Hepatic Lectin Receptors in Glycoprotein Homeostasis

描述（由申请人提供）：40多年前，吉尔伯特阿什韦尔和阿纳托尔莫雷尔的经典研究报告了第一个细胞受体和第一个哺乳动物凝集素的鉴定和分离。他们的研究结果表明，肝细胞的去唾液酸糖蛋白受体，现在被称为Ashwell-Morell受体（AMR），结合并内化外源性给药的循环糖蛋白，这些糖蛋白已被神经氨酸酶（唾液酸酶）活性重塑。神经氨酸酶对糖蛋白的这种重塑从所连接的聚糖链的末端去除唾液酸，从而暴露潜在的半乳糖。所得暴露的半乳糖键有助于AMR配体的形成。AMR介导的这种脱唾液酸糖蛋白清除的高容量和快速动力学表明，这种功能已经发展到从循环中消除潜在有害的血液成分。然而，自AMR发现以来的几十年中，内源性配体仍然不确定，并且没有确定AMR的生理目的。最近，主要研究者的实验室鉴定出血小板表达AMR的内源性配体。这一发现包括在肺炎链球菌（SPN）诱导的脓毒症过程中，肝细胞通过AMR依赖性血小板清除引起血小板减少症。此外，AMR依赖性血小板减少症与细菌的NanA神经氨酸酶引起的血小板去唾液酸化有关。本文提出的研究将确定血小板减少症对AMR功能在调节小鼠中SPN败血症的凝血病和致死性方面提供的保护的贡献。使用AMR依赖性和AMR非依赖性血小板清除机制，重组NanA神经氨酸酶和抗CD 41治疗将在比较SPN脓毒症期间凝血功能障碍和死亡率的结局中类似地建立血小板减少症。此外，初步数据表明，在SPN脓毒症过程中，NanA和抗CD 41在给药后具有显著的治疗活性。因此，这些研究将同时表征治疗脓毒症的新型且潜在有效的未来治疗方法。这些研究将进一步解决AMR在SPN脓毒症期间重组NanA和抗CD 41给药的治疗活性中的作用。假设AMR-血小板受体-配体相互作用需要高度唾液酸化的血小板GpIba蛋白。因此，本文提出的研究将在GpIb？缺乏血小板，并将进一步确定是否血小板减少症由于GpIb？在SPN脓毒症的过程中也是保护性的。AMR在哺乳动物中高度保守并进化出快速诱导血小板减少症的能力的原因表明，血小板在某些情况下可能成为致病性的，例如在脓毒症期间产生的血小板中诱导颗粒酶-B表达。粒酶-B引起细胞死亡和组织损伤，导致败血症中的致死性。因此，本提案将测试AMR依赖性血小板减少症的保护作用是否可归因于表达颗粒酶B的血小板的消除。 公共卫生相关性：包括Ashwel-Morell受体（AMR）的肝凝集素结合并从由神经氨酸酶活性重塑的血液循环组分中消除。脓毒症期间血液中神经氨酸酶活性急性升高，从而诱导AMR介导的清除，作为减少凝血病和死亡率的手段。本文提出的研究将确定血小板清除这种保护性反应的贡献，揭示血小板Gp 1b的作用？作为AMR配体，并确定AMR介导的表达颗粒酶B的细胞毒性血小板的清除是否是脓毒症期间AMR保护的基础。

DESCRIPTION (provided by applicant): The classical studies of Gilbert Ashwell and Anatol Morell over 40 years ago reported the identification and isolation of the first cellular receptor ad the first mammalian lectin. Their findings revealed that the asialoglycoprotein receptor of hepatocytes, now termed the Ashwell-Morell receptor (AMR), binds and internalizes exogenously administered circulating glycoproteins that have been remodeled by neuraminidase (sialidase) activity. This remodeling of glycoproteins by neuraminidase removes sialic acid from the termini of the attached glycan chains, unmasking the underlying galactose. The resulting exposed galactose linkages contribute to the formation of AMR ligands. The high capacity and rapid kinetics of AMR-mediated clearance of such asialoglycoproteins suggests that this function has evolved to eliminate potentially deleterious blood components from circulation. However, in the decades since the discovery of the AMR, endogenous ligands remained undefined and no physiological purpose for the AMR was identified. Recently, the Principal Investigator's laboratory identified platelets as expressing endogenous ligands of the AMR. This discovery included the finding that hepatocytes cause thrombocytopenia by AMR-dependent platelet clearance in the course of sepsis induced by Streptococcal pneumoniae (SPN). In addition, AMR-dependent thrombocytopenia was linked to platelet de-sialylation by the bacterium's NanA neuraminidase. Research proposed herein will determine the contribution of thrombocytopenia to the protection afforded by AMR function in moderating the coagulopathy and lethality of SPN sepsis in the mouse. Using AMR-dependent and AMR-independent platelet clearance mechanisms, recombinant NanA neuraminidase and anti-CD41 treatment will similarly establish thrombocytopenia in comparing the outcomes on coagulopathy and mortality during SPN sepsis. Moreover, preliminary data indicates substantial therapeutic activity of NanA and anti-CD41 upon their administration during the course of SPN sepsis. Therefore these studies will be simultaneously characterizing a novel and potentially effective future therapeutic approach to treat sepsis. These studies will further resolve the role of the AMR in the therapeutic activities of recombinant NanA and anti-CD41 administration during SPN sepsis. The AMR-platelet receptor-ligand interaction has been hypothesized to require the highly sialylated platelet GpIba protein. Therefore, research proposed herein will test this hypothesis among GpIb?-deficient platelets, and will further determine whether thrombocytopenia due to GpIb? deficiency in also protective in the course of SPN sepsis. The reason why the AMR is highly conserved among mammals and evolved an ability to rapidly induce thrombocytopenia suggests that platelets may become pathogenic in some circumstances, such as the induction of Granzyme-B expression among platelets produced during sepsis. Granzyme-B causes cell death and tissue damage leading to lethality in sepsis. Therefore this proposal will test whether the protective effect of AMR-dependent thrombocytopenia can be ascribed to the elimination of platelets expressing Granzyme-B. PUBLIC HEALTH RELEVANCE: Hepatic lectins that include the Ashwel-Morell receptor (AMR) bind and eliminate from blood circulation components remodeled by neuraminidase activity. An acute elevation of neuraminidase activity occurs in the blood during sepsis, thereby inducing AMR-mediated clearance as a means to diminish coagulopathy and the rate of mortality. The research proposed herein will define the contribution of platelet clearance to this protective response, reveal the role of platelet Gp1b? as an AMR ligand, and determine whether AMR- mediated clearance of Granzyme B-expressing cytotoxic platelets is the basis of AMR protection during sepsis.