Lopez M, Tetaert D, Juliant S, Gazon M, Cerutti M, Verbert A, Delannoy P
Laboratoire de Chimie Biologique, Unité de Glycobiologie Structurale et Fonctionnelle, UMR CNRS no. 8576, Université des Sciences et Technologies de Lille, F-59655, Villeneuve d'Ascq, France.
Biochim Biophys Acta. 1999 Mar 14;1427(1):49-61. doi: 10.1016/s0304-4165(98)00176-7.
The enzyme activities involved in O-glycosylation have been studied in three insect cell lines, Spodoptera frugiperda (Sf-9), Mamestra brassicae (Mb) and Trichoplusia ni (Tn) cultured in two different serum-free media. The structural features of O-glycoproteins in these insect cells were investigated using a panel of lectins and the glycosyltransferase activities involved in O-glycan biosynthesis of insect cells were measured (i.e., UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, UDP-Gal:core-1 beta1, 3-galactosyltransferase, CMP-NeuAc:Galbeta1-3GalNAc alpha2, 3-sialyltransferase, and UDP-Gal:Galbeta1-3GalNAc alpha1, 4-galactosyltransferase activities). First, we show that O-glycosylation potential depends on cell type. All three lepidopteran cell lines express GalNAcalpha-O-Ser/Thr antigen, which is recognized by soy bean agglutinin and reflects high UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase activity. Capillary electrophoresis and mass spectrometry studies revealed the presence of at least two different UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases in these insect cells. Only some O-linked GalNAc residues are further processed by the addition of beta1,3-linked Gal residues to form T-antigen, as shown by the binding of peanut agglutinin. This reflects relative low levels of UDP-Gal:core-1 beta1,3-galactosyltransferase in insect cells, as compared to those observed in mammalian control cells. In addition, we detected strong binding of Bandeiraea simplicifolia lectin-I isolectin B4 to Mamestra brassicae endogenous glycoproteins, which suggests a high activity of a UDP-Gal:Galbeta1-3GalNAc alpha1, 4-galactosyltransferase. This explains the absence of PNA binding to Mamestra brassicae glycoproteins. Furthermore, our results substantiated that there is no sialyltransferase activity and, therefore, no terminal sialic acid production by these cell lines. Finally, we found that the culture medium influences the O-glycosylation potential of each cell line.
在两种不同的无血清培养基中培养的三种昆虫细胞系，即草地贪夜蛾（Sf - 9）、甘蓝夜蛾（Mb）和粉纹夜蛾（Tn）中，研究了参与O - 糖基化的酶活性。使用一组凝集素研究了这些昆虫细胞中O - 糖蛋白的结构特征，并测量了参与昆虫细胞O - 聚糖生物合成的糖基转移酶活性（即UDP - GalNAc：多肽N - 乙酰半乳糖胺基转移酶、UDP - Gal：核心 - 1 β1,3 - 半乳糖基转移酶、CMP - NeuAc：Galβ1 - 3GalNAc α2,3 - 唾液酸转移酶和UDP - Gal：Galβ1 - 3GalNAc α1,4 - 半乳糖基转移酶活性）。首先，我们表明O - 糖基化潜力取决于细胞类型。所有三种鳞翅目细胞系均表达GalNAcα - O - Ser/Thr抗原，该抗原可被大豆凝集素识别，反映出高UDP - GalNAc：多肽N - 乙酰半乳糖胺基转移酶活性。毛细管电泳和质谱研究表明，这些昆虫细胞中存在至少两种不同的UDP - GalNAc：多肽N - 乙酰半乳糖胺基转移酶。只有一些O - 连接的GalNAc残基通过添加β1,3 - 连接的Gal残基进一步加工形成T抗原，这通过花生凝集素的结合得以证明。与在哺乳动物对照细胞中观察到的情况相比，这反映了昆虫细胞中UDP - Gal：核心 - 1 β1,3 - 半乳糖基转移酶的相对低水平。此外，我们检测到单叶豆凝集素 - I同工凝集素B4与甘蓝夜蛾内源性糖蛋白的强结合，这表明UDP - Gal：Galβ1 - 3GalNAc α1,4 - 半乳糖基转移酶具有高活性。这解释了花生凝集素与甘蓝夜蛾糖蛋白不结合的原因。此外，我们的结果证实这些细胞系不存在唾液酸转移酶活性，因此也不产生末端唾液酸。最后，我们发现培养基会影响每个细胞系的O - 糖基化潜力。
