Consequences of C-terminal domains and N-terminal signal peptide deletions on LEKTI secretion, stability, and subcellular distribution
Consequences of C-terminal domains and N-terminal signal peptide deletions on LEKTI secretion, stability, and subcellular distribution
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DOI：
10.1016/j.abb.2004.12.012
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发表时间：
2005-03-01
影响因子：
3.9
通讯作者：
Clayman, GL
中科院分区：
文献类型：
--
作者：
Jayakumar, A;Kang, Y;Clayman, GL
分泌型淋巴上皮型抑制因子(Lekti)是一种含有N端信号肽和15个潜在抑制结构域的前体lekti蛋白。这种抑制剂具有特殊的意义，因为它对严重的先天性疾病Netherton综合征具有重要的病理生理学意义。我们发现Lekti是一种参与细胞外基质重塑的丝氨酸蛋白酶家族的有效抑制剂，它在头颈部鳞状细胞癌中的表达下调。我们构建了lekti基因的缺失突变体，在HEK 293T细胞中表达，并对其分泌行为、稳定性和亚细胞分布进行了分析，以评价其C末端结构域和N末端信号肽在lekti分泌中的作用。蛋白水解酶抑制功能。Pro-lekti被加工并分泌到培养液中。根据N末端部分测序和免疫印迹分析，切割产物的氨基-羧基末端大小顺序为：37、40、60 kDa。呋喃西林的抑制剂能促进未加工的lekti的分泌，这表明分泌不需要加工。原Lekti的N末端信号肽的缺失改变了Lekti从内质网(ER)到细胞质的分布，并显著降低了其稳定性，这与其无法分泌到介质中的情况一致。有趣的是。当我们删除C-末端结构域时，稳定的部分lekti(LD-1-6)积累，并仍与ER结合，但不分泌。重组LD-1-6特异性抑制胰酶活性。我们认为，Lekti进入内质网需要N-端的信号肽，C-端区的元件可能对lekti的分泌起到调节作用。2004 Elsevier Inc.保留所有权利。
The secretory lympho-epithelial Kazal-type-inhibitor (LEKTI) is synthesized as a pro-LEKTI protein containing an N-terminal signal peptide and 15 potentially inhibitory domains. This inhibitor is of special interest because of its pathophysiological importance for the severe congenital disease Netherton syndrome. We showed that LEKTI is a potent inhibitor of a family of serine proteinases involved in extracellular matrix remodeling and its expression is downregulated in head and neck squamous cell carcinomas. To assess the role of C-terminal domains and N-terminal signal peptide in LEKTI secretion, we constructed deletion mutants of LEKTI, expressed them in HEK 293T cells, and analyzed their secretion behavior, stability, subcellular distribution. and proteinase inhibitory function. Pro-LEKTI is processed and secreted into the medium. On the basis of partial N-terminal sequencing and immunoblotting, the cleavage products are ordered front amino- to carboxy-terminal as follows: 37, 40, and 60 kDa. Inhibitors of furin lead to enhanced secretion of unprocessed LEKTI, suggesting that processing was not required for secretion. Deletion of the N-terminal signal peptide of pro-LEKTI caused altered distribution of LEKTI from endoplasmic reticulum (ER) to cytoplasm and markedly reduced its stability, consistent with its failure to become secreted into the medium. Interestingly. when we deleted the C-terminal domains, stable partial LEKTI (LD-1-6) accumulated and still retained its association with ER but was not secreted. Recombinant LD-1-6 specifically inhibited the trypsin activity. We conclude that N-terminal signal peptide is required for LEKTI import into ER and elements present in C-terminal domains may have a role in regulating LEKTI secretion. 2004 Elsevier Inc. All rights reserved.