Characterization of protease processing sites during conversion of rat profilaggrin to filaggrin.
Characterization of protease processing sites during conversion of rat profilaggrin to filaggrin.
复制标题
大鼠丝聚合蛋白原转化为丝聚合蛋白过程中蛋白酶加工位点的表征。
DOI：
10.1021/bi00089a020
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发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
Walsh,KA
中科院分区：
文献类型：
--
作者：
Resing,KA;Johnson,RS;Walsh,KA
原丝聚蛋白是角化上皮细胞的一种中间趋化相关蛋白。它由多个拷贝的相似的聚丝蛋白结构域连接肽接头区域;在表皮的终末分化，接头区域被处理掉，以调节的方式。为了表征大鼠聚丝蛋白原中蛋白水解的位点，通过反相HPLC分离聚丝蛋白和聚丝蛋白原的胰蛋白酶肽，并通过雾化辅助电喷雾电离质谱分析HPLC级分。通过串联质谱法确认或校正肽序列;在几种情况下，这是通过碰撞活化质量超过3 kDa的肽的多电荷配体来实现的。胰蛋白酶肽占所有的序列预测的部分cDNA序列，除了6个精氨酸或二肽。虽然cDNA序列预测的聚丝蛋白结构域之间的异质性的8个网站，只有一个被观察到。还观察到一个额外的非预期异质性位点。比较来自聚丝蛋白的肽与聚丝蛋白原的肽，揭示了聚丝蛋白特有的几种肽，特别是在新的氨基和羧基末端，其由聚丝蛋白原的接头区的蛋白水解加工产生。氨基端和羧基端都是“不规则的”，表明加工过程可能涉及初始内肽酶裂解后的外肽酶作用。通过电喷雾质谱法测定该丝蛋白混合物的平均质量为42 452 Da，与末端序列的质谱分析预测的结果合理一致。发现大鼠聚丝蛋白原的连接肽有两种形式，其不同之处仅在于丝氨酸22的磷酸化状态。哺乳动物表皮终末分化中发生的最后事件之一是聚丝蛋白原的合成和聚丝蛋白原加工成聚丝蛋白[综述参见Resing和Dale（1991）]。聚丝蛋白原的合成涉及几种激酶的广泛磷酸化以及次晶颗粒（透明角质蛋白颗粒）细胞质中的沉积。聚丝蛋白原具有不寻常的重复结构，聚丝蛋白的每个结构域通过在加工过程中去除的接头肽连接（Mckinley-Grant et al.，1989; Resing等人，1989年）。聚丝蛋白原的单体重复单元在动物物种内显示出彼此高度的相似性（在小鼠中95%的同一性，在人中85%），但在物种之间变化很大（小鼠和人之间为30-42%）（Gan等人，1990; Haydock & Dale，1990; Rothnagel & Steinert，1990）。当细胞终末分化时，随着聚丝蛋白原在多步过程中被去磷酸化和蛋白水解以产生聚丝蛋白，颗粒分散（Resing等人，1989年）。
Revised Manuscript Received July 9, 1993· abstract: Profilaggrin is an intermediate filament-associated protein of cornifiedepithelia. It consists of multiple copies of similar filaggrin domains joined by peptide linker regions; during terminal differentiation of the epidermis, the linker regions are processed away in a regulated manner. In order to characterize the sites of proteolysis in rat profilaggrin, tryptic peptides of filaggrin and profilaggrin were fractionated by reverse-phase HPLC, and the HPLC fractions were analyzed by nebulization-assisted electrospray ionization mass spectrometry. Peptide sequences were confirmed or corrected by tandem mass spectrometry; in several cases, this was achieved by collisionalactivation of multiply charged precursorions of peptides exceeding 3 kDa in mass. The tryptic peptides accounted for all of the sequence predicted by a partial cDNA sequence, with the exception of six arginines or dipeptides. Although the cDNA sequence predicted eight sites of heterogeneity among the filaggrin domains, only one of these was observed. An additional unpredicted site of heterogeneity was also seen. Comparison of the peptides from filaggrin with those of profilaggrin revealed several peptides unique to filaggrin, specifically at the new amino-and carboxyltermini, that result from proteolytic processing of the linker region of profilaggrin. Both the amino-and carboxyl-termini were “ragged”, suggesting that processing may involve exopeptidase actionafter an initial endopeptidase cleavage. The average mass of this mixture of filaggrins was determined by electrospray mass spectrometry to be 42 452 Da, in reasonable agreement with that predicted from the mass spectrometric analysis of the terminal sequences. The linker peptide of rat profilaggrin was found in two forms, which differed only in the phosphorylation state of serine 22.One of the last events occurring in terminal differentiation of mammalian epidermis is the synthesis of profilaggrin and processing of profilaggrinto filaggrin [for a review, see Resing and Dale (1991)]. Synthesis of profilaggrin involves extensive phosphorylation byseveral kinases and deposition in the cytosol in a paracrystalline granule (the keratohyalin granule). Profilaggrin has an unusual repeating structure, with each domain of filaggrin joined by a linker peptide that is removed during processing (Mckinley-Grant et al., 1989; Resing et al., 1989). The monomer repeat units of profilaggrin display a high degree of similarity to one another within an animal species (95% identity in mouse, 85% in human), but vary greatly between species (30-42% between mouse and human)(Gan et al., 1990; Haydock & Dale, 1990; Rothnagel & Steinert, 1990). As a cell terminally differentiates, the granules disperse as the profilaggrin is both dephosphorylated and proteolyzed in a multistep process to yield filaggrin (Resing et al., 1989).