聚唾液酸与唾液酸的研究进展

唾液酸是一族神经氨酸(Neuraminic acid)的衍生物。聚唾液酸(Polysialic acid)是唾液酸(Sialic acid)单体以α-2,8或α-2,9键连接的直链同聚物,是一些哺乳动物细胞中糖蛋白的组成部分和少数几种细菌的胞外多糖组分。综述了唾液酸和聚唾液酸的结构、性质、生物学功能、生物合成和生产应用。     

Polysialic acid: Biosynthesis,novel functions and applications

Abstract

As an anti-adhesive, a reservoir for key biological molecules, and a modulator of signaling, polysialic acid (polySia) is critical for nervous system development and maintenance, promotes cancer metastasis, tissue regeneration and repair, and is implicated in psychiatric diseases. In this review, we focus on the biosynthesis and functions of mammalian polySia, and the use of polySia in therapeutic applications. PolySia modifies a small subset of mammalian glycoproteins, with the neural cell adhesion molecule, NCAM, serving as its major carrier. Studies show that mammalian polysialyltransferases employ a unique recognition mechanism to limit the addition of polySia to a select group of proteins. PolySia has long been considered an anti-adhesive molecule, and its impact on cell adhesion and signaling attributed directly to this property. However, recent studies have shown that polySia specifically binds neurotrophins, growth factors, and neurotransmitters and that this binding depends on chain length. This work highlights the importance of considering polySia quality and quantity, and not simply its presence or absence, as its various roles are explored. The capsular polySia of neuroinvasive bacteria allows these organisms to evade the host immune response. While this “stealth” characteristic has made meningitis vaccine development difficult, it has also made polySia a worthy replacement for polyetheylene glycol in the generation of therapeutic proteins with low immunogenicity and improved circulating half-lives. Bacterial polysialyltransferases are more promiscuous than the protein-specific mammalian enzymes, and new studies suggest that these enzymes have tremendous therapeutic potential, especially for strategies aimed at neural regeneration and tissue repair.     

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。     

Polysialic acid limits choline acetyltransferase activity induced by brain-derived neurotrophic factor

Choline acetyltransferase (ChAT), the enzyme synthesizing acetylcholine, is known to be activated by brain derived neurotrophic factor (BDNF). We found that the specific removal of the carbohydrate polysialic acid (PSA) significantly increased BDNF-induced ChAT-activity in embryonic septal neurons. Using a p75 neurotrophin receptor (p75(NTR)) function-blocking antibody and K252a, a-pan tropomyosin related kinase (Trk) inhibitor, we demonstrate that BDNF-induced ChAT activity requires the stimulation of p75(NTR) and TrkB. PSA removal drastically increased radioactive iodinated ([(125)I])BDNF's maximal binding capacity (Bmax), derived from concentrations of [(125)I]BDNF ranging from 1 pM to 3.2 nM. In the presence of unlabeled nerve growth factor to prevent the binding of [(125)I]BDNF to p75(NTR) sites, the impact of PSA removal on the binding capacity of [(125)I]BDNF was greatly reduced. In conclusion, PSA limits BDNF-induced ChAT activity and BDNF-receptor interactions. BDNF-induced ChAT activity is TrkB and p75(NTR) dependent, and upon PSA removal the additional binding of BDNF to its receptors, especially p75(NTR), likely contributes to the maximal ChAT activity observed. In vivo, the ontogenetic loss of PSA in the postnatal period may allow more interactions between BDNF and its receptors to increase ChAT activity and assure the proper development of the cholinergic septal neurons.     

Nonyloxytryptamine mimics polysialic acid and modulates neuronal and glial functions in cell culture

Polysialic acid (PSA) is a major regulator of cell–cell interactions in the developing nervous system and in neural plasticity in the adult. As a polyanionic molecule with high water‐binding capacity, PSA increases the intercellular space generating permissive conditions for cell motility. PSA enhances stem cell migration and axon path finding and promotes repair in the lesioned peripheral and central nervous systems, thus contributing to regeneration. As a next step in developing an improved PSA‐based approach to treat nervous system injuries, we searched for small organic compounds that mimic PSA and identified as a PSA mimetic 5‐nonyloxytryptamine oxalate, described as a selective 5‐hydroxytryptamine receptor 1B (5‐HT_1B) agonist. Similar to PSA, 5‐nonyloxytryptamine binds to the PSA‐specific monoclonal antibody 735, enhances neurite outgrowth of cultured primary neurons and process formation of Schwann cells, protects neurons from oxidative stress, reduces migration of astrocytes and enhances myelination in vitro. Furthermore, nonyloxytryptamine treatment enhances expression of the neural cell adhesion molecule (NCAM) and its polysialylated form PSA‐NCAM and reduces expression of the microtubule‐associated protein MAP2 in cultured neuroblastoma cells. These results demonstrate that 5‐nonyloxytryptamine mimics PSA and triggers PSA‐mediated functions, thus contributing to the repertoire of molecules with the potential to improve recovery in acute and chronic injuries of the mammalian peripheral and central nervous systems.

