Medda S, Chemelli R M, Martin J L, Pohl L R, Swank R T
Roswell Park Memorial Institute, Department of Molecular and Cellular Biology, Buffalo, New York 14263.
J Biol Chem. 1989 Sep 25;264(27):15824-8.
The proenzyme form of beta-glucuronidase is compartmentalized in large quantities within the endoplasmic reticulum by binding to the esterase, egasyn. Also, the propeptide of the proenzyme form of beta-glucuronidase is likely located at the carboxyl terminus. We have, therefore, tested if this carboxyl-terminal peptide is important in binding to egasyn. A polyclonal antibody to a 30-mer synthetic peptide, corresponding to the carboxyl-terminal 30 amino acids of pro-beta-glucuronidase, provided evidence that egasyn binds to the carboxyl terminus of beta-glucuronidase. This antibody interacted with proenzyme beta-glucuronidase-egasyn complexes in which one, two, or three egasyn molecules were bound to the beta-glucuronidase tetramer, but not with those complexes (M4) which contained four egasyn molecules. We interpret these results as indicating that all available carboxyl termini of the beta-glucuronidase proenzyme tetramer are shielded by egasyn in the M4 complexes. The same antibody did not recognize the mature lysosomal form of beta-glucuronidase, indicating that only the proenzyme form of microsomal beta-glucuronidase contains the original carboxyl terminus. Also, the synthetic 30-mer was found to be a specific and potent inhibitor (50% inhibition at 1.3 microM) of the esterase activity of purified egasyn but exhibited little inhibitory activity toward other purified esterases including a rat trifluoroacetylated esterase or egasyn esterase from another species. Together, these data describe a potent interaction of the exposed carboxyl terminus of precursor glucuronidase with the esterase catalytic site of egasyn, which in turn results in the specific localization of glucuronidase within the lumen of the endoplasmic reticulum.
β-葡萄糖醛酸酶的酶原形式通过与酯酶egasyn结合而大量分隔在内质网中。此外，β-葡萄糖醛酸酶酶原形式的前肽可能位于羧基末端。因此，我们测试了该羧基末端肽在与egasyn结合中是否重要。针对与前β-葡萄糖醛酸酶的羧基末端30个氨基酸对应的30聚体合成肽的多克隆抗体，提供了egasyn与β-葡萄糖醛酸酶的羧基末端结合的证据。该抗体与其中一个、两个或三个egasyn分子结合到β-葡萄糖醛酸酶四聚体上的酶原β-葡萄糖醛酸酶-egasyn复合物相互作用，但不与包含四个egasyn分子的那些复合物（M4）相互作用。我们将这些结果解释为表明β-葡萄糖醛酸酶酶原四聚体的所有可用羧基末端在M4复合物中被egasyn屏蔽。相同的抗体不识别β-葡萄糖醛酸酶的成熟溶酶体形式，表明只有微粒体β-葡萄糖醛酸酶的酶原形式含有原始羧基末端。此外，发现合成的30聚体是纯化的egasyn酯酶活性的特异性强效抑制剂（在1.3μM时50%抑制），但对包括大鼠三氟乙酰化酯酶或来自另一个物种的egasyn酯酶在内的其他纯化酯酶几乎没有抑制活性。总之，这些数据描述了前体葡萄糖醛酸酶暴露的羧基末端与egasyn的酯酶催化位点之间的强效相互作用，这反过来导致葡萄糖醛酸酶在内质网腔内的特异性定位。
