神经细胞粘附分子

神经细胞粘附分子（ＮＣＡＭ）是一种糖蛋白,能介导细胞与细胞及细胞与细胞外基质间相互作用,它在细胞的识别及转移、肿瘤的浸润与生长、神经再生、跨膜信号的传导、学习和记忆等方面均起着一定的作用,本文对神经细胞粘附分子的结构、表达和功能加以概述,以增加对其的了解。     

The 180-kD component of the neural cell adhesion molecule N-CAM is involved in cell-cell contacts and cytoskeleton-membrane interactions

Summary N-CAM₁₈₀, the molecular form of the three neural cell adhesion molecules (N-CAM) with the largest cytoplasmic domain, is accumulated at sites of cell-cell contact (cell bodies, neurites, growth cones) in cultures of neuroblastoma and cerebellum. At these sites the cytoskeletonmembrane linker protein brain spectrin and actin are also accumulated. Brain spectrin copurifies with N-CAM₁₈₀ by immunoaffinity chromatography and binds specifically to N-CAM₁₈₀ but not to N-CAM₁₄₀ or N-CAM₁₂₀ in a solid-phase binding test. These observations indicate an association of N-CAM₁₈₀ with the cytoskeleton in vivo. This association may underlie the reduced lateral mobility of N-CAM₁₈₀ in the surface membrane compared to N-CAM₁₄₀ (Pollerberg et al. 1986). Together with the fact that N-CAM₁₈₀ is only expressed after termination of neuron migration in vivo (Persohn and Schachner, unpublished) these results suggest a role for N-CAM₁₈₀ in stabilization of cell contacts.     

神经细胞粘附分子结构特征和生理功能

神经细胞粘附分子是一类调节细胞与细胞、细胞与细胞外基质间粘附作用的膜表面糖蛋白,主要有NCAM-180、NCAM-140、NCAM-120三种形式,多与PSA结合在一起。在神经系统中,NCAM的表达具有时间和空间特异性,最主要的作用为调节神经系统的可塑性,这种作用可能是通过PSA-NCAM对AMPA的调节作用,主要是通过调节蛋白激酶的表达和细胞内Ca^2 浓度来实现的。     

ShcA regulates neurite outgrowth stimulated by neural cell adhesion molecule but not by fibroblast growth factor 2: evidence for a distinct fibroblast growth factor receptor response to neural cell adhesion molecule activation

Homophilic binding in trans of the neural cell adhesion molecule (NCAM) mediates adhesion between cells and leads, via activation of intracellular signaling cascades, to neurite outgrowth in primary neurons as well as in the neuronal cell line PC12. NCAM mediates neurite extension in PC12 cells by two principal routes of signaling: NCAM/Fyn and NCAM/fibroblast growth factor receptor (FGFR), respectively. Previous studies have shown that activation of mitogen-activated protein kinases is a pivotal point of convergence in NCAM signaling, but the mechanisms behind this activation are not clear. Here, we investigated the involvement of adaptor proteins in NCAM and fibroblast growth factor 2 (FGF2)-mediated neurite outgrowth in the PC12-E2 cell line. We found that both FGFR substrate-2 and Grb2 play important roles in NCAM as well as in FGF2-stimulated events. In contrast, the docking protein ShcA was pivotal to neurite outgrowth induced by NCAM, but not by FGF2, in PC12 cells. Moreover, in rat cerebellar granule neurons, phosphorylation of ShcA was stimulated by an NCAM mimicking peptide, but not by FGF2. This activation was blocked by inhibitors of both FGFR and Fyn, indicating that NCAM activates FGFR signaling in a manner distinct from FGF2 stimulation, and regulates ShcA phosphorylation by the concerted efforts of the NCAM/FGFR as well as the NCAM/Fyn signaling pathway.     

