McGwire B S, Chang K P
Department of Microbiology/Immunology, University of the Health Sciences/Chicago Medical School, North Chicago, Illinois 60064, USA.
J Biol Chem. 1996 Apr 5;271(14):7903-9. doi: 10.1074/jbc.271.14.7903.
Leishmanolysin (EC 3.4.24.36) (gp63) is a HEXXH metalloprotease, encoded by multicopied genes in Leishmania and implicated in the infectivity of these parasitic protozoa. We examined posttranslational regulation of gp63 expression by site-specific mutagenesis of the predicted catalytic/zinc-binding sites in the H264EXXH motif, the potential sites of N-glycosylation and glycosyl phosphatidylinositol addition. Mutant and wild-type genes were cloned into a Leishmania-specific vector for transfecting a deficient variant, which produced gp63 approximately 20-fold less than wild-type cells. The selective conditions chosen fully restored this deficiency in transfectants with the wild-type gene. Under these conditions, all transfectants were found comparable in both the plasmid copy number per cell and elevation of gp63 transcripts. Mutant and wild-type products in the transfectants were then compared quantitatively and qualitatively by specific immunologic and protease assays. The results indicate the following. 1) Glu-265 in the HEXXH motif is indispensable for the catalytic activity of gp63. The propeptide of the inactive mutant products was cleaved, suggestive of a non-intramolecular event. 2) Substitution of either His residue in HEXXH leads to apparent intracellular degradation of the mutant products, pointing to a role for zinc binding in in vivo stability of gp63. 3) The three potential sites of N-glycosylation at Asn-300, Asn-407, and Asn-534 are all utilized and contribute to intracellular stability of gp63. 4) Substitution of Asn-577 causes release of all mutant products, indicative of its specificity as a glycosyl phosphatidylinositol addition site for membrane anchoring of gp63. It is suggested that expression of gp63 as a functional protease is regulated by these posttranslational modification pathways.
利什曼原虫溶血素（EC 3.4.24.36）（gp63）是一种HEXXH金属蛋白酶，由利什曼原虫中的多拷贝基因编码，并与这些寄生原生动物的感染性有关。我们通过对H264EXXH基序中预测的催化/锌结合位点、潜在的N-糖基化位点和糖基磷脂酰肌醇添加位点进行定点诱变，研究了gp63表达的翻译后调控。将突变型和野生型基因克隆到利什曼原虫特异性载体中，用于转染一个缺陷变体，该变体产生的gp63比野生型细胞少约20倍。所选择的选择性条件完全恢复了野生型基因转染子中的这种缺陷。在这些条件下，发现所有转染子在每个细胞的质粒拷贝数和gp63转录本的升高方面都具有可比性。然后通过特异性免疫和蛋白酶测定对转染子中的突变型和野生型产物进行定量和定性比较。结果表明如下：1）HEXXH基序中的Glu-265对于gp63的催化活性是必不可少的。无活性突变产物的前肽被切割，提示这是一个非分子内事件。2）HEXXH中任一His残基的取代导致突变产物在细胞内明显降解，表明锌结合在gp63的体内稳定性中起作用。3）Asn-300、Asn-407和Asn-534处的三个潜在N-糖基化位点均被利用，并有助于gp63的细胞内稳定性。4）Asn-577的取代导致所有突变产物释放，表明其作为gp63膜锚定的糖基磷脂酰肌醇添加位点的特异性。提示gp63作为一种功能性蛋白酶的表达受这些翻译后修饰途径的调控。
