CALRETICULIN--A SECRETED PROTEIN IN IXODID TICK SALIVA

钙网蛋白——蜱虫唾液中的一种分泌蛋白

• 批准号: 2068720
• 负责人: GLEN R NEEDHAM
• 金额: $ 21.27万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-01 至 1998-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2068720
• 关键词: Ixodes; arthropod borne communicable disease; calcium binding protein; communicable disease transmission; complementary DNA; eating; electroporation; host organism interaction; human tissue; immunoelectron microscopy; in situ hybridization; nucleic acid probes; nucleic acid sequence; polymerase chain reaction; protein biosynthesis; protein sequence; radionuclides; salivary glands; secretory protein; western blottings

Ticks transmit more types of microorganisms and infect more people with disease than any other arthropod in the U.S. Lyme disease is responsible for a resurgence of concern about tick, although Rocky Mountain spotted fever, Colorado tick fever and relapsing fever remain a continuous threat to the human population on a regional basis. The focus of our proposal is on the tick's salivary glands that facilitate disease transmission and produce t he antihemostasis, antiinflammatory and immunosuppressive agents, which control the bite site so the parasite can feed. Our overall objective is to characterize the primary salivary components that mediate the feeding-site environment. Understanding heir roles should provide fundamental insights into the disease transmission process and how the host's responses to the parasite are neutralized or even used to the tick's advantage. Until recently, progress towards characterization of organic salivary components was only modest, and based upon responses of in vitro assays to saliva or gland extracts. The primary rate- limiting steps has been that of tissue (salivary gland) abundance. Advances in molecular genetics technology now provide the opportunity to overcome this limitation. We have identified a form of calreticulin in two medically important tick species (feeding Amblyomma americanum & Dermacentor variabilis females), based upon complete sequencing of a cDNA, (69% deduced amino acid homology with rabbit skeletal muscle calreticulin) and positive immunoblotting of gland extracts and saliva with goat anti-rabbit calreticulin. Calreticulin is a highly conserved Ca2+ binding protein within non-muscle cell endoplasmic reticulum and smooth muscle sarcoplasmic reticulum. That the protein within non-muscle cell endoplasmic reticulum and smooth muscle sarcoplasmic reticulum. That the protein is likely to be synthesized for secretion is supported by sequence data as the ER retention signal (KDEL) is apparently not present; and, it is found in the saliva. Light and ultrastructural level immunolocalization of the protein and in situ hybridization studies of glands during morphogenesis will help correlate protein secretion with host-related responses, and determine where the protein is synthesized and located within the cell. Hemostasis (platelet aggregation) and vascular (smooth muscle contraction) assays will provide valuable information about the role of the gland protein as a secreted product. Ca2+-dependent and independent processes will be investigated. Characterization of calreticulin using these methods will present a significant advancement in describing when and where its is secreted, while beginning the process of defining its physiological significance in the feeding process. This understanding will be critical in understanding the parasite-host relationship and possibly disease transmission by ixodid ticks.

蜱虫传播更多类型的微生物并感染更多人 莱姆病是美国最常见的节肢动物疾病 尽管落基山发现了蜱虫，但人们对蜱虫的担忧再次出现 发烧、科罗拉多蜱热和回归热仍然是持续的威胁 以区域为基础的人口。 我们提案的重点 位于蜱的唾液腺上，促进疾病传播 产生抗止血、抗炎和免疫抑制作用 药剂，控制叮咬部位，以便寄生虫可以进食。 我们的 总体目标是表征主要唾液成分 调节饲喂现场环境。 了解继承人的角色应该 提供对疾病传播过程的基本见解 宿主对寄生虫的反应如何被中和甚至习惯 蜱虫的优势。 直到最近，表征方面取得了进展 有机唾液成分的含量很低，并且基于反应 对唾液或腺体提取物进行体外测定。 初级费率- 限制步骤是组织（唾液腺）丰度。 分子遗传学技术的进步现在提供了机会 克服这个限制。 我们已经鉴定出一种形式的钙网蛋白 两种具有重要医学意义的蜱虫（以 Amblyomma americanum 为食和 Dermacentor variabilis 雌性），基于完整测序 cDNA，（推断与兔骨骼肌氨基酸同源性为 69% 钙网蛋白）和腺体提取物和唾液的阳性免疫印迹 与山羊抗兔钙网蛋白。 钙网蛋白是一种高度保守的 非肌细胞内质网内的 Ca2+ 结合蛋白和 平滑肌肌浆网。 非肌肉内的蛋白质 细胞内质网和平滑肌肌浆网。 支持该蛋白质可能被合成用于分泌 通过序列数据，因为 ER 保留信号 (KDEL) 显然不是 展示;并且，它存在于唾液中。 光和超微结构水平 蛋白质的免疫定位和原位杂交研究 形态发生过程中的腺体将有助于将蛋白质分泌与 宿主相关反应，并确定蛋白质的合成位置 并位于细胞内。 止血（血小板聚集）和 血管（平滑肌收缩）检测将提供有价值的信息 有关腺体蛋白作为分泌产物的作用的信息。 将研究 Ca2+ 依赖性和独立过程。 使用这些方法对钙网蛋白进行表征将呈现出 在描述其分泌时间和地点方面取得了重大进展， 同时开始定义其生理意义的过程 在饲养过程中。 这种理解对于以下方面至关重要： 了解寄生虫与宿主的关系以及可能的疾病 通过 ixodid 蜱传播。