Identification of serum factor inducing ectodomain shedding of proHB-EGF and studies of noncleavable mutants of proHB-EGF
Identification of serum factor inducing ectodomain shedding of proHB-EGF and studies of noncleavable mutants of proHB-EGF
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DOI：
10.1006/bbrc.2001.4879
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发表时间：
2001-05-18
影响因子：
3.1
通讯作者：
Mekada, E
中科院分区：
文献类型：
--
作者：
Hirata, M;Umata, T;Mekada, E
跨膜形式的HB-EGF的胞外区(proHB-EGF)在细胞表面被蛋白酶切割，产生一种可溶性的生长因子。包括TPA在内的许多刺激都会加速这种分裂。然而，在正常培养条件下，proHB-EGF在没有任何特殊刺激的情况下被结构性地脱落。我们在这里证明，结构性分裂主要是由添加的FCS中所含的因子(S)在培养基中引起的。对血清因素的分析，包括酶消化、薄层层析分离和纯化磷脂的脱落试验，表明溶血磷脂酸(LPA)是FCS刺激ProHB-EGF脱落的主要因素。我们还研究了两种突变形式的proHB-EGF的胞外结构域脱落，这两种突变形式在假定的切割位点附近有一个氨基酸替换。这些突变形式都表现出对TPA和LPA刺激的抗性，表明ProHB-EGF在TPA和LPA刺激下在相似的位置被切割。(C)2001年学术出版社。
The ectodomain of the transmembrane form of HB-EGF (proHB-EGF) is cleaved at the cell surface by proteases, yielding a soluble growth factor. A number of stimuli, including TPA, accelerate this cleavage. However, proHB-EGF is shed constitutively under normal culture conditions without any particular stimuli. We demonstrate here that constitutive cleavage resulted largely from factor(s) contained in supplemented FCS in a culture medium. Analysis of serum factors, including digestion with enzymes, separation by thin layer chromatography, and shedding assay with purified phospholipids, revealed that lysophosphatidic acid (LPA) is a major factor in FCS for stimulation of proHB-EGF shedding. We also studied here ectodomain shedding of two kinds of mutant form of proHB-EGF which have a single amino acid substitution around the putative cleavage sites. These mutant forms showed resistance to stimuli of both TPA and LPA, suggesting that proHB-EGF is cleaved at the similar site by stimulation with TPA and LPA. (C) 2001 Academic Press.