N-linked glycosylation enzymes in the diatom Thalassiosira oceanica exhibit a diel cycle in transcript abundance and favor for NXT-type sites.

N-linked glycosylation is a posttranslational modification affecting protein folding and function. The N-linked glycosylation pathway in algae is poorly characterized, and further knowledge is needed to understand the cell biology of algae and the evolution of N-linked glycosylation. This study investigated the N-linked glycosylation pathway in Thalassiosira oceanica, an open ocean diatom adapted to survive at growth-limiting iron concentrations. Here we identified and annotated the genes coding for the essential enzymes involved in the N-linked glycosylation pathway of T. oceanica. Transcript levels for genes coding for calreticulin, oligosaccharyltransferase (OST), N-acetylglucosaminyltransferase (GnT1), and UDP-glucose glucosyltransferase (UGGT) under high- and low-iron growth conditions revealed diel transcription patterns with a significant decrease of calreticulin and OST transcripts under iron-limitation. Solid-phase extraction of N-linked glycosylated peptides (SPEG) revealed 118 N-linked glycosylated peptides from cells grown in high- and low-iron growth conditions. The identified peptides had 81% NXT-type motifs, with X being any amino acids except proline. The presence of N-linked glycosylation sites in the iron starvation-induced protein 1a (ISIP1a) confirmed its predicted topology, contributing to the biochemical characterization of ISIP1 proteins. Analysis of extensive oceanic gene databases showed a global distribution of calreticulin, OST, and UGGT, reinforcing the importance of glycosylation in microalgae.