Crystal structure of the Ig1 domain of the neural cell adhesion molecule NCAM2 displays domain swapping

The crystal structure of the first immunoglobulin (Ig1) domain of neural cell adhesion molecule 2 (NCAM2/OCAM/RNCAM) is presented at a resolution of 2.7 Å. NCAM2 is a member of the immunoglobulin superfamily of cell adhesion molecules (IgCAMs). In the structure, two Ig domains interact by domain swapping, as the two N-terminal β-strands are interchanged. β-Strand swapping at the terminal domain is the accepted mechanism of homophilic interactions amongst the cadherins, another class of CAMs, but it has not been observed within the IgCAM superfamily. Gel-filtration chromatography demonstrated the ability of NCAM2 Ig1 to form dimers in solution. Taken together, these observations suggest that β-strand swapping could have a role in the molecular mechanism of homophilic binding for NCAM2.     

ldlD-14细胞表达NCAM并控制其N-聚糖合成的细胞模型的建立

神经细胞黏附分子(Neural cell adhesion molecule,NCAM)是一类表达于神经元、胶质细胞、骨骼细胞以及自然杀伤细胞表面的糖蛋白。NCAM在细胞-细胞黏附及神经细胞迁移等过程中起着重要作用,也是用来研究多聚唾液酸(Polysialic acid,PSA)的模式蛋白。将来源于小鼠乳腺上皮细胞NMuMG中的NCAM基因克隆到真核表达载体pcDNA3.1(+),转染至中国仓鼠卵巢细胞突变株ldlD-14细胞中,通过抗生素G418筛选及蛋白质印迹法检测,得到过表达NCAM的永久转染细胞株。利用ldlD-14细胞的特性,通过在无血清的基本培养基中添加半乳糖与否可以轻易操纵NCAM分子上糖链的修饰,为后期研究糖基化对NCAM分子功能的影响提供工作基础。     

The neural cell adhesion molecule binds to fibroblast growth factor receptor 2

The neural cell adhesion molecule (NCAM) can bind to and activate fibroblast growth factor receptor 1 (FGFR1). However, there are four major FGFR isoforms (FGFR1-FGFR4), and it is not known whether NCAM also interacts directly with the other three FGFR isoforms. In this study, we show by surface plasmon resonance analysis that NCAM can bind to FGFR2 with an affinity similar to that for the NCAM-FGFR1 interaction. However, the kinetic parameters for the NCAM-FGFR2 binding are different from those of the NCAM-FGFR1 binding. Both receptors were shown to cycle relatively fast between the NCAM bound and unbound states, although FGFR2 cycling was clearly faster (13 times) than the FGFR1 cycling. Moreover, ATP was more effective in inhibiting the binding of NCAM to FGFR1 than to FGFR2, indicating that the binding sites in NCAM for the two receptors are similar, but not identical.     

Structure of the tandem fibronectin type 3 domains of neural cell adhesion molecule

Activation of the fibroblast growth factor receptor (FGFR) by neural cell adhesion molecule (NCAM) is essential for NCAM-mediated neurite outgrowth. Previous peptide studies have identified two regions in the fibronectin type 3 (FN3)-like domains of NCAM as being important for these activities. Here we report the crystal structure of the NCAM FN3 domain tandem, which reveals an acutely bent domain arrangement. Mutation of a non-conserved surface residue (M610R) led to a second crystal form showing a substantially different conformation. Thus, the FN3 domain linker is highly flexible, suggesting that it corresponds to the hinge seen in electron micrographs of NCAM. The two putative FGFR1-binding segments, one in each NCAM FN3 domain, are situated close to the domain interface. They form a contiguous patch in the more severely bent conformation but become separated upon straightening of the FN3 tandem, suggesting that conformational changes within NCAM may modulate FGFR1 activation. Surface plasmon resonance experiments demonstrated only a very weak interaction between the NCAM FN3 tandem and soluble FGFR1 proteins expressed in mammalian cells (dissociation constant > 100 μM). Thus, the NCAM-FGFR1 interaction at the cell surface is likely to depend upon avidity effects due to receptor clustering.     

