Processing of proenkephalin-A in bovine chromaffin cells. Identification of natural derived fragments by N-terminal sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry.

A large variety of proenkephalin-A-derived peptides (PEAPs) are present in bovine adrenal medulla secretory granules that are cosecreted with catecholamines upon stimulation of chromaffin cells. In the present paper, after reverse phase high performance liquid chromatography of intragranular soluble material, PEAPs were immunodetected with antisera raised against specific proenkephalin-A (PEA) sequences (PEA63-70 and PEA224-237) and analyzed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Thirty PEAPs were characterized in addition to enkephalins and whole PEA, indicating that preferential proteolytic attacks occurred at both N- and C-terminal regions. A similar approach was used to characterize PEA-derived fragments exocytotically released into the extracellular space that showed five additional minor PEAPs. Among all these naturally generated peptides, enkelytin, the antibacterial bisphos- phorylated C-terminal peptide (PEA209-237), was predominantly generated, as shown by MALDI-TOF mass spectrometry analysis, which constituted an efficient method for its identification. Finally, the data on PEA intragranular and extracellular processing in adrenal medulla are discussed in regard to the known enzymatic processing mechanisms. We note the high conservation of the cleavage points in evolutionarily diverse organisms, highlighting an important biological function for the released PEAPs.

Secretory granules in the bovine adrenal medullary chromaffin cells contain a complex mixture of secretory products that are co-released with catecholamines into the circulation in response to splanchnic nerve stimulation (1,2) . Among the high molecular mass water-soluble proteins, proenkephalin-A (PEA) 1 and a family of acidic proteins, named chromogranins, constitute the major constituents of the chromaffin granules. These proteins are considered as protein precursors and are actively processed to low molecular weight peptides. Previously, we have characterized the intragranular and extracellular processing of chromogranin A and B (3,4) in chromaffin granules from bovine adrenal medulla. PEA generates enkephalins, other opioid peptides, and other derived peptides (PEAPs), which display various biological effects, including antinociception and immunomodulation (5). Because large amounts of enkephalins and enkephalin-containing peptides are found in adrenal medullary chromaffin granules, these organelles represent an excellent model to study the intragranular processing of PEA (6 -8). Several opioid peptides, including Met-enkephalin and Leu-enkephalin in the ratio 4:1, the opioid heptapeptide (Met-enkephalin-Arg-Phe), the opioid octapeptide (Met-enkephalin-Arg-Gly-Leu) and C-or N-terminally extended variants of these peptides are liberated by cleavage of the PEA at pairs of basic residues (6,9). In addition, processing of PEA has been analyzed in stably transfected mouse anterior pituitary tumor (AtT-20) cells (10), showing that PEA processing proceeds through an orderly series of steps. Similarly to other precursors, PEA processing appears to start with the removal of the bovine C-terminal peptide B (PEA209 -239) (10,11).
Since 1995, we have shown that antibacterial activities are present within the intragranular chromaffin granule matrix and are recovered in the extracellular medium following secretion. This activity has been assigned to chromogranin A and B fragments and PEAPs (4,(12)(13)(14)(15)(16)(17). Among the intragranular matrix components, enkelytin, an antibacterial peptide corresponding to the bisphosphorylated fragment PEAP209 -237, was identified (14). The antibacterial spectrum of enkelytin shows that this peptide is active in the micromolar range against several Gram-positive bacteria including Staphylococcus aureus, but it is not able to inhibit Gram-negative bacterial growth nor to lyse erythrocytes (15). In addition, the two antibacterial C-terminal PEAPs, enkelytin (PEAP209 -237) and the bisphosphorylated peptide B (PEAP209 -239) are secreted from stimulated cultured chromaffin cells and are immunodetectable in wound fluids and polymorphonuclear neutrophil secretions (15). The co-release of enkelytin and PEAP209 -239 with catecholamines suggests that in stressful situations, these peptides may play an important role in defense mechanisms. Recently, we have described the release of the antibacterial form of the peptide B in the hemolymph of leech (Theromyzon tessulatum) and mussel (Mytilus edulis) following lipopolysaccaride stimulation, surgical trauma, and electrical stimulation of the neural tissues (18). Thus, the antibacterial C-terminal fragments PEA209 -237/239 that are expressed in the nervous and immune systems appear to be highly conserved during evolution and may provide a basis for neuroimmune interactions (5,15).
