Presence of putative histidine-rich proteins in the amphibian epidermis.

Lorenzo Alibardi, Enzo Spisni, Mattia Toni
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引用次数: 11

Abstract

In amphibian epidermis mucus is thought to constitute the matrix material that links keratin filaments present in cells of the corneous layer. As contrast in mammals, and perhaps in all amniotes, histidine-rich proteins form the matrix material. In order to address the study of matrix molecules in the epidermis of the first tetrapods, the amphibians, an autoradiographic and electrophoretic study has been done after administration of tritiated histidine. Histological analysis of amphibian epidermis shows that histidine is taken up in the upper intermediate and replacement layers beneath the corneous layer. Ultrastructural autoradiographic analysis reveals that electron-dense interkeratin material is labeled after administration of tritiated histidine. Electrophoretic analysis of the epidermis shows labeled proteic bands at 58-61, 50-55, 40-45, and some only weakly labeled at 30 and 24-25 kDa at 4-48 hours after injection of tritiated histidine. Keratin markers show that bands at 40-61 kDa contain keratins. Most histidine is probably converted into other amino acids such as glutamate and glutamine that are incorporated into newly synthetized keratins. However, non-keratin histidine-incorporating proteins within the keratin range could also be formed. The bands at 30 and 24-25 kDa suggest that these putative histidine-rich proteins are not keratins. In fact, their molecular weigh is below the range of that for keratins. In contrast with the mammalian condition, but resembling reports for lizard epidermis, putative histidine-rich proteins in amphibians have no high molecular weight precursor. Although filaggrin is not detectable by immunofluorescence in sections of amphibian epidermis, protein extraction, electrophoresis and immunoblotting are more sensitive. In the epidermis of toad and frog, but only occasionally in that of newt, filaggrin cross-reactive proteic bands are seen at 50-55, 40-45, and sometimes at 25 kDa. This suggests that after extraction and unmasking of reactive sites in the epidermis of more terrestrial amphians (anurans), some HRPs with filaggrin-like cross-reactivity are present. The overlap that exists at 50-55 kDa between filaggrin-positive and AE2-positive keratins, but not that at 40-45 kDa further indicate that non-keratin, filaggrin-like proteins may be present in anuran epidermis. The present study suggests for the first time that very small amounts of histidine-rich proteins are produced among keratin filaments in upper intermediate, replacement and corneous layers of amphibian epidermis. Although the molecular composition of these proteins is unknown, precluding understanding of their relationship to those of mammals and reptiles, these cationic proteins might have originated in conjunction with the formation of a horny layer during the adaptation to land during the Carboniferous and were possibly refined later in the epidermis of amniotes.

两栖动物表皮中假定的富含组氨酸的蛋白质的存在。
两栖动物表皮粘液被认为是连接角质层细胞中角蛋白丝的基质物质。与哺乳动物相反,也许在所有羊膜动物中,富含组氨酸的蛋白质形成基质物质。为了研究最早的四足动物——两栖动物表皮中基质分子的结构,在给药后进行了氚化组氨酸的放射自显影和电泳研究。对两栖动物表皮的组织学分析表明,组氨酸在角质层以下的上、中间和替代层中被吸收。超微结构放射自显像分析显示,经氚化组氨酸处理后,电子致密的角间蛋白被标记。电泳分析显示,在注射氚化组氨酸后4-48小时,表皮在58-61、50-55、40-45处有标记的蛋白带,部分蛋白带在30和24-25 kDa处只有弱标记。角蛋白标记显示40-61 kDa的条带含有角蛋白。大多数组氨酸可能转化为其他氨基酸,如谷氨酸和谷氨酰胺,并与新合成的角蛋白结合。然而,角蛋白范围内的非角蛋白组氨酸结合蛋白也可以形成。30和24-25 kDa的条带表明这些假定的富含组氨酸的蛋白质不是角蛋白。事实上,它们的分子量低于角蛋白的分子量范围。与哺乳动物的情况相反,但类似于蜥蜴表皮的报道,两栖动物中假定的富含组氨酸的蛋白质没有高分子量的前体。虽然聚丝蛋白在两栖动物表皮的切片中不能被免疫荧光检测到,但蛋白质提取、电泳和免疫印迹法更敏感。在蟾蜍和青蛙表皮中,聚丝蛋白交叉反应蛋白条带在50- 55,40 -45,有时在25 kDa处可见,但在蝾螈表皮中偶有。这表明,在提取和揭露更多陆生两栖动物表皮的反应位点后,存在一些具有聚丝蛋白样交叉反应性的hrp。聚丝蛋白阳性和ae2阳性角蛋白在50-55 kDa处存在重叠,但在40-45 kDa处没有重叠,这进一步表明非角蛋白、聚丝蛋白样蛋白可能存在于无尾蜥蜴表皮中。本研究首次表明,在两栖动物表皮的上中间层、替代层和角质层的角蛋白丝中产生了极少量的富含组氨酸的蛋白质。尽管这些蛋白质的分子组成尚不清楚,因此无法理解它们与哺乳动物和爬行动物的关系,但这些阳离子蛋白质可能起源于石炭纪适应陆地过程中角质层的形成,并可能后来在羊膜动物的表皮中被精制。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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