Roles of macrophage C-type lectin in cellular trafficking of normal and malignant cells

本项目的目的是阐明小鼠巨噬细胞C-型凝集素特异性半乳糖和N-乙酰半乳糖胺（MMGL）的生物学作用，并研究这种分子对正常和恶性细胞的细胞运输的可能贡献。为了建立MMGL阳性巨噬细胞定位的地形信息，我们制备了特异性针对MMGL的大鼠单克隆抗体（mAb）。我们系统地筛选这些单克隆抗体的MMGL表达的网站，通过免疫印迹和免疫组织学分析，并发现选择性定位的MMGL阳性的巨噬细胞在结缔组织中。我们还检测到MMGL阳性的巨噬细胞浸润在肺转移性小鼠卵巢肿瘤细胞产生的转移结节中。此外，我们还证明了MMGL负责识别肿瘤细胞上展示的截短O-连接糖链。为了研究MMGL作为细胞受体对细胞运输的可能贡献，我们将编码MMGL的cDNA转染到CTLL-2小鼠T细胞系中，并询问细胞运输的性质是否可以改变。使用单克隆抗体通过免疫印迹和流式细胞术证实MMGL分子的表达。我们发现优先定位的MMGL转染的细胞内的转移结节的小鼠卵巢肿瘤细胞注射后，这些细胞标记的荧光探针。

Aim of this project is to elucidate biological roles of mouse macrophage C-type lectin specific for galactose and N-acetylgalactosamine (MMGL) and to study a possible contribution of this molecule on cellular trafficking of normal and malignant cells. In order to establish topographical information on localization of MMGL-positive macrophages, we produced rat monoclonal antibodies (mAbs) specific for MMGL.We developed a novel carbohydrate binding assay based on an ELISA format to facilitate mAb screening. We systematically screened for sites of expression of MMGL using these mAbs by means of immunoblot and immunohistological analyzes, and found selective localization of MMGL-positive macrophages in connective tissue. We also detected MMGL-positive macrophages infiltrating in the metastatic nodules produced by metastatic mouse ovarian tumor cells in lung. Furthermore, we also demonstrated that MMGL is responsible for the recognition of truncated O-linked sugar chains displayd on tumor cells. In order to study possible contribution of MMGL to cellular trafficking as a cellular receptor, we transfected cDNA encoding MMGL to CTLL-2 mouse T cell line and asked whether properties of cellular trafficking could be changed. The expression of MMGL molecules was confirmed by immunoblot and flow-cytometry using mAbs. We found preferential localization of the MMGL-transfected cells within the metastatic noduled of mouse ovarian tumor cells upon injection of these cells labeled with fluorescent probes.