Yasunori Tawaragi

Gene and precursor structures of human C-type natriuretic peptide

We have isolated the gene for human C-type natriuretic peptide (CNP) from a human genomic library using a cloned porcine CNP genomic DNA fragment as probe. Human CNP gene consists of at least two coding blocks and an intron, and encodes a 126-residue CNP precursor (human prepro-CNP). From a comparison of the amino acid sequences of porcine and rat prepro-CNPs, human prepro-CNP is found to be processed to generate 22-and 53-residue peptides (human CNP-22 and human CNP-53, respectively) as major endogenous CNPs in human. Interestingly, human CNP-53 has two amino acid substitutions as compared to the porcine and rat CNP-53s, whereas human CNP-22 is identical to the porcine and rat CNP-22s. Intravenous injection of human CNP-53 into anesthetized rats induces diuretic-natriuretic and hypotensive activities with same potencies as porcine CNP-53 does, although these activities were considerably lower (about 1/100) than those of human alpha-ANP.

Structure of dog and rabbit precursors of atrial natriuretic polypeptides deduced from nucleotide sequence of cloned cDNA

The structure of precursors of dog and rabbit atrial natriuretic polypeptides was determined by nucleotide sequence analysis of cloned cDNA of mRNA encoding the peptides. The dog and rabbit precursors are 149 and 153 residues long having 23- and 25-residue putative signal peptides at their N-termini respectively. The 28-residue peptide with identical sequence to that of human, which has potent natriuretic activity, is located at the C-terminus of the dog precursor. The 28-residue peptide of identical sequence to that of mouse/rat is located at C-terminus of rabbit precursor followed by additional Arg-Arg sequence which is also found in rat/mouse precursors and is apparently removed during processing.

Inhibition of in vitro angiogenesis by lymphotoxin and interferon-γ

The effects of lymphotoxin (LT) and interferon (IFN)-gamma on the capillary formation was examined using an in vitro angiogenesis model system. Both LT and IFN-gamma inhibited the capillary formation in a dose dependent manner. To elucidate the mode of action, effects of the lymphokines on endothelial and myofibroblastic cells were studied. We found that the lymphokines inhibited not only the growth of endothelial cells but also the production of collagen by myofibroblastic cells. These results suggest that the pleiotropic effects of the lymphokines on different types of cells might result in the inhibition of the capillary formation.

Gene and precursor structure of porcine C-type natriuretic peptide

Recently we isolated from porcine brain two related peptides, a 22-residue peptide (CNP-22) and its N-terminally elongated peptide (CNP-53; 53-residue), which belong to the third type of mammalian natriuretic peptide designated C-type natriuretic peptide family (CNP) (1,2). To elucidate the structure of their precursor form, we have now isolated the gene for this porcine CNP and prepared its cDNA from COS-1 cells transfected with the gene. Nucleotide sequence analyses have revealed that the gene consists of a least two exons and an intron and encodes the 126-residue CNP precursor (porcine prepro-CNP), in which a putative signal peptide and the CNP-53 sequence are located at the N- and C-terminus, respectively. The C-terminal cysteine codon of CNP-53 is directly followed by a termination codon, indicating that the C-terminus of porcine CNP is generated per se.

Structural requirements of C-type natriuretic peptide for elevation of cyclic GMP in cultured vascular smooth muscle cells

C-type natriuretic peptide (CNP), which was recently found to be a selective ligand for one of the two known natriuretic peptide receptor guanylyl cyclases (NPR-B), potently stimulates cGMP production in cultured rat vascular smooth muscle cells (VSMC) and exerts potent antiproliferative effects on the cells. To investigate the structural requirements of CNP for stimulation of cGMP accumulation via NPR-B, we prepared CNP analogs and tested them on cultured rat VSMC. Our results indicate that only the ring portion of CNP with a disulfide bond (CNP(6-22)) participates in stimulation of cGMP accumulation, especially the sequence Leu9-Lys10-Leu11 in the ring portion executes essential roles for both elevation of cGMP and selectivity of the ligand for NPR-B. We also found a good correlation between the activities of the CNP analogs for stimulation of cGMP accumulation and inhibition of DNA synthesis.