title: Characterization of the βγ-crystallin domains of βγ-CAT, a non-lens βγ-crystallin and trefoil factor complex, from the skin of the toad Bombina maxima.
abstract: βγ-CAT is a naturally existing 72-kDa complex of a non-lens βγ-crystallin (α-subunit, CAT-α) and a trefoil factor (β-subunit, CAT-β) that contains a non-covalently linked form of αβ(2) and was isolated from the skin secretions of the toad Bombina maxima. The N-terminal region of CAT-α (CAT-αN, residues 1-170) contains two βγ-crystallin domains while the C-terminal region (CAT-αC) has sequence homology to the membrane insertion domain of the Clostridium perfringens epsilon toxin. To examine the biochemical characteristics of the βγ-crystallin domains of βγ-CAT, CAT-αN, CAT-αC and CAT-β were expressed in Escherichia coli. Co-immunoprecipitation of the naturally assembled βγ-CAT confirmed that the CAT-α and CAT-β complex always exists. Furthermore, recombinant CAT-β bound recombinant CAT-αN. Ca(2+)-binding motifs were identified in CAT-αN, and recombinant CAT-αN was able to bind the calcium probe terbium. However, the conformation of CAT-αN was not significantly altered upon Ca(2+) binding. βγ-CAT possesses strong hemolytic activity toward human erythrocytes, and treatment of erythrocytes with βγ-CAT resulted in a rapid Ca(2+) influx, eventually leading to hemolysis. However, in the absence of extracellular Ca(2+), no significant hemolysis was detected, even though the binding and oligomerization of βγ-CAT in the erythrocyte membrane was observed. Our data demonstrate the binding of CAT-β (a trefoil factor) to CAT-αN (βγ-crystallin domains) and provide a basis for the formation of a βγ-crystallin and trefoil factor complex in vivo. Furthermore, the βγ-crystallin domains of βγ-CAT are able to bind Ca(2+), and βγ-CAT-induced hemolysis is Ca(2+) dependent.
authors.affiliation: Key Laboratory of Animal Models and Human Disease Mechanisms of the Chinese Academy of Sciences, Kunming Institute of Zoology, The Chinese Academy of Sciences, 32 East Jiao Chang Road, Kunming 650223, China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Gao
authors.forename: Yang
authors.initials: Y
authors.lastname: Xiang
authors.forename: Lin
authors.initials: L
authors.lastname: Zeng
authors.forename: Xu Tong
authors.initials: XT
authors.lastname: Ma
authors.forename: Wen Hui
authors.initials: WH
authors.lastname: Lee
authors.forename: Yun
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002025:Bufonidae / Q000378:metabolism*; D002942:Circular Dichroism; D003459:Crystallins / Q000737:chemistry* / Q000378:metabolism*; D005434:Flow Cytometry; D047468:Immunoprecipitation; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012867:Skin / Q000378:metabolism*; D000071164:Trefoil Factor-2
chemical_list: D003459:Crystallins; D010455:Peptides; D000071164:Trefoil Factor-2