     

Retinoic acid treatment and cell aggregation independently regulate alternative splicing in P19 cells during neural differentiation

To induce neural differentiation of P19 cells, two different treatments, RA (retinoic acid) and cell aggregation, are required. However, there has been no report that RA treatment alone or cell aggregation alone could control alternative splicing regulation in P19 cells. Therefore, we focused on alternative splicing effects by neural induction (RA treatment and/or cell aggregation) in P19 cells. We analysed the splicing patterns of several genes, including 5‐HT3R‐A (5‐hydroxytryptamine receptor), Actn1 (actinin alpha1), CUGBP2 (CUG‐binding protein) and PTB (polypyrimidine track‐binding protein), which showed different responses during the early neural induction of P19 cells. We show here that RA treatment alone changes the alternative splice mechanism of 5‐HT3R‐A. Cell aggregation alone controls alternative splicing regulation of Actn1. Both treatments (RA and cell aggregation) compensate and regulate the alternative splicing mechanism of CUGBP2. However, PTB is independent of RA and cell aggregation. Taken together, our results suggest that RA treatment and cell aggregation independently regulate the alternative splicing mechanism in the early stage of P19 cells during neural differentiation.     

Deficits of olfactory interneurons in polysialyltransferase‐ and NCAM‐deficient mice

The neurogenic niche of the anterior subventricular zone (SVZ) persistently generates neuroblasts, which migrate along the rostral migratory stream (RMS) into the olfactory bulb (OB), where they differentiate into granule and periglomerular cells. Loss of the neural cell adhesion molecule NCAM or its post‐translational modification polysialic acid (polySia) impairs migration causing accumulations of cells in the proximal RMS and decreased OB volume. Polysialylation of NCAM is implemented by two polysialyltransferases, ST8SIA2 and ST8SIA4, with overlapping functions. Here, we used mice with Ncam1 and polysialyltransferase deletions to analyze how partial or complete loss of polySia synthesis or a combined loss of polySia and NCAM affects the RMS and the interneuron composition in the OB. Numerous calretinin (CR)‐positive cells were detected dispersed around the RMS in Ncam1 knockout, St8sia2, St8sia4 double‐knockout, and St8sia2, St8sia4, Ncam1 triple‐knockout mice, as well as in St8sia2 ^?/? but not in St8sia4 ^?/? mice. These changes were not reflected by reductions of CR‐positive cells in the granule or glomerular layer of the OB. Instead, calbindin‐positive periglomerular interneurons were strongly reduced in all polySia‐NCAM negative mice and slightly attenuated in St8sia2 ^?/? as well as in the St8sia4 ^?/? mice, which were devoid of ectopic CR‐positive cells along the RMS. Consistent with the early developmental generation of calbindin‐ as compared with CR‐positive OB interneurons, this phenotype was fully developed at postnatal day 5. Together, these results demonstrate that the early development of calbindin‐positive periglomerular interneurons depends on the presentation of polySia on NCAM and requires the activity of both polysialyltransferases. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 421–433, 2016     

Targeting epileptogenesis-associated induction of neurogenesis by enzymatic depolysialylation of NCAM counteracts spatial learning dysfunction but fails to impact epilepsy development

