Nguyen T T, Qasim M A, Morris S, Lu C C, Hill D, Laskowski M, Sakanari J A
Department of Pathology, University of California, V.A. Medical Center, San Francisco, USA.
Mol Biochem Parasitol. 1999 Jul 30;102(1):79-89. doi: 10.1016/s0166-6851(99)00088-2.
Two elastase inhibitors, ASPI-1 and ASPI-2, from the parasitic nematode Anisakis simplex, have been isolated and characterized. Because these inhibitors are similar in size (60 amino acids in length) and primary sequence (52 and 47% identical) to the Ascaris suum chymotrypsin/elastase inhibitor-1 (AsC/E-1), we suggest that these Anisakis elastase inhibitors belong to the same unique class of canonical inhibitors formed by the family of Ascaris inhibitors (Huang K, Strynadka NCJ, Bernard VD, Peanasky RJ, James MG. Structure 1994;2:679-689). To compare ASPI-1 with AsC/E-1, we expressed both inhibitors in Pichia pastoris and found that: (1) the association constant of rASPI-1 with porcine pancreatic elastase (PPE) is similar to native inhibitor (Ka = 4.5 x 10(9) and 6.4 x 10(9) M(-1), respectively); (2) rASPI-1 is a potent inhibitor of PPE and human leukocyte elastase (Ka = 1.6 x 10(9) M(-1)); and (3) it is only a very weak inhibitor of chymotrypsin (CHYM) (Ka = 1.2 x 10(6) M(-1)). In contrast to the Anisakis inhibitor, however, rAsC/E inhibitor-1 is a very strong inhibitor of both PPE (Ka = 3.5 x 10(10) M(-1)) and CHYM (Ka = 3.6 x 10(12) M(-1)). We also found that the determined reactive sites (P1-P'1) of rASPI-1 and rAsC/E-1, as recognized by PPE, are Ala 28-Met 29 and Leu 31-Met 32, respectively. These P1-P'1 residues of AsC/E-1 constitute the same reactive site as that also recognized by CHYM (Peanasky RJ, Bentz Y, Homandberg GA, Minor ST, Babin DR. Arch Biochem Biophys 1994;232:135-142). The difference in specificities of ASPI-1 and AsC/E-1 toward their cognate serine proteases may be attributed to the P1 and P'3 residues in the inhibitors. Elastase, which recognizes both alanine and leucine, canaccommodate both ascarid inhibitors, whereas chymotrypsin, which prefers bulky, hydrophobic residues, only recognizes the Ascaris C/E inhibitor-1.
已从寄生线虫简单异尖线虫中分离并鉴定出两种弹性蛋白酶抑制剂ASPI - 1和ASPI - 2。由于这些抑制剂在大小（长度为60个氨基酸）和一级序列（分别有52%和47%相同）上与猪蛔虫胰凝乳蛋白酶/弹性蛋白酶抑制剂-1（AsC/E - 1）相似，我们认为这些异尖线虫弹性蛋白酶抑制剂属于由蛔虫抑制剂家族形成的同一独特类别的典型抑制剂（Huang K，Strynadka NCJ，Bernard VD，Peanasky RJ，James MG。Structure 1994；2：679 - 689）。为了将ASPI - 1与AsC/E - 1进行比较，我们在毕赤酵母中表达了这两种抑制剂，发现：（1）重组ASPI - 1与猪胰弹性蛋白酶（PPE）的结合常数与天然抑制剂相似（Ka分别为4.5×10⁹和6.4×10⁹ M⁻¹）；（2）重组ASPI - 1是PPE和人白细胞弹性蛋白酶的有效抑制剂（Ka = 1.6×10⁹ M⁻¹）；（3）它只是胰凝乳蛋白酶（CHYM）的非常弱的抑制剂（Ka = 1.2×10⁶ M⁻¹）。然而，与异尖线虫抑制剂不同，重组AsC/E抑制剂-1对PPE（Ka = 3.5×10¹⁰ M⁻¹）和CHYM（Ka = 3.6×10¹² M⁻¹）都是非常强的抑制剂。我们还发现，PPE识别的重组ASPI - 1和重组AsC/E - 1的确定反应位点（P1 - P'1）分别是Ala 28 - Met 29和Leu 31 - Met 32。AsC/E - 1的这些P1 - P'1残基构成了与CHYM识别的相同反应位点（Peanasky RJ，Bentz Y，Homandberg GA，Minor ST，Babin DR。Arch Biochem Biophys 1994；232：135 - 142）。ASPI - 1和AsC/E - 1对其同源丝氨酸蛋白酶特异性的差异可能归因于抑制剂中的P1和P'3残基。识别丙氨酸和亮氨酸的弹性蛋白酶可以容纳这两种蛔虫抑制剂，而更喜欢大的疏水残基的胰凝乳蛋白酶只识别蛔虫C/E抑制剂-1。
