Tomoko Shimojo

cDNA Cloning of Human Calpastatin: Sequence Homology Among Human, Pig, and Rabbit Calpastatins

cDNA of human calpastatin, an inhibitor protein specific for calpain (EC 3.4.22.17; Ca2(+)-dependent cysteine proteinase) was isolated by screening of a library prepared from human liver mRNA with pig calpastatin cDNA fragment as a probe. The primary structure of human calpastatin was deduced from the nucleotide sequence of the cDNA and compared with that of pig and rabbit calpastatins already reported. Human calpastatin consisted of 673 amino acid residues and had 78% and 77% identity to pig or rabbit calpastatins, respectively. Human calpastatin had a domain structure with four internally repetitive sequences and one N-terminal non-homologous sequence like the other calpastatins. Human calpastatin had two deletions, 22 and 13 residues long in domain L and domain 1, respectively, compared to pig or rabbit calpastatins.

Characterization of a functional domain of human calpastatin

Expression plasmids were constructed from the cDNA of human calpastatin to examine the contribution to the inhibition of calpain of highly conserved sequences in each of four repetitive domains. A series of deletion derivatives of domain 1 proteins, truncated at either the amino or carboxy terminus, were produced in E. coli. Deletion from the amino terminus past the amino terminal conserved sequence decreased the inhibition. When the middle conserved sequence, the M-sequence, was further deleted, no inhibition was detected, but deletion from the carboxy terminus past the carboxy terminal conserved sequence did not decrease the inhibition until the M-sequence was reached. Nuclear magnetic resonance and circular dichroism spectra showed that domain 1 has an unfolded structure. Peptides that contained the M-sequence and some neighboring sequences were synthesized to measure the minimum size of the inhibitory peptide, which was the M-sequence with the next six residues on the amino terminal side.

Inhibition of Lung Metastasis by Synthetic and Recombinant Fragments of Human Fibronectin with Functional Domains

We have investigated the antimetastatic effect of synthetic or recombinant peptides containing the functional domains of fibronectin on experimental and spontaneous lung metastases of murine tumor cells. CS1 peptide which is present within type III homology connecting segment (IIICS) as well as C‐274 (cell‐binding domain) were able to inhibit experimental lung metastasis when co‐injected intravenously (iv) with B16‐BL6 melanoma cells, while H‐271 (heparin‐binding domain) could not. In the spontaneous metastasis model, multiple iv administrations of CS1 or C‐274 after surgical excision of primary tumors caused a significant reduction of metastatic colonies in the lung. Both CS1 and C‐274 significantly inhibited cell adhesion and migration to fibronectin‐coated substrates when added freely in solution. CS1 peptide also inhibited the cell adhesion and migration to laminin‐coated substrates, but C‐274 did not. H‐271 did not have any inhibitory effect on cell adhesion or migration to either of the substrates. Similarly, CS1 inhibited tumor invasion to both Matrigel/fibronectin‐ and Matrigel/laminin‐coated filters, whereas C‐274 inhibited the invasion to only Matrigel/fibronectin‐coated filter. These results indicate that CS1 peptide of fibronectin, lacking the Arg‐Gly‐Asp‐containing domain, actively inhibits tumor metastases in spontaneous and experimental metastasis models. The use of such a peptide might offer a promising therapeutic approach for combatting or preventing cancer metastasis.

Construction and characterization of a fusion protein with epidermal growth factor and the cell-binding domain of fibronectin

An efficient expression system was constructed for C‐EGF, a fusion protein made of a fragment of the cell‐binding domain of human fibronectin (FN) bound with epidermal growth factor (EGF), C‐EGF was produced in Escherichia coli HB101 cells carrying the recombinant plasmid pCE102 as inclusion bodies, which were solubilized and refolded after purification. C‐EGF had both cell‐adhesive and EGF activities, so it might be more effective than EGF in therapeutic applications. This fusion system would be useful for the construction of a recombinant drug delivery system for cells that have fibronectin receptors (integrins).