3,4-dihydroxy-L-phenylalanine as a cell adhesion molecule in serum-free cell culture

In this article, we examined the feasibility of using 3,4‐dihydroxy‐L ‐phenylalanine (DOPA) as a cell adhesion molecule in serum‐free cultures of anchorage‐dependent mammalian cells. DOPA is a critical, functional element in mussel adhesive proteins and is known to bind strongly to various natural or synthetic materials. DOPA coating on culture plates was confirmed using X‐ray photoelectron spectroscopy and energy‐dispersive spectroscopy. Human dermal fibroblasts (HDFs) were cultured on DOPA‐coated, fibronectin‐coated, or no material‐coated culture plates in serum‐free medium. HDFs cultured on DOPA showed the highest cell adhesion ratio, spreading, and viability but the lowest apoptotic activity. Therefore, DOPA may be a useful cell‐adhesion molecule for serum‐free culture. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 1055–1060, 2012     

Laminin alpha 5 mediates ectopic adhesion of hepatocellular carcinoma through integrins and/or Lutheran/basal cell adhesion molecule

Laminins are a diverse group of α/β/γ heterotrimers formed from five α, three β and three γ chains; they are major components of all basal laminae (BLs). One laminin chain that has garnered particular interest due to its widespread expression pattern and importance during development is laminin α5. Little is known, however, about the expression and function of laminins containing the α5 chain in human hepatocellular carcinoma (HCC). Here, using a specific antibody, we examined the expression of laminin α5 in normal liver and in HCCs. In normal liver, although laminin α5 was observed in hepatic BLs underlying blood vessels and bile ducts, it was absent from the parenchyma, which may be the origin of HCC. On the other hand, laminin α5 deposition was observed throughout all HCCs tested, regardless of tumor grade. In well-differentiated HCCs, it localized along the trabecules of the tumor. In poorly-differentiated HCCs, it was present in surrounding tumor nodules. In HCC cell lines, laminin α5 heterotrimerized with β and γ chains and was secreted into the culture media. To attempt to understand the function of laminins containing α5, the expression of its receptors in HCCs was also determined. In this regard, α3β1/α6β1 integrins and Lutheran/basal cell adhesion molecule (Lu/B-CAM) were expressed in HCC cells. In vitro studies showed that HCC cells readily attached to laminin containing the α5 chain, more so than did primary hepatocytes. In addition to α3β1/α6β1 integrins and Lu/B-CAM, laminin α5 was recognized by integrin α1β1, which also was expressed in HCC cells. These results suggest that laminins containing α5 serve as functional substrates regulating progression of HCC.     

Localization of neural cell adhesion molecule (N-CAM) immunoreactivity in adult rat tissues

Neural cell adhesion molecule (N-CAM) mediates homophilic adhesion between cells and heterophilic adhesion between cells and extracellular matrix in a Ca²⁺-independent manner. N-CAM is widely expressed during development and plays a crucial role in cell division, migration, and differentiation, but its expression is restricted in adults. The distribution of N-CAM immunoreactivity in adult rat tissues was investigated in the present study. N-CAM immunoreactivity was present in the nervous system in the molecular layer of the cerebellum, ependymal cells surrounding the central canal, axons of the white matter, and in Lamina X of the gray matter of the spinal cord. N-CAM immunoreactivity also was found in autonomic nerves. In the digestive system, N-CAM immunoreactivity was found in the stratified squamous epithelium and nerve plexus of the esophagus, glandular cells of the stomach and pylorus, lamina propria, and epithelium of the villi of the duodenum, jejunum, and ileum. N-CAM immunoreactivity was demonstrated in the secretory cells of the adenohypophysis, islets of Langerhans, and acinar cells of the exocrine pancreas. Alveolar cells of the lung were also N-CAM immunoreactive. In the urinary system, N-CAM immunoreactivity was seen in the proximal convoluted tubules of the kidney. In the male reproductive system, N-CAM immunoreactivity was demonstrated in the nerve plexus around the urethral epithelium and in the nerve fibers around the smooth muscle cells of the corpus cavernosum penis. In the visual system, N-CAM immunoreactivity was seen in the epithelial cells of the corpus ciliaris. Cornea and lens epithelium also showed positive immunoreactivity. Our results suggest that cells in many tissues and organs of the adult rat synthesize N-CAM.     

