Biotoxin Units，Key Laboratory of Animal Models and Human Disease Mechanisms，Kunming Institute of Zoology，the Chinese Academy of Sciences，Kunming Yunnan 650223，China；2. Graduate School of the Chinese Academy of Sciences，Beijing 100049，China；3. Department of Anesthesiology，the First Affiliated Hospital of Kunming Medical College，Kunming Yunnan 650032，China
bg-CAT is a naturally existing protein complex of non-lens bg-crystallin and trefoil factor, purified from Bombina maxima skin secretions. In HUVECs, bg-CAT can be rapidly endocytosed via intracellular vacuole formation and translocated to the nucleus to regulate cell fuction. In this paper, we found that it contains conserved Walker B motifs (IILYDEPS, residues 6-13) and Walker A motifs (GQSLSGKS, residues 96-103) in the bg-CAT a-subunit sequence. bg-CAT showed potential NTP-binding and weak GTPase/ATPase activities in vitro. Through Western blotting analysis, we found that the a- and b-subunits of bg-CAT participated in a 150 kDa SDS-stable protein complex formation, which also contained positive ubiquitination signals in the bg-CAT treated HUVEC. Furthermore，under confocal microscopy, the immunofluorescence signals of ubiquitin and bg-CAT subunits were co-localized in the vacuoles that were distributed in the cytoplasm and nucleus. In addition, bg-CAT could induce several tumor cell’s detachment and apoptosis, and selectively kill tumor cells. These findings provide a clue to understand the mechanism of bg-CAT endocysis and nuclear transport, and give an insight to investigate the possible occurrence of similar molecule’s cellular functions and action mechanisms of non-lens bg-crystallins and trefoil factors in mammals.