In the present work, we used reverse phase HPLC, peptide sequencing and matrix-assisted laser desorption ionizationtime of flight (MALDI-TOF) mass spectrometry to identify all the PEAPs (in addition to Leu-and Met-enkephalins and whole PEA) present in chromaffin granules and chromaffin cell secretions. Thirty PEAPs have been characterized, indicating that preferential proteolytic attacks are located at the N-and Cterminal regions (PEA1-116 and PEA209 -239, respectively), as well as 16 new proteolytic cleavage sites. Among all the naturally released PEAPs, enkelytin, the antibacterial C-terminal fragment corresponding to the bisphosphorylated form (Ser 221 and Ser 223 ) of PEA209 -237 was a predominant component. In addition, MALDI-TOF mass spectrometry is a very efficient tool for the identification of peptides in biological materials.

Preparation of Soluble Material Present in Chromaffin Granules-
Secretory granules were isolated from bovine adrenal medulla according to Smith and Winkler (19), except that sucrose solutions were buffered with 10 mM Hepes (pH 7). Granules were lysed by successive freezing and thawing in 10 mM Hepes (pH 7). Granule membranes were sedimented by centrifugation at 100,000 ϫ g for 30 min (20). The supernatant was collected, and aliquots were stored at Ϫ20°C. In some experiments, protease inhibitors were included in solutions, but their presence did not modify the pattern of proteolytic fragments.
Isolation of Proteins Released from Stimulated Cultured Chromaffin Cells-Chromaffin cells were isolated from fresh bovine adrenal glands and cultured as described previously (3). Cells were plated at a density of 10 7 cells/50 mm in plastic Petri dishes. After 3 days in culture, the medium was removed, and cells were washed four times with Locke's solution (140 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO 4 , 2.5 mM CaCl 2 , 11 mM glucose, 0.5 mM ascorbic acid, 15 mM Hepes, pH 7.5) and subsequently stimulated for 10 min with Locke's solution containing 10 M nicotine. External medium was carefully collected, completed with trifluoroacetic acid up to 0.1%, lyophilized, and stored at Ϫ20°C.
Purification of PEAPs by Reverse Phase HPLC-PEAPs were isolated using the Applied Biosystems HPLC system 140 B. Chromatography was performed on a Macherey Nagel Nucleosil 300-5C18 column (4 ϫ 250 mm; particle size, 5 mm). Absorbance was monitored at 214 nm, and the solvent system consisted of 0.05% (v/v) trifluoroacetic acid in water (solvent A) and 0.05% (v/v) trifluoroacetic acid in acetonitrile (solvent B). Elution was performed at a flow rate of 700 l/min using gradient as shown on chromatogram.
Sequence Analysis of PEAPs-The sequence of purified peptides was determined in our laboratory by automatic Edman degradation on an Applied Biosystems 473A microsequencer. Samples purified by HPLC were loaded on polybrene-treated and precycled glass fiber filters (3). PTH-derivatives (Pth-Xaa) were identified by chromatography on a C-18 column (PTH C-18, 2.1 ϫ 200 mm).
Mass Spectra Analysis-Mass determination was carried out in our laboratory on a Brucker BIFLEX TM MALDI-TOF equipped with the SCOUT TM system, High Resolution Optics with X-Y multisample probe, a gridless reflector, and the HIMAS TM linear detector. This instrument has a maximum accelerating potential of 30 kV and may be operated either in the linear or reflector mode. Ionization was accomplished with a 337-nm beam from a nitrogen laser with a repetition rate of 3 Hz. The output signal from the detector was digitized at a sampling rate of 250 MHz in linear mode and 500 MHz in reflector mode using a 1 GHz digital oscilloscope (Lecroy model). The instrument control and data processing were accomplished with software supplied by Bruker using a Sun Sparc workstation. These studies were realized using as the matrix ␣-cyano-4-hydroxycinnamic acid (Sigma) prepared as a satured solution in acetone. Aliquots (1-2 l) of the sample matrix solution were deposited onto probe tips and air dried. After fast spreading and fast evaporation of the solvent, we obtained a thin layer of matrix crystals (21). A micromolar analyte solution was applied to the matrix and allowed to dry under moderate vacuum. This preparation was washed by applying 0.7 l of a 0.5% trifluoroacetic acid in water solution and then flushed after a few seconds. This cleaning procedure often allows an increase in sensitivity and mass accuracy by removing the remaining cations.

