Cloning and sequence analysis of cnaA gene encoding the catalytic subunit of calcineurin from Aspergillus oryzae.

Calcineurin has been implicated in ion-homeostasis, stress adaptation in yeast and for hyphal growth in filamentous fungi. Genomic DNA and cDNA encoding the catalytic subunit of calcineurin (cnaA) were isolated from Aspergillus oryzae. The cnaA open reading frame extended to 1727 bp and encoded a putative protein of 514 amino acids. Comparative analysis of the nucleotide sequence of cnaA genomic DNA and cDNA confirmed the presence of three introns and a highly conserved calmodulin binding domain. The deduced amino acid sequence was homologous to calcineurin A from Aspergillus nidulans (92%), Neurospora crassa (84%), human (67%), Saccharomyces cerevisiae (58%) and Schizosaccharomyces pombe (54%). Further, A. oryzae cnaA cDNA complemented S. cerevisiae calcineurin disruptant strain (Deltacmp1 Deltacmp2), which was not viable in the presence of high concentrations of NaCl (1.2 M) and at alkaline pH 8.5.

Ouabain and substrate affinities of human Na(+)-K(+)-ATPase alpha(1)beta(1), alpha(2)beta(1), and alpha(3)beta(1) when expressed separately in yeast cells.

Human Na(+)-K(+)-ATPase alpha(1)beta(1), alpha(2)beta(1), and alpha(3)beta(1) heterodimers were expressed individually in yeast, and ouabain binding and ATP hydrolysis were measured in membrane fractions. The ouabain equilibrium dissociation constant was 13-17 nM for alpha(1)beta(1) and alpha(3)beta(1) at 37 degrees C and 32 nM for alpha(2)beta(1), indicating that the human alpha-subunit isoforms have a similar high affinity for cardiac glycosides. K(0.5) values for antagonism of ouabain binding by K(+) were ranked in order as follows: alpha(2) (6.3 +/- 2.4 mM) > alpha(3) (1.6 +/- 0.5 mM) approximately alpha(1) (0.9 +/- 0.6 mM), and K(0.5) values for Na(+) antagonism of ouabain binding to all heterodimers were 9.5-13.8 mM. The molecular turnover for ATP hydrolysis by alpha(1)beta(1) (6,652 min(-1)) was about twice as high as that by alpha(3)beta(1) (3,145 min(-1)). These properties of the human heterodimers expressed in yeast are in good agreement with properties of the human Na(+)-K(+)-ATPase expressed in Xenopus oocytes (G Crambert, U Hasler, AT Beggah, C Yu, NN Modyanov, J-D Horisberger, L Lelievie, and K Geering. J Biol Chem 275: 1976-1986, 2000). In contrast to Na(+) pumps expressed in Xenopus oocytes, the alpha(2)beta(1) complex in yeast membranes was significantly less stable than alpha(1)beta(1) or alpha(3)beta(1), resulting in a lower functional expression level. The alpha(2)beta(1) complex was also more easily denatured by SDS than was the alpha(1)beta(1) or the alpha(3)beta(1) complex.

Orientation of cecropin A helices in phospholipid bilayers determined by solid-state NMR spectroscopy.

The orientation of the insect antibiotic peptide cecropin A (CecA) in the phospholipid bilayer membrane was determined using (15)N solid-state NMR spectroscopy. Two peptide samples, each specifically labeled with (15)N at Val(11) or Ala(27), were synthesized by solid phase techniques. The peptides were incorporated into phospholipid bilayers, prepared from a mixture of dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol, and oriented on glass slides. The (15)N chemical shift solid-state NMR spectra from these uniaxially oriented samples display a single (15)N chemical shift frequency for each labeled residue. Both frequencies are near the upfield end of the (15)N chemical shift powder pattern, as expected for an alpha-helix with its long axis in the plane of the membrane and the NH bonds perpendicular to the direction of the magnetic field. These results support a mechanism of action in which CecA binds to and covers the membrane surface, thereby causing a general destabilization and leakiness of the lipid bilayer membrane. The data are discussed in relation to a proposed mechanism of membrane lysis and bacterial killing via an ion channel activity of CecA.

Structure-activity studies of normal and retro pig cecropin-melittin hybrids.

