Glycosylation analysis of two cysteine proteinase inhibitors from Atlantic salmon 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 the skin of Atlantic salmon (Salmo salar L.), named salmon kininogen and salarin. 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 carry biantennary alpha2,3-sialylated N-glycans. A very high amount of O-acetylated 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 sialic acids in the N-glycans. A small number of Neu5Acalpha2-8Neu5Ac structures were observed in the N-glycans as well. O-glycans from both proteins were recovered by reductive beta-elimination and were identified by mass spectrometric methods as mono- and disialylated core type 1 tri- and tetrasaccharides. The method used for O-glycan isolation prevented the identification of possible O-acetylation in the O-glycan-bound sialic acids, but O-acetylation was observed in one O-glycosylated peptide isolated from trypsin digest of salarin. The chemical nature of the sialic acid modifications was further studied by liquid chromatography tandem mass spectrometry of 1,2-diamino-4,5-methylenedioxybenzene-derivatized sialic acids, revealing 7-, 8-, and 9- but no 4-O-acetylation. To our knowledge, these are the first observations of sialic acid O-acetylation in N-glycans on fish species and represent clearly the most extensive N-glycan O-acetylation described on any species.
Ylönen
A
A
Institute of Biotechnology, Protein Chemistry Laboratory, P.O. Box 56 (Viikinkaari 9), University of Helsinki, FIN-00014 University of Helsinki, Finland.
Kalkkinen
N
N
Saarinen
J
J
Bøgwald
J
J
Helin
J
J
D000107:Acetylation; D000595:Amino Acid Sequence; D000818:Animals; D002240:Carbohydrate Sequence; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism*; D029941:Fish Proteins; D006023:Glycoproteins / Q000378:metabolism; D006031:Glycosylation; D007704:Kininogens / Q000378:metabolism; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D012474:Salmon; D012867:Skin / Q000737:chemistry / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D015853:Cysteine Proteinase Inhibitors; D029941:Fish Proteins; D006023:Glycoproteins; D007704:Kininogens; D011134:Polysaccharides; C403188:salarin protein, Salmo salar