GalNAcβ1,3-linked paragloboside carries the epitope of a sperm maturation-related glycoprotein that is recognized by the monoclonal antibody MC121
GalNAcβ1,3-linked paragloboside carries the epitope of a sperm maturation-related glycoprotein that is recognized by the monoclonal antibody MC121
复制标题
DOI：
10.1016/j.bbrc.2011.02.019
复制
发表时间：
2011-03-18
影响因子：
3.1
通讯作者：
Kiyokawa, Nobutaka
中科院分区：
文献类型：
--
作者：
Katagiri, Yohko U.;Sato, Ban;Kiyokawa, Nobutaka
哺乳动物在附睾转运过程中精子的功能成熟伴随着由于蛋白质的结合或去除或与附睾中存在的蛋白酶、糖苷酶和糖基转移酶的相互作用而导致的质膜的变化。为了研究精子在附睾成熟过程中表面的变化，我们之前建立了几种针对小鼠附睾精子尾部54 kDa唾液酸糖蛋白的单克隆抗体，这些抗体在附睾运输过程中逐渐增加了抗原决定簇的表达。其中一种单克隆抗体 MC121 在糖蛋白去唾液酸化后与聚偏二氟乙烯膜上的小鼠精子糖蛋白发生反应，并且用 β-N-乙酰己糖胺酶处理去唾液酸化的精子糖蛋白大大降低了抗原决定簇的表达。此外还与小鼠附睾精子尾部发生反应。 MC121 与人红细胞 (hRBC) 发生反应。 MC121 诱导唾液酸酶处理的 hRBC 发生凝集，并且用福尔马林蒸气固定的 hRBC 染色比用甲醇固定的 hRBC 染色严重得多。使用 MC121 对唾液酸酶处理的 hRBC 脂质进行薄层色谱 (TLC) 免疫染色，表明带有表位的分子是鞘糖脂 (GSL)，并且 MC121 与五糖 GSL 发生反应 通过 TLC 印迹矩阵辅助激光解吸电离飞行时间质谱分析唾液酸酶处理的 GSL (MALDI TOF MS)显示MC121结合的GSL是[HexNAc][HexNAc + Hex][Hex][Hex]-Cer。用 mAb TH2 染色的脂质条带对 GSL、GalNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-神经酰胺具有特异性。这些结果表明，MC121 结合的表位存在于新乳糖系列 GSL、IV(3)GalNAc beta-nLc(4)Cer(2) 序列中。 (C) 2011 Elsevier Inc. 保留所有权利。
The functional maturation of spermatozoa during epididymal transit in mammals accompanies the changes in their plasma membrane due to the binding or removal of proteins or interactions with the proteases, glycosidases and glycosyltransferases present in the epididymis. In order to study the surface changes in spermatozoa during their maturation in the epididymis, we previously established several monoclonal antibodies against the 54 kDa sialoglycoprotein of mouse cauda epididymal spermatozoa, which gradually increased the expression of antigenic determinants during epididymal transit. One of these monoclonal antibodies, MC121, reacted with mouse sperm glycoproteins on a polyvinylidene fluoride membrane after desialylation of the glycoproteins, and the treatment of the desialylated sperm glycoproteins with beta-N-acetylhexosaminidase greatly decreased the expression of the antigenic determinants. In addition to reacting with mouse cauda epididymal spermatozoa. MC121 reacted with human red blood cells (hRBCs). MC121 induced agglutination of sialidase-treated hRBCs and stained hRBCs fixed with formalin vapor much more heavily than it stained hRBCs fixed with methanol. The thin layer chromatography (TLC) immunostaining of the sialidase-treated lipids of hRBCs with MC121 suggested that the epitope-bearing molecule is a glycosphingolipids (GSL), and that MC121 reacts with a pentaose-GSL Analysis of sialidase-treated GSLs by TLC-Blot-Matrix Assisted Laser Desorption Ionization Time-of-Flight mass spectrometry (MALDI TOF MS) revealed that the GSL bound by MC121 was [HexNAc][HexNAc + Hex][Hex][Hex]-Cer. The lipid band stained with mAb TH2, which is specific for a GSL, GalNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-ceramide. These results indicated that the epitope to which MC121 binds is present in a neolacto-series GSL, IV(3)GalNAc beta-nLc(4)Cer(2) sequence. (C) 2011 Elsevier Inc. All rights reserved.