Chimeric analogs of cecropin P1 and melittin with normal and retro sequences were synthesized to explore the effect of sequence, amide bond direction (helical dipole), charge, amphipathicity and hydrophobicity on their antibacterial activity and channel-forming ability. When viewed from the opposite end by rotation in the plane 180 degrees retro analogs have the same sequence as the parent with reversed amide bond and helical dipole directions. The expected activities were related to the important structural features and a series of assumptions were made. Retro analogs are expected to be inactive if both sequence and amide bond direction make critical contributions to the activity. CP1(1-10)M(2-9) amide, (SWLSKTAKKLIGAVLKVL), showed a broad antibacterial spectrum with high activity against the two Gram-negative and three Gram-positive bacteria tested. Retro-CP1(1-10)M(2-9) was less active compared to its normal peptide. CP1(1-9)M(1-8) and CP1(1-9)M(2-8) amides were found to be active against Gram-negative Escherichia coli and also Gram-positive Streptococcus pyogenes, but inactive against the other test organisms. The corresponding retro analogs were inactive against all the five bacteria tested. These results suggest that both sequence and amide bond direction (helix dipole) are important structural requirements for the activity of CP1-M hybrids. Acetylation of the N-terminal amine in both normal and retro analogs lowered their activity, indicating the contribution of free amine to the activity. These analogs form ion-conducting channels in lipid bilayers. The action of the peptides may be explained by self-aggregation and formation of ion-conducting pores across bacterial membranes. Conformational analysis obtained from CD measurements showed that all analogs form amphipathic alpha-helices in presence of 12-20% hexafluoro isopropanol. The retro CP1(1-10)M(2-9) amide showed higher helicity and is more potent compared to other retro analogs synthesized. These studies show the effect of small sequence modifications on the biological activity of the peptides and on their alpha-helical conformation in HFIP, the structure-inducing organic solvent.

7Li and 23Na NMR studies of transmembrane cation transport mediated by ionophore lasalocid A.

Ion transport across phospholipid vesicles was studied by 7Li and 23Na-NMR using an aqueous anionic paramagnetic shift reagent, dysprosium nitrilotriacetate [Dy(NTA)2](3-), mediated by ionophores, lasalocid A and A23187. The intra- and extracellular 7Li and 23Na-NMR signals were well separated (20 Hz) at mM concentration of the shift reagent. The observed data on the rate constant for lithium transport across DPPC vesicles at various concentrations of the ionophores indicated that lasalocid A is a more efficient carrier for lithium ion compared with the sodium ion transport by this ionophore, while A23187 was not specific to either of the ions (Li or Na).

Synthesis and study of normal, enantio, retro, and retroenantio isomers of cecropin A-melittin hybrids, their end group effects and selective enzyme inactivation.

In our effort to understand the structural requirements for the antimicrobial activity of cecropin A (CA) and melittin (M), we synthesized the normal, enantio, retro and retroenantio hybrid analogs; we related activity to their sequence, chirality, amide bond direction (helix dipole) and end group charges. To compare the effect of the end groups, each of these analogs was synthesized both with an acid and an amide C-terminus and also with and without an N alpha-acetyl N-terminus. The all-L- and all-D-enantiomers of several cecropin-melittin hybrids were previously found to be equally potent against several bacterial species, and no chiral effect was observed. This general rule has now been confirmed and extended. However, two exceptions have been found. All-L-CA(1-13)M(1-13) acid was 5 times and 9 times less potent than the all-D-analog, respectively, toward gram-positive Staphylococcus aureus and gram-negative Pseudomonas aeruginosa. All-L-CA(1-7)M(2-9) acid was 5 times and 14 times less active against S. aureus and P. aeruginosa, respectively, than its all-D acid isomer. The corresponding D- and L-retro analogs differed only marginally. A role for proteolytic enzymes has been implicated as a cause for these differences in the activities of L- and D-enantiomers. In all cases, blocking the alpha-amine by acetylation had no significant effect on potency. The retro and retroenantio analogs of CA(1-13)M(1-13) acid were as potent as their normal and enantio analogs against all the test bacteria. The C-terminal amides also showed similar potency against four test bacteria. It should be noted that the negative end of the helix dipole of a normal peptide points toward the C-terminus, whereas it points away in the case of a retro derivative when viewed in the direction of the normal sequence.

Synthesis and antibacterial action of cecropin and proline-arginine-rich peptides from pig intestine.

Two antimicrobial peptides, cecropin P1 (CP1), with a C-terminal carboxyl group, and PR-39, with an amidated, C-terminus, are found in the small intestine of the pig. Each is active against both Gram-positive and Gram-negative bacteria. We have synthesized these peptides and several analogs, including the D-enantiomers and the retro sequences, each with a free or acetylated amino terminus. The CP1 amide was also prepared. The retro CP1 peptides were much less active than the parent CP1 peptide, confirming the importance of sequence or the amide bond and helix dipole direction, and the N alpha-acetyl peptides were also less active, indicating that a free amino terminus is essential for high activity. The ratio of the lethal concentration of L/D isomers of CP1 is less than 1 for Gram-negative, but greater than 1 for Gram-positive bacteria. PR-39 showed no significant chiral selectivity toward Escherichia coli, Bacillus subtilis and Streptococcus pyogenes, but the L/D ratio was high for Pseudomonas aeruginosa (66), and very high for Staphylococcus aureus (> 1000). In the latter case the lethal concentration for the D-isomer was 0.57 microM, whereas this organism was quite resistant to the L-isomer (> 600 microM). Thus the enantiomers of CP1 and PR-39 are not equally active for all species. In a plate assay with a very small log-phase inoculum of Staph aureus, D-PR-39 produced a clear zone of killing surrounded by a zone of stimulated growth. After prolonged incubation the two zones became one clear zone. Addition of D-PR-39 to the wells of a dense turbid plate of growing cells showed a cleared zone for each of the test organisms, indicating that PR-39 lyses the bacteria rather than simply inhibiting their multiplication.