Processing of PEA in bovine chromaffin granules
Direct MALDI-TOF Analysis of HPLC Fractions from Soluble Intragranular Material-Immediately after the preparation of soluble material from bovine chromaffin granules, a separation by chromatography on a reverse phase C-18 column was performed (Fig. 1A). PEAPs were recovered in fractions labeled 1-3, according to the immunoreactivity with antisera FIG. 1. Isolation, Western blot analysis, and direct MALDI-TOF mass spectrometry of PEAPs from bovine chromaffin intragranular matrix. A, soluble proteins from chromaffin granules were separated on a Macherey Nagel reverse phase Nucleosil 300-5C18 column (4 ϫ 250 mm). Absorbance was monitored at 214 nm, and elution was performed at a flow rate of 700 l/min with the gradient indicated on the chromatogram. PEAPs were identified in fractions 1-3. B, Western blot analysis of fractions 1-3 was performed using polyclonal antibodies directed against PEA63-70 and PEA224 -237. C, characterization of intragranular PEAPs included in fractions 1 and 2, using direct MALDI-TOF mass spectrometry. P, phosphorylation. To characterize all the PEAPs present in the other fractions ( Fig. 1A), sequencing and mass spectrometry analysis were performed on the fractions eluted before and after peaks a and c, respectively. Only, Met-and Leu-enkephalins were found in the peaks preceding the fraction 1, whereas entire PEA was found in the peak eluted at 40% acetonitrile (data not shown).
Repurification and Analysis by Sequencing and MALDI-TOF Mass Spectrometry of Intragranular PEAPs-To complete this study, additional purifications by reverse phase HPLC were performed to (i) further identify the complex peptide mixture present in fraction 1, (ii) characterize the immunodetected N-terminal fragments (antisera 63-70) present in fractions 2 and 3 (Fig. 1B), and (iii) elucidate the structure of median PEAPs located within the PEA core . Thus, one of the repurification steps of fraction 2 is shown in Fig. 3A. Five fractions (a-e) were recovered and analyzed by the combination of sequencing and MALDI-TOF analysis (Fig. 3B). Five different PEAPs were identified, corresponding to sequences located in the core of the protein (80 -105, 80 -106, 116 -155); two peptides are located in the N-terminal moiety (1-68 and 1-74). These data are in accordance with the Western blot results (Fig. 1B). Two distinct fractions (d and e) contain an unique sequence corresponding to PEAP1-74, which suggests the presence of a thin difference in the spatial conformation of the peptides that may induce a difference in the elution of the peptide. For instance, this fragment includes three glutamine and one asparagine residues that may be partially deamidated during preparation, introducing additional negative charges to the molecule. The analysis of peak a (Fig. 1A) shows the presence of PEAP80 -105 and 80 -106, which corresponds, respectively, to the amidorphin and its extended form. Finally, analysis of PEAPs included in fraction 1 allowed us to identify three N-terminal fragments (PEA1-68, PEA1-74, and PEA1-77), a median peptide (PEA116 -155), and five C-terminal fragments corresponding to PEA209 -232 (non-, mono-, and bis-phosphorylated), PEA209 -235, and the phosphorylated form of PEA211-237, whereas the study of fraction 3 led to the characterization of PEA1-165 and PEA116 -196. Thus, this complete analysis of the soluble intragranular material shows the presence of 25 different PEAPs (Table I) as the result of a complex proteolytic mechanism occurring along the polypeptide chain of PEA. These data are summarized in Fig. 4.

