Human Glandular Kallikrein 2
Human Glandular Kallikrein 2
复制标题
人腺激肽释放酶2
DOI：
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发表时间：
1999
期刊：
影响因子：
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通讯作者：
J. Lövgren
中科院分区：
文献类型：
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作者：
J. Lövgren
人腺激肽释放酶2（hK 2）是一种丝氨酸蛋白酶，其主要由前列腺表达，并且在精浆中以6 mg/ml的平均水平发现。它与前列腺特异性抗原（PSA或hK 3）具有79%的氨基酸序列同一性。本研究的目的是利用重组表达技术制备hK 2，研究其生物学和生化特性以及与PSA免疫检测的交叉反应。在哺乳动物和昆虫细胞中与PSA平行产生重组hK 2。这两种蛋白质进行纯化，表征和显示是免疫交叉反应。重组PSA以高水平表达并作为无活性酶原（proPSA）回收。重组hK 2的表达水平较低，并且hK 2总是以具有酶活性的成熟形式回收。hK 2的前肽的突变导致hK 2作为失活酶原的恢复，并且表达水平增加15至40倍。我们已经表明，proPSA可以激活hK 2在生理PSA与hK 2的比例，使hK 2在前列腺液中的PSA的可能的激活剂。从产生PSA和hK 2的LNCaP细胞的废培养基中纯化的PSA仅部分转化为成熟形式。这可能是由于培养基中的缓慢活化过程。HK 2切割凝胶蛋白semenogelin I和II，以及某些单和双丝氨酸羧基端侧的三肽底物。我们还研究了几种细胞外蛋白酶抑制剂对hK 2的调节，其中只有蛋白C抑制剂PCI能够快速抑制hK 2。锌在前列腺中以极高的浓度存在，并且锌离子显示能够以微摩尔水平抑制hK 2。（减）
Human glandular kallikrein 2 (hK2) is a serine protease, which is predominantly expressed by the prostate gland and found in seminal plasma at mean levels of 6 mg/ml. It has 79% amino acid sequence identity with prostate-specific antigen (PSA or hK3). The aim of this work was to produce hK2 by recombinant expression technologies in order to study its biochemical and biological properties and its cross-reaction with PSA immunoassays. Recombinant hK2 was produced in parallel with PSA in mammalian and insect cells. The two proteins were purified, characterized and shown to be immunologically cross-reactive. Recombinant PSA was expressed at high levels and recovered as an inactive zymogen (proPSA). The expression levels of recombinant hK2 were low and hK2 was always recovered in an enzymatically active mature form. Mutation of the propeptide of hK2 resulted in recovery of hK2 as an inactive zymogen, and a 15 to 40 fold increase in the expression levels. We have shown that proPSA can be activated by hK2 at physiological PSA to hK2 ratios, making hK2 a likely activator of PSA in prostatic fluid. PSA purified from the spent medium of LNCaP cells, which produce both PSA and hK2, was only partially converted into mature form. This could be due to a slow activation process in culture medium. HK2 cleaves the gel-proteins semenogelin I and II, and tripeptide substrates on the carboxy-terminal side of certain single and double arginines. We also studied the regulation of hK2 by several extracellular protease inhibitors of which only protein C inhibitor, PCI, was able to rapidly inhibit hK2. Zinc is present at extremely high concentrations in the prostate, and zinc ions were shown to be able to inhibit hK2 at micromolar levels. (Less)