Berg D T, Grinnell B W
Cardiovascular Research, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285.
Biochem Biophys Res Commun. 1991 Sep 30;179(3):1289-96. doi: 10.1016/0006-291x(91)91713-m.
We have explored the heterogeneity in the proteolytic processing of the N-terminus of human tissue plasminogen activator. We demonstrate that normal propeptide processing occurs following Arg-4, preceding the sequence Gly-Ala-Arg-Ser+1. Generation of the previously designated Ser+1 occurs via secondary proteolysis following secretion. By site-directed mutagenesis, we have eliminated this cleavage site resulting in a derivative containing the propeptide sequence. N-terminal sequence analysis of this form indicated that signal peptide cleavage occurs following Ser-13. The pro-tPA derivative had near normal serine protease and plasminogen activating activities, and could be stimulated by fibrin. An additional derivative, containing the tribasic sequence from the human protein C propeptide preceding Ser+1, was secreted with full processing of the propeptide. Our data have defined the cleavages for the signal peptide and propeptide and demonstrate that a tribasic sequence can be used to eliminate N-terminal heterogeneity in this molecule. In addition, we demonstrate that, unlike several other serine proteases, a propeptide sequence does not alter the activity of this enzyme.
我们研究了人组织纤溶酶原激活物N端蛋白水解加工过程中的异质性。我们证明正常的前肽加工发生在精氨酸-4之后，甘氨酸-丙氨酸-精氨酸-丝氨酸+1序列之前。先前指定的丝氨酸+1的产生是在分泌后通过二次蛋白水解发生的。通过定点诱变，我们消除了这个切割位点，从而得到了一个包含前肽序列的衍生物。对这种形式的N端序列分析表明，信号肽的切割发生在丝氨酸-13之后。前组织纤溶酶原激活物衍生物具有接近正常的丝氨酸蛋白酶和纤溶酶原激活活性，并且可以被纤维蛋白刺激。另一种衍生物包含位于丝氨酸+1之前的人蛋白C前肽的三元序列，其前肽得到了完全加工并被分泌。我们的数据确定了信号肽和前肽的切割位点，并证明三元序列可用于消除该分子中的N端异质性。此外，我们证明，与其他几种丝氨酸蛋白酶不同，前肽序列不会改变这种酶的活性。
