VSIG1 is a transmembrane protein localized on plasma membrane of glandular

The immunohistochemical analysis showed that VSIG1 protein is localised on the basolateral plasma membrane of the epithelial cells. The localization of VSIG1 to cell-cell contacts suggested that VSIG1 plays diverse roles in cell-cell-cell-cell adhesion.

Adhesion molecules play essential roles in the overall tissue organization and the proper physiological function of organs by establishing and organizing cell-cell contacts (Aurrand-Lions et al., 2000). Most of the members of CTX/JAM subfamily are junctional adhesion molecule localizing on the plasma membrane of the cell-cell adhesion (Ebnet et al., 2003).

Epithelial cells have distinct apical and basolateral membrane domains that differ in protein and lipid composition. These two domains are separated by tight junctions (TJs), where the certain leaflets of plasma membrane of adjacent cells appear as series of fusions, the so called TJ-strands (Tsukita et al., 2001). These fusion points restrict the free diffusion of lipids and integral membrane proteins between the two compartments (fence junction). Tjs, therefore, are crucial in the generation and maintains of cellular polarity in vertebrate endothelial and epithelial cells (Yeaman et al., 1999). Three types of tight junction proteins have identified so far. These are occludin, claudins (Furuse at al., 1998), and several immunoglobulin (Ig) subfamily members such as JAM-1, (Martin-Pudura et al., 1998; Ebnet et al., 2003), ESAM (Nasdala et al., 2002) and CAR (Cohen et al., 2001). Among these, occludin and claudins seem to form the molecular basis of tight junction strand, as antibodies against occludin exclusively label Tj strands (Saitou et al., 1997). To determine whether VSIG1 is localized to TJs and/or adhesion junction of the basolateral membrane, co-immunolocalization of VSIG1 and occludin and/or JAM-1 must be done.

The intercellular adhesion is mediated through interaction of adhesion molecules between two apposing cells. Several members of immunoglobulin superfamily are involved in the cell-cell adhesion. Nectins are cell adhesion proteins and belong to immunoglobulin superfamily. The extracellular regions of nectins homophilically and heterophilically interact in trans with each other; (Takahashi et al.,1999; Satoh-Horikawa et al., 2000; Reymond et al., 2001). Two nectin molecules at the surface of the same cell first form cis-dimers, followed by trans-interaction by the cis-dimers on apposing cells (Miyahara et al., 2000) (Fig. 4.3). When this trans-interaction occurs between two apposing cells, the cells adhere to each other (Fig. 4.3). In contrast to the nectins, the extracellular regions of cadherins only homophilically interact in trans with each other (Takeichi., 1995) (Fig 4.3). The trans-interaction between cadherin molecules is Ca²⁺-dependent, whereas that between nectin molecules is Ca²⁺ -independent.

Figure 4.3 Schematic illustration of the cis- and trans-interactions of nectins (Takai et al., 2008). Two nectin molecules of the same plasma membrane first form dimers and then form a trans-interaction by the cis-dimers on apposing cells through each first Ig-like loop or in a zipper-like fashion with all three Ig-like loops.

To determine the role of Vsig1 in cell-cell adhesion, we have established a stable transfected HepG2 cell line, in which the Vsig1 is overexpressed.

Immunohistochemical analysis revealad that the VSIG1 protein is localized at the plasma membrane of the stable transfected HepG2 cells. To check the role of VSIG1 in cell-cell adhesion, we performed adhesion assay. The adhesion assay showed that the aggregation activity of VSIG1-expressing cells is weaker than that of control cell line, which does not express VSIG1. These results reveal that the overexpression of VSIG1 in HepG2 cells decreases the cell adhesion. These results can be explained by several reasons.

The cell adhesion molecules make trans-interaction by homophilic and heterophilic manner. Some molecules make the trans-interaction by both of the manner and some molecules make it by only homophilic and heterophilic manner, respectively. The VSIG1 may mediate cell-cell adhesion by homophilic and heterophilic interaction. The homophilic interaction of VSIG1↔VSIG1 alone could not support the cell adhesion.

Therefore, the absence of the heterotypic partner of VSIG1 in HepG1 cell line may be the cause for the low affinity of cell-cell adhesion. Similar results were obtained by the experiments on the Necl-5-overexpressing cell line (Aoki et a., 1997; Ikeda et al., 2003).

The other possible explanation is that the cell-cell adhesion activity of VSIG1 is Ca²⁺ -dependent. E-cadherin is one of the well characterized cell adhesion molecules and can

be clear example of Ca²⁺-dependent cell-cell adhesion. Kitajima et al (1996) did experiments to check whether the E-cadherin mediated cell-cell aggregation is Ca² -dependent. They produced stable transfected hepatocytes, which overexpressed E-cadherin (PC12/tax). They did 2 parallel aggregation assays with the PC12/tax cell line.

One assay was performed with medium containing 1mM CaCl2 and another assay containing 1mM EDTA. The results of aggregation assay showed that PC12/tax cells are aggregating in the presence of 1 mM CaCl2, whereas the 1 mM EDTA treated PC12/tax cells failed to aggregate (Kitajima et al., 1996). These findings revealed that the cell-cell adhesion mediated by E-cadherin is significantly dependent on the presence of Ca²⁺.

The third possible explanation is the absence of an interaction partner, which is interacting with intracellular domain of the VSIG1 in the HepG2 cell line. The C-terminal end of JAM-2 contains a PDZ binding domain, which is responsible for interacting with cytoplasmic interaction partners such as PAR-3, ZO-1 and ZO-2. JAM-2-overexpressing CHO cell line can aggregate and grow as multilayer. Deletion of the PDZ binding domain disrupts the interaction of the JAM-2 protein with its cytoplasmic partners. Cells, which are overexpresing mutated JAM-2 have low affinity to aggregate and they grow as monolayer (Ebnet et al., 2003). This result indicates that the interaction of the intracellular domain of adhesion molecules with their interacting protein is crucial for cell-cell adhesion. Based on the above finding, it can be assumed that the absence of VSIG1-cytoplasmic partners in VSIG1-overexpressing HepG2 cell is one of the possible reasons for the low capacity of the VSIG1-overexpressing cells to aggregate.