Polysialylation is a post-translational modification of the neural cell adhesion molecule (NCAM), which in the adult brain promotes structural changes in regions of neurogenesis and neuroplasticity. Because a variety of plastic changes including neurogenesis have been suggested to be functionally involved in the pathophysiology of epilepsies, it is of specific interest to define the impact of the polysialic acid (PSA)-NCAM system on development of this disease and associated comorbidities. Therefore, we studied the impact of transient enzymatic depolysialylation of NCAM on the pathophysiology in an electrically induced rat post-status epilepticus (SE) model. Loss of PSA counteracted the SE-induced increase in neurogenesis in a significant manner. This effect of endoneuraminidase (endoN) treatment on hippocampal neurogenesis did not impact the subsequent development of spontaneous seizures. In contrast, transient lack of PSA during SE and in the early phase of epileptogenesis exhibited a cognition sparing effect as revealed in the Morris water maze paradigm. In conclusion, our data do not support a central role of neurogenesis in the development of a hyperexcitable epileptic network. However, in view of the cognition-sparing effect, the transient modulation of the PSA-NCAM system seems to allow beneficial long-term disease modification, which might be mediated by the partial normalization of neurogenesis.     

Retinoic acid induced repression of AP-1 activity is mediated by protein phosphatase 2A in ovarian carcinoma cells

In previous studies we have shown that all-trans retinoic acid (atRA)-treatment of the atRA-sensitive ovarian carcinoma cell line CA-OV3 repressed AP-1 activity by about 50%, while a similar effect was not observed in the atRA-resistant ovarian carcinoma cell line, SK-OV3. These results suggested that the repression of AP-1 activity may be one of the mechanisms by which atRA inhibits the growth of atRA-sensitive CA-OV3 cells. In the present studies, we investigated further the molecular mechanism by which AP-1 activity is repressed by atRA. We show that the repression of AP-1 activity correlates with an increase in JunB protein expression and a decrease in N-terminal phosphorylation of c-Jun. The decrease in N-terminal phosphorylation of c-Jun does not appear to be modulated by JNK or ERK, since their protein expression patterns and kinase activity do not correlate with the repression of AP-1 activity following treatment with atRA. However, the activity of the protein phosphatase PP2A was found to increase 24 h following atRA treatment in CA-OV3 cells. Moreover, the catalytic subunit of PP2A was found to associate with c-Jun in vivo following atRA treatment. Since the inhibition of AP-1 activity following atRA treatment of CA-OV3 cells was abolished in the presence of specific PP2A inhibitors, it is likely that PP2A plays an important role in the atRA-induced repression of AP-1.     

Retinoic acid induces myoblasts transdifferentiation into premeiotic Stra8-positive cells

Spermatogonia and sperm-like cells can be derived in vitro via the addition of RA (retinoic acid) to pluripotent ES and EG cells. At present, however, these cells have not been derived from unipotent cells. Here, we have generated premeiotic Stra8-positive cells from C2C12 myoblasts following treatment with 10 μM all-trans-RA for 8 days. The differentiated C2C12 cells exhibited spherical morphology similar to spermatogonia, and they expressed gene markers of premeiosis, meiosis and postmeiosis. In addition, some of the transdifferentiated Stra8-positive cells had a tail-like phenotype. Flow cytometry results indicated that up to 20% of RA-induced C2C12 cells were Stra8-positive. Mvh (mouse vasa homologue) protein, a germ cell-specific ATP-dependent RNA helicase and Prm1 (protamine 1) were detected in transdifferentiated cells. The DNA content in induced C2C12 cells showed that Stra8-positive cells were diploid, suggesting that the myoblast transdifferentiation was in the premeiotic stage of spermatogenesis. The derivation of Stra8-positive cells from C2C12 myoblasts has important implications for studying unipotent cell differentiation. Furthermore, C2C12 myoblasts may provide a useful in vitro cell model to study signal transduction and transdifferentiation during RA treatments.     

Retinoic acid stimulates growth hormone synthesis in human somatotrophic adenoma cells: Characterization of its nuclear receptors

In order to gain a better understanding on the possible role of retinoic acid (RA) on human GH secretion, we have characterized the expression of its nuclear receptors in somatotropic adenoma cell extracts. By immunoblotting with rabbit polyclonal antibodies directed against RARα, β, and γ and RXRα and β, we could only detect the presence of RARα and RXRα proteins. The predominant expression of RXRα was confirmed at the mRNA level by Northern and slot-blot analysis. When then investigated the effect of RA on GH synthesis in cell culture of adenomatous somatotrophs. In cultured cells, RA (1 μM) stimulated GH secretion, increased intracellular GH content and GH mRNA levels within 72 h, suggesting a modulation of GH synthesis by RA. J. Cell. Biochem 65:25–31. © 1997 Wiley-Liss, Inc.     

