Sihlbom Carina, van Dijk Härd Iris, Lidell Martin E, Noll Thomas, Hansson Gunnar C, Bäckström Malin
Department of Medical Biochemistry, Institute of Biomedicine, University of Gothenburg, 405 30 Gothenburg, Sweden.
Glycobiology. 2009 Apr;19(4):375-81. doi: 10.1093/glycob/cwn144. Epub 2008 Dec 18.
MUC1 is a mucin glycoprotein containing multiple tandem repeats of 20 amino acids, with five serines and threonines that can be O-glycosylated. Here, we investigated the O-glycosylation site occupancy in MUC1 glycoproteins produced in two mutant CHO cell lines, Lec3.2.8.1 and ldlD. We found that the average site occupancy was higher in MUC1 from Lec3.2.8.1 than from ldlD and that the occupancy increased with the number of tandem repeats in the protein and also depended on the culture conditions used for production. Moreover, we describe the successful use of electron-capture dissociation (ECD) fragmentation, coupled to online liquid chromatography mass spectrometry, to determine the glycosylation of individual sites in recombinant MUC1 proteins with 16 tandem repeats. We analyzed MUC1 tandem repeat peptides with 1-5 GalNAc residues by ECD fragmentation and found that the first site to be glycosylated was either Ser-5 or Thr-6, with the addition of a second GalNAc at Thr-14. For peptides with three GalNAc residues, several different variants of glycopeptides were found, indicating a heterogeneous order of glycosylation at this stage. In contrast, only one variant was found for peptides with four GalNAc residues, where Thr-19 in the PDTR motif was left unglycosylated, indicating that this site is glycosylated last. The results gave novel insight into the order of GalNAc substitution in MUC1 in vivo.
MUC1是一种粘蛋白糖蛋白，包含20个氨基酸的多个串联重复序列，有5个可进行O-糖基化的丝氨酸和苏氨酸。在此，我们研究了在两种突变的CHO细胞系Lec3.2.8.1和ldlD中产生的MUC1糖蛋白的O-糖基化位点占据情况。我们发现，Lec3.2.8.1细胞系中MUC1的平均位点占据率高于ldlD细胞系，且位点占据率随蛋白质中串联重复序列的数量增加而升高，还取决于用于生产的培养条件。此外，我们描述了成功运用电子捕获解离（ECD）碎裂技术并结合在线液相色谱质谱法，来确定具有16个串联重复序列的重组MUC1蛋白中各个位点的糖基化情况。我们通过ECD碎裂分析了带有1 - 5个N-乙酰半乳糖胺（GalNAc）残基的MUC1串联重复肽段，发现第一个被糖基化的位点是Ser-5或Thr-6，在Thr-14处添加第二个GalNAc。对于带有三个GalNAc残基的肽段，发现了几种不同的糖肽变体，表明在此阶段糖基化顺序存在异质性。相比之下，对于带有四个GalNAc残基的肽段，仅发现一种变体，其中PDTR基序中的Thr-19未被糖基化，这表明该位点最后被糖基化。这些结果为体内MUC1中GalNAc取代的顺序提供了新的见解。
