Watkinson A
Unilever Research, Cell Biology and Physiology Unit, Sharnbrook, Bedford, UK.
Arch Dermatol Res. 1999 May;291(5):260-8. doi: 10.1007/s004030050406.
Proteolytic enzymes play crucial roles in the formation of the stratum corneum barrier tissue and in its subsequent maturation. Despite this, the proteases involved in stratum corneum physiology are not well characterized. Hence, studies were performed to identify these proteolytic enzymes present in the peripheral layers of this tissue using a combination of tape stripping and zymography. Using this approach, a novel human cysteine protease was identified and characterized, and named stratum corneum thiol protease (SCTP). Gelatin zymography revealed that SCTP is composed of two variants with apparent molecular weights of 34 and 35 kDa which do not correspond to any previously described stratum corneum protease. Mechanistically SCTP belongs to the cysteine proteinase class as shown by: (1) acid protease activity, (2) a requirement for mild reducing conditions, and (3) the specific inhibition of activity by E64 and Z-phe-ala-diazomethylketone. Further analysis using concanavalin A affinity chromatography demonstrated that the two 34 and 35 kDa variants are both glycoproteins, which, after removal of the oligosaccharide sidechains with the specific enzyme N-glycopeptidase F, reveal a single active core protease of 32 kDa. SCTP did not crossreact with antibodies raised against the lysosomal cysteine proteases cathepsins B, H or L, thereby distinguishing it from the classical cysteine cathepsins. Localization studies revealed that SCTP is present at all depths in the stratum corneum, thereby precluding microbial contamination as the enzyme source. Moreover, it was also present at all body sites investigated, except for the hyperkeratotic palmoplantar stratum corneum. SCTP was found to be a product of late differentiation in cultured human keratinocytes; the enzyme was synthesized by differentiated calcium-switched cells and secreted into the medium, whereas nondifferentiated basal keratinocytes did not produce this protease. Moreover, human fibroblast cultures did not produce the enzyme, suggesting that SCTP is not produced by the dermis and hence is epidermal specific. The function of SCTP is unknown, but the observed gelatinolytic activity coupled with its secretion into the medium by cultured keratinocytes indicates that physiologically it is responsible for the degradation of extracellular structural proteins. Furthermore, the optimal activity at acid pH suggests that it can function in the acidic environment of the stratum corneum. It remains to be elucidated whether this enzyme has a role in desquamation.
蛋白水解酶在角质层屏障组织的形成及其随后的成熟过程中发挥着关键作用。尽管如此，参与角质层生理过程的蛋白酶尚未得到充分表征。因此，我们采用胶带剥离和酶谱分析相结合的方法，对该组织外周层中存在的这些蛋白水解酶进行了研究。通过这种方法，我们鉴定并表征了一种新型的人半胱氨酸蛋白酶，并将其命名为角质层硫醇蛋白酶（SCTP）。明胶酶谱分析显示，SCTP由两种变体组成，其表观分子量分别为34 kDa和35 kDa，这与之前描述的任何角质层蛋白酶均不对应。从机制上讲，SCTP属于半胱氨酸蛋白酶类，这表现在以下几个方面：（1）酸性蛋白酶活性；（2）需要温和的还原条件；（3）E64和Z-苯丙氨酸-丙氨酸-重氮甲基酮对其活性具有特异性抑制作用。使用伴刀豆球蛋白A亲和色谱进行的进一步分析表明，这两种34 kDa和35 kDa的变体均为糖蛋白，在用特异性酶N-糖肽酶F去除寡糖侧链后，显示出一个单一的32 kDa活性核心蛋白酶。SCTP与针对溶酶体半胱氨酸蛋白酶组织蛋白酶B、H或L产生的抗体不发生交叉反应，从而将其与经典的半胱氨酸组织蛋白酶区分开来。定位研究表明，SCTP存在于角质层的所有深度，从而排除了微生物污染作为酶源的可能性。此外，除了角化过度的掌跖角质层外，它也存在于所有研究的身体部位。研究发现SCTP是培养的人角质形成细胞晚期分化的产物；该酶由分化的钙转换细胞合成并分泌到培养基中，而未分化的基底角质形成细胞不产生这种蛋白酶。此外，人成纤维细胞培养物不产生该酶，这表明SCTP不是由真皮产生的，因此是表皮特异性的。SCTP的功能尚不清楚，但观察到的明胶溶解活性及其通过培养的角质形成细胞分泌到培养基中表明，在生理上它负责细胞外结构蛋白的降解。此外，在酸性pH下的最佳活性表明它可以在角质层的酸性环境中发挥作用。这种酶是否在脱屑过程中起作用还有待阐明。
