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Endocrinology, doi:10.1210/en.2006-0105
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Endocrinology Vol. 147, No. 7 3457-3461
Copyright © 2006 by The Endocrine Society

Molecular Evolution of Proadrenomedullin N-Terminal 20 Peptide (PAMP): Evidence for Gene Co-Option

Alfredo Martínez, José Antonio Bengoechea and Frank Cuttitta

Department of Neuroanatomy and Cell Biology (A.M.), Instituto Cajal, Consejo Superior de Investigaciones Científicas, 28002 Madrid, Spain; Program Infection and Immunity (J.A.B.), Fundación Caubet-Cimera, 07110 Bunyola, Spain; and Angiogenesis Core Facility (F.C.), National Cancer Institute, National Institutes of Health, Bethesda, Maryland 28092

Address all correspondence and requests for reprints to: Alfredo Martínez, Department of Neuroanatomy and Cell Biology, Instituto Cajal, Consejo Superior de Investigaciones Científicas, Avenida Doctor Arce 37, 28002 Madrid, Spain. E-mail: amartinez{at}cajal.csic.es.


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Posttranslational processing of proadrenomedullin generates two biologically active peptides, adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP). Sequence comparison of homologous proadrenomedullin genes in vertebrate evolution shows a high degree of stability in the reading frame for AM, whereas PAMP sequence changes rapidly. Here we investigate the functional significance of PAMP phylogenetic variation studying two of PAMP’s better characterized physiological activities, angiogenic potential and antimicrobial capability, with synthetic peptides carrying the predicted sequence for human, mouse, chicken, and fish PAMP. All tested peptides induced angiogenesis when compared with untreated controls, but chicken and fish PAMP, which lack terminal amidation, were apparently less angiogenic than their human and mouse homologs. Confirming the role of amidation in angiogenesis, Gly-extended and free acid variants of human PAMP produced responses similar to the natural nonamidated peptides. In contrast, antimicrobial activity was restricted to human PAMP, indicating that this function may have been acquired at a late time during the evolution of PAMP. Interestingly, free acid human PAMP retained antimicrobial activity whereas the Gly-extended form did not. This fact may reflect the need for maintaining a tightly defined structural conformation in the pore-forming mechanism proposed for these antimicrobial agents. The evolution of PAMP provides an example of an angiogenic peptide that developed antimicrobial capabilities without losing its original function.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PROADRENOMEDULLIN N-terminal 20 peptide (PAMP) is a biologically active molecule that, together with adrenomedullin (AM), is produced by posttranslational processing of a longer prohormone (1). Expression of the proadrenomedullin gene has been found in most organs and tissues, including the central nervous system, endocrine glands, integument, cardiovascular system, and many others (2). Several functions have been ascribed to AM (3), but less is known about the physiological implications of PAMP. Among the functions of PAMP, its antimicrobial activity (4) and its influence in promoting angiogenesis (5) are among the best characterized. Other actions include vasodilatation (6), bronchodilatation (7), inhibition of neural transmission at peripheral sympathetic nerve endings (8), growth regulation (9), hormone secretion modulation (10, 11, 12), and regulation of intestinal absorption (13).

With the growing number of available whole-genome sequences, comparisons of the reading frames for AM and PAMP among different vertebrate species can be accomplished (14). A careful analysis of these sequences shows that AM changed modestly through vertebrate evolution, whereas the region coding for PAMP experienced wide variations (Table 1Go). These changes include the acquisition of an amidation motif at the carboxy end of PAMP, which is not present in fish or birds but is found in all mammals so far studied. In clear contrast, the AM peptide is amidated in all known vertebrate species (Table 1Go).


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  		TABLE 1. Sequence comparison of mature peptides of the proadrenomedullin gene in different vertebrates

 
Interestingly, five proadrenomedullin-like genes are found in fish, whereas only two (AM and AM2) remain in higher vertebrates (15). Of these two genes, only the first one contains the PAMP sequence. The different number of paralogous genes of the AM family seems to be another example of the multiple duplications that were experienced by the ancestral fish lineage (16). Of the five fish proadrenomedullin genes, only one (TrAM-1) contains a PAMP-like region and an exon/intron structure similar to its tetrapod homolog.

