Characterisation and cellular localisation of a GPEET procyclin precursor in Trypanosoma brucei insect forms
Characterisation and cellular localisation of a GPEET procyclin precursor in Trypanosoma brucei insect forms
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DOI：
10.1016/s0166-6851(01)00398-x
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发表时间：
2002-01-01
影响因子：
1.5
通讯作者：
Roditi, I
中科院分区：
文献类型：
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作者：
Bütikofer, P;Vassella, E;Roditi, I
原环素代表布氏锥虫昆虫形式的主要表面分子，由两类蛋白质组成，其特征在于内部串联二肽（EP）或五肽重复序列（GPEET），并通过复杂的糖基化糖基磷脂酰肌醇（GPI）锚连接到膜上。两种不同形式的GPEET可以通过其与抗GPEET抗体的不同反应性来区分。22-32 kDa的主要组分被结合磷酸化形式的GPEET的单克隆抗体识别，20 kDa的次要组分被针对合成GPEET肽产生的多克隆抗血清识别。两种形式之间的关系是通过（i）富集20 kDa形式，并使用MALDI-TOF质谱法确定其精确质量;（ii）研究两种形式在多形T同步分化过程中的表达。布氏杆菌，血流形式的前循环形式;（iii）通过免疫荧光显微镜分析其亚细胞分布;和（iv）脉冲追踪标记使用氚化GPI。前体结果表明，20 kDa形式代表GPEET的生物合成前体，其刚刚开始接收GPI锚的聚-N-乙酰乳糖胺重复的组分。(C)2002 Elsevier Science B. V.保留所有权利。
The procyclins represent the major surface molecules of Trypanosoma brucei insect forms and consist of two classes of proteins that are characterised by internal tandem dipeptide (EP) or pentapeptide repeats (GPEET) and are attached to the membrane by a complex glycosylated glycosylphosphatidylinositol (GPI) anchor. Two different forms of GPEET can be distinguished by their differential reactivity with anti-GPEET antibodies. A major component of 22-32 kDa is recognised by a monoclonal antibody which binds to the phosphorylated form of GPEET, and a minor component of 20 kDa is recognised by a polyclonal antiserum which was raised against a synthetic GPEET peptide. The relationship between the two forms was established by (i) enriching for the 20 kDa form and determining its precise mass using MALDI-TOF mass spectrometry; (ii) studying the expression of the two forms during synchronous differentiation of pleomorphic T. brucei, bloodstream forms to procyclic forms; (iii) analysing their sub-cellular distribution by immunofluorescence microscopy; and (iv) pulse-chase labelling using tritiated GPI. precursors. The results indicate that the 20 kDa form represents a biosynthetic precursor of GPEET, which has just started to receive components of the poly-N-acetyllactosamine repeat of the GPI anchor. (C) 2002 Elsevier Science B.V. All rights reserved.