Rauvala H, Merenmies J, Pihlaskari R, Korkolainen M, Huhtala M L, Panula P
Department of Medical Chemistry, University of Helsinki, Finland.
J Cell Biol. 1988 Dec;107(6 Pt 1):2293-305. doi: 10.1083/jcb.107.6.2293.
A membrane-bound adhesive protein that promotes neurite outgrowth in brain neurons has been isolated from rat brain (Rauvala, H., and R. Pihlaskari. 1987. J. Biol. Chem. 262:16625-16635). The protein is an immunochemically distinct molecule with a subunit size of approximately 30 kD (p30). p30 is an abundant protein in perinatal rat brain, but its content decreases rapidly after birth. In the present study the amino-terminal sequence of p30 was determined by automated Edman degradations. A single amino-terminal sequence was found, which is not present in previously studied adhesive molecules. This unique sequence has a cluster of five positive charges within the first 11 amino acid residues: Gly-Lys-Gly-Asp-Pro-Lys-Lys-Pro-Arg-Gly-Lys. Antisynthetic peptide antibodies that recognize this sequence were produced in a rabbit, purified with a peptide affinity column, and shown to bind specifically to p30. The antipeptide antibodies were used, together with anti-p30 antibodies, to study the localization of p30 in brain cells and in neuroblastoma cells as follows. (a) Immunofluorescence and immunoelectron microscopy indicated that p30 is a component of neurons in mixed cultures of brain cells. The neurons and the neuroblastoma cells expressed p30 at their surface in the cell bodies and the neurites. In the neurites p30 was found especially in the adhesive distal tips of the processes. In addition the protein was detected in ribosomal particles and in intracellular membranes in a proportion of cells. (b) The antibodies immobilized on microtiter wells enhanced adhesion and neurite growth indicating that p30 is surface exposed in adhering neural cells. (c) Immunoblotting showed that p30 is extracted from suspended cells by heparin suggesting that a heparin-like structure is required for the binding of p30 to the neuronal cell surface. A model summarizing the suggested interactions of p30 in cell adhesion and neurite growth is presented.
一种促进脑神经元轴突生长的膜结合黏附蛋白已从大鼠脑中分离出来（劳瓦拉，H.，和R. 皮尔拉斯卡里。1987.《生物化学杂志》262:16625 - 16635）。该蛋白是一种免疫化学上不同的分子，亚基大小约为30 kD（p30）。p30是围产期大鼠脑中的一种丰富蛋白，但出生后其含量迅速下降。在本研究中，通过自动埃德曼降解法测定了p30的氨基末端序列。发现了一个单一的氨基末端序列，该序列在先前研究的黏附分子中不存在。这个独特的序列在前11个氨基酸残基内有一簇五个正电荷：甘氨酸 - 赖氨酸 - 甘氨酸 - 天冬氨酸 - 脯氨酸 - 赖氨酸 - 赖氨酸 - 脯氨酸 - 精氨酸 - 甘氨酸 - 赖氨酸。识别该序列的抗合成肽抗体在兔体内产生，用肽亲和柱纯化，并显示能特异性结合p30。抗肽抗体与抗p30抗体一起用于如下研究p30在脑细胞和神经母细胞瘤细胞中的定位。（a）免疫荧光和免疫电子显微镜表明，p30是脑细胞混合培养物中神经元的一个组成部分。神经元和神经母细胞瘤细胞在其细胞体和轴突的表面表达p30。在轴突中，p30尤其在突起的黏附远端末端被发现。此外，在一部分细胞的核糖体颗粒和细胞内膜中也检测到了该蛋白。（b）固定在微量滴定板孔上的抗体增强了黏附作用和轴突生长，表明p30在黏附的神经细胞表面暴露。（c）免疫印迹显示，肝素可从悬浮细胞中提取p30，这表明p30与神经元细胞表面结合需要类似肝素的结构。提出了一个总结p30在细胞黏附和轴突生长中推测相互作用的模型。
