Elsevier

Peptides

Volume 38, Issue 2, December 2012, Pages 427-436
Peptides

Structural and activity changes in three bioactive anuran peptides when Asp is replaced by isoAsp

https://doi.org/10.1016/j.peptides.2012.10.001Get rights and content

Abstract

The Asp and isoAsp isomers of three bioactive peptides, Crinia angiotensin 11 [APGDRIYHPF(OH)], uperin 1.1 [pEADPNAFYGLM(NH2)] and citropin 1.1 [GLFDVIKKVASVIGGL(NH2)] were tested for changes in (i) susceptibility towards proteolytic cleavage, (ii) activity (smooth muscle activity for Crinia angiotensin 11 and uperin 1.1 isomers, and antimicrobial activity for the two isomers of citropin 1.1), and (iii) 3D structures in water, trifluoroethanol-d3/water (1:1) and DPC micelles as determined by 2D nuclear magnetic resonance spectroscopy. Proteolytic cleavage with trypsin was identical for each pair of Asp/isoAsp isomers. Cleavage with chymotrypsin was the same for the Crinia angiotensin and uperin 1.1 isomeric pairs, but different for the two Asp/isoAsp citropin 1.1 isomers. Chymotrypsin cleaved at Phe3 (adjacent to Asp4) for citropin 1.1, but not at Phe3 (adjacent to isoAsp4) for isoAsp citropin 1.1. The smooth muscle activity of the isoAsp isomer of Crinia angiotensin 11 was less than that of the Asp isomer. The smooth muscle activity of isoAsp3-uperin 1.1 is greater than that of the Asp isomer at low concentration (<10−9 M) but no different from the Asp isomer at concentrations > 10−9 M. Citropin 1.1 is a wide-spectrum antibiotic against Gram positive organisms, while the isoAsp isomer is inactive against the test pathogens Staphylococcus aureus and Bacillus subtilis. The observed changes in activity are accompanied by changes in the 3D structures of isomers as determined by 2D nuclear magnetic resonance spectroscopy.

Highlights

► The Asp (and Asn) to isoAsp interconversion is a natural phenomenon. ► In the amphibian peptides studied it affects both their structure and bioactivity. ► The effects are unpredictable, with each system responding differently. ► isoAsp also perturbs protease recognition and consequently proteolysis.

Introduction

Peptides and proteins may undergo a number of spontaneous reactions at physiological pH and temperature. One of these involves the conversion of Asp (and Asn) to isoAsp through a five-membered succinimide intermediate (Scheme 1). This occurs by the nucleophilic attack of the amide NH of the C-flanking residue at the side chain carbonyl of Asp (or Asn) to form a succinimide which is then hydrolyzed to form Asp and isoAsp in an approximate 1:3 ratio. Formation of isoAsp results in the CH2 of the Asp (or Asn) side chain inserting into the backbone of the protein/peptide [2], [11], [13], [23], [45], [47]. The most favorable residue on the C-terminal side of Asp (Asn) to enable this conversion is Gly, but Ser and His are also known to assist the reaction [2], [11], [13], [23], [45], [47]. A large hydrophobic side chain on the residue following Asp (or Asn) generally disfavors the isomerization, although an example of Asp and isoAsp cyclic peptides occurring together with Leu following Asp has recently been reported. However, interconversion between the isomers was not reported [31]. The residue preceding Asp/Asn has been found to have little effect on this isomerization [11], [23]. It is also possible for isoAsp to be methylated by isoAsp methyltransferase enzymes, and the resultant methyl esters can subsequently convert to Asp [23], [28]. In contrast, other methyl esters are stable, especially when isoAsp is followed by a bulky amino acid residue [34], [37].

The replacement of Asp by isoAsp changes the nature of the peptide/protein backbone and may also change the 3D shape of the molecule. The following select examples of implications of Asp/isoAsp isomerizations have been reported: (i) the presence of isoAsp may perturb protease recognition and subsequently reduce peptide/protein degradation [2], [5], [15], [28], [39], (ii) isoAsp formation at a reactive center may effect a change (either an increase or decrease depending on the specific example) in function of a protein, enzyme or receptor active site, [2], [9], [12], [15], [16], [49] and (iii) in Alzheimer's disease, the extent of conversion of Asp (Asn) to isoAsp in plaques formed from the peptide amyloid β (1–42) is a function of the age of the plaque; i.e. an indication of an ageing phenomenon [21], [46].

