PMID: 21053369

 

    Legend: Sugar

Title : Glycosylation analysis of interleukin-23 receptor : elucidation of glycosylation sites and characterization of attached glycan structures

Abstract :
  1. Interleukin-23 ( IL-23 ) is a heterodimeric cytokine, a central factor in chronic/autoimmune inflammation
  2. It signals through a heterodimeric receptor consisting of IL-23r , which is heavily glycosylated
  3. The structural characterization of IL-23r has not been reported
  4. In this work, glycosylation profiles of soluble recombinant human IL-23r ( rhIL-23r ) were established using mass spectrometry (MS), which included defining glycosylation sites , degree of glycosylation occupancy of each site and structure of attached oligosaccharides
  5. Specifically, precursor ion scan of oxonium ion protonated N-acetylglucosamine (GlcNAc(+)) (m/z 204) was performed using a triple quadrupole MS instrument to locate the retention time of glycopeptides
  6. Both the glycopeptides and their corresponding deglycosylated forms in each collected HPLC fraction were studied by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (LTQ-Orbitrap) for glycosylation site profiling
  7. The attached glycan structures were elucidated by collision-induced dissociation ( CID ) fragmentation of target glycopeptides in combination with accurate mass measurement
  8. Eight glycosylation sites were identified on IL-23r ( Asn24, Asn209, Asn239, Asn157, Asn118, Asn250, Asn58 and Asn6 )
  9. Most of the glycosylation sites were > 95% occupied except Asn250 and Asn6
  10. Those two sites were 88% and 45% occupied by estimation from trypsin digestion and were 55% and 42% occupied from LysC digestion
  11. Multiple glycoforms were observed in IL-23r
  12. Most of them were bi-, tri- or tetra-antennary complex type structures with fucose and sialic acid.
  13. High mannose and hybrid type glycans were only observed on Asn157
  14. The structural characterization on IL-23r glycosylation provides useful information for better understanding of the biological function of IL-23r