Regulation of spinal neurons through proteolytic actions by serine proteases.

为了了解我们先前从脑中分离到的一种丝氨酸蛋白酶--胃动素/神经胰蛋白酶(PRSS12)的生理功能，制备了缺失胃动素基因的小鼠。我们还对重组Motopsin的酶学性质进行了表征，并对Motopsin在中枢神经系统的定位进行了分析。在本项目刚开始时就有报道称motopsin基因突变会导致智力低下，我们将motopsin集中在大脑皮层1)mmotopsin靶向小鼠的制备将小鼠motopsin基因的第一个外显子替换到pGKneo盒中。用Southern杂交、Northern杂交和抗Motopsin抗体免疫组织化学方法证实了基因靶向性。在光束行走和脚印测试中，突变小鼠没有表现出运动缺陷。明暗箱试验和高架加迷宫试验显示突变小鼠的焦虑程度比野生型小鼠低。有趣的是，树突棘在扣带回皮质和hi…的锥体神经元减少。海马区CA1区较多，Motopsin在该区域优先表达。我们的结果表明，Motopsin对情绪的发展具有重要的作用，而不是对运动行为的作用。2)Motopsin的特征我们去年发现Motopsin在体外激活了组织纤溶酶原激活物(Wa)。胃动素与组织型纤溶酶原激活剂的共存当Motopsin和tPA在HEK293细胞中共表达时，这两种酶都定位于分泌面。免疫组织化学技术显示，生后第10天，Motopsin和tPA在扣带回和海马区的锥体神经元中有明显的定位。此外，通过酵母双杂交系统分离到12个与Motopsin相互作用的候选基因。其中，一些基因的mRNAs在motopsin表达的细胞中被检测到。3)脊柱的表达调控通过选择性剪接产生了四种类型的脊柱蛋白亚型。类型1和类型2缺乏跨膜结构域，而类型3和类型4是类型II跨膜蛋白。有趣的是，虽然1型和2型mRNAs在包括中枢神经系统在内的各种组织中都有表达，但在脊髓中只检测到了3型和4型mRNAs。较少

In order to understand physiological function of a serine protease, motopsin/neurotrypsin (PRSS12), which we previously isolated from brain cDNA, mice lacking motopsin gene were prepared. We also characterized enzymatic property of recombinant motopsin and analyzed the localization of motopsin in the central nervous system. After mutation of motopsin gene has been reported to cause mental retardation just on starting this project, we focused motopsin in the cerebral cortex.1)Preparation of motopsin targeting miceThe first exon of mouse motopsin gene was replaced to pGKneo cassette. Gene targeting was confirmed by Southern hybridization, Northern hybridization and immunohistochemistry using anti-motopsin IgG. The mutant mice showed no deficit on motor on beam walking and foot print tests. Light-dark box test and elevated plus maze showed tendency that mutant mice had less anxiety than wild type. Interestingly, dendritic spine was decreased at pyramidal neurons of cingulate cortex and hi … More ppocampal CA1 region, at which motopsin was preferentially expressed. Our results suggest that motopsin have significant function for the development of emotion rather than motor behavior.2)Characterization of motopsinWe found that motopsin activates tissue plasminogen activator (WA) in vitro last year. The co-localization of motopsin and tPA. When motopsin and tPA were co-expressed in HEK293 cells, both proteases were localized in secretary vesides. Immunohistochemical technique dearly showed oo-localiation of motopsin and tPA at pyramidal neurons of cingulate cortex and hippcampus at postnatal day 10. Further, 12 candidate genes which interact with motopsin were isolated by yeast two hybrid system. Among them, mRNAs for a few genes were detected at motopsin-expressing cells.3)Regulation of the expression of spinesinFour types of spinesin isoforms were generated by alternative splicing. Type 1 and 2 lack the transmembrane domain, whereas type 3 and 4 are type II transmembrane proteins. Interestingly, only type 3 and 4 mRNAs were detected in spinal cord although type 1 and 2 mRNAs were expressed in various tissues including the CNS. Less