Mandell Kenneth J, McCall Ingrid C, Parkos Charles A
Epithelial Pathobiology Research Unit, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
J Biol Chem. 2004 Apr 16;279(16):16254-62. doi: 10.1074/jbc.M309483200. Epub 2004 Jan 28.
Junctional adhesion molecule-1 (JAM1) is a tight junction-associated immunoglobulin superfamily protein implicated in the regulation of tight junctions and leukocyte transmigration. The structural basis for the function of JAM1 has yet to be determined. Here we provide evidence that JAM1 homodimer formation is important for its function in epithelial cells. Experiments were conducted to determine the effects of a panel of JAM1 monoclonal antibodies on epithelial barrier recovery after transient disruption by calcium switch. Two monoclonal antibodies were observed to inhibit barrier recovery in contrast to another monoclonal antibody that had no effect. Epitope mapping by phage display revealed that both inhibitory antibodies bind to a region of JAM1 located within the N-terminal Ig-like loop (residues 111-123). Competition experiments with synthetic peptides and site-directed mutagenesis confirmed the location of this epitope. Analysis of the crystal structure of JAM1 revealed that this epitope includes residues within the putative homodimer interface, and one of the two inhibitory antibodies was then shown to block JAM1 homodimer formation in vitro. Finally, mutations within the homodimer interface were shown to prevent enrichment of JAM1 at points of cell contact, presumably by interference with homophilic interactions. These findings suggest that homodimer formation may be important for localization of JAM1 at tight junctions and for regulation of epithelial barrier function.
连接粘附分子1（JAM1）是一种与紧密连接相关的免疫球蛋白超家族蛋白，参与紧密连接和白细胞迁移的调节。JAM1功能的结构基础尚未确定。在此，我们提供证据表明JAM1同源二聚体的形成对其在上皮细胞中的功能很重要。进行实验以确定一组JAM1单克隆抗体对钙切换瞬时破坏后上皮屏障恢复的影响。与另一种无作用的单克隆抗体相比，观察到两种单克隆抗体抑制屏障恢复。通过噬菌体展示进行的表位作图显示，两种抑制性抗体均与位于N端免疫球蛋白样环内（第111 - 123位氨基酸）的JAM1区域结合。用合成肽进行的竞争实验和定点诱变证实了该表位的位置。对JAM1晶体结构的分析表明，该表位包括假定同源二聚体界面内的氨基酸残基，然后显示两种抑制性抗体之一在体外可阻断JAM1同源二聚体的形成。最后，同源二聚体界面内的突变显示可阻止JAM1在细胞接触点富集，推测是通过干扰同源相互作用。这些发现表明同源二聚体的形成可能对JAM1在紧密连接处的定位以及上皮屏障功能的调节很重要。
