Proteolytic inactivation of the leukocyte C5a receptor by proteinases derived from Porphyromonas gingivalis
Proteolytic inactivation of the leukocyte C5a receptor by proteinases derived from Porphyromonas gingivalis
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DOI：
10.1128/iai.64.6.1984-1991.1996
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发表时间：
1996-06-01
影响因子：
3.1
通讯作者：
Hugli, TE
中科院分区：
文献类型：
--
作者：
Jagels, MA;Travis, J;Hugli, TE
厌氧细菌牙龈卟啉单胞菌被认为是成人牙周炎的主要病原体，这种细菌会产生几种蛋白酶，这表明它们有助于引起牙龈卟啉单胞菌感染的毒力和局部组织损伤。具有切割精氨酸x或赖氨酸x肽键特异性的半胱氨酸蛋白酶（即牙龈蛋白酶）已被表征为与从该细菌表面脱落的囊泡相关的主要酶。最近有研究表明，这些蛋白酶能够降解血液补体成分C5，从而产生具有生物活性的C5a。通过使用亲和纯化的兔抗体对C5a受体(C5aR；CD88)，我们证明了与牙龈卟啉卟啉囊泡相关的非半胱氨酸蛋白酶可以切割人中性粒细胞上的C5aR，来自牙龈卟啉囊泡的酶对C5aR的蛋白水解攻击被TPCK（甲基磺酰基苯丙酰氯甲基酮）、PMSF（苯基甲基磺酰氟）和二氯异香豆素抑制，表明丝氨酸蛋白酶主要负责这种降解活性。纯化的囊泡蛋白酶Lys-gingipain也能切割人中性粒细胞C5aR的n端区域，而Arg-gingipain则不能。Lys-gingipain的活性基本上对这些抑制剂具有抗性，但被TLCK （N α -对- toyl -l -赖氨酸氯甲基酮）和碘乙酰胺抑制。一种模拟C5aR n端区域的合成肽（残基9至29；PDYGHY DDKDTLDLNTPVDKT）很容易被胰凝乳蛋白酶切割，但不被胰蛋白酶切割，尽管存在两个潜在的胰蛋白酶（即赖氨酸- x）切割位点。用质谱法测定了C5aR 9-29肽的裂解位点。该分析表明，C5aR肽在两个潜在的Lys-gingipain位点（即17和18残基之间[K-D]和28和29残基之间[K-T]）和两个chymotrypsin位点（14和15残基之间[Y-D]和20和21残基之间[L-D]）上都容易被切割。这些研究表明，P， gingivalis含有至少两种能够切割C5aR的酶，Lys-gingipain和第二种不同于Arg-或Lys-gingipain的非色氨酸丝氨酸蛋白酶。
The anaerobic bacterium Porphyromonas gingivalis has been implicated as a primary causative agent in adult periodontitis, Several proteinases are produced by this bacterium, and it is suggested that they contribute to virulence and to local tissue injury resulting from infection by P. gingivalis. Cysteine proteinases with specificities to cleave either Arg-X or Lys-X peptide bonds (i.e., gingipains) have been characterized as predominant enzymes associated with vesicles shed from the surface of this bacterium. It has recently been demonstrated that these proteinases are capable of degrading the blood complement component C5, resulting in the generation of biologically active C5a. By using an affinity-purified rabbit antibody raised against residues 9 to 29 of the C5a receptor (C5aR; CD88), we demonstrate that noncysteinyl proteinases associated with vesicles obtained from P. gingivalis cleave the C5aR on human neutrophils, Proteolytic attack of the C5aR by enzymes from the P. gingivalis vesicles was inhibited by TPCK (tolylsulfonyl phenylalanyl chloromethyl ketone), PMSF (phenylmethylsulfonyl fluoride), and dichloroisocoumarin, suggesting that serine proteinases are primarily responsible for this degradative activity. The purified vesicle proteinase Lys-gingipain but not Arg-gingipain also cleaved the N-terminal region of the C5aR on the human neutrophils. Lys-gingipain activity was essentially resistant to these inhibitors but was inhibited by TLCK (N alpha-p-tosyl-L-lysine chloromethyl ketone) and iodoacetamide. A synthetic peptide that mimics the N-terminal region of C5aR (residues 9 to 29; PDYGHY DDKDTLDLNTPVDKT) was readily cleaved by chymotrypsin but not by trypsin, despite the presence of two potential trypsin (i.e., lysyl-X) cleavage sites. The specific sites of cleavage in the C5aR 9-29 peptide were determined by mass spectroscopy for both chymotrypsin and Lys-gingipain digests. This analysis demonstrated that the C5aR peptide is susceptible to cleavage at both potential Lys-gingipain sites (i.e., between residues 17 and 18 [K-D] and 28 and 29 [K-T]) and at two chymotrypsin sites (between residues 14 and 15 [Y-D] and 20 and 21 [L-D]), respectively. These studies suggest that P, gingivalis contains at least two enzymes capable of cleaving the C5aR, Lys-gingipain and a second nontryptic serine proteinase that is distinct from either Arg- or Lys-gingipain.