Hepatic Lectin Receptors in Glycoprotein Homeostasis

描述(申请人提供)：40多年前Gilbert Ashwell和Anatol Morell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。他们的发现表明，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将唾液酸从连接的糖链末端移除，从而暴露了潜在的半乳糖。由此产生的暴露的半乳糖连接有助于AMR配体的形成。AMR介导的这种去唾液酸糖蛋白清除的高容量和快速动力学表明，这种功能已经进化到从循环中消除潜在的有害血液成分。然而，自发现AMR以来的几十年里，内源性配体仍然没有确定，也没有确定AMR的生理目的。最近，首席调查员的实验室发现，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依赖的血小板清除导致血小板减少。此外，依赖AMR的血小板减少与细菌的NaNA神经氨酸酶对血小板的去唾液作用有关。本文提出的研究将确定血小板减少对AMR功能在缓和小鼠SPN脓毒症的凝血障碍和致死性方面所起的保护作用。使用AMR依赖和AMR非依赖的血小板清除机制，重组NANA神经氨酸酶和抗CD41治疗在比较SPN脓毒症期间凝血障碍和死亡率的结果中，同样会建立血小板减少症。此外，初步数据表明，在SPN脓毒症过程中给予NaNA和抗CD41后，具有显著的治疗活性。因此，这些研究将同时表征一种新的、潜在有效的未来治疗脓毒症的方法。这些研究将进一步明确AMR在SPN脓毒症期间重组NANA和抗CD41治疗活性中的作用。AMR-血小板受体-配体的相互作用被认为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？缺陷的血小板中检验这一假说，并将进一步确定GPIB是否导致血小板减少？在SPN脓毒症的病程中也有保护作用。AMR在哺乳动物中高度保守，并进化出快速诱导血小板减少的能力的原因表明，在某些情况下，血小板可能是致病的，例如在脓毒症期间诱导血小板产生颗粒酶-B。颗粒酶-B导致细胞死亡和组织损伤，导致脓毒症的致命性。因此，这项提议将检验AMR依赖型血小板减少症的保护作用是否可以归因于消除表达颗粒酶-B的血小板。 公共卫生相关性：包括Ashwel-Morell受体(AMR)在内的肝脏凝集素结合和消除由神经氨酸酶活性重塑的血液循环成分。脓毒症期间血液中神经氨酸酶活性急剧升高，从而诱导AMR介导的清除，作为减少凝血障碍和死亡率的一种手段。本文提出的研究将确定血小板清除对这种保护性反应的贡献，揭示血小板GP1b？作为AMR的配体，并确定AMR介导的颗粒酶B表达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.