During the past two decades, in excess of 200 bioactive peptides have been isolated and identified from the skin glands of Australian anurans. The activities of these peptides include immunomodulators, smooth muscle active peptides, opioids, neuropeptides, nNOS inhibitors, and membrane active peptides [including anticancer, antimicrobial, antifungal and antiviral (for viruses with envelopes, e.g. HIV and Herpes) peptides] [1], [44]. In only two cases has the Asp/isoAsp interconversion been reported during this research [51], [52], [for caeridin 1.1, GLLDGLLGTLGL(NH2) and caeridin 1.4, GLLDGLLGGLGL(NH2)]. Both examples have an Asp Gly moiety and show no biological activity in our testing regime.

From the data presented above, there is a possibility that changing Asp for isoAsp may decrease the ability of proteases to degrade some bioactive peptides. Secondly, there is a possibility that replacing Asp by isoAsp will change the bioactivity of a peptide. If protease degradation of the peptide is reduced, and the activity enhanced, this could improve any possible pharmaceutical potential of that bioactive peptide.

For this project, we have chosen to study the structures and activities of three highly potent Asp-containing bioactive peptides from anurans and their isoAsp synthetic modifications. The first two systems to be studied are the peptides, Crinia-angiotensin 11 (from Crinia georgiana [19]) and uperin 1.1 (a tachykinin type peptide from Uperoleia inundata [6]), both of which are potent smooth-muscle contractors. Activities of several other tachykinin peptides related to uperin 1.1 [namely uperolein (from Uperoleia rugosa [48]) and physalaemin (from Physalamus bilogonigerus [18]) will also be compared with the activities of the uperin 1.1 isomers. The third major system for study, citropin 1.1 (from Litoria citropa [53]) is a wide-spectrum α-helical antibiotic that is active at micromolar concentrations against Gram positive pathogens. The three systems chosen for study are peptides which have residues next to Asp which do not favor isomerization to isoAsp. Thus we can study the isomeric Asp and isoAsp peptides without being concerned about possible isomerization of the peptides occurring under the conditions of the experiments. The experiments are three-fold and involve a comparison of (i) the extent of proteolytic cleavage of each isomer using Asp-N, trypsin and chymotrypsin, (ii) the smooth muscle or antimicrobial activities (as appropriate) for Asp and isoAsp isomers, and (iii) 3D structures, as indicated by 2D nuclear magnetic resonance experiments of the Asp and isoAsp isomers measured in both water and membrane mimicking solvents.

Section snippets

Methods and techniques

This work conforms with the Code of Practice for the Care and Use of Animals for Scientific Purposes (1990) and the Prevention of Cruelty to Animals Act (1985), and was approved by The University of Adelaide Animal Ethics Committee.

Results

Results are presented in three sections: 3.1, the Crinia angiotensin 11 isomers; 3.2, the uperin 1.1 isomers and other tachykinin peptides, and 3.3, the citropin 1.1 isomers. Each section contains (i) either the smooth muscle activity or antibiotic activities (as appropriate) and (ii) the results of the 2D NMR experiments. With regard to NMR experiments, all peptides studied show random conformations when the NMR spectra are measured in water (data not presented here). Structures are also

The Crinia angiotensin 11 isomers

Crinia angiotensin 11 has three more residues (APG) at the N-terminal end of the peptide than human angiotensin 11 [DRVYIHPF(OH)] [19]. Pharmacological testing of Crinia angiotensin 11 has shown that it has a similar spectrum of activities as angiotensin [17], [20]. Angiotensin 11 is a component of the renin-angiotensin system, regulating plasma volume, sympathetic nervous activity, thirst responses, blood pressure and muscle contraction [17]. It acts through the G-protein coupled receptors

Conclusions

Replacing Asp with isoAsp in these small peptides does not change the extent of degradation of the isomeric peptides by the proteolytic enzyme trypsin. However, chymotrypsin does not cleave next to Phe3 of isoAsp4-citropin 1.1. This observation needs further investigation for cognate peptides, but is beyond the scope of the present investigation.

There is no clear trend, for the systems investigated, that changing Asp by isoAsp will change the activity of the peptide in a particular direction.

Acknowledgements

We thank the Australian Research Council for funding our amphibian peptide research programme. ANC acknowledges the award of an Australian Postgraduate Award Ph.D. scholarship. L.Z. is an Awardee of the National Distinguished Young Scholar Program in China.

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