Schechter N M, Wang Z M, Blacher R W, Lessin S R, Lazarus G S, Rubin H
Department of Dermatology, University of Pennsylvania, Philadelphia 19104.
J Immunol. 1994 Apr 15;152(8):4062-9.
This study establishes the primary structure of human skin chymase and provides further evidence for the presence of a cathepsin G-like proteinase within human mast cells. The amino acid sequence of human skin chymase was established by protein methods and by analysis of PCR amplification products obtained with cDNA-derived from urticaria pigmentosa (UP) lesions. UP is a disease characterized by skin lesions containing high numbers of mast cells. Proteolytic digests of human chymase purified from normal skin yielded 10 resolvable peptides that were sequenced by automated Edman degradation. The amino acid sequences for these peptides combined with the sequence obtained for the protein's NH2-terminal region (35 residues) accounted for 137 residues of the human skin chymase sequence. This partial amino acid sequence corresponded to the sequence of human heart chymase, a proteinase isolated from heart tissue with immunologic and hydrolytic properties similar to skin chymase. PCR amplification of UP-derived cDNA with primers based on the cDNA structure of heart chymase demonstrated a single amplification product of expected size which was subcloned and sequenced. The amino acid sequence (135 residues) deduced from this product was identical to that of heart chymase in the region between the primers. This sequence, along with that established for the purified protein, constituted 99% of the heart chymase primary structure, strongly indicating that human skin and heart chymases have identical primary structures. Amplification of the same UP-cDNA with primers coding for the NH2- and COOH-terminal sequences of human neutrophil cathepsin G also produced a specific amplification product which was sequenced. The deduced amino acid sequence between the primers was identical to that reported for neutrophil cathepsin G, indicating that the protein of cutaneous mast cells previously shown to be immunologically cross-reactive with neutrophil cathepsin G has a comparable amino acid sequence. UP-cDNA demonstrating amplification products for cathepsin G did not demonstrate amplification products for human neutrophil elastase, suggesting that the cathepsin G PCR amplification product was not derived from neutrophils or monocytes possibly contaminating the lesion. These studies provide further evidence that human skin mast cells contain two different chymotrypsin-like proteinases.
本研究确定了人皮肤糜酶的一级结构，并为人类肥大细胞中存在组织蛋白酶G样蛋白酶提供了进一步证据。通过蛋白质方法以及对从色素性荨麻疹（UP）皮损中获得的cDNA进行PCR扩增产物分析，确定了人皮肤糜酶的氨基酸序列。UP是一种以含有大量肥大细胞的皮肤损害为特征的疾病。从正常皮肤中纯化的人糜酶的蛋白水解消化产物产生了10个可分辨的肽段，通过自动Edman降解对其进行测序。这些肽段的氨基酸序列与该蛋白氨基末端区域（35个残基）获得的序列相结合，构成了人皮肤糜酶序列的137个残基。这个部分氨基酸序列与人心糜酶的序列相对应，人心糜酶是一种从心脏组织中分离出来的蛋白酶，其免疫和水解特性与皮肤糜酶相似。基于心糜酶的cDNA结构，用引物对UP来源的cDNA进行PCR扩增，得到了预期大小的单一扩增产物，将其亚克隆并测序。从该产物推导的氨基酸序列（135个残基）在引物之间的区域与心糜酶的序列相同。这个序列，连同为纯化蛋白确定的序列，构成了心糜酶一级结构的99%，有力地表明人皮肤和心糜酶具有相同的一级结构。用编码人中性粒细胞组织蛋白酶G氨基末端和羧基末端序列的引物对同一UP-cDNA进行扩增，也产生了一个经过测序的特异性扩增产物。引物之间推导的氨基酸序列与报道的中性粒细胞组织蛋白酶G的序列相同，表明先前显示与中性粒细胞组织蛋白酶G有免疫交叉反应的皮肤肥大细胞蛋白具有可比的氨基酸序列。显示组织蛋白酶G扩增产物的UP-cDNA未显示人中性粒细胞弹性蛋白酶的扩增产物，这表明组织蛋白酶G的PCR扩增产物不是来自可能污染皮损的中性粒细胞或单核细胞。这些研究提供了进一步证据，证明人皮肤肥大细胞含有两种不同的类胰凝乳蛋白酶。
