Horikoshi T, Arany I, Rajaraman S, Chen S H, Brysk H, Lei G, Tyring S K, Brysk M M
Department of Dermatology, University of Texas Medical Branch, Galveston 77555, USA.
Biochimie. 1998 Jul;80(7):605-12. doi: 10.1016/s0300-9084(98)80013-8.
Cathepsin D is an ubiquitously expressed lysosomal aspartic proteinase, with well-determined structural and chemical properties but a less clearly defined biological role. In stratified epithelia, the chronology of cathepsin D activation and degradation can be connected with stages of cellular differentiation. We partially purified cathepsin D from human epidermis and from separated stratum corneum by standard biochemical procedures, monitored by SDS-PAGE and Western blotting, and verified its identity as to molecular mass, pH optimum, N-terminal sequencing, reactivity with the specific antibody, inhibition by pepstatin A, and specific enzyme activity. It had hemoglobin-degrading activity over the acid range, with maximum at pH 3. It also degraded bovine serum albumin, human keratins, and stratum corneum extracts at pH 4. We discerned all three isoforms of human cathepsin D (the 52 kDa proenzyme and the active forms at 48 kDa and 33 kDa) in the epidermis; both active forms were also seen in the stratum corneum, but the proenzyme was not. Gene expression of cathepsin D in epidermal keratinocytes resembled that of suprabasal structural proteins (involucrin, keratin K10, transglutaminase) in its response to the calcium switch. An antibody to the 33 kda isoform immunolocalized to the granular layer and the stratum corneum (whereas antibodies to the 48 kDa isoform have been reported to stain mainly the upper spinous and granular layers). A plausible hypothesis to harmonize these results is that cathepsin D is first expressed as the proenzyme in the upper spinous layer, is activated in the lysosomes in the granular layer to the 48 kDa form, and is degraded to the 33 kDa form in the transition zone between the granular layer and the stratum corneum. As the stratum corneum is an acid environment, with an ambient pH of approximately 4.5, cathepsin D is available and suited to contribute to desquamation.
组织蛋白酶D是一种广泛表达的溶酶体天冬氨酸蛋白酶，其结构和化学性质已明确，但生物学作用尚不清楚。在复层上皮中，组织蛋白酶D的激活和降解过程与细胞分化阶段相关。我们采用标准生化方法从人表皮和分离的角质层中部分纯化了组织蛋白酶D，通过SDS-PAGE和蛋白质免疫印迹进行监测，并从分子量、最适pH值、N端测序、与特异性抗体的反应性、胃蛋白酶抑制剂A的抑制作用以及特异性酶活性等方面对其进行了鉴定。它在酸性范围内具有血红蛋白降解活性，在pH 3时活性最高。在pH 4时，它还能降解牛血清白蛋白、人角蛋白和角质层提取物。我们在表皮中识别出了人组织蛋白酶D的所有三种同工型（52 kDa的酶原以及活性形式的48 kDa和33 kDa）；在角质层中也发现了两种活性形式，但未发现酶原。表皮角质形成细胞中组织蛋白酶D的基因表达在对钙转换的反应方面与基底上层结构蛋白（内披蛋白、角蛋白K10、转谷氨酰胺酶）相似。一种针对33 kDa同工型的抗体免疫定位在颗粒层和角质层（而据报道，针对48 kDa同工型的抗体主要染色上棘层和颗粒层）。一个协调这些结果的合理假说是，组织蛋白酶D首先在上棘层以酶原形式表达，在颗粒层的溶酶体中被激活为48 kDa形式，并在颗粒层和角质层之间的过渡区降解为33 kDa形式。由于角质层是酸性环境，环境pH约为4.5，组织蛋白酶D可发挥作用并有助于脱屑。
