Bitoun Emmanuelle, Micheloni Alessia, Lamant Laurence, Bonnart Chrystelle, Tartaglia-Polcini Alessandro, Cobbold Christian, Al Saati Talal, Mariotti Feliciana, Mazereeuw-Hautier Juliette, Boralevi Franck, Hohl Daniel, Harper John, Bodemer Christine, D'Alessio Marina, Hovnanian Alain
Wellcome Trust Centre for Human Genetics, University of Oxford, UK.
Hum Mol Genet. 2003 Oct 1;12(19):2417-30. doi: 10.1093/hmg/ddg247. Epub 2003 Jul 29.
SPINK5, encoding the putative multi-domain serine protease inhibitor LEKTI, was recently identified as the defective gene in the severe autosomal recessive ichthyosiform skin condition, Netherton syndrome (NS). Using monoclonal and polyclonal antibodies, we show that LEKTI is a marker of epithelial differentiation, strongly expressed in the granular and uppermost spinous layers of the epidermis, and in differentiated layers of stratified epithelia. LEKTI expression was also demonstrated in normal differentiated human primary keratinocytes (HK) through detection of a 145 kDa full-length protein and a shorter isoform of 125 kDa. Both proteins are N-glycosylated and rapidly processed in a post-endoplasmic reticulum compartment into at least three C-terminal fragments of 42, 65 and 68 kDa, also identified in conditioned media. Processing of the 145 and 125 kDa precursors was prevented in HK by treatment with a furin inhibitor. In addition, in vitro cleavage of the recombinant 145 kDa precursor by furin generated C-terminal fragments of 65 and 68 kDa, further supporting the involvement of furin in LEKTI processing. In contrast, LEKTI precursors and proteolytic fragments were not detected in differentiated HK from NS patients. Defective expression of LEKTI in skin sections was a constant feature in NS patients, whilst an extended reactivity pattern was observed in samples from other keratinizing disorders, demonstrating that loss of LEKTI expression in the epidermis is a diagnostic feature of NS. The identification of novel processed forms of LEKTI provides the basis for future functional and structural studies of fragments with physiological relevance.
SPINK5基因编码假定的多结构域丝氨酸蛋白酶抑制剂LEKTI，最近被确定为严重常染色体隐性鱼鳞病样皮肤病—— Netherton综合征（NS）中的缺陷基因。我们使用单克隆抗体和多克隆抗体表明，LEKTI是上皮分化的标志物，在表皮的颗粒层和最上层棘层以及复层上皮的分化层中强烈表达。通过检测145 kDa的全长蛋白和125 kDa的较短异构体，在正常分化的人原代角质形成细胞（HK）中也证实了LEKTI的表达。这两种蛋白都进行了N-糖基化，并在内质网后区室中迅速加工成至少三个C端片段，分别为42 kDa、65 kDa和68 kDa，在条件培养基中也能鉴定到。用弗林蛋白酶抑制剂处理HK可阻止145 kDa和125 kDa前体的加工。此外，弗林蛋白酶对重组145 kDa前体的体外切割产生了65 kDa和68 kDa的C端片段，进一步支持了弗林蛋白酶参与LEKTI加工的观点。相比之下，在NS患者的分化HK中未检测到LEKTI前体和蛋白水解片段。LEKTI在皮肤切片中的表达缺陷是NS患者的一个恒定特征，而在其他角化障碍的样本中观察到了扩展的反应模式，表明表皮中LEKTI表达的缺失是NS的一个诊断特征。LEKTI新加工形式的鉴定为未来对具有生理相关性的片段进行功能和结构研究提供了基础。
