Parry Simon, Wong Nyet-Kui, Easton Richard L, Panico Maria, Haslam Stuart M, Morris Howard R, Anderson Peggy, Klotz Kenneth L, Herr John C, Diekman Alan B, Dell Anne
Division of Molecular Biosciences, Imperial College London, London SW7 2AZ, UK.
Glycobiology. 2007 Oct;17(10):1120-6. doi: 10.1093/glycob/cwm076. Epub 2007 Jul 19.
CD52 is composed of a 12 amino acid peptide with N-linked glycans bound to the single potential glycosylation site at position 3, and a glycosylphosphatidylinositol-anchor attached at the C-terminus. Some glycoforms of this molecule expressed in the male reproductive tract are recognized by complement-dependent sperm-immobilizing antibodies in infertile patients making this antigen an important target for immunocontraception and fertility studies. Although the amount of posttranslational modification is already remarkable for such a small polypeptide, O-glycosylation of CD52 has additionally been implicated by several studies, but never rigorously characterized. In this report, we show clear evidence for the presence of O-glycans in CD52 preparations immunopurified using the murine S19 monoclonal antibody generated against sperm agglutination antigen-1 (SAGA-1), a male reproductive tract specific form of CD52. The O-glycans have been characterized by MALDI-TOF and tandem mass spectrometry after reductive elimination and permethylation. The data indicate that the major SAGA-1 O-glycans are core 1 and 2 mucin-type structures, with and without sialic acid (NeuAc(0-2)Hex(1-3)HexNAc(1-2)HexNAcitol). Minor fucosy- lated O-glycans are also present including some struc- tures with putative Le(y) epitopes (NeuAc(0-1)Fuc(1-3)Hex(1-2) HexNAc(0-1)HexNAcitol). Analysis of O-glycopeptides by tandem mass spectrometry provided an additional level of support for the O-glycosylation of SAGA-1. Elucidation of the O-glycosylation of SAGA-1 adds to the complexity of this molecule and may help to explain its biological activity.
CD52由一个12个氨基酸的肽段组成，该肽段在第3位的单个潜在糖基化位点上结合有N-连接聚糖，并且在C末端连接有糖基磷脂酰肌醇锚定物。在男性生殖道中表达的该分子的一些糖型被不育患者中依赖补体的精子固定抗体识别，使得该抗原成为免疫避孕和生育研究的重要靶点。尽管对于如此小的多肽，翻译后修饰的量已经很显著，但几项研究还表明CD52存在O-糖基化现象，但从未进行过严格的表征。在本报告中，我们提供了明确的证据，证明使用针对精子凝集抗原-1（SAGA-1，一种男性生殖道特异性形式的CD52）产生的鼠源S19单克隆抗体免疫纯化的CD52制剂中存在O-聚糖。在还原消除和全甲基化后，通过基质辅助激光解吸电离飞行时间质谱（MALDI-TOF）和串联质谱对O-聚糖进行了表征。数据表明，主要的SAGA-1 O-聚糖是核心1和2粘蛋白型结构，有或没有唾液酸（NeuAc(0-2)Hex(1-3)HexNAc(1-2)HexNAcitol）。还存在少量岩藻糖基化的O-聚糖，包括一些具有假定的Le(y)表位（NeuAc(0-1)Fuc(1-3)Hex(1-2)HexNAc(0-1)HexNAcitol）的结构。通过串联质谱对O-糖肽进行分析，为SAGA-1的O-糖基化提供了额外的支持。对SAGA-1的O-糖基化的阐明增加了该分子的复杂性，并可能有助于解释其生物学活性。
