Yamauchi Y, Stevens J W, Macon K J, Volanakis J E
Department of Medicine, University of Alabama at Birmingham 35294.
J Immunol. 1994 Apr 1;152(7):3645-53.
We have expressed a full-length cDNA clone encoding human factor D by using a baculovirus expression system. The purified recombinant protein reacted with Ab against native factor D, but was hemolytically inactive and slightly larger than factor D. These results suggested that the recombinant protein was the elusive zymogen of factor D. Amino acid sequencing demonstrated that the recombinant factor D consisted of two proenzyme forms with respective activation peptides, AAPPRGR and APPRGR. Catalytic amounts of trypsin converted recombinant profactor D to its enzymatically active form, exhibiting SDS-PAGE mobility and specific hemolytic activity similar to those of native factor D. About 90% of trypsin-activated recombinant profactor D had the same NH2-terminus as factor D. Human thrombin, kallikrein, and plasmin could also activate recombinant profactor D, but relatively high concentrations of these enzymes were required and the specific hemolytic activity of the "activated" profactor D was about one-third that of native factor D. Trypsin-activatable profactor D was also purified from the urine of a patient with Fanconi's syndrome. This native profactor D represented less than 1.0% of the total antigenic factor D in the patient's urine and had a Gly-Arg dipeptide as the activation peptide. Apparently, urine profactor D was produced by cleavage of pre-profactor D at Arg-(-3) by a serine protease with trypsin-like specificity, which probably is different from the putative leader peptidase that produces the recombinant profactor D. Urine profactor D was inhibited by diisopropyl fluorophosphate although the recombinant proenzyme was resistant to this inhibitor.
我们利用杆状病毒表达系统表达了编码人D因子的全长cDNA克隆。纯化的重组蛋白与抗天然D因子的抗体发生反应，但无溶血活性，且略大于D因子。这些结果表明，重组蛋白是难以捉摸的D因子酶原。氨基酸测序表明，重组D因子由两种分别带有激活肽AAPPRGR和APPRGR的酶原形式组成。催化量的胰蛋白酶将重组前D因子转化为其酶活性形式，其SDS-PAGE迁移率和特异性溶血活性与天然D因子相似。约90%经胰蛋白酶激活的重组前D因子与D因子具有相同的NH2末端。人凝血酶、激肽释放酶和纤溶酶也能激活重组前D因子，但需要相对较高浓度的这些酶，且“激活”的前D因子的特异性溶血活性约为天然D因子的三分之一。可被胰蛋白酶激活的前D因子也从一名范科尼综合征患者的尿液中纯化得到。这种天然前D因子占患者尿液中总抗原性D因子的比例不到1.0%，其激活肽为甘氨酰-精氨酸二肽。显然，尿液前D因子是由前前D因子在精氨酸-(-3)处被具有胰蛋白酶样特异性的丝氨酸蛋白酶切割产生的，这可能与产生重组前D因子的假定前导肽酶不同。尽管重组酶原对二异丙基氟磷酸有抗性，但尿液前D因子却被其抑制。
