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.

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.

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.

Conformational properties of the guanine-binding site of ribonuclease T1 inferred from the X-ray structure and protein engineering.

Recognition by ribonuclease T1 of guanine bases via multidentate hydrogen bonding and stacking interactions appears to be mediated mainly by a short peptide segment formed by one stretch of a heptapeptide, Tyr42-Asn43-Asn44-Tyr45-Glu46-Gly47- Phe48. The segment displays a unique folding of the polypeptide chain--consisting of a reverse turn, Asn44-Tyr45-Glu46-Gly47, stabilized by a hydrogen-bond network involving the side chain of Asn44, the main-chain atoms of Asn44, Gly47 and Phe48 and one water molecule. The segment is connected to the C terminus of a beta-strand and expands into a loop region between Asn43 and Ser54. Low values for the crystallographic thermal parameters of the segment indicate that the structure has a rigidity comparable to that of a beta-pleated sheet. Replacement of Asn44 with alanine leads to a far lower enzymatic activity and demonstrates that the side chain of Asn44 plays a key role in polypeptide folding in addition to a role in maintaining the segment structure. Substitution of Asn43 by alanine to remove a weak hydrogen bond to the guanine base destabilized the transition state of the complex by 6.3 kJ/mol at 37 degrees C. In contrast, mutation of Glu46 to alanine to remove a strong hydrogen bond to the guanine base caused a destabilization of the complex by 14.0 kJ/mol. A double-mutant enzyme with substitutions of Asn43 by a histidine and Asn44 by an aspartic acid, to reproduce the natural substitutions found in ribonuclease Ms, showed an activity and base specificity similar to that of the wild-type ribonuclease Ms. The segment therefore appears to be well conserved in several fungal ribonucleases.

Cloning of cDNA encoding a new peptide C-terminal alpha-amidating enzyme having a putative membrane-spanning domain from Xenopus laevis skin.

A cDNA clone encoding a precursor of a peptide C-terminal alpha-amidating enzyme (AE-I) from Xenopus laevis skin was recently isolated and sequenced in our laboratory. In this study, by using the restriction fragment of this clone as a hybridization probe, we have identified the cDNA encoding another new peptide C-terminal alpha-amidating enzyme (tentatively named AE-II) distinct from AE-I. The cDNA encodes a polypeptide of 875 amino acid residues, which contains a region extensively homologous to AE-I precursor at N-terminus. The encoded protein characteristically has a putative membrane-spanning domain near C-terminus. Our results indicate that C-terminal alpha-amide formation of peptides in Xenopus skin is regulated by at least two distinct alpha-amidating enzymes.

Two histidine residues are essential for ribonuclease T1 activity as is the case for ribonuclease A.

Ribonuclease T1 (RNase T1, EC 3.1.27.3) is a guanosine-specific ribonuclease that cleaves the 3',5'-phosphodiester linkage of single-stranded RNA. It is assumed that the reaction is generated by concerted acid-base catalysis between residues Glu-58 and His-92 or His-40. From the results of chemical modification and NMR studies, it appeared that the residue Glu-58 was indispensable for nucleolytic activity. However, we have recently demonstrated that Glu-58 is an important but not an essential residue for catalytic activity, using the methods of genetic engineering to change Glu-58 to Gln-58 etc [Nishikawa, S., Morioka, H., Fuchimura, K., Tanaka, T., Uesugi, S., Ohtsuka, E., & Ikehara, M. (1986) Biochem. Biophys. Res. Commun. 138, 789-794]. In the present paper, we report that mutants of RNase T1 with residue Ala-40 or Ala-92 have almost no activity, while mutants that contain Ala-58 retain considerable activity. These results show that the two histidine residues, His-40 and His-92, but not Glu-58, are indispensable for the catalytic activity of the enzyme. We propose a revised reaction mechanism in which two histidine residues play a major role, as they do in the case of RNase A.

Cloning and sequence of cDNA encoding a peptide C-terminal alpha-amidating enzyme from Xenopus laevis.

The C-terminal alpha-amide formation of the peptides is one of the most important events of prohormone processing. We have recently isolated an alpha-amidating enzyme, AE-I, from Xenopus laevis skin, which is the only enzyme ever purified to homogeneity. In this study, we report cloning and sequence of cDNA encoding AE-I. Our results indicate that enzyme AE-I is initially synthesized as a precursor with 400 amino acid residues, which is further processed to the mature enzyme consisting of 344 residues. Preliminary expression in E. coli of the cDNA corresponding to AE-I was found to produce an enzyme with appreciable alpha-amidating activity.

Modification of Glu 58, an amino acid of the active center of ribonuclease T1, to Gln and Asp.

Glu 58 is one of the amino acids which participates in its catalytic action of ribonuclease T1. We mutated this residue to Gln 58 or Asp 58 by genetic engineering using chemically synthesized genes. The mutant enzymes were expressed in E. coli as fused proteins and purified to homogeniety on SDS-PAGE after cleavage with cyanogen bromide. Their activities in hydrolyzing pGpC were reduced to 10% in the Asp 58 mutant and about 1% in the Gln 58 mutant compared to that of the wild-type enzyme. These results suggest that Glu 58 is important but not essential for catalysis of ribonuclease T1.