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型凝集素对半乳糖和N-乙酰半乳糖胺(MMGL)的特异性生物学作用，并研究该分子在正常和恶性细胞的细胞转运中的可能作用。为了建立MMGL阳性巨噬细胞定位的地形图信息，我们制备了针对MMGL的大鼠单抗(MAbs)。我们发展了一种基于ELISA法的糖类结合试验，以便于mAb的筛选。我们用这些单抗通过免疫印迹和免疫组织学分析系统地筛选MMGL的表达部位，发现MMGL阳性的巨噬细胞选择性地定位于结缔组织中。我们还检测到MMGL阳性的巨噬细胞在肺转移的小鼠卵巢癌细胞产生的转移结节中渗透。此外，我们还证明了MMGL负责识别肿瘤细胞上显示的截短的O-连接糖链。为了研究MMGL作为一种细胞受体在细胞转运中的可能作用，我们将编码MMGL的cDNA导入CTLL-2小鼠T细胞系，并探讨是否可以改变MMGL的转运特性。免疫印迹和单抗流式细胞术检测MMGL分子的表达。我们发现，当注射荧光探针标记的小鼠卵巢癌细胞时，MMGL转基因细胞在转移结节内有较好的定位。

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.