Kapron J T, Hilliard G M, Lakins J N, Tenniswood M P, West K A, Carr S A, Crabb J W
W. Alton Jones Cell Science Center, Lake Placid, New York 12946, USA.
Protein Sci. 1997 Oct;6(10):2120-33. doi: 10.1002/pro.5560061007.
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 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. 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.
聚集素是一种普遍存在的异源二聚体糖蛋白，具有多种可能的功能，这些功能可能受糖基化影响。已通过质谱分析和埃德曼降解法对人血清聚集素中的寡糖连接位点进行了鉴定，并对寡糖进行了结构表征分析。基质辅助激光解吸电离质谱分析显示了全聚集素的两种分子量形式（58,505±250和63,507±200）。质谱分析还揭示了与聚集素的α和β亚基相关的分子异质性，这与多种糖型的存在一致。数据表明，聚集素按重量计含有17 - 27%的碳水化合物，α亚基含有0 - 30%的碳水化合物，β亚基含有27 - 30%的碳水化合物。采用具有阶梯碰撞能量扫描的液相色谱电喷雾质谱法选择性地鉴定并制备性分离胰蛋白酶糖肽。然后使用埃德曼序列分析来确认糖肽的身份，并确定每个肽段内的连接位点。总共鉴定出6个N - 连接糖基化位点，3个在α亚基中（α64N、α81N、α123N），3个在β亚基中（β64N、β127N和β147N）。通过质谱鉴定出7种不同的可能寡糖结构类型，包括：一种单唾液酸二天线结构、不带或带有一个岩藻糖的双唾液酸二天线结构、不带或带有一个岩藻糖的三唾液酸三天线结构，以及可能带有两个岩藻糖的三唾液酸三天线结构和/或四唾液酸三天线结构。β64N位点的糖基化多样性最低，检测到两种寡糖类型，而β147N位点的多样性最高，检测到五或六种寡糖类型。总体而言，检测到的最丰富糖型是不带岩藻糖的双唾液酸二天线结构，最不丰富的是单唾液酸二天线结构、带有两个岩藻糖的三唾液酸三天线结构和/或四唾液酸三天线结构。通过对分离出的α和β亚基的分析，鉴定出了占一级结构99%的聚集素肽段，包括所有含丝氨酸和苏氨酸的肽段。未发现存在O - 连接或硫酸化寡糖的证据。这些结果为更好地理解聚集素的结构 - 功能关系以及聚集素糖基化在生理功能中所起的作用提供了分子基础。
