Ranalexin. A novel antimicrobial peptide from bullfrog (Rana catesbeiana) skin, structurally related to the bacterial antibiotic, polymyxin.

D.P Clark,S Durell,W.L Maloy,M Zasloff

doi:10.1016/s0021-9258(17)34136-4

D.P Clark, S Durell + Show 2 more

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https://doi.org/10.1016/s0021-9258(17)34136-4

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Abstract

Antimicrobial peptides comprise a diverse class of molecules used in host defense by plants, insects, and animals. In this study we have isolated a novel antimicrobial peptide from the skin of the bullfrog, Rana catesbeiana. This 20 amino acid peptide, which we have termed Ranalexin, has the amino acid sequence: NH2-Phe-Leu-Gly-Gly-Leu-Ile-Lys-Ile-Val-Pro-Ala-Met-Ile-Cys-Ala-Val-Thr- Lys-Lys - Cys-COOH, and it contains a single intramolecular disulfide bond which forms a heptapeptide ring within the molecule. Structurally, Ranalexin resembles the bacterial antibiotic, polymyxin, which contains a similar heptapeptide ring. We have also cloned the cDNA for Ranalexin from a metamorphic R. catesbeiana tadpole cDNA library. Based on the cDNA sequence, it appears that Ranalexin is initially synthesized as a propeptide with a putative signal sequence and an acidic amino acid-rich region at its amino-terminal end. Interestingly, the putative signal sequence of the Ranalexin cDNA is strikingly similar to the signal sequence of opioid peptide precursors isolated from the skin of the South American frogs Phyllomedusa sauvagei and Phyllomedusa bicolor. Northern blot analysis and in situ hybridization experiments demonstrated that Ranalexin mRNA is first expressed in R. catesbeiana skin at metamorphosis and continues to be expressed into adulthood.

Highlights

Antimicrobialpeptidescompriseadiverseclassof this class include the magaininwsh,ich were firstisolated from molecules used in host defense by plants, insects,and amphibian skin (7), and laterfrom amphibian stomach (8).A animals
Basedon the cDNA in hemocytes and the hemolymph of insects (14, 51). It appears that Ranalexinis initially synthe- disulfidbeond-containing antimicrobipael ptides include sized as a propeptide with a putative signal sequence tachyplesin, a 17 amino acid peptide with two intramolecular and an acidic amino acid-rich regiointsaatmino-termi- disulfidebondsfrom the horseshoe crab hemocyte
The putative signal sequence of bactenecin, a cyclic dodecapeptide with a single intramolecular the Ranalexin cDNA is strikingly similar to the signal disulfide bond, from bovine neutrophils (16).Additional classes sequence of opioid peptide precursors isolated from thoef antimicrobial molecules include the proline-rich peptides, skinof the South American frPohgsyllomedusa sauvagei which include Bac5 and Bac7 from bovine neutrophils (17), andPhyllomedusa bicolor.Northern blot analysis ainnd apidaecin, from insect hemolymph (18),and PRP 39from porsitu hybridization experiments demonstrated that Ra- cine intestine(19).In addition,a tryptophan-rich peptidefrom nalexin mRNA is first expressed inR. catesbeianaskin bovine neutrophils termed indolicidin has been described (20)

Summary

MATERIALS AND METHODS

Class consists of small, basic peptides (21-26 residues) which Peptide Purification-R. catesbeiana tadpoles were purchased from can beconfigured a s single, amphipathic a-helices. T/G)GC-3' (correspondingto amino acids Ile-6 through Ala-11) and aT7 amide, 4 x SSC, 50p g / d salmon sperm DNA, 25 pg/mtlotal yeast RNA, 17-merprimer (Stratagene); 2) 5'-CA(T/C)'IT(T/C)'IT(AICIT/G)GT(AIC1/x Denhardt's solution, 10%dextran sulfate, 10 rndithiothreitol, and. Each polymerase chain reaction used ing conditions wereas follows: 2 x SSC, 0.1% BME for 10 min at 55 "C the previously describedR.catesbeiana tadpole cDNA library as a tem- x 3, followed byincubation for 30min at 37 "Cin asolution of RNase A plate. Northern Blot Analysis-Total cellular RNA was prepared from tadpole and adult R.catesbeiana (Carolina Biological Supply Co.) by the guanidine isothiocyanate method (26) and separated by electrophoresis on1.2% agarose gels containing 6.7% formaldehyde and 1 x MOPS buffer (20 m~ MOPS, 5 m~ sodium acetate, and 1rn Na,EDTA,pH 7.0). The RNA was transferred by capillary action onto Zetabind nylon

RESULTS

TABLEI Minimal inhibitory concentrationsof Ranalexin and deletion analogs

SignaSl equenceAcidicRegion

Ranalexin Is a New Member of a Known Peptide Family in