Relationship between an animal lectin VIP36 and the cytoskeletal proteins in quality control mechanism

36 kDa囊泡整合蛋白(VIP36)是一种与高甘露糖糖基化的动物细胞内凝集素结合蛋白和脂类。利用这笔资金，我们利用合成肽NGSLSYDHSKDGRWS(氨基酸185-199，鉴定为大鼠VIP36的CRD的一部分)、QKRQERNKRFY(348-358，VIP36的胞浆结构域)和NFLKSPKDNVDDPTGNFR(299-316，茎结构域)，制备了针对其三个蛋白结构域的多克隆和单克隆抗体。我们获得了抗体，并阐明了以下三点：(1)我们发现VIP36在大鼠肝细胞中高表达。VIP36存在于高尔基体中，在内质网和高尔基体中以不同的形式定位。针对VIP36不同结构域的不同抗体证实了VIP36的异质性分布。这有力地表明，VIP36的异质性可能在细胞分泌蛋白的分类中起到一定的作用。(2)我们通过免疫电子显微镜发现，VIP36可能在高尔基体后分泌途径中起到分类蛋白的作用，位于VIP36阳性囊泡结构中，通过高尔基体和质膜之间的循环运输被鉴定为内体成分。(3)VIP36在肝细胞中高表达，我们现在建立了夹心酶联免疫吸附试验(ELISA)系统来定量这些水平。我们不能完全阐明调控肥胖Zucker大鼠VIP36增加的途径，但我们目前的数据表明，VIP36水平与肝脏代谢之间存在关联。因此，我们认为VIP36可能在脂肪肝的形成中起一定作用，其测定可能用于未来对肥胖等代谢性疾病的诊断和预后。

Vesicular integral protein of 36kDa (VIP36) is an intracellular animal lectin binding protein and lipids that is glycosylated with high-mannose glycans. By using this grant we generated polyclonal and monoclonal specific antibodies against three of its protein domains using synthetic peptides, NGSLSYDHSKDGRWS (amino acids 185-199, identified as a part of CRD of rat VIP36), QKRQERNKRFY (348-358, cytosolic domain of VIP36) and NFLKSPKDNVDDPTGNFR (299-316, stalk domain). We have obtained the antibodies and clarified the following three points.(1)We have found that VIP36 is highly expressed in rat hepatocytes. Two types of VIP36 exist in the Golgi apparatus and localize in heterogeneous form in the ER and the Golgi apparatus. The heterogeneous distribution was confirmed by the different antibodies against different domains of VIP36. This strongly suggests that the heterogeneity of VIP36s may play some roles in the sorting of secretory proteins in cells.(2)We have found by immunoelectron microscopy that VIP36 may function as sorting protein in the post-Golgi secretory pathway, located in the VIP36-positive vesicular structures identified as endosomal component through the recycling trafficking between the Golgi apparatus and the plasma membrane.(3)VIP36 is highly expressed in hepatocytes and we have now developed the sandwich enzyme-linked immunosorbent assay (ELISA) system to quantify these levels. We could not fully clarify the pathways that regulate the increase in VIP36 in obese Zucker rats but our present data suggest that there is an association between the VIP36 levels and liver metabolism. We contend therefore that VIP36 may play some roles in the formation of a fatty liver, and that its measurement may be used for the future diagnosis and prognosis of metabolic disorders such as obesity.