NOVEL ENDOTHELIAL LECTINS--FUNCTION AND REGULATION

新型内皮凝集素——功能与调节

• 批准号: 6138689
• 负责人: J. Michael Pierce
• 金额: $ 22.54万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-01-01 至 2002-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6138689
• 关键词: Xenopus oocyte; cell line; embryogenesis; genetically modified animals; glycoproteins; immunocytochemistry; laboratory mouse; laboratory rabbit; lectin; ligands; molecular cloning; protein biosynthesis; protein localization; protein signal sequence; protein structure function; secretory protein; solubility; vascular endothelium

We have discovered two novel carbohydrate binding proteins, or lectins, in human endothelial cells, termed EL-1 and -2. Our long term goal is to understand the function of these lectins and how their biosynthesis and release are regulated. These lectins are quite similar in structure to a lectin we cloned from Xenopus oocyte cortical granules, XL35, which functions in the block to polyspermy. XL35 shows a monomer size of 45 kDa, forms putative decamers in solution, requires calcium to bind its oligosaccharide ligand, is a secreted, soluble protein, and displays no homology to lectins other than EL-1 and -2. EL-1 mRNA is expressed in a unique set of tissues: heart, small intestine, colon, thymus, lymph node, spleen, and a few others, but not in brain, kidney, or lung. By contrast, EL-2 mRNA is only expressed in small intestine. Immunohistological examination of a number of these tissues using antiserum against XL35 showed exclusive localization of EL-1 in blood vessel endothelia. Two cultured endothelial cells express EL-1 mRNA and protein that cross-reacts with anti-XL35 antibody: a transformed mouse lymph node endothelial cell line and a human aortic primary cell line. The chromosomal localization of both lectins is 1q23, the same locus at which the selectins are encoded, as well as other endothelial cell adhesion molecules. Our aims are: 1) To understand the mechanisms for the unique tissue expression patterns for EL-1 and -2, to describe the biosynthetic pathway and localization of EL-1 in cultured cells, and to determine the signals that cause the release of the lectins; 2) To determine the nature and structure of the glycoconjugate and oligosacchande ligand(s) for EL-1 and EL-2, how their expression is regulated, and the consequences of EL-glycoconjugate recognition and binding; 3) To determine if the mouse homologs of the lectins are expressed during embryogenesis and to investigate their function by constructing transgenic mice with homozygous deletions of the EL genes. Our hypothesis is that there is a signal, perhaps a cytokine or hormone, that causes the extracellular release of the stored EL, much as fertilization and calcium influx stimulates release of XL35 from the oocyte cortical granules. The released lectins can then recognize and bind their targets, perhaps on circulating cells, initiating adhesion, either among these target cells or between the target cells and endothelia or substrata. The structure of the lectins, their chromosomal localization, and presence in the secretory pathway of a cultured cell type suggest that they function in unique cell adhesion events mediated by endothelial cell present in an unusual set of tissues. Endothelia mediate many medically relevant adhesion events, including initial events in atherosclerosis, inflammation, and metastasis. Understanding the function of these novel lectins, therefore, may well have important medical applications.

我们发现了两种新的碳水化合物结合蛋白，或凝集素， 在人内皮细胞中，称为EL-1和EL-2。 我们的长期目标是 了解这些凝集素的功能以及它们的生物合成 和释放是有规律的。 这些凝集素在结构上非常相似 我们从爪蟾卵母细胞皮质颗粒XL 35中克隆了一种凝集素， 在块中的函数多精子。 XL 35显示单体尺寸为45 kDa在溶液中形成推定的十聚体，需要钙结合其 寡糖配体，是一种分泌的可溶性蛋白质， 与除EL-1和EL-2以外的凝集素同源。 IL-1 mRNA在人乳腺癌细胞中表达， 一组独特的组织：心脏、小肠、结肠、胸腺、淋巴 淋巴结，脾脏和其他一些，但不是在脑，肾，或肺。 通过 而EL-2 mRNA仅在小肠表达。 免疫组织学检查的一些这些组织使用 抗XL 35的抗血清显示EL-1在血液中的专一性定位 血管内皮 两种培养的内皮细胞表达EL-1 mRNA， 与抗XL 35抗体交叉反应的蛋白质：转化小鼠 淋巴结内皮细胞系和人主动脉原代细胞系。 这两种凝集素的染色体定位是1 q23， 选择素编码的细胞，以及其他内皮细胞 粘附分子 我们的目标是：（1）了解 EL-1和EL-2的独特组织表达模式，以描述 EL-1在培养细胞中的生物合成途径和定位，以及 确定引起凝集素释放的信号; 2） 确定糖缀合物的性质和结构， EL-1和EL-2的寡糖配体，它们的表达如何 调节，和EL-糖缀合物识别的后果， 3）确定凝集素的小鼠同源物是否是 在胚胎发生过程中表达，并通过 构建EL基因纯合缺失的转基因小鼠。 我们的假设是有一种信号，可能是细胞因子或激素， 导致细胞外释放储存的EL， 受精和钙离子内流刺激XL 35从细胞中释放， 卵母细胞皮质颗粒 然后释放的凝集素可以识别并 结合它们的目标，也许在循环细胞上，启动粘附， 或者在这些靶细胞之间或者在靶细胞之间， 内皮或基质。凝集素的结构，它们的染色体 定位和存在于培养细胞的分泌途径中 表明它们在介导的独特细胞粘附事件中起作用 由一组不寻常的组织中的内皮细胞引起。内皮 介导许多医学相关粘连事件，包括初始 动脉粥样硬化、炎症和转移中的事件。 理解 因此，这些新型凝集素的功能可能具有重要的 医疗应用。