the precise function of these N-glycoproteins and are beneficial for future designing the microalga to produce the functional humanized biopharmaceutical N-glycoproteins for the clinical therapeutics.

Highlights

  • IntroductionIn the molecular function class, it is found that most N-glycosylated proteins are involved in the catalytic activity and binding

  • 69 N-glycans corresponding to 59 N-glycoproteins were identified and classified into high mannose and hybrid type N-glycans

  • The enriched N-glycopeptides were subsequently analyzed by LC-MS/MS with MaxQuant to classify the N-glycopeptide sequence with a maximum false discovery rate of 1% (Figure 1A)

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Summary

Introduction

In the molecular function class, it is found that most N-glycosylated proteins are involved in the catalytic activity and binding. It is shown that 639 N-glycoproteins were putatively located to eight subcellular organelles, such as chloroplast, cytoplasm, nucleus, plasma membrane, mitochondria, vacuolar membrane, extracellular, and ER. Most N-glycoproteins were targeted to chloroplast (294 N-glycoproteins) and cytoplasm (103 N-glycoproteins) (Figure 3C)

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