Glycosylation analysis of two cysteine proteinase inhibitors from Atlanticsalmon skin: di-O acetylated sialic acids are the major sialic acid species on N-glycans

We have recently identified two novel cysteine proteinase inhibitors from t he skin of Atlantic salmon (Salmo salar L,), named salmon kininogen and sal arin, In preliminary experiments, the proteins were found to be both N- as well as O-glycosylated, In the present study we show that both proteins car ry biantennary alpha2,3-sialylated N-glycans, A very high amount of O-acety lated Neu5Ac units are present in the N-glycans, comprising about 60% di-O- acetylated species. Non-O-acetylated Neu5Ac make up less than 5% of the sia lic acids in the N-glycans, A small number of Neu5Ac alpha2-8Neu5Ac structu res were observed in the N-glycans as well. O-glycans from both proteins we re recovered by reductive beta-elimination and were identified by mass spec trometric methods as mono- and disialylated core type 1 tri- and tetrasacch arides. The method used for O-glycan isolation prevented the identification of possible O-acetylation in the O-glycan-bound sialic acids, but O-acetyl ation was observed in one O-glycosylated peptide isolated from trypsin dige st of salarin, The chemical nature of the sialic acid modifications was fur ther studied by liquid chromatography tandem mass spectrometry of 1,2-diami no-4,5-methylenedioxybenzene-derivatized sialic acids, revealing 7-, 8-, an d 9- but no 4-O-acetylation, To our knowledge, these are the first observat ions of sialic acid O-acetylation in N-glycans on fish species and represen t clearly the most extensive N-glycan O-acetylation described on any specie s.