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

36kDa的水疱积分蛋白（VIP36）是一种细胞内的动物凝集素结合蛋白和脂质，由高甘露糖聚糖糖基化。利用该基金，我们合成了NGSLSYDHSKDGRWS（氨基酸185-199，鉴定为大鼠VIP36的CRD的一部分）、QKRQERNKRFY （348-358， VIP36的胞质结构域）和NFLKSPKDNVDDPTGNFR （299-316， stalk结构域）三个蛋白域的多克隆和单克隆特异性抗体。我们获得了抗体，并澄清了以下三点。(1)我们发现VIP36在大鼠肝细胞中高表达。两种类型的VIP36存在于高尔基体中，并以异质形式定位于内质网和高尔基体。针对VIP36不同结构域的不同抗体证实了这种异质性分布。这强烈提示vip36的异质性可能在细胞分泌蛋白的分选中起一定作用。(2)我们通过免疫电镜发现，VIP36可能在高尔基体后分泌途径中发挥分选蛋白的作用，该蛋白位于VIP36阳性的囊泡结构中，通过高尔基体和质膜之间的再循环运输被鉴定为内体成分。(3)VIP36在肝细胞中高度表达，我们现在开发了三明治酶联免疫吸附测定（ELISA）系统来量化这些水平。我们无法完全阐明肥胖Zucker大鼠中调节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发挥了一定的作用，其测量可能用于肥胖症等代谢性疾病的未来诊断和预后。

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.