Kallapur S G, Akeson R A
Division of Basic Science Research, Children's Hospital Research Foundation, Cincinnati, OH 45229-2899.
J Neurosci Res. 1992 Dec;33(4):538-48. doi: 10.1002/jnr.490330406.
The neural cell adhesion molecule (NCAM) has been strongly implicated in several aspects of neural development. NCAM mediated adhesion has been proposed to involve a homophilic interaction between NCAMs on adjacent cells. The heparin binding domain (HBD) is an amino acid sequence within NCAM and has been shown to be involved in NCAM mediated adhesion but the relationship of this domain to NCAM segments mediating homophilic adhesion has not been defined. In the present study, a synthetic peptide corresponding to the HBD has been used as a substrate to determine its role in NCAM mediated adhesion. A neural cell line expressing NCAM (B35) and its derived clone which does not express NCAM (B35 clone 3) adhered similarly to plates coated with HBD peptide. A polyclonal antiserum to NCAM inhibited B35 cell-HBD peptide adhesion by only 10%, a value not statistically different from inhibition caused by preimmune serum. Both these experiments suggested no direct NCAM-HBD interactions. To test whether the HBD peptide bound to cell surface heparan sulfate proteoglycans (HSPG), HSPG synthesis was inhibited using beta-D-xyloside. After treatment, B35 cell adhesion to the HBD peptide, but not to control substrates, was significantly decreased. B35 cell adhesion to the HBD peptide could be inhibited by 10(-7) M heparin but not chondroitin sulfate. Preincubation of the substrate (HBD peptide) with heparin caused dramatic reduction of B35 cell-HBD peptide adhesion whereas preincubation of B35 cells with heparin caused only modest reductions in cell-HBD adhesion. Furthermore, inhibition of HSPG sulfation with sodium chlorate also decreased the adhesion of B35 cells to the HBD peptide. These results strongly suggest that, within the assay system, the NCAM HBD does not participate in homophilic interactions but binds to cell surface heparan sulfate proteoglycan. This interaction potentially represents an important mechanism of NCAM adhesion and further supports the view that NCAM has multiple structurally independent binding sites.
神经细胞黏附分子（NCAM）在神经发育的多个方面都有着重要作用。有研究表明，NCAM介导的黏附作用涉及相邻细胞上NCAM之间的嗜同性相互作用。肝素结合结构域（HBD）是NCAM内部的一段氨基酸序列，已被证明与NCAM介导的黏附有关，但该结构域与介导嗜同性黏附的NCAM片段之间的关系尚未明确。在本研究中，一段与HBD对应的合成肽被用作底物，以确定其在NCAM介导的黏附过程中的作用。一种表达NCAM的神经细胞系（B35）及其不表达NCAM的衍生克隆（B35克隆3）对包被有HBD肽的平板的黏附情况相似。一种针对NCAM的多克隆抗血清仅使B35细胞与HBD肽的黏附减少了10%，这一数值与免疫前血清所导致的抑制作用在统计学上并无差异。这两个实验均表明不存在直接的NCAM-HBD相互作用。为了检测HBD肽是否与细胞表面硫酸乙酰肝素蛋白聚糖（HSPG）结合，使用β-D-木糖苷抑制HSPG的合成。处理后，B35细胞对HBD肽的黏附显著降低，但对对照底物的黏附不受影响。10⁻⁷ M的肝素可抑制B35细胞与HBD肽的黏附，但硫酸软骨素则无此作用。将底物（HBD肽）与肝素预孵育会导致B35细胞与HBD肽的黏附显著减少，而将B35细胞与肝素预孵育只会使细胞与HBD的黏附略有减少。此外，用氯酸钠抑制HSPG硫酸化也会降低B35细胞与HBD肽的黏附。这些结果有力地表明，在该检测系统中，NCAM的HBD不参与嗜同性相互作用，而是与细胞表面硫酸乙酰肝素蛋白聚糖结合。这种相互作用可能代表了NCAM黏附的一种重要机制，并进一步支持了NCAM具有多个结构独立的结合位点这一观点。
