Reverter D, Ventura S, Villegas V, Vendrell J, Avilés F X
Departament de Bioquímica i Biologia Molecular, Unitat de Ciències and the Institut de Biologia Fonamental, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain.
J Biol Chem. 1998 Feb 6;273(6):3535-41. doi: 10.1074/jbc.273.6.3535.
The cDNA of human procarboxypeptidase A2 has been overexpressed in the methylotrophic yeast Pichia pastoris and secreted into the culture medium by means of the alpha-mating factor signal sequence, yielding a major protein of identical size and N-terminal sequence as the wild-type form. Two other forms containing the proenzyme have also been overexpressed: one of them resulted from an incomplete processing of the signal peptide, whereas the other was a glycosylated derivative. Recombinant procarboxypeptidase A2 was purified to homogeneity, and it was shown that its mature active form displays functional properties similar to those of the enzyme directly isolated from human pancreas. The overall yield was approximately 250 mg of proenzyme or 180 mg of mature enzyme/liter of cell culture. The proteolysis-promoted activation process of the recombinant proenzyme has been studied in detail. During maturation by trypsin, the increase in activity of the enzyme is a rapid and monotonic event, which reflects the rate of the proteolytic release of the inhibitory pro-segment and the weaker nature of its interactions with the enzyme moiety compared with procarboxypeptidases of the A1 type. Three main forms of the pro-segment (96, 94, and 92 amino acids), with no inhibitory capability in the severed state, and a single mature carboxypeptidase A2 are produced during this process. No further proteolysis of these pro-segments by the generated carboxypeptidase A2 occurs, in contrast with observations made in other procarboxypeptidases (A1 and B). This differential behavior is a result of the extreme specificity of carboxypeptidase A2.
人羧肽酶原A2的cDNA已在甲基营养型酵母毕赤酵母中过表达，并通过α-交配因子信号序列分泌到培养基中，产生一种大小和N端序列与野生型形式相同的主要蛋白质。另外两种含有酶原的形式也已过表达：其中一种是信号肽加工不完全导致的，而另一种是糖基化衍生物。重组羧肽酶原A2被纯化至同质，结果表明其成熟活性形式具有与直接从人胰腺分离的酶相似的功能特性。总产率约为每升细胞培养物250毫克酶原或180毫克成熟酶。已详细研究了重组酶原的蛋白水解促进激活过程。在胰蛋白酶成熟过程中，酶活性的增加是一个快速且单调的事件，这反映了抑制性前肽段蛋白水解释放的速率以及与A1型羧肽酶原相比，其与酶部分相互作用较弱的性质。在此过程中产生了三种主要形式的前肽段（96、94和92个氨基酸），在切断状态下无抑制能力，以及单一的成熟羧肽酶A2。与在其他羧肽酶原（A1和B）中观察到的情况相反，生成的羧肽酶A2不会对这些前肽段进行进一步的蛋白水解。这种差异行为是羧肽酶A2极端特异性的结果。