Hydrophobic effects on antibacterial and channel-forming properties of cecropin A-melittin hybrids.

The design of cecropin-melittin hybrid analogues is of interest due to the similarities in the structure of the antimicrobial peptides cecropin and melittin but differences in their lytic properties. We suspected that a hydrophobic residue in position 2 of milittin (Ile8 in the hybrid) plays an important role in the activity of the 15-residue hybrid, KWKLFKKIGAVLKVL-NH2, [CA(1-7)M(2-9)NH2] and have now examined its role in the analogue toward five test bacteria. Deletion of Ile8 reduced activity, and it was not restored by lengthening to 15 residues by addition of another threonine at the C-terminus. Replacement of Ile8 by a hydrophobic leucine maintained good activity and Ala8 was equally active for four organisms, although less active against Staphylococcus aureus. Replacement by the hydrophilic Ser8 strongly reduced potency against all five organisms. Deletion of Leu15 decreased activity, but addition of Thr16 maintained good activity. The presence of hydrophobic residues appears to have a significant effect on the process of antibacterial activity. These peptide analogues showed voltage-dependent conductance changes and are capable of forming ion-pores in planar lipid bilayers. The antibacterial action of the peptides is thought to be first an ionic interaction with the anionic phosphate groups of the membrane followed by interaction with the hydrocarbon core of the membrane and subsequent reorientation into amphipathic alpha-helical peptides that form pores (ion-channels), which span the membrane. The analogue also showed an increase in alpha-helicity with an increase in hexafluoro 2-propanol concentration.

Retro and retroenantio analogs of cecropin-melittin hybrids.

Hybrid analogs of cecropin A (CA) and melittin (M), which are potent antibacterial peptides, have been synthesized. To understand the structural requirements for this antibacterial activity, we have also synthesized the enantio, retro, and retroenantio isomers of two of the hybrids and their N-terminally acetylated derivatives. All analogs of CA(1-13)M(1-13)-NH2 were as active as the parent peptide against five test bacterial strains, but one bacterial strain was resistant to the retro and retroenantio derivatives. Similarly, all analogs of CA(1-7)M(2-9)-NH2 were active against four strains, while two strains were resistant to the retro and retroenantio analogs containing free NH3+ end groups, but acetylation restored activity against one of them. From these data it was concluded that chirality of the peptide was not a critical feature, and full activity could be achieved with peptides containing either all L- or all D-amino acids in their respective right-handed or left-handed helical conformations. For most of the bacterial strains, the sequence of these peptides or the direction of the peptide bonds could be critical but not both at the same time. For some strains, both needed to be conserved.

Design and synthesis of antimicrobial peptides.

The cecropins are a group of potent antimicrobial peptides, initially discovered in insects but later found in other animals including mammals. Synthetic peptide chemistry has played an important role in establishing their primary sequences, as well as the steps in the processing of the biosynthetic preprocecropins. Solid-phase peptide synthesis has been the method of choice. Synthetic chimeric peptides have led to more active products and a better understanding of their mode of action. The structural requirements for high activity include a basic amphipathic N-terminus, a short central flexible sequence and a hydrophobic helical C-terminus. Cecropin-melittin hybrids as small as 15 residues are highly active. In planar lipid bilayers the cecropins form pores which pass ions and carry a current under a voltage gradient. Synthetic D-enantiomers of several antibacterial peptides carry the same current as the natural all-L-peptides and are equally active against several test bacteria. Therefore, the activity is not dependent on chiral interactions between the peptides and the lipid bilayers or the bacterial membranes. Recent examination of retro and retroenantio peptides has further defined the limits of the structural requirements of these peptides. Some of the hybrid peptides are active against Plasmodium falciparum and Mycobacterium smegmatis.

Bradykinin and angiotensin II analogs containing a conformationally constrained proline analog.

Three analogs of bradykinin and one of angiotensin II have been prepared in which the naturally occurring proline residues have been replaced by the bicyclic amino acid, 2,4-methanoproline (2,4-MePro). The relative binding affinities for these analogs were determined to be significantly reduced in the cases of the three bradykinin analogs; [2,4-MePro3]-BK retains 1.3%, [2,4-MePro7]-BK retains 0.3% and [2,4-MePro2]-BK retains 0.021% of the binding affinity of bradykinin. Results from other modification at positions three and seven indicate preference for the trans-amide bond preceding these residues implying that other factors, either steric or conformational, are responsible for the decreased affinity for the receptor seen with 2,4-MePro substitution. The retention of significant binding affinity (26%) in the case of [Ile5,2,4-MePro7]-angiotensin II gives direct evidence that the trans-conformation of the proline amide bond is the one recognized by the AII receptor. Only significant retention of activity can be interpreted unambiguously with the use of this proline analog because of its known conformational differences from Pro as well as its increased steric requirements at the receptor.