Processing of PEA in Material Released from Nicotine-stimulated Cultured Chromaffin Cells
After stimulation of cultured chromaffin cells with 10 M nicotine, PEAPs present in the extracellular medium were separated by HPLC on a reverse phase C-18 column (Fig. 5A). Analysis of peaks 1-3 by sequencing and MALDI-TOF mass spectrometry (Fig. 5B) revealed that peaks 1 and 2 contain C-terminal fragments PEA209 -237/239 with one, two, and three phosphate groups, whereas peak 3 contains a mixture of N-and C-terminal fragments PEA1-69/70/72 and PEA230 -237. It is important to note that the predominant fragments exocytotically released from nicotine-stimulated cultured chromaffin cells were located in the N-terminal domain (PEA1-72, synenkephalin) and also in the C-terminal domain (PEA209 - FIG. 2. 3200 -3900-Da window of the MALDI-TOF spectra of the peptidic material included in fraction 1 (Fig. 1). Among the detected molecules, peaks with m/z values of 3355.7, 3434.3, 3513.6 (enkelytin), and 3594.0 were respectively attributed to the non-, mono-, bis-, and tri-phosphorylated forms of PEAP209 -237. fraction 2 (Fig. 1A). A, chromatography on a Macherey Nagel reverse phase Nucleosil 300-5C18 column (2 ϫ 125 mm). Absorbance was monitored at 214 nm, and elution was performed at a flow rate of 300 l/min with the gradient indicated on the chromatogram. B, characterization by sequencing and MALDI-TOF mass spectrometry of the PEAPs included in fractions a-e. 239) of the precursor (Fig. 4). Furthermore, the three cleavage sites Leu-Ala (69 -70), Ala-Lys (70 -71), and Met-Glu (229 -230) correspond to new proteolytic attacks that have not yet been identified. Interestingly, the mono-and bis-phosphorylated forms of PEA209 -239 are concomitantly recovered in fractions 1 and 2. Because three phosphorylation sites (Ser 215 , Ser 221 , and Ser 223 ; Fig. 4) are present in PEA, the fragments recovered in fractions 1 and 2 are not phosphorylated at identical positions, leading to different elution times. Taking together, the analysis of the PEAPs released in the extracellular space upon nicotine cell stimulation led to the identification of five new fragments, resulting from extracellular processing. DISCUSSION It is well documented that pro-hormones and pro-neuropeptides are synthesized as inactive large precursors that are processed during their intracellular transport and their storage to generate active peptides (25,26). Conversion of these precursors to smaller biologically active peptides requires specific proteolytic cleavages. Thus, several endopeptidases (cleaving inside the protein sequence) and exopeptidases (removing amino acids from the NH 2 -and COOH-ends) are involved in PEA maturation (6). The knowledge of pro-hormone and proneuropeptide processing and of its relationship to the secretory pathway has significantly progressed during the last 5 years. In the trans-Golgi network, pro-hormones are thought to be sorted with processing enzymes into regulated secretory vesicles (27). Within these organelles, pro-hormone processing is dependent on condensation-induced protein organization, on calcium concentration that may reach several mM (i.e. 20 mM in chromaffin granules), and on pH that is below 6.0 in most endocrine and neuroendocrine secretory granules (27,28). Prohormone cleavage, occurring mainly at specific paired basic sites, takes place in a strict temporal order and is likely to involve a variety of endoproteases. This processing is completed in some cases by the combined action of amino-and carboxy-peptidases.

FIG. 3. Purification and characterization by sequencing and MALDI-TOF mass spectrometry of several PEAPs present in
In the present study we have identified 30 PEAPs generated during the intragranular and extracellular processing of PEA. Some of these peptides have never been described up to now. These data were made possible by the combination of sensitive methods, in particularly MALDI-TOF mass spectrometry, which allows for low level peptide analysis.
An initial screening, based on the expected molecular mass of three immunoreactive fractions, resulting from the first HPLC purification of the soluble intragranular material led to the characterization of eight peptides. Analysis of fractions eluted before and after the peaks of interest indicated the presence of enkephalins and whole PEA. Then several additional purifications were crucial for the structural characterization of minor but numerous PEAPs present in immunoreactive fractions ( Figs. 1 and 2). Further analyses identified 25 intragranular PEAPs in fractions 1-3 (Table I)  of Arg 163 residue. Natural processing of Met-enkephalin was also observed at positions 74 -75 (Tyr/Gly-Gly/Phe/Met) and 235-236 (Tyr/Gly/Gly-Phe/Met). Additional endogenous splitting points were characterized along the polypeptide chain in position outside the basic residues such as 138 -139 (Asp-Gln/ Arg) and 196 -197 (Met-Asp/Tyr/Gln/Lys), involving carboxypeptidases in combination with different enzymes specific to basic residues and present in chromaffin granules. Processing of the C-terminal moiety of PEA in positions 208 -209, 210 -211, 229 -230, 232-233, 235-236, and 237-238 generated a complex mixture of peptides including non-, mono-, bis-, and triphosphorylated forms. Synenkephalin (PEAP1-72) was hardly detectable in the soluble intragranular material, whereas it was described previously as a major component (6). This was not due to further degradation occurring during purification or due to storage conditions, because the addition of protease inhibitors did not modify peptide patterns. However, we have detected synenkephalin in the extracellular medium after stimulation of cultured cells, indicating that this peptide is generated by proteolysis of PEAP1-74 and actively processed to PEAP1-68.
