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Identification and characterization of glycosylation sites in human serum clusterin
Author(s) -
Kapron James T.,
Hilliard George M.,
Lakins Johnathon N.,
Tenniswood MARTIN P. R.,
West Karen A.,
Carr Steven A.,
Crabb John W.
Publication year - 1997
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560061007
Subject(s) - fucose , glycosylation , clusterin , chemistry , oligosaccharide , edman degradation , mass spectrometry , glycoprotein , biochemistry , protein subunit , peptide sequence , chromatography , apoptosis , gene
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 holoclusterin (58,505 + 250 and 63,507 + 200). Mass spectrometry also revealed molecular heterogeneity associated with both the α and β subunits of clusterin, consistent with the presence of multiple glycoforms. The data indicate that clusterin contains 17‐27% carbohydrate by weight, the α subunit contains 0‐30% carbohydrate and the β 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 α subunit (α4N, αy 81 N, α 123 N) and three in the β subunit (β 64 N, β 127 N, and β 147 N). Seven different possible types of oligosaccharide structures were identified by mass including: a monosialo‐biantennary 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 β 64 N exhibited the least glycosylation diversity, with two detected types of oligosaccharides, and site β 147 N 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, trisialotriantennay with two fucose and/or tetrasialotriantennary. Clusterin peptides accounting for 99% of the primary structure were identified from analysis of the isolated α and β subunits, including all 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.