Binding of neural cell adhesion molecules (N-CAMs) to the cellular prion protein
Binding of neural cell adhesion molecules (N-CAMs) to the cellular prion protein
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DOI：
10.1006/jmbi.2000.5183
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发表时间：
2001-12-14
影响因子：
5.6
通讯作者：
Prusiner, SB
中科院分区：
文献类型：
--
作者：
Schmitt-Ulms, G;Legname, G;Prusiner, SB
为了确定细胞朊病毒蛋白（PrPC）的分子相互作用伙伴，我们试图应用原位交联方法来维持PrPC的微环境。对易受朊病毒感染的小鼠神经母细胞瘤细胞（N2a）进行轻度甲醛交联，发现PrPC存在于200至225 kDa的高分子质量（HMM）蛋白复合物中。LC/MS/MS分析鉴定出三种小鼠神经细胞粘附分子（N-CAM）剪接变异体，分离出具有小泡样结构域（CLDs）的复合物。酶去除n连接的糖部分不会破坏复合物，认为PrP与N-CAM的相互作用是通过氨基酸侧链发生的。此外，在N2a和朊病毒感染的N2a （ScN2a）细胞中发现相似水平的PrP/N-CAM复合物。利用N-CAM特异性肽库，确定了prp结合位点由两个连续的纤维连接蛋白III型（FNIII）模块中的β -链C和C'组成，这些模块位于N-CAM的膜附着位点附近。通过对PrP缺失突变体的原位交联发现，结合位点的PrP面由N端、螺旋A（残基144154）和邻近的PrP环区组成。N-CAM缺陷（N-CAM(-/-)）小鼠被痒病朊病毒脑内攻击后，平均潜伏期为122 （+/-4.1，SEM）天，这表明N-CAM不参与PrPSc的复制。我们的发现提出了N-CAM可能与PrPC一起执行一些尚未确定的生理细胞功能的可能性。(C) 2001学术出版社。
To identify molecular interaction partners of the cellular prion protein (PrPC), we sought to apply an in situ crosslinking method that maintains the microenvironment of PrPC. Mild formaldehyde crosslinking of mouse neuroblastoma cells (N2a) that are susceptible to prion infection revealed the presence of PrPC in high molecular mass (HMM) protein complexes of 200 to 225 kDa. LC/MS/MS analysis identified three murine splice-variants of the neural cell adhesion molecule (N-CAM) in the complexes, which isolate with caveolae-like domains (CLDs). Enzymatic removal of N-linked sugar moieties did not disrupt the complexes, arguing that the interaction of PrP with N-CAM occurs through amino acid side-chains. Additionally, similar levels of PrP/N-CAM complexes were found in N2a and prion-infected N2a (ScN2a) cells. With the use of an N-CAM-specific peptide library, the PrP-binding site was determined to comprise beta-strands C and C' within the two consecutive fibronectin type III (FNIII) modules found in proximity of the membrane-attachment site of N-CAM. As revealed by in situ crosslinking of PrP deletion mutants, the PrP face of the binding site is formed by the N terminus, helix A (residues 144154) and the adjacent loop region of PrP. N-CAM-deficient (N-CAM(-/-)) mice that were intracerebrally challenged with scrapie prions succumbed to disease with a mean incubation period of 122 (+/-4.1, SEM) days, arguing that N-CAM is not involved in PrPSc replication. Our findings raise the possibility that N-CAM may join with PrPC in carrying out some as yet unidentified physiologic cellular function. (C) 2001 Academic Press.