Global Profiling of N-Glycoproteins and N-Glycans in the Diatom Phaeodactylum tricornutum

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.

Endoplasmic reticulum-quality control pathway and endoplasmic reticulum-associated degradation mechanism regulate the N-glycoproteins and N-glycan structures in the diatom Phaeodactylum tricornutum

“…Algal cells were collected on the 24th hour of tunicamycin stress. 1.5 mg fresh algal cells were grinded by liquid nitrogen into cell powder and used for the protein extraction described in our previous paper [29]. The concentration of protein was measured via BCA kit according to the instructions of manufacturer (ab102536, Abcam, China).…”

“…The digested glycopeptides were washed by C18 ZipTips and lyophilized for liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. The procedure and data setting were carried out according to method stated by [21], and [29] Compared to that, in the wild type, N-glycopeptides with ratio ≥ 1.5 and p value < 0.05 were defined as differentially expressed N-glycopeptides.…”

“…The N-glycan structures in diatom P. tricornutum were previously reported to be mannose-type ranging from Man-5 to Man-9 via MALDI-TOF MS, IMS-MS and ESI-MS n fragmentation patterns data [1,7,17]. Subsequently, addition to the mannose-type, a hybrid N-glycan with a terminal GlcNAc residue was identified in P. tricornutum via C18-RPLC-MS/MS (HCD) analysis [29]. In this study, mannose-type with 3 to 9 mannose residues was identified as the primarily N-glycan structure through the high throughput method.…”

“…Studies on protein glycosylation pathway, especially on the protein N-glycosylation pathway have received very little attention so far. The pathway of protein N-glycosylation modification was only recently proposed in P. tricornutum [14,29]. In N-glycan processing, a Man5GlcNAc2-oligosaccharide intermediate was first synthesized and connected to a dolichol pyrophosphate on the cytosolic side of the endoplasmic reticulum (ER) [19].…”

“…Additionally, it was also reported that the blocking of protein N-glycosylation modification by tunicamycin resulted in ER stress which caused oxidative stress in cell [2]. So far, N-glycan structures were widely analyzed in mammal's tissueand cell-type-specific levels, however, the relative information is very limited in plants, especially in algae [19,29]. The analysis of N-glycan will not only help unravel the N-linked oligosaccharides structure of proteins, but also provide information for studying the function of the proteins.…”