Identification of N-linked Glycoproteins in Silkworm Serum Using Con A Lectin Affinity Chromatography and Mass Spectrometry.

Glycosylation is one of the most common post-translational modifications to occur during protein biosynthesis, but remains poorly understood in insects. In this study, we collected serum proteins from two silkworm developmental stages, namely day 7 of the fifth instar larval stage and day 2 of the pupal stage. Results of SDS-PAGE and periodic acid-Schiff staining revealed that most serum proteins with high abundance were putative glycoproteins. LC-MS/MS identified 149 larval and 303 pupal serum proteins in the Con A lectin-enriched fractions. GO analysis revealed that many serum proteins were involved in the proteolysis and carbohydrate metabolic process. 82 N-linked glycoproteins with at least one glycosylation site were identified. N-Linked glycosylation occurred at the sequon, Asn-X-Ser/Thr, and the proportions of Ser and Thr glycosylation at the hydroxy position were found 39.6% and 60.3%, respectively. The N-glycan structures found in serum glycoproteins were mainly Man2FucGlcNAc2 (67.9%). Since storage protein 1 and transferrin had a relatively high abundance in the serum and could be significantly enriched by Con A lectin, their glycosylation was analyzed in detail. Glycoside hydrases, serine proteases and serpins were found to form three interacting glycoprotein networks using the website STRING. This study provides important clues for the understanding of the function of N-linked glycosylation in metabolism, immunity, and metamorphosis.