Burke Robert D, Osborne Lisa, Wang Diana, Murabe Naoyuki, Yaguchi Shunsuke, Nakajima Yoko
Department of Biology and Biochemistry, University of Victoria, Victoria, British Columbia V8W 3N5, Canada.
J Comp Neurol. 2006 May 10;496(2):244-51. doi: 10.1002/cne.20939.
Interest in chordate evolution has emphasized a need for a better understanding of the comparative neuroanatomy of invertebrate deuterostomes. However, molecular and genetic approaches to neurobiological studies in these groups are hampered by a lack of neuron-specific molecular markers. A monoclonal antibody, 1E11, is neuron specific and is useful in identification of neural structures in larvae and adults of echinoderms, hemichordates, and urochordates. To identify a neuron-specific gene product, we have characterized the antigen recognized by 1E11. In immunoblots and immunoprecipitations of neural tissue from adult Strongylocentrotus purpuratus, 1E11 recognizes a 57-kDa band. Tandem mass spectrometry of trypsin digests of the 57-kDa band permitted peptide mass mapping and sequencing of five peptides. All of the sequenced peptides, and 12 additional mass-mapped peptides, are found within the open reading frame of a cDNA encoding synaptotagmin B (Sp-SynB). In situ RNA hybridizations with synaptotagmin B probes with S. purpuratus larvae reveal a pattern of expression that is similar to that revealed by the antibody 1E11. Antibodies produced against a bacterially expressed Sp-SynB protein recognize a 57-kDa protein and colocalize with 1E11. When a full-length Sp-SynB cDNA is expressed in chicken embryonic cells, the cells become immunoreactive to 1E11. We conclude that synaptotagmin B is a gene expressed in neurons that has conserved epitopes in other invertebrate deuterostomes.
对脊索动物进化的研究兴趣凸显了更好地理解无脊椎后口动物比较神经解剖学的必要性。然而，这些类群中神经生物学研究的分子和遗传方法因缺乏神经元特异性分子标记而受到阻碍。一种单克隆抗体1E11具有神经元特异性，可用于识别棘皮动物、半索动物和尾索动物幼虫及成体中的神经结构。为了鉴定一种神经元特异性基因产物，我们对1E11识别的抗原进行了表征。在对成年紫球海胆神经组织的免疫印迹和免疫沉淀实验中，1E11识别出一条57 kDa的条带。对该57 kDa条带的胰蛋白酶消化产物进行串联质谱分析，实现了五个肽段的肽质量图谱绘制和测序。所有测序的肽段以及另外12个质量图谱绘制的肽段，都在编码突触结合蛋白B（Sp-SynB）的cDNA开放阅读框内被发现。用突触结合蛋白B探针与紫球海胆幼虫进行原位RNA杂交，揭示出一种与抗体1E11所揭示的表达模式相似的模式。针对细菌表达的Sp-SynB蛋白产生的抗体识别一种57 kDa的蛋白，并与1E11共定位。当全长Sp-SynB cDNA在鸡胚胎细胞中表达时，这些细胞对1E11产生免疫反应。我们得出结论，突触结合蛋白B是一种在神经元中表达的基因，在其他无脊椎后口动物中具有保守的表位。