The present experimental data also demonstrate that in chromaffin granules two predominant C-terminal fragments are generated, the bisphosphorylated forms of PEA209 -237 (enkelytin) and of PEA209 -239 (peptide B), which were described both to display antibacterial activity (14, 15). As described previously (11), the processing of PEA may start at the C-terminal end in chromaffin granules. Interestingly, during the processing of PEA, enkelytin is one of the major peptides generated and complements the results obtained on invertebrates, demonstrating the occurrence of a similar mechanism in animals 500 million years divergent from man in evolution (18). The direct detection of enkelytin by MALDI-TOF analysis further demonstrates that this new tool is useful and efficient for peptide detection and analysis in biological fluids.
Since the first study of Udenfriend and Kilpatrick in 1984 (29) on PEA processing, numerous groups looked for the presence of proteolytic enzymes in the intragranular matrix and in the membranes of chromaffin granules. The prohormones convertases 1/3 and 2 have been identified in chromaffin cells and shown to be involved in PEA maturation (30). These endopeptidases are members of the subtilisin-like enzyme family and were identified because of their sequence homology with the yeast endopeptidase Kex2 gene (31)(32)(33)(34)(35). These enzymes cleave the polypeptidic chains at the C-terminal side of dibasic amino acids and at the N-terminal side of a single arginine residue. In this regard, Lys-Arg sites were mainly cleaved, whereas Lys-Lys or Arg-Lys sites were weakly cleaved (30,35,36). In chromaffin granules, 7B2, a protein related to the chromogranin/ secretogranin family, inhibits prohormone convertase 2 and plays a role in its chaperoning (33,37,38). The prohormone thiol-protease is a multicatalytic cysteine protease complex detected in chromaffin granules as a major PEA processing enzyme (35, 39 -42). Prohormone thiol-protease cleaves the polypeptidic chain at the N-terminal side of dibasic residues or between the Arg-Arg, Lys-Lys, and Lys-Arg sites or at the N-terminal side of a single arginine residue (Gly-Arg) (41,43,FIG. 4. Schematic representation of PEA processing. Cleavage sites resulting from intragranular (black arrows) and extracellular processing (dotted arrows) are shown along the bovine PEA sequence (24). The proteolytic splitting points inducing Leu-and Met-enkephalin are not indicated. Arrows located above the sequence represent proteolytic cleavages previously reported, whereas arrows located under the sequence represent new proteolytic cleavages determined in the present work. Basic residues (Lys and Arg) are in bold type. Leu-and Metenkephalins sequences are underlined, and the three phosphorylated sites are indicated with black dots.

FIG. 5. Purification and characterization by sequencing and MALDI-TOF mass spectrometry of PEAPs in extracellular medium after secretion of stimulated chromaffin cells.
A, secreted proteins were separated on a Macherey Nagel reverse phase Nucleosil 300-5C18 column (4 ϫ 250 mm). Absorbance was monitored at 214 nm, and elution was performed at a flow rate of 700 l/min with the gradient indicated on the chromatogram. B, characterization of PEAPs included in fractions 1-3 by sequencing and MALDI-TOF mass spectrometry. P, phosphorylation; X, cysteine residue. 44). Adrenorphin-Gly-generating enzyme was characterized to be present in the adrenal medulla as an endopeptidase cleaving at the N-terminal side of single arginine residues and at dibasic sites (45). The proopiomelanocortin-converting enzyme, also named 70-kDa Asp-protease, is present in chromaffin cells and is mainly involved in the maturation of propiomelanocortin and PEA (35,46). This enzyme cleaves between two basic residues and at C-terminal side of the dibasic residue Lys-Arg and Arg-Lys sites (pH optimum of 5.5). Furthermore, several serine proteases (trypsin-like activity) have been described in chromaffin cells of adrenal medulla and proposed to be involved in PEA maturation (47); this group comprises an enzyme that cleaves mainly the Lys-Arg bond (48), a serine protease of 30 kDa (47,49,50), as well as two different enzymes with respective molecular masses of 76 and 30 kDa (51). In addition, an endooligopeptidase A-like protein activity has been identified in secretory granules of PC-12 cells (52). This enzyme of 71 kDa is implicated in the conversion of enkephalin-containing peptides into enkephalins. In addition, the presence of peptidylglycine-␣-amidating monooxygenase within the chromaffin granules has been well documented (53). This enzyme performs the C-terminal amidation of peptide ending with a C-terminal glycine residue. Therefore, the complete processing of PEA is obtained after the combination of numerous proteolytic activities of endopeptidases and of several potential exopeptidases, such as the aminopeptidases (acting at the N-terminal end) and the carboxypeptidases (acting at the C-terminal end; Table  II). The aminopeptidