Direct Interaction of CASK/LIN-2 and Syndecan Heparan Sulfate Proteoglycan and Their Overlapping Distribution in Neuronal Synapses

CASK, the rat homolog of a gene (LIN-2) required for vulval differentiation in Caenorhabditis elegans, is expressed in mammalian brain, but its function in neurons is unknown. CASK is distributed in a punctate somatodendritic pattern in neurons. By immunogold EM, CASK protein is concentrated in synapses, but is also present at nonsynaptic membranes and in intracellular compartments. This immunolocalization is consistent with biochemical studies showing the presence of CASK in soluble and synaptosomal membrane fractions and its enrichment in postsynaptic density fractions of rat brain. By yeast two-hybrid screening, a specific interaction was identified between the PDZ domain of CASK and the COOH terminal tail of syndecan-2, a cell surface heparan sulfate proteoglycan (HSPG). The interaction was confirmed by coimmunoprecipitation from heterologous cells. In brain, syndecan-2 localizes specifically at synaptic junctions where it shows overlapping distribution with CASK, consistent with an interaction between these proteins in synapses. Cell surface HSPGs can bind to extracellular matrix proteins, and are required for the action of various heparin-binding polypeptide growth/differentiation factors. The synaptic localization of CASK and syndecan suggests a potential role for these proteins in adhesion and signaling at neuronal synapses.     

Cell Surface Heparan Sulfate Proteoglycan Syndecan-2 Induces the Maturation of Dendritic Spines in Rat Hippocampal Neurons

Dendritic spines are small protrusions that receive synapses, and changes in spine morphology are thought to be the structural basis for learning and memory. We demonstrate that the cell surface heparan sulfate proteoglycan syndecan-2 plays a critical role in spine development. Syndecan-2 is concentrated at the synapses, specifically on the dendritic spines of cultured hippocampal neurons, and its accumulation occurs concomitant with the morphological maturation of spines from long thin protrusions to stubby and headed shapes. Early introduction of syndecan-2 cDNA into immature hippocampal neurons, by transient transfection, accelerates spine formation from dendritic protrusions. Deletion of the COOH-terminal EFYA motif of syndecan-2, the binding site for PDZ domain proteins, abrogates the spine-promoting activity of syndecan-2. Syndecan-2 clustering on dendritic protrusions does not require the PDZ domain-binding motif, but another portion of the cytoplasmic domain which includes a protein kinase C phosphorylation site. Our results indicate that syndecan-2 plays a direct role in the development of postsynaptic specialization through its interactions with PDZ domain proteins.     

Syndecan-1 and Syndecan-4 Are Independent Indicators in Breast Carcinoma

Syndecan proteoglycans may be key regulators of tumor invasion and metastasis because this four-member family of transmembrane receptors regulates cell adhesion, proliferation, and differentiation. Their expression can also serve as prognostic markers. In breast carcinomas, syndecan-1 overexpression correlates with poor prognosis and aggressive phenotype. Syndecan-4 is expressed in most breast carcinoma cell lines, but its role in malignancy is unclear. A possible relationship between syndecan-1 and syndecan-4 expression and established prognostic factors in breast carcinomas was examined. Duplicate samples of 114 benign and malignant breast disease cases were stained for the two syndecans. Clinicopathological information was available for all cases. Syndecan-1 was detected in 72.8% of cases, with significant association between its expression and histological tumor type (p<0.05) and high grade tumors (p<0.05). Syndecan-4 was expressed in 66.7% of cases; expression correlated significantly with positive estrogen (p<0.01) and progesterone (p<0.01) receptor status. Independent expression of the two syndecans was noted from an analysis of single and double positive cases. There was a statistical relationship between syndecan-1 presence in high-grade tumors and absence of syndecan-4, whereas syndecan-4 presence in cases positive for estrogen and progesterone receptor associated with syndecan-1 absence. These syndecans may, therefore, have distinct roles in regulating breast carcinoma cell behavior.     

硫酸乙酰肝素蛋白聚糖的功能机制研究进展

硫酸乙酰肝素蛋白聚糖是由核心蛋白和与之相连的硫酸乙酰肝素糖链组成,广泛分布于细胞膜与细胞外基质中。其中多配体蛋白聚糖（syndecan）和糖基磷脂酰肌醇锚定蛋白聚糖（glypican）存在与细胞膜上,而串珠蛋白聚糖（perlecan）和组合蛋白聚糖（agrin）表达在细胞外基质中。该类蛋白在生理与病理历程中,如发育、伤口愈合、肿瘤发生发展、感染、免疫应答等过程中担任重要作用,这些功能是其核心蛋白和糖链共同作用的结果。概述硫酸乙酰肝素蛋白聚糖的功能及其机制研究进展,同时强调其在作为药物靶标和临床诊断研究中的应用。     

Amino Acid Sequence and Antimicrobial Activity of Chitin-Binding Peptides,Pp-AMP 1 and Pp-AMP 2, from Japanese Bamboo Shoots (Phyllostachys pubescens)

Two novel chitin-binding peptides, designated Pp-AMP 1 and Pp-AMP 2, which had antimicrobial activity against pathogenic bacteria and fungi, were purified from Japanese bamboo shoots (Phyllostachys pubescens) by a simple procedure based on chitin affinity chromatography. They had the common structural features of the plant defensin family, but they could not be grouped in any type of that family. They showed a high degree of homology to mistletoe toxins.     

Purification,Characterization, and Sequencing of a Novel Type of Antimicrobial Peptides,Fa-AMP1 and Fa-AMP2, from Seeds of Buckwheat (Fagopyrum esculentum Moench.)

Novel antimicrobial peptides (AMP), designated Fa-AMP1 and Fa-AMP2, were purified from the seeds of buckwheat (Fagopyrum esculentum Moench.) by gel filtration on Sephadex G75, ion-exchange HPLC on SP COSMOGEL, and reverse-phase HPLC. They were basic peptides having isoelectric points of over 10. Fa-AMP1 and Fa-AMP2 had molecular masses of 3,879 Da and 3,906 Da on MALDI-TOF MS analysis, and their extinction coefficients in 1% aqueous solutions at 280 nm were 42.8 and 38.9, respectively. Half of all amino acid residues of Fa-AMP1 and Fa-AMP2 were cysteine and glycine, and they had continuous sequences of cysteine and glycine. The concentrations of peptides required for 50% inhibition (IC₅₀) of the growth of plant pathogenic fungi, and Gram-positive and -negative bacteria were 11 to 36 μg/ml. The structural and antimicrobial characteristics of Fa-AMPs indicated that they are a novel type of antimicrobial peptides belonging to a plant defensin family.     

核糖核酸酶5在宿主防御中的作用及机制

抗菌肽是机体天然免疫的重要组成部分。核糖核酸酶5（ribonuclease5,RNASE5;又名angiogenin）属于核糖核酸酶A超家族,是一个分泌型小分子蛋白质,广泛分布于机体需要抵御外界病原微生物的组织中。RNASE5对病毒、细菌以及真菌都存在抑制效应,具有广谱抗菌特点,但其表达和活性受到宿主生理状态和外界环境多层次的调控。RNASE5存在多样的抗微生物作用机制,其带正电荷的理化特性破坏微生物细胞壁,而其核糖核酸酶活性则是杀伤真菌所必须的。除了直接作用于微生物外,RNASE5还可作为重要因子调节宿主免疫功能,参与多种病理过程。本文综述了RANSE5的结构、生物活性与功能、作用特点与机制,并讨论了在其研究中存在的问题,以期为今后的研究提供新思路和新方向。     

In search of a function of Manduca sexta hemolymph protease-1 in the innate immune system

Extracellular serine protease cascades mediate immune signaling and responses in insects. In the tobacco hornworm Manduca sexta, nearly 30 serine proteases (SPs) and their homologs (SPHs) are cloned from hemocytes and fat body. Some of them participate in prophenoloxidase (proPO) activation and proSpätzle processing. Here we report the cDNA cloning of hemolymph protease-1b (HP1b), which is 90% identical and 95% similar to HP1a (formerly HP1). The HP1a and HP1b mRNA levels in hemocytes was down- and up-regulated after an immune challenge, respectively. Quantitative real-time polymerase chain reactions revealed their tissue-specific and development-dependent expression, mostly in hemocytes of the feeding larvae. We isolated HP1 precursor (proHP1) from larval hemolymph and observed micro-heterogeneity caused by N-linked glycosylation. Supplementation of the purified proHP1 to plasma samples from naïve larvae or induced ones injected with bacteria caused a small PO activity increase, much lower than those elicited by recombinant proHP1a/b, but no proteolytic cleavage was detected in the zymogens. Incubation of proHP1a/b or their catalytic domains with a cationic detergent, cetylpyridinium chloride, induced an amidase activity that hydrolyzed LDLH-p-nitroanilide. Since LDLH corresponds to the P4–P1 region before the proteolytic activation site of proHP6, we propose that the active but uncleaved proHP1 may cut proHP6 to generate HP6 that in turn activates proPAP1 and proHP8. The catalytic domain of HP1a/b, which by itself does not activate purified proHP6 or hydrolyze LDLH-p-nitroanilide, somehow generated active HP6, HP8, PAP1 and PO in plasma. Together, these results indicate that proHP1 participates in the proPO activation system, although detailed mechanism needs further exploration.