Endothelial intercellular cell adhesion molecule 1 contributes to cell aggregate formation in CHO cells cultured in serum-free media

Chinese hamster ovary (CHO) cells have been adapted to grow in serum-free media and in suspension culture to facilitate manufacturing needs. Some CHO cell lines, however, tend to form cell aggregates while being cultured in suspension. This can result in reduced viability and capacity for single cell cloning (SCC) via limiting dilution, and process steps to mitigate cell aggregate formation, for example, addition of anti-cell-aggregation agents. In this study, we have identified endothelial intercellular cell adhesion molecule 1 (ICAM-1) as a key protein promoting cell aggregate formation in a production competent CHO cell line, which is prone to cell aggregate formation. Knocking out (KO) the ICAM-1 gene significantly decreased cell aggregate formation in the culture media without anti-cell-aggregation reagent. This trait can simplify the process of transfection, selection, automated clone isolation, and so on. Evaluation in standard cell line development of ICAM-1 KO and wild-type CHO hosts did not reveal any noticeable impacts on titer or product quality. Furthermore, analysis of a derived nonaggregating cell line showed significant reductions in expression of cell adhesion proteins. Overall, our data suggest that deletion of ICAM-1 and perhaps other cell adhesion proteins can reduce cell aggregate formation and improve clonality assurance during SCC.     

Proteolytic cleavage of the neural cell adhesion molecule by ADAM17/TACE is involved in neurite outgrowth

The transmembrane and multidomain neural cell adhesion molecule (NCAM) plays important functional roles in the developing and adult nervous system. NCAM is proteolytically processed and appears in soluble forms in the cerebrospinal fluid and in serum under normal and pathological conditions. In this report, we present evidence that the metalloprotease a disintegrin and a metalloprotease (ADAM)17/tumour necrosis factor alpha converting enzyme (TACE) cleaves the polysialylated as well as the non-polysialylated transmembrane isoforms of NCAM, whereas the glycophosphatidylinositol-linked isoform of NCAM is not proteolytically cleaved. A truncated, enzymatically inactive mutant of TACE did not result in release of the NCAM110 cleavage product. Proteolytic cleavage was enhanced by a calmodulin-specific inhibitor and the actin-destabilizing agents cytochalasin D and latrunculin B. In contrast, the microtubule-stabilizing agent colchicine or microtubule-destabilizing agent paclitaxel did not affect the release of the 110-kDa fragment of NCAM. Neurite outgrowth from cerebellar microexplants was inhibited in the presence of the metalloprotease inhibitor GM 6001 on substrate-coated NCAM, but not on poly-l-lysine. Upon transfection of hippocampal neurones with an enzymatically inactive mutant of TACE, NCAM-stimulated neurite outgrowth was inhibited without affecting neurite outgrowth on poly-l-lysine, showing that proteolytic processing of NCAM by the metalloprotease TACE is involved in NCAM-mediated neurite outgrowth.     

Gicerin,a cell adhesion molecule,participates in the histogenesis of retina

Gicerin is a novel cell adhesion molecule that belongs to the immunoglobulin superfamily. Gicerin protein adheres to neurite outgrowth factor (NOF), an extracellular matrix protein in the laminin family, and also exhibits homophilic adhesion. Heterophilic adhesion of gicerin to NOF is thought to play an active role in neurite outgrowth of developing retinal cells in vitro. In this study, we examined the adhesion activity of gicerin during the retinal development of Japanese quail using an antibody directed against gicerin, to elucidate the biological importance of gicerin in retinal histogenesis. Immunohistochemical and Western blot analysis showed that gicerin was highly expressed in the developing retina but suppressed in the mature retina. The aggregation of neural retinal cells from 5-day embryonic quail retina was significantly inhibited when incubated with a polyclonal antibody to gicerin, suggesting that gicerin protein participates in the adhesion of neural retinal cells of the developing retina. Furthermore, histogenesis of retina both in the organ cultures and in ovo embryos was severely disrupted by incubation with a gicerin antibody. These findings provide evidence that gicerin plays an important role in retinal histogenesis. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 769–780, 1997     

Baculovirus expression and bioactivity of a soluble 140 kDa extracellular cleavage fragment of L1 neural cell adhesion molecule

