IDENTIFICATION AND CHARACTERIZATION OF GLYCOSYLATION SITES IN HUMAN SERUM CLUSTERIN

Clusterin is a ubiquitous, heterodimeric glycoprotein with multiple po ssible functions that are likely influenced by glycosylation. Identifi cation of oligosaccharide attachment sites and structural characteriza tion of oligosaccharides in human serum clusterin has been performed b y mass spectrometry and Edman degradation. Matrix-assisted laser desor ption ionization mass spectrometry revealed two molecular weight speci es of holoclusterin (58,505 +/- 250 and 63,507 +/- 200). Mass spectrom etry also revealed molecular heterogeneity associated with both the al pha and beta subunits of clusterin, consistent with the presence of mu ltiple glycoforms. The data indicate that clusterin contains 17-27% ca rbohydrate by weight, the alpha subunit contains 0-30% carbohydrate an d 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 trypti c glycopeptides. Edman sequence analysis was then used to confirm the identities of the glycopeptides and to define the attachment sites wit hin each peptide. A total of six N-linked glycosylation sites were ide ntified, three in the alpha subunit (alpha(64)N, alpha(81)N, alpha(123 )N) and three in the beta subunit (beta(64)N, beta(127)N, and beta(147 )N). Seven different possible types of oligosaccharide structures were identified by mass including: a monosialobiantennary structure, bisia lobiantennary structures without or with one fucose, trisialotriantenn ary structures without or with one fucose, and possibly a trisialotria ntennary structure with two fucose and/or a tetrasialotriantennary str ucture. Site beta(64)N exhibited the least glycosylation diversity, wi th two detected types of oligosaccharides, and site beta(147)N exhibit ed the greatest diversity, with five or six detected types of oligosac charides. Overall, the most abundant glycoforms detected were bisialob iantennary without fucose and the least abundant were monosialobianten nary, trisialotriantennary with two fucose and/or tetrasialotriantenna ry. Clusterin peptides accounting for 99% of the primary structure wer e identified from analysis of the isolated alpha and beta subunits, in cluding all Ser-and Thr-containing peptides. No evidence was found for the presence of O-linked or sulfated oligosaccharides. The results pr ovide a molecular basis for developing a better understanding of clust erin structure-function relationships and the role clusterin glycosyla tion plays in physiological function.