Volkmer H, Hassel B, Wolff J M, Frank R, Rathjen F G
Zentrum für Molekulare Neurobiologie, Hamburg, Germany.
J Cell Biol. 1992 Jul;118(1):149-61. doi: 10.1083/jcb.118.1.149.
The chick axon-associated surface glycoprotein neurofascin is implicated in axonal growth and fasciculation as revealed by antibody perturbation experiments. Here we report the complete cDNA sequence of neurofascin. It is composed of four structural elements: At the NH2 terminus neurofascin contains six Ig-like motifs of the C2 subcategory followed by four fibronectin type III (FNIII)-related repeats. Between the FNIII-like repeats and the plasma membrane spanning region neurofascin contains a domain 75-amino acid residues-long rich in proline, alanine and threonine which might be the target of extensive O-linked glycosylation. A transmembrane segment is followed by a 113-amino acid residues-long cytoplasmic domain. Sequence comparisons indicate that neurofascin is most closely related to chick Nr-CAM and forms with L1 (Ng-CAM) and Nr-CAM a subgroup within the vertebrate Ig superfamily. Sequencing of several overlapping cDNA probes reveals interesting heterogeneities throughout the neurofascin polypeptide. Genomic Southern blots analyzed with neurofascin cDNA clones suggest that neurofascin is encoded by a single gene and its pre-mRNA might be therefore alternatively spliced. Northern blot analysis with domain specific probes showed that neurofascin mRNAs of about 8.5 kb are expressed throughout development in embryonic brain but not in liver. Isolation of neurofascin by immunoaffinity chromatography results in several molecular mass components. To analyze their origin the amino-terminal sequences of several neurofascin components were determined. The NH2-terminal sequences of the 185, 160, and 110-135 kD components are all the same as the NH2 termini predicted by the cDNA sequence, whereas the other neurofascin components start with a sequence found in a putative alternatively spliced segment between the Ig- and FNIII-like part indicating that they are derived by proteolytic cleavage. A combination of enzymatic and chemical deglycosylation procedures and the analysis of peanut lectin binding reveals O- and N-linked carbohydrates on neurofascin components which might generate additional heterogeneity.
抗体干扰实验表明，鸡轴突相关表面糖蛋白神经束蛋白与轴突生长和束状化有关。在此，我们报道神经束蛋白的完整cDNA序列。它由四个结构元件组成：在NH2末端，神经束蛋白包含六个C2亚类的免疫球蛋白样基序，随后是四个纤连蛋白III型（FNIII）相关重复序列。在FNIII样重复序列和跨质膜区域之间，神经束蛋白包含一个由75个氨基酸残基组成的富含脯氨酸、丙氨酸和苏氨酸的结构域，该结构域可能是广泛O-连接糖基化的靶点。一个跨膜片段之后是一个由113个氨基酸残基组成的胞质结构域。序列比较表明，神经束蛋白与鸡Nr-CAM关系最为密切，并与L1（Ng-CAM）和Nr-CAM在脊椎动物免疫球蛋白超家族中形成一个亚组。对几个重叠cDNA探针的测序揭示了神经束蛋白多肽中有趣的异质性。用神经束蛋白cDNA克隆分析基因组Southern印迹表明，神经束蛋白由单个基因编码，因此其前体mRNA可能存在可变剪接。用结构域特异性探针进行Northern印迹分析表明，约8.5 kb的神经束蛋白mRNA在胚胎脑发育过程中均有表达，但在肝脏中不表达。通过免疫亲和层析分离神经束蛋白会产生几种分子量组分。为了分析它们的来源，测定了几种神经束蛋白组分的氨基末端序列。185、160和110 - 135 kD组分的NH2末端序列与cDNA序列预测的NH2末端相同，而其他神经束蛋白组分则以在Ig样和FNIII样部分之间的一个假定可变剪接片段中发现的序列开始，这表明它们是通过蛋白水解切割产生的。酶促和化学去糖基化程序的组合以及花生凝集素结合分析揭示了神经束蛋白组分上的O-连接和N-连接碳水化合物，这可能会产生额外的异质性。
