Robinson N A, LaCelle P T, Eckert R L
Department of Physiology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4970, USA.
J Invest Dermatol. 1996 Jul;107(1):101-7. doi: 10.1111/1523-1747.ep12298323.
Involucrin (hINV) is an important structural component of the keratinocyte cornified envelope that is expressed early in the keratinocyte differentiation process and is thought to be a component of the initial envelope scaffolding. We have previously shown that cyanogen bromide (CNBr) cleavage of cornified envelopes isolated from cultured foreskin keratinocytes releases several discrete involucrin-immunoreactive peptides. In this study, we compare the pattern of release of immunoreactive hINV fragments from envelopes prepared from human breast skin and foreskin, and from spontaneous and induced envelopes prepared from cultured keratinocytes. We also identify one of the released products. Envelopes prepared from human breast skin or foreskin, or spontaneous or induced envelopes prepared from cultured cells differ significantly in structure. The envelopes isolated from epidermis appear to be structurally complete, whereas spontaneous envelopes appear less complete and the induced envelopes appear to be the least complete. In spite of these structural differences, CNBr cleavage releases an identical quartet of hINV-immunoreactive peptides migrating between 68 and 81 kDa from each preparation. Immunoblots indicate that the quantity of hINV-immunoreactive material released per microg of envelope protein is as follows: induced > spontaneous > foreskin > breast skin. The fastest migrating peptide (68 kDa) comigrates with a peptide that is released after CNBr cleavage of bacterially produced-recombinant hINV. Amino-terminal amino acid sequencing of this peptide from recombinant hINV and from the cornified envelopes yields the sequence G-Q-L-K-H-L-E-Q-Q-E-G-Q-P-K-H. These results suggest that this fragment is the 275-amino acid segment of hINV beginning at G311 and extending to K585, and that this peptide is not crosslinked to another protein. These results indicate that a population of the envelope-associated hINV present in cultured and in vivo keratinocytes is crosslinked in the amino-terminal half. It is possible that this species represents an early intermediate in the involucrin crosslinking process.
兜甲蛋白（hINV）是角质形成细胞角化包膜的重要结构成分，在角质形成细胞分化过程早期表达，被认为是初始包膜支架的组成部分。我们之前已经表明，从培养的包皮角质形成细胞中分离出的角化包膜经溴化氰（CNBr）裂解后会释放出几种离散的兜甲蛋白免疫反应性肽段。在本研究中，我们比较了从人乳房皮肤和包皮制备的包膜以及从培养的角质形成细胞制备的自发和诱导包膜中免疫反应性hINV片段的释放模式。我们还鉴定了其中一种释放产物。从人乳房皮肤或包皮制备的包膜，或从培养细胞制备的自发或诱导包膜在结构上有显著差异。从表皮分离的包膜在结构上似乎是完整的，而自发包膜看起来不太完整，诱导包膜似乎是最不完整的。尽管存在这些结构差异，但CNBr裂解从每种制备物中释放出一组相同的4种hINV免疫反应性肽段，其迁移率在68至81 kDa之间。免疫印迹表明，每微克包膜蛋白释放的hINV免疫反应性物质的量如下：诱导包膜＞自发包膜＞包皮包膜＞乳房皮肤包膜。迁移最快的肽段（68 kDa）与细菌产生的重组hINV经CNBr裂解后释放的一种肽段迁移位置相同。对来自重组hINV和角化包膜的该肽段进行氨基末端氨基酸测序，得到的序列为G-Q-L-K-H-L-E-Q-Q-E-G-Q-P-K-H。这些结果表明，该片段是hINV从G311开始延伸至K585的275个氨基酸段，并且该肽段未与另一种蛋白质交联。这些结果表明，培养的和体内角质形成细胞中存在的与包膜相关的hINV群体在氨基末端一半区域发生了交联。这种类型的hINV有可能代表兜甲蛋白交联过程中的早期中间体。
