A Novel Non-Lens βγ-Crystallin and Trefoil Factor Complex from Amphibian Skin and Its Functional Implications

Background: In vertebrates, non-lens beta gamma-crystallins are widely expressed in various tissues, but their functions are unknown. The molecular mechanisms of trefoil factors, initiators of mucosal healing and being greatly involved in tumorigenesis, have remained elusive. Principal Findings: A naturally existing 72-kDa complex of non-lens beta gamma-crystallin (alpha-subunit) and trefoil factor (beta-subunit), named beta gamma-CAT, was identified from frog Bombina maxima skin secretions. Its alpha-subunit and beta-subunit (containing three trefoil factor domains), with a non-covalently linked form of alpha beta(2), show significant sequence homology to ep37 proteins, a group of non-lens beta gamma-crystallins identified in newt Cynops pyrrhogaster and mammalian trefoil factors, respectively. beta gamma-CAT showed potent hemolytic activity on mammalian erythrocytes. The specific antiserum against each subunit was able to neutralize its hemolytic activity, indicating that the two subunits are functionally associated. beta gamma-CAT formed membrane pores with a functional diameter about 2.0 nm, leading to K(+) efflux and colloid-osmotic hemolysis. High molecular weight SDS-stable oligomers (>240-kDa) were detected by antibodies against the alpha-subunit with Western blotting. Furthermore, beta gamma-CAT showed multiple cellular effects on human umbilical vein endothelial cells. Low dosages of beta gamma-CAT (25-50 pM) were able to stimulate cell migration and wound healing. At high concentrations, it induced cell detachment (EC(50) 10 nM) and apoptosis. beta gamma-CAT was rapidly endocytosed via intracellular vacuole formation. Under confocal microscope, some of the vacuoles were translocated to nucleus and partially fused with nuclear membrane. Bafilomycin A1 (a specific inhibitor of the vacuolar-type ATPase) and nocodazole (an agent of microtuble depolymerizing), while inhibited beta gamma-CAT induced vacuole formation, significantly inhibited beta gamma-CAT induced cell detachment, suggesting that beta gamma-CAT endocytosis is important for its activities. Conclusions/Significance: These findings illustrate novel cellular functions of non-lens beta gamma-cyrstallins and action mechanism via association with trefoil factors, serving as clues for investigating the possible occurrence of similar molecules and action mechanisms in mammals.