Retinoic acid abolishes the calcitonin gene-related peptide autocrine system in F9 teratocarcinoma cells

Calcitonin gene-related peptide (CGRP), expressed predominantly in F9 embryonal carcinoma cells, is both a potent chemotactic agent and an autocrine growth factor for these cells. We analyzed the effect of retinoic acid (RA)-induced differentiation of F9 cells into primitive parietal endoderm-like cells, on CGRP production and the CGRP responsiveness of these cells. Poly(A) RNA extracted from F9 cells and analysed by Northern blotting and hybridization with a CGRP probe showed a specific band of about 1200 bases corresponding to mature CGRP mRNA. This band was not detected in F9 cells treated for 6 days with RA (differentiated primitive parietal endoderm-like cells) or in PYS cells (established parietal endoderm-like cell line). During RA-induced differentiation of F9 cells, CGRP mRNA levels fell within 24 h after treatment and were almost undetectable after 2 days. RA treatment also reduced CGRP secretion by F9 cells; the effect was maximal at 3 days and remained stable thereafter. Similarly, RA rapidly reduced adenylate cyclase responsiveness to chicken CGRP (cCGRP) and human CGRP (hCGRP). An 80% fall in cAMP release into the culture medium in the presence of CGRP was observed after 24 h of RA treatment. These results demonstrate that RA rapidly abolishes the CGRP autocrine system involved in the proliferation of F9 cells, at the same time inducing their differentiation into primitive parietal endoderm. They point to the interaction between retinoic acid and growth factors in the regulation of cell proliferation and differentiation. J. Cell. Biochem. 64:447–457. © 1997 Wiley-Liss, Inc.     

Expression of the Wnt inhibitor Dickkopf-1 is required for the induction of neural markers in mouse embryonic stem cells differentiating in response to retinoic acid

Cultured mouse D3 embryonic stem (ES) cells differentiating into embryoid bodies (EBs) expressed several Wnt isoforms, nearly all isotypes of the Wnt receptor Frizzled and the Wnt/Dickkopf (Dkk) co-receptor low-density lipoprotein receptor-related protein (LRP) type 5. A 4-day treatment with retinoic acid (RA), which promoted neural differentiation of EBs, substantially increased the expression of the Wnt antagonist Dkk-1, and induced the synthesis of the Wnt/Dkk-1 co-receptor LRP6. Recombinant Dkk-1 applied to EBs behaved like RA in inducing the expression of the neural markers nestin and distal-less homeobox gene (Dlx-2). Recombinant Dkk-1 was able to inhibit the Wnt pathway, as shown by a reduction in nuclear beta-catenin levels. Remarkably, the antisense- or small interfering RNA-induced knockdown of Dkk-1 largely reduced the expression of Dlx-2, and the neuronal marker beta-III tubulin in EBs exposed to RA. These data suggest that induction of Dkk-1 and the ensuing inhibition of the canonical Wnt pathway is required for neural differentiation of ES cells.     

The intact parasympathetic nerve promotes submandibular gland regeneration through ductal cell proliferation

ObjectivesSalivary gland regeneration is closely related to the parasympathetic nerve; however, the mechanism behind this relationship is still unclear. The aim of this study was to evaluate the relationship between the parasympathetic nerve and morphological differences during salivary gland regeneration.Materials and MethodsWe used a duct ligation/deligation‐induced submandibular gland regeneration model of Sprague‐Dawley (SD) rats. The regenerated submandibular gland with or without chorda lingual (CL) innervation was detected by haematoxylin–eosin staining, real‐time PCR (RT‐PCR), immunohistochemistry and Western blotting. We counted the number of Ki67‐positive cells to reveal the proliferation process that occurs during gland regeneration. Finally, we examined the expression of the following markers: aquaporin 5, cytokeratin 7, neural cell adhesion molecule (NCAM) and polysialyltransferases.ResultsIntact parasympathetic innervation promoted submandibular gland regeneration. The process of gland regeneration was significantly repressed by cutting off the CL nerve. During gland regeneration, Ki67‐positive cells were mainly found in the ductal structures. Moreover, the expression of NCAM and polysialyltransferases‐1 (PST) expression in the innervation group was significantly increased during early regeneration and decreased in the late stages. In the denervated submandibular glands, the expression of NCAM decreased during regeneration.ConclusionsOur findings revealed that the regeneration of submandibular glands with intact parasympathetic innervation was associated with duct cell proliferation and the increased expression of PST and NCAM.