PMID: 9336835

 

    Legend: Sugar

Title : Identification and characterization of glycosylation sites in human serum clusterin

Abstract :
  1. Clusterin is a ubiquitous, heterodimeric glycoprotein with multiple possible functions that are likely influenced by glycosylation
  2. Identification of oligosaccharide attachment sites and structural characterization of oligosaccharides in human serum clusterin has been performed by mass spectrometry and Edman degradation
  3. Matrix-assisted laser desorption ionization mass spectrometry revealed two molecular weight species of holo clusterin (58,505 +/- 250 and 63,507 +/- 200)
  4. Mass spectrometry also revealed molecular heterogeneity associated with both the alpha and beta subunits of clusterin , consistent with the presence of multiple glycoforms
  5. The data indicate that clusterin contains 17-27% carbohydrate by weight, the alpha subunit contains 0-30% carbohydrate and the beta subunit contains 27-30% carbohydrate
  6. Liquid chromatography electrospray mass spectrometry with stepped collision energy scanning was used to selectively identify and preparatively fractionate tryptic glycopeptides
  7. Edman sequence analysis was then used to confirm the identities of the glycopeptides and to define the attachment sites within each peptide
  8. A total of six N-linked glycosylation sites were identified, three in the alpha subunit (alpha 64N, alpha 81N, alpha 123N) and three in the beta subunit (beta 64N, beta 127N, and beta 147N)
  9. Seven different possible types of oligosaccharide structures were identified by mass including: a monosialobiantennary structure, bisialobiantennary structures without or with one fucose, trisialotriantennary structures without or with one fucose, and possibly a trisialotriantennary structure with two fucose and/or a tetrasialotriantennary structure
  10. Site beta 64N exhibited the least glycosylation diversity, with two detected types of oligosaccharides , and site beta 147N exhibited the greatest diversity, with five or six detected types of oligosaccharides
  11. Overall , the most abundant glycoforms detected were bisialobiantennary without fucose and the least abundant were monosialobiantennary, trisialotriantennary with two fucose and/or tetrasialotriantennary
  12. Clusterin peptides accounting for 99% of the primary structure were identified from analysis of the isolated alpha and beta subunits , including all Ser- and Thr-containing peptides. Ser- and Thr-containing peptides
  13. No evidence was found for the presence of O-linked or sulfated oligosaccharides
  14. The results provide a molecular basis for developing a better understanding of clusterin structure-function relationships and the role clusterin glycosylation plays in physiological function