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

Abstract

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.

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update covering the period 2001–2002

“…These include membranes such as those having a low molecular weight cut-off that can be used for drop dialysis (Schilling et al, 2001(Schilling et al, , 2002aYlönen et al, 2001;Post et al, 2002); resins, such as SP20SS (Huang et al, 2002), OASIS cartridges from Waters (Charlwood et al, 2000(Charlwood et al, , 2001aCharlwood, Skehel, & Camilleri, 2001b) (for 3-acetamido-6-aminoacridine (AA-Ac, 2/5) derivatives), H þ Dowex AG 50W X8 (for acidic xylo-oligosaccharides) (Kabel, Schols, & Voragen, 2002b) or Amberlite IR-120 (H þ ) (Janusch et al, 2002), anion-exchange ZipTips (for O-deacylated lipooligosaccharides) (Schilling et al, 2002a), or C18 ZipTips (Charlwood et al, 2001a;Charlwood, Skehel, & Camilleri, 2001b) from Millipore for AA-Ac-derivatized N-linked glycans. Various investigators have desalted lipopolysaccharides using Dowex 50W (Schilling et al, 2001(Schilling et al, , 2002aJones et al, 2002).…”

Purification and characterization of novel kininogens from spotted wolffish and Atlantic cod

“…The permethylated O‐glycan pool was analysed by MALDI‐TOF MS, and the spectrum revealed one major [M + Na] + signal at m / z 1256.63 (not shown). The mass is appropriate for the composition (Neu5Ac) 2 (Hex) 1 ‐ (HexNAc‐ol) 1 , suggesting that wolffish kininogen carries disialylated core type‐1 O‐glycans similar to those previously identified from salmon kininogen [14].…”

“…Digestions with Newcastle disease virus (NDV) neuraminidase and Streptococcus pneumoniae β1,4‐galactosidase (Oxford Glycosciences, Abingdon, UK) were carried out as described previously [14]. Digestion with jack bean β‐galactosidase (Glyko, Novato, CA, USA) was carried out in 10 µL of 100 m m sodium citrate, pH 4.5, at 37 °C.…”

“…Two major clusters of ions are observed, and within both clusters adducts of +42 or +80 Da can be revealed. We have recently identified from salmon kininogen N-glycans very high levels of sialic acid O-acetylation [14], and it is probable that the +42 Da adducts in the cod kininogen N-glycans represent this modification as well. This is supported by the higher number of acetyl (Ac) groups in those glycans that carry more sialic acid residues.…”

Exploration of the Sialic Acid World

“…452,453 But also (α2!3) linkages with Gal have been reported for Neu5,7Ac 2 , Neu5,8Ac 2 , Neu5,7,8Ac 3 , and Neu5,8,9Ac 3 . 452 Furthermore, [Neu5Ac(α2!8)] n , [Neu5Gc(α2!8)] n , and [Neu5Ac/5Gc(α2!8)] n sequences with 4Ac, 7Ac, 9Ac, or 9Lt substituents, 455 a Neu5Ac8S(α2!9)[Neu5Ac(α2!9)] n sequence, 456 and the [Neu5Gc(α2!O5)] n sequence (linkage between Neu5Gc C2 and the N-glycolyl OH function of the next Neu5Gc residue; Fig. 26) should be mentioned.…”