Substrate specificity of human kallikrein 6 - Salt and glycosaminoglycan activation effects
Substrate specificity of human kallikrein 6 - Salt and glycosaminoglycan activation effects
复制标题
DOI：
10.1074/jbc.m510096200
复制
发表时间：
2006-02-10
影响因子：
4.8
通讯作者：
Juliano, MA
中科院分区：
文献类型：
--
作者：
Angelo, PF;Lima, AR;Juliano, MA
人激肽释放酶6（hK 6）在中枢神经系统中大量表达，并且与脱髓鞘疾病有关。这项研究提供了生化数据的底物特异性和激活hK 6的糖胺聚糖和亲液盐，这遵循Hofmeister系列。衍生自Abz-KLRSSKQ-EDDnp的荧光共振能量转移（FRET）肽家族的筛选导致发现Abz-AFRFSQ- EDDnp（其中Abz是邻氨基苯甲酸，EDDnp是N-[2，4-二硝基苯基]乙二胺）是迄今为止针对hK 6描述的最佳合成底物（k（cat）/K-m = 38，667 s（-1）mM（-1））。值得注意的是，AFRFS序列被发现作为一个基序的氨基端结构域的7个人离子型谷氨酸受体亚基。我们还检测了hK 6对FRET肽的水解活性，所述FRET肽来源于人髓鞘碱性蛋白、A β淀粉样肽的前体、α（1）-抗胰凝乳蛋白酶的反应中心环、纤溶酶原以及hK 6的成熟和失活切割位点，所述hK 6的成熟和失活切割位点先前被描述为hK 6的天然底物。最佳底物来源于髓鞘碱性蛋白。hK 6成熟裂解位点水解不良，没有发现证据支持先前报道的两步自激活过程。最后，我们分析了FRET肽衍生自跨越蛋白酶激活受体（PAR）1 - 4激活的切割位点的序列，只有底物与PAR 2序列被水解。这些结果进一步支持了hK 6在中枢神经系统中表达的假设，即hK 6参与正常的髓鞘更新/脱髓鞘过程，但它不太可能自我激活。本研究还提示hK 6可能调节离子型谷氨酸受体和激活PAR 2。
Human kallikrein 6 ( hK6) is abundantly expressed in the central nervous system and is implicated in demyelinating disease. This study provided biochemical data about the substrate specificity and activation of hK6 by glycosaminoglycans and by kosmotropic salts, which followed the Hofmeister series. The screening of fluorescence resonance energy transfer ( FRET) peptide families derived from Abz-KLRSSKQ-EDDnp resulted in the finding that Abz-AFRFSQ- EDDnp ( where Abz is ortho-aminobenzoic acid and EDDnp is N-[2,4-dinitrophenyl]ethylenediamine)) is the best synthetic substrate described so far for hK6 ( k(cat)/K-m = 38,667 s(-1)mM(-1)). It is noteworthy that the AFRFS sequence was found as a motif in the amino-terminal domain of seven human ionotropic glutamate receptor subunits. We also examined the hK6 hydrolytic activity on FRET peptides derived from human myelin basic protein, precursor of the A beta amyloid peptide, reactive center loop of alpha(1)-antichymotrypsin, plasminogen, and maturation and inactivation cleavage sites of hK6, which were described earlier as natural substrates for hK6. The best substrates were derived from myelin basic protein. The hK6 maturation cleavage site was poorly hydrolyzed, and no evidence was found to support a two-step self-activation process reported previously. Finally, we assayed FRET peptides derived from sequences that span the cleavage sites for activation of protease-activated receptors ( PAR) 1 - 4, and only the substrate with the PAR 2 sequence was hydrolyzed. These results further supported the hypothesis that hK6 expressed in the central nervous system is involved in normal myelin turnover/demyelination processes, but it is unlikely to self-activate. This report also suggested the possible modulation of ionotropic glutamate receptors and activation of PAR 2 by hK6.