The rapid changes experienced by PAMP during vertebrate evolution, and the number of functions played by this molecule, make PAMP a perfect system to study how peptides co-opt new physiological properties. Here we use synthetic PAMP peptides, belonging to different vertebrate groups, and study their antimicrobial and angiogenic properties. Although all the peptides induce angiogenesis, only human PAMP is able to kill microbes. We conclude that natural selection made an antimicrobial peptide out of PAMP while preserving and even improving its original angiogenic properties.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Synthetic peptides
The following peptides were synthesized by Princeton Biomolecules Corp. (Langhorne, PA) and analyzed by HPLC to confirm purity higher than 95%: P1, human PAMP, ARLDVASEFRKKWNKWALSR-NH2; P2, human PAMP-Gly extended, ARLDVASEFRKKWNKWALSRG; P3, human PAMP free acid, ARLDVASEFRKKWNKWALSR; P4, mouse PAMP, AGPDTPSQFRKKWNKWALSR-NH2; P5, chicken PAMP, ARVDVATEFKRKWTSWVLSRA; and P6, Fugu PAMP, VEFDAKPQLKKRLNILLRNRL. All peptides were highly soluble in water. Note that monkey PAMP has been shown to be identical to human PAMP (17).

Angiogenesis assay
The directed in vivo angiogenesis assay was used as described (18, 19, 20). Briefly, the different PAMP molecules were diluted in growth factor-reduced Matrigel (BD Biosciences, Bedford, MA) to a final concentration of 1 nM, and the silicone angioreactors were filled up with this Matrigel solution. Control implants contained Matrigel alone. Once the matrix had solidified, the angioreactors were implanted under the skin of anesthetized nude mice (Harland, Barcelona, Spain) and left there for 11 d. Ten minutes before being killed, fluorescein isothiocyanate-dextran (25 mg/ml, 100 µl/mouse) was injected into the tail vein. After removing the angioreactors, the matrix was digested with dispase and the amount of fluorescence quantified in a plate reader (Titertek Multiskan PLUS; Cultek, Madrid, Spain). All experiments were performed in a blinded fashion and were approved by the proper welfare animal committees.

Radial diffusion antimicrobial assay
The antimicrobial activities of the peptides were assayed using the radial diffusion method as previously described (21). Briefly, Escherichia coli C600 was grown in Luria broth and collected in the exponential phase of growth. An underlay gel that contained 1% (wt/vol) agarose (SeaKem LE agarose; Cambrex, Walkersville, MD), 2 mM HEPES (pH 7.2), and 0.3 mg trypticase soy broth powder ml21 was equilibrated at 50 C and inoculated with the bacteria to a final concentration of 105 cfu/ml of molten gel. This gel was poured into standard petri dishes, and after polymerization, small wells of 10-µl capacity were carved. Aliquots of 5 µl of the different peptides were added and allowed to diffuse for 2 h at 37 C. After that, a 10-ml overlay gel composed of 1% agarose and 6% TSB powder in water was poured on top of the previous one, and the plates were incubated overnight at 37 C. The next day, the diameters of the inhibition halos were measured to the nearest 0.1 mm, and after subtracting the diameter of the well, were expressed in inhibition units (10 U = 1 mm). All experiments were performed in triplicate on two separate occasions.

Outer membrane permeability to 1-N-phenyl-napthylamine (NPN)
NPN is an uncharged hydrophobic fluorescent probe whose quantum yield suddenly increases when transferred from a hydrophilic to a hydrophobic environment. When NPN is added to cells, it fluoresces weakly because it is unable to breach the outer membrane (OM) barrier and is a substrate for efflux pumps. However, upon OM destabilization and in the presence of an energy inhibitor, NPN partitions into the membrane, emitting a bright fluorescence (22, 23, 24).

A suspension of exponentially growing cells of E. coli C600 (OD600 = 0.5) was prepared in 2 mM HEPES (pH 7.5); 1 ml of this suspension was transferred to 1-cm fluorometric cuvettes, and NPN was added (final concentration, 10 µM). When the effect of the peptides was tested, they were added 10 sec before NPN. Fluorescence was monitored with a spectrofluorophotometer (RF-5301PC; Shimadzu, Kyoto, Japan) set as follows: excitation, 350 nm; emission, 420 nm; and slit width, 3 nm. Results are expressed as relative fluorescence units. Measurements were recorded as ASCII files and exported to a personal computer for plotting. All measurements were done in duplicate on two separate occasions.

