Biochemical analysis of the novel tumor suppressor gene product RECK

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RECK is a novel tumor suppressor gene which reverses normally the activated-ras-transformed mouse fibroblast cells. In malignant transformed cells, its expression is suppressed. Forced exogenous expression of the gene in the cancer cells suppresses colony formation in soft agar and inhibits tumor metastasis. We characterized the gene product as follows :(1) Partial cDNA was expressed in E.coli, and the purified recombinant protein was subjected to immunize mice for preparation of monoclonal antibodies. Hybridomas were screened with ELISA method. (2) RECK cDNA was expressed in insect cells using baculovirus system. Western analysis using the monoclonal antibodies revealed that the molecular mass of the expressed protein was about 100 kDa. It agreed well with the value estimated from the amino acid sequence and suggested low glycosylation. (3) RECK contains signal sequence at N-terminus and hydrophobic sequence at C-terminus and is transported onto the membrane outside of the cell. Deletion of the C-terminal hydrophobic region caused secretion of RECK into the culture medium. We established a method of affinity purification of the secreted type of RECK by substituting the C-terminal sequence with a tag of histidine hexamer. (4) Potential reactive site residue (P1) of the Kazal domain of RECK is Asp and it suggests caspase-like proteases as target enzymes. RECK did not inhibit caspase-1. It did not interact with granzyme B,either.