Title : Identification and characterization of glycosylation
sites in human serum
clusterin
Abstract :
- Clusterin is a ubiquitous, heterodimeric glycoprotein with multiple possible functions that are likely influenced by glycosylation
- Identification of oligosaccharide attachment sites and structural characterization of oligosaccharides in human serum clusterin has been performed by mass spectrometry and Edman degradation
- Matrix-assisted laser desorption ionization mass spectrometry revealed two molecular weight species of holo clusterin (58,505 +/- 250 and 63,507 +/- 200)
- Mass spectrometry also revealed molecular heterogeneity associated with both the alpha and beta subunits of clusterin , consistent with the presence of multiple glycoforms
- 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
- Liquid chromatography electrospray mass spectrometry with stepped collision energy scanning was used to selectively identify and preparatively fractionate tryptic glycopeptides
- Edman sequence analysis was then used to confirm the identities of the glycopeptides and to define the attachment sites within each peptide
- 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)
- 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
- 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
- Overall , the most abundant glycoforms detected were bisialobiantennary without fucose and the least abundant were monosialobiantennary, trisialotriantennary with two fucose and/or tetrasialotriantennary
- 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
- No evidence was found for the presence of O-linked or sulfated oligosaccharides
- 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