Statistical analysis
Data were analyzed by two-tailed Student’s t test and are represented as the mean and SD of at least six independent measurements.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Angiogenesis assay
All peptides tested produced a statistically significant increase in angiogenesis when compared with the negative control (Matrigel alone) (Fig. 1Go). As previously reported (5), mature human PAMP (P1) elicited an intense proangiogenic response (P < 0.01). Interestingly, both the immature Gly-extended version of human PAMP (P2) and the artificial free-acid form (P3) induced a weaker response at the same concentration, suggesting that the amide group is important for this activity. Mouse PAMP, which shares with human PAMP the amide group, had an activity comparable to its human counterpart at 1 nM. The chicken and fish molecules, which lack terminal amidation, had intermediate angiogenic effects somewhat similar to the immature human peptides (Fig. 1Go).


Figure 1
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  		FIG. 1. Angiogenesis assay (directed in vivo angiogenesis assay) for the six synthetic PAMP peptides. The peptides were diluted in Matrigel at a final concentration of 1 nM and implanted under the skin of nude mice for 11 d. After iv injection of fluorescein isothiocyanate-dextran, the implants were removed and the amount of fluorescence trapped in the Matrigel quantified. Each bar represents the mean ± SD of eight independent measurements. Statistically significant differences with Matrigel control (or as indicated by brackets) are indicated as follows: *, P < 0.05; **, P < 0.01. R.F.U., Relative fluorescent units.

 
Antimicrobial activity
Of the six peptides tested, only P1 (mature human PAMP) and P3 (free-acid human PAMP) had any detectable killing effect on E. coli (Fig. 2Go). Of the two, P3 was more efficient than P1 at equimolar concentrations and was able to kill bacteria at lower concentrations (12.5 µM) than the mature peptide. No antimicrobial activity was observed for the other peptides, even at higher peptide concentrations (results not shown). It has been suggested that the antimicrobial mechanism of action for PAMP implies punching holes in the bacterial outer membrane (25). To ensure that our peptides were working through this mechanism and to provide additional proof of their differential antimicrobial behavior, E. coli was exposed to the peptides in the presence of a fluorescent probe. In agreement with the results obtained by the radial diffusion assay, P1 and P3 were very efficient in increasing outer membrane permeability, in a similar way to the positive control, polymyxin B (Fig. 3Go). Polymyxin B is one of most potent permeabilizers of the OM (22, 23, 24). All the other peptides, including P2, did not show any activity, and the data are not included in Fig. 3Go to avoid cluttering the graph.


Figure 2
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  		FIG. 2. Antimicrobial activity of the six synthetic peptides on E. coli. Small wells in an agar plate were filled with the indicated peptide concentrations, and the size of the inhibition haloes was measured. Values are presented as radial diffusion units (R.D.U.) where 1 mm = 10 RDU. Points represent mean ± SD of six independent measurements. Only P1 and P3 had measurable antimicrobial activity in this assay.

 

Figure 3
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  		FIG. 3. OM permeability assay on E. coli. Bacteria were exposed to the synthetic peptides (20 µM) in the presence of a fluorescent probe (NPN). An increase in fluorescence, indicated as relative fluorescence units (R.F.U.), indicates elevated permeability. Polymyxin B (1 µM) was used as a positive control. The negative control (Control) are bacteria without any addition. Peptides P2, P4, P5, and P6 elicited a response undistinguishable from the negative control and are not represented here to avoid clutter. These data are representative of two separate experiments.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
New biological properties might arise through different mechanisms. These include gene duplication whereby one of the copies retains the original function, whereas the other can mutate freely without deleterious consequences (26, 27). More rare are the examples of genes that maintain an activity while developing a new one (28, 29). Additional mechanisms include variations in the alternative splicing of a particular gene (30).

