Biochemical Characteristics And Mechanisms Of Non-lens βγ-crystallin And Trefoil Factor Complex

βγ-CAT is a naturally existing 72-kDa complex of non-lensβγ-crystallin (α-subunit, CAT-α) and trefoil factor (β-subunit, CAT-β), with a non-covalently linked form ofαβ₂, identified from frog Bombina maxima skin secretions. The N-terminal part (CAT-αN, 1-170 residues) of CAT-αare twoβγ-crystallin domains, while the rest C-terminal part (CAT-αC) shows sequence homology to membrane insertion domain of Clostridium perfringens epsilon toxin. To examine the biochemical characteristics of theβγ-crystallin domains ofβγ-CAT, CAT-αN, CAT-αC and CAT-βwere expressed in Escherichia coli, respectively. Co-immunoprecipitation of naturally purifiedβγ-CAT comfirmed the constant existence of CAT-αand CAT-βcomplex. Pull-down assays showed that recombinant CAT-βcould bind to recombinant CAT-αN that is composed of twoβγ-crystallin domains. Ca²⁺ -binding motifs were found in CAT-αΝthat folds mainly inβ-sheet conformation. Recombinant CAT-αΝwas able to bind the calcium probe terbium, and the conformation of the protein is not significantly altered upon binding Ca²⁺ .In recent years, it has been reported that apoptosis may occur in platelets and play a role in the clearance of effete platelets.βγ-CAT caused several in vivo toxic effects on mammals. Through determined hematological parameters of rabbits, it has been found thatβγ-CAT significantly reduced the number of platelets in a time-dependent manner. Here, in order to explore the effect ofβγ-CAT on platelet, washed platelets were incubated with various concentrations ofβγ-CAT for 30 min. We found thatβγ-CAT induced several apoptosis events in human platelets, including caspase-3 activation, phosphatidylserine (PS) exposure, depolarization of mitochondrial inner transmembrane potential (ΔΨm), cytochrome c release and strong expression of pro-apoptotic Bax and Bak proteins. However,βγ-CAT did not significantly induce platelet activation as detected by P-selectin surface expression, GPIIb/IIIa activation and platelet aggregation.βγ-CAT protein possesses strong hemolytic activity on human erythrocytes. Treatment of the erythrocytes withβγ-CAT (1 nM) resulted in rapid Ca²⁺ influx in the cells and eventually led to hemolysis. However, in the absence of extracellular Ca²⁺ (in the presence of 20 mM EGTA), though the binding and oligomerization ofβγ-CAT in erythrocyte membranes were observed, no significant hemolysis could be detected. In addition, we observed thatβγ-CAT-induced PS exposure andΔΨm depolarization in platelets are Ca²⁺ -dependent. Taken together, our data reveal the binding capacity of CAT-β(a trefoil factor) to CAT-αN (βγ-crystallin domains), providing a basis for the formation ofβγ-crystallin and trefoil factor complex in vivo. Furthermore, theβγ-crystallin domains ofβγ-CAT are able to bind Ca²⁺ , andβγ-CAT-induced hemolysis and platelet apoptosis are Ca²⁺ -dependent.