Structure-function studies on inhibitory activity of Bungarus multicinctus protease inhibitor-like protein on matrix metalloprotease-2, and invasion and migration of human neuroblastoma SK-N-SH cells.
In view of the findings that several Kunitz-type protein inhibitors suppress tumor invasion and metastasis, the aim of the present study is to explore whether Bungarus multicinctus protease inhibitor-like protein-2 (PILP-2) and PILP-3 exhibit anti-tumor activity. Although approximately 28% of amino acid substitutions occurred between PILP-2 and PILP-3, molecular modeling suggested that PILP-2 and PILP-3 shared similar folded structures. Unlike PILP-2, PILP-3 showed a notable activity in abolishing migration and invasion of human neuroblastoma SK-N-SH cells. The ability of PILP-3 to inhibit matrix metalloprotease-2 (MMP-2) activity was higher than that of PILP-2. Pull-down assay revealed protein-protein interaction between PILP-3 and MMP-2. In contrast to mutation on N-terminal region, replacement of amino acids at C-terminus attenuated notably the ability of PILP-3 to inhibit cell invasion, cell migration and MMP-2 activity as well as the binding capability of PILP-3 with MMP-2. Molecular docking showed that N-terminal region of PILP-2 and PILP-3 fitted into the cleft around the active site of MMP-2 catalytic domain. In contrast to that of PILP-2, C-terminal region of PILP-3 was suggested to be in close contact with catalytic domain of MMP-2. Collectively, our data indicate that PILP-3 is a MMP-2 inhibitor and shows an activity in inhibiting migration and invasion of neuroblastoma, and suggest that intact C-terminus is crucial to the activities of PILP-3.
Chou
Wen-Min
WM
Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung 804, Taiwan.
Liu
Wen-Hsin
WH
Chen
Ku-Chung
KC
Chang
Long-Sen
LS
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001932:Brain Neoplasms / Q000473:pathology*; D017838:Bungarus / Q000502:physiology*; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects*; D004546:Elapid Venoms / Q000737:chemistry / Q000201:enzymology* / Q000494:pharmacology*; D006801:Humans; D061965:Matrix Metalloproteinase Inhibitors; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000502:physiology; D009361:Neoplasm Invasiveness / Q000473:pathology*; D009447:Neuroblastoma / Q000473:pathology*; D011480:Protease Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D014945:Wound Healing / Q000187:drug effects
D004546:Elapid Venoms; D061965:Matrix Metalloproteinase Inhibitors; D011480:Protease Inhibitors