Wilson D J, Kim D S, Clarke G A, Marshall-Clarke S, Moss D J
Department of Human Anatomy and Cell Biology, Liverpool University, UK.
J Cell Sci. 1996 Dec;109 ( Pt 13):3129-38. doi: 10.1242/jcs.109.13.3129.
We have previously identified a glycosylphosphatidylinositol-linked glycoprotein of 55 kDa (GP55) which inhibits neurite outgrowth. We now provide evidence that GP55, isolated from adult chick brain, consists of at least two bands, both of which are active, i.e., block outgrowth of neurites from chick dorsal root ganglion neurons. An antiserum raised against the adult proteins reverses the inhibition and preliminary experiments suggest that GP55 is restricted to the nervous system, increases during development from very low levels at embryonic day 10 and is most abundant after hatching. Immunofluorescence reveals that GP55 is expressed on neurons cultured from an embryonic day 14 chick brain but is barely detectable on embryonic day 10 dorsal root ganglion neurons or embryonic day 8 forebrain neurons; the neurons which respond to substrate-bound GP55. Peptide sequencing revealed considerable homology with OBCAM, a protein previously identified on the basis of binding opiates. Nested polymerase chain reaction using primers to the OBCAM sequence and internal primers to GP55 peptides produced two different polymerase chain reaction fragments with homology to OBCAM. A full length clone (E19S) corresponding to one polymerase chain reaction product and a partial length clone (E14S) corresponding to the second have been isolated from an embryonic chick brain library. Both are members of the immunoglobulin superfamily and have (or are expected to have) three C2 domains. E19S has 90% homology with LAMP at the amino acid level. This sequence only partially matches the peptides from the adult protein and hence is probably not a major component of the adult proteins. E14S (GP55-A) has 83% homology to OBCAM at the amino acid level over the region sequenced. The sequence matches several of the peptides from the adult protein and is hence likely to correspond to a major component of the adult proteins. Thus members of the GP55 family are related to OBCAM, neurotrimin, LAMP and a recently discovered chick protein CEPU-1. Our results suggest molecules within this family are capable of acting as cell adhesion molecules and inhibitors of neurite outgrowth.
我们之前鉴定出一种55 kDa的糖基磷脂酰肌醇连接糖蛋白（GP55），它能抑制神经突生长。我们现在提供证据表明，从成年鸡脑中分离出的GP55至少由两条带组成，两条带均具有活性，即能阻止鸡背根神经节神经元的神经突生长。针对成年蛋白产生的抗血清可逆转这种抑制作用，初步实验表明，GP55局限于神经系统，在发育过程中从胚胎第10天的极低水平开始增加，在孵化后最为丰富。免疫荧光显示，GP55在从胚胎第14天鸡脑中培养的神经元上表达，但在胚胎第10天的背根神经节神经元或胚胎第8天的前脑神经元上几乎检测不到；这些神经元对与底物结合的GP55有反应。肽序列分析显示与OBCAM有相当高的同源性，OBCAM是一种先前基于与阿片类药物结合而鉴定出的蛋白质。使用针对OBCAM序列的引物和针对GP55肽的内部引物进行巢式聚合酶链反应，产生了两个与OBCAM具有同源性的不同聚合酶链反应片段。从胚胎鸡脑文库中分离出了与一个聚合酶链反应产物对应的全长克隆（E19S）和与第二个产物对应的部分长度克隆（E14S）。两者都是免疫球蛋白超家族的成员，并且具有（或预期具有）三个C2结构域。E19S在氨基酸水平上与LAMP有90%的同源性。该序列仅与成年蛋白的肽部分匹配，因此可能不是成年蛋白的主要成分。E14S（GP55 - A）在测序区域的氨基酸水平上与OBCAM有83%的同源性。该序列与成年蛋白的几个肽匹配，因此可能对应于成年蛋白的主要成分。因此，GP55家族的成员与OBCAM、神经纤连蛋白、LAMP以及最近发现的鸡蛋白CEPU - 1相关。我们的结果表明，该家族中的分子能够作为细胞粘附分子和神经突生长抑制剂发挥作用。