In the case of the proadrenomedullin gene, the regions coding for AM and PAMP seem to have been subjected to different selective pressure with AM being relatively well conserved throughout evolution and PAMP varying more rapidly. There are mathematical models to analyze whether the sequence changes observed in a genetic system are random (neutral evolution) or intended by Darwinian selection to either remove a character (purifying selection) or establish a new function (positive selection) (31, 32). When we tried to apply these mathematical approaches to the proadrenomedullin gene, we did not obtain any meaningful result, because of the low number of available sequences and the long time intervals among them. Nevertheless, other peptide families with antimicrobial or angiogenic activity have evolved through positive selection (adaptive evolution) (33, 34, 35). It will be interesting to repeat the analysis once more sequences become available.

Human PAMP has been shown recently as a surprisingly potent angiogenic factor acting through specific receptors in endothelial cells (5). Here we confirm that those results apply also to PAMP molecules of different species. Although more complex studies including dose-dependent curves are needed to properly quantify angiogenic potential, analysis of naturally occurring or artificial variants of PAMP lacking the amide group at the carboxy end suggest the relevance of this terminal amide for the angiogenic potential of the molecule. Amidation seems to improve the activity of the peptides carrying it by protecting them from proteolysis and thus prolonging their half-life (36). Therefore, the acquisition of the amidation motif by PAMP somewhere between birds and mammals may have improved the angiogenic phenotype elicited by this peptide. One caveat of our angiogenic analysis is that it was done in a single species (mouse) where the mammalian receptors for PAMP may require amidation. It is possible that fish and chicken PAMP molecules may be more efficient in their corresponding systems.

Antimicrobial analysis, using two different approaches, revealed that only primate PAMP was able to prevent bacterial growth, indicating that this activity may have appeared late in the phylogenetic tree. Surprisingly, stark differences were found when comparing the three human forms of the peptide, which vary in only one amino acid or less. The free-acid form was more efficient than the mature peptide, whereas the Gly-extended immature form was not active at all. The ability of PAMP and AM to kill bacteria seems to be related to their capability of inserting themselves in the OM, creating pores that disturb bacterial integrity in a receptor-independent fashion (25), and here we confirmed these observations with an OM permeability assay. A similar mechanism has been demonstrated for other antimicrobial peptides (37, 38), and even a single amino acid change may introduce conformational modifications that result in a loss of activity (39). A recent conformational analysis of PAMP shows that its structure is compatible with a pore-forming mechanism (40). Repeating such structural analysis for Gly-extended PAMP may explain why this form does not kill bacteria. In addition, this marked difference between the immature and the mature forms of PAMP, which is similar to what happens with AM, highlights the importance of the amidating enzymes, as has been recently shown by a knockout of peptidylglycine {alpha}-amidating monooxygenase, which results in embryonic lethality (41).

The innate immune response is composed, among other molecules, by antimicrobial peptides present in the integument and the mucosal surfaces acting as a first line of protection against invading microorganisms (42). Both AM and PAMP are present in those locations, and at least in humans, they seem to be part of this system (4, 43). Although traditional distinctions are beginning to blur in vertebrates (44), it is well understood that the innate immune system is phylogenetically more primitive than adaptive hematopoietic immunity (45). In contrast with this view, we describe here the co-option of antimicrobial activity by a preexisting molecule at a relatively late time, indicating that individual components of the innate system may have appeared at different time periods. This fact may reflect the continuous need to generate new antimicrobial molecules to counteract the fast rate of bacterial mutation and adaptation (46).

In summary, we describe the appearance of a new functional attribute in an angiogenic molecule, together with an improvement in angiogenic potential.


   Footnotes
 
A.M. is supported by a grant from the Spanish Ministry of Science and Education, BFU2004-02838.

Disclosure summary: A.M. and F.C. are inventors on the antimicrobial properties (U.S. Patent No. 09/931,700) and the angiogenic potential (Worldwide Patent No. WO2004/0433383 A2) of PAMP. J.A.B. has nothing to declare.

First Published Online March 30, 2006

Abbreviations: AM, Adrenomedullin; NPN, 1-N-phenyl-napthylamine; OM, outer membrane; PAMP, proadrenomedullin N-terminal 20 peptide.

Received January 25, 2006.

Accepted for publication March 20, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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