L1 neural cell adhesion molecule is the founding member of the L1 subfamily of the immunoglobulin superfamily and plays an important role in the overall development of both the central and peripheral nervous systems, making it an attractive candidate for promoting neural regeneration following injury. Currently, L1 used for experimental studies is primarily mammalian-derived; however, the insect cell expression system described here provides an alternative source of recombinant L1 with equivalent bioactivity. A 140 kDa L1 fragment based on a physiological plasmin cleavage site in the extracellular domain was cloned and expressed with a C-terminal 6x histidine tag. Recombinant insect cell-derived L1 was analyzed by Western blot using an antibody to human L1 to confirm immunogenicity and to optimize infection conditions for recombinant L1 production. The recombinant protein was secreted by insect cells, efficiently purified under non-denaturing conditions using dialysis followed by metal affinity chromatography, and analyzed by SDS-PAGE to produce a single band of the expected approximate 140 kDa size. The bioactivity of insect cell-derived L1 was compared to mammalian-derived L1-Fc and poly-L-lysine (PLL) using chick embryonic forebrain neurons. The results show comparable, robust neurite outgrowth at 24h on insect cell-derived L1 and mammalian-derived L1-Fc, with significantly longer neurites than those observed on PLL. Future studies will examine the immobilization of L1 to biomaterial surfaces in physiologically appropriate orientation via the C-terminal 6x histidine tag and will investigate their application in promoting axonal regeneration in the injured nervous system.     

Identification and characterization of a novel neural cell adhesion molecule (NCAM)-associated protein from quail myoblasts: relationship to myotube formation and induction of neurite-like protrusions

Abstract We identified a novel neural cell adhesion molecule (NCAM)-associated protein, myo genesis-related and N CAM- a ssociated p rotein (MYONAP), the expression of which increases during the formation of myotubes in quail myoblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (QM-RSV cells). MYONAP shares homology with PL48 in human cytotrophoblasts and KIAA0386 in human brain. Excess expression of MYONAP in presumptive QM-RSV myoblasts induced long protrusions like neurites in cooperation with microtubules. Suppression of MYONAP by antisense cDNA prevented myotubes from forming in spite of the expression of myogenin, creatine kinase, and myosin, and rendered myoblast membranes resistant to fusion. Yeast two-hybrid screening showed that MYONAP interacted with NCAM specifically. Deletion of the NCAM-associated domain resulted in a loss of the function that induces neurite-like protrusions to form and disturbed the elongation of microtubules. The results suggested that MYONAP influenced the functions of microtubules and was involved in the formation of myotubes via its interaction with NCAM.     

IgLON cell adhesion molecules regulate synaptogenesis in hippocampal neurons

IgLON cell adhesion molecules (CAMs) belonging to the immunoglobulin superfamily comprise of LAMP, neurotrimin (Ntm), OBCAM, and Kilon. In the present study, we performed the single and double transfection of IgLON gene constructs into hippocampal neurons in vitro and evaluated synaptic number. The quantitative analysis showed that the single over‐expression of LAMP or OBCAM increased synaptic number, while the over‐expression of Kilon reduced synaptic number and Ntm had no effects. The double over‐expression of Kilon‐Ntm, Kilon‐OBCAM, LAMP‐Ntm, and Ntm‐OBCAM decreased synaptic number and that of Kilon‐LAMP and LAMP‐OBCAM had no effect. These results suggest that IgLON CAMs participate in regulating synapse formation in hippocampal neurons. Copyright © 2009 John Wiley & Sons, Ltd.     

1H and 15N resonance assignment of the second fibronectin type III module of the neural cell adhesion molecule

We report here the NMR assignment of the second fibronectin type III module of the neural cell adhesion molecule (NCAM). This module has previously been shown to interact with the fibroblast growth factor receptor (FGFR), and the FGFR-binding site was mapped by NMR to the FG-loop region of the module. The FG-loop region also contains a putative nucleotide-binding motif, which was shown by NMR to interact with ATP. Furthermore, ATP was demonstrated to inhibit binding of the second F3 module of NCAM to FGFR.