Role of the lysosomal membrane protein LIMP-2 as a transport receptor for Glucocerebrosidase in disease

酸性水解酶是降解溶酶体内大分子的必需酶。溶酶体水解酶功能失调引起的大量疾病凸显了这一过程的生理作用。一般来说，这些酶是通过甘露糖-6-磷酸途径转运到溶酶体的，甘露糖-6-磷酸途径是除酶和组织特有的例外情况下常见的转运途径。其中一个例外是葡萄糖脑苷酶(GCase)，它的突变会导致高谢病(GD)。我们已经证明，GCase利用溶酶体膜蛋白(LIMP-2)运输溶酶体，并且GCase的溶酶体功能与LIMP-2密切相关。编码LIMP-2基因的零突变导致肌阵挛肾衰竭综合征，一种常染色体隐性遗传性溶酶体储存障碍(LSD)，合并肾衰竭和进行性肌阵挛癫痫。一些患者还会出现脱髓鞘周围神经病变和听力损失。LIMP-2基因缺陷小鼠具有非常相似的表型，这使得该小鼠模型成为分析AMRF导致零突变后果的有价值的工具。通过解决LIMP-2胞外结构域的结构，我们发现LIMP-2可能是双功能的。它由一个对脂质结合和转运很重要的部分β-Barrel核心和一个介导与GCase相互作用的三螺旋束组成。LIMP-2/GCase相互作用是溶酶体将GCase从内质网转运到溶酶体所必需的。AMRF零突变导致的LIMP-2缺陷影响了GCase的溶酶体脂质转运和溶酶体转运。正如已经提到的，编码GCase、GD的基因会导致GBA1的突变，GCase是最普遍的LSD，其特征是肝脾肿大、骨骼异常，在极少数情况下会出现中枢神经系统表现，如共济失调或癫痫。最近又发现了SCARB2和GBA1基因变异，它们被认为是帕金森氏病(PD)和路易体痴呆(DLB)的危险因素。特别是，GCase突变是PD和DLB的主要遗传因素，在散发性PD脑中也发现GCase活性降低。为了分析LIMP-2作为GCase的溶酶体转运受体和作为脂质/胆固醇转运蛋白的生理作用，我们建立了一个表达LIMP-2突变体的KI小鼠模型，该模型在GCase结合部位携带一个点突变。我们的初步结果表明，Ki小鼠表达GCase结合缺陷的LIMP-2，导致肝脏溶酶体GCase水平显著降低，这与LIMP-2缺陷小鼠的水平相当。我们的初步结果有力地表明，LIMP-2KI小鼠提供了一种独特的工具，可以在不干扰LIMP-2表达的情况下分析溶酶体GCase活性显著下降的生理后果。这里提出的实验将确定LIMP-2缺乏所介导的病理是否至少部分依赖于GCase。

Acid hydrolases are essential for the degradation of macromolecules within lysosomes. The physiological role of this process is highlighted by a large number of disorders caused by dysfunctional lysosomal hydrolases. In general are these enzymes transported to lysosomes by the mannose-6-phosphat pathway, which is the common trafficking pathway with enzyme and tissue specific exceptions. One of such an exception is glucocerebrosidase (GCase), in which mutations cause Gaucher Disease (GD). We have demonstrated that GCase utilizes the lysosomal membrane protein (LIMP-2) for lysosomal transport and that lysosomal function of GCase is intimately connected to LIMP-2. Null-mutations in the gene encoding LIMP-2 cause Action Myoclonus-Renal Failure syndrome, an autosomal-recessive lysosomal storage disorder (LSD) with the combination of kidney failure and progressive myoclonus epilepsy. Some patients also develop a demyelinating peripheral neuropathy and hearing loss. LIMP-2-deficient mice have a very similar phenotype, making this mouse model a valuable tool to analyze consequences of AMRF causing null-mutations.By solving the structure of the LIMP-2 ectodomain, we found that LIMP-2 is likely bifunctional. It consists of a partial β-barrel core, important for lipid binding and transport and a three helix bundle that mediates interaction with GCase. LIMP-2/GCase interaction is necessary for lysosomal transport of GCase from the Endoplasmic Reticulum to lysosomes. LIMP-2 deficiency, caused by AMRF null-mutations is affecting both, lysosomal lipid transport and lysosomal trafficking of GCase. As already mentioned cause mutations in GBA1, the gene encoding GCase, GD, the most prevalent LSD, characterized by hepatosplenomegaly, bone abnormalities and in rare cases central nervous system manifestations, such as ataxia or epilepsy. More recent have been variants in SCARB2 and GBA1 identified as risk factors for Parkinson`s disease (PD) and Dementia with Lewy Bodies (DLB). In particular, mutations in GCase are a major genetic contributor to PD and DLB and decreased GCase activity has also been found in sporadic PD brains. To dissect the physiological role of LIMP-2 as a lysosomal trafficking receptor for GCase and as a lipid/cholesterol transporter, we have generated a knockin (KI) mouse model, expressing a LIMP-2 mutant, carrying a point mutation in the GCase binding site. Our preliminary results show that KI mice are expressing GCase binding deficient LIMP-2, resulting in significantly reduced lysosomal GCase levels in liver, which are in a comparable range of LIMP-2 deficient mice.Our preliminary results strongly suggest that the LIMP-2 KI mice provide a unique tool to analyze the physiological consequences of a significant depletion of lysosomal GCase activity without disrupting LIMP-2 expression. The experiments proposed here will establish whether the pathology mediated by LIMP-2 deficiency may at least be in part dependent of GCase.