Mutational analysis of the C-terminus in ion transport peptide (ITP) expressed in Drosophila Kc1 cells.
Mutational analysis of the C-terminus in ion transport peptide (ITP) expressed in Drosophila Kc1 cells.
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DOI：
10.1002/1520-6327(200011)45:3
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发表时间：
2000-11
影响因子：
2.2
通讯作者：
Y. J. Wang;Y. Zhao;J. Meredith;J. Phillips;D. Theilmann;H. Brock
中科院分区：
文献类型：
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作者：
Y. J. Wang;Y. Zhao;J. Meredith;J. Phillips;D. Theilmann;H. Brock
离子转运肽(ITP)能刺激血吸虫体内的氯离子转运(以短路电流I(Sc)表示)和液体重吸收。我们报道了转基因的果蝇Kc1细胞分泌一种多肽(KcITP(75))，虽然在N端被正确切割，但与天然的ITP(ScgITP)和合成的ITP(SynITP)相比，对回肠I(Sc)的刺激活性降低了10倍。我们提供的证据表明，KcITP(75)活性的降低是由于C-末端序列LGKK(KcITP(75))未被加工成L-酰胺所致。为了支持这一点，甘氨酸延伸的ITP(即KcITP(73)以LG结尾)而不是KcITP(75)(以LGKK结尾)的体外酰胺化显著增加了生物测定中的比活性。C末端参与的进一步证据包括截短突变体(例如，KcITP(71)缺乏LGKK)和一个突变体(其中丙氨酸取代KcITP(73)中的末端甘氨酸)完全失去刺激。此外，Kc1细胞表达的天然同源物(KcITP-L，仅在C-端序列不同)不刺激回肠I(Sc)。相反，KcITP-L是一个弱的ITP拮抗剂，截短突变体KcITP(71)也是如此。KcITP(70)无拮抗作用。C末端的一个短的合成肽片段(面纱-酰胺)不能刺激回肠I(Sc)，这表明ITP的其他区域也是生物学活动所必需的。拱门。
Ion transport peptide (ITP) stimulates Cl(-) transport (measured as short-circuit current, I(sc)) and fluid reabsorption in Schistocerca gregaria ilea. We report that Drosophila Kc1 cells transfected with preproITP cDNA secrete a peptide (KcITP(75)) that, while cleaved correctly at the N-terminus, had reduced (10-fold) stimulatory activity on ileal I(sc) compared to both native ITP (ScgITP) and synthetic ITP (synITP). We provide evidence that the reduced activity of KcITP(75) is due to incomplete processing of the C-terminal sequence LGKK (KcITP(75)) to L-amide. In support of this, in vitro amidation of glycine extended ITP (i.e., KcITP(73) ending in LG) but not KcITP(75) (ending in LGKK) significantly increased specific activity in the bioassay. Further evidence for C-terminus involvement includes complete loss of stimulation by truncated mutants (e.g., KcITP(71) which lacks LGKK) and a mutant in which alanine is substituted for the terminal glycine in KcITP(73). Moreover a natural homologue (KcITP-L, which differs only in the C-terminal sequence) expressed by Kc1 cells does not stimulate ileal I(sc). Rather KcITP-L acts as a weak ITP antagonist, as does the truncated mutant KcITP(71). KcITP(70) has no antagonistic effect. A short synthetic peptide fragment of the C-terminus (VEIL-amide) does not stimulate ileal I(sc), indicating that other regions of ITP are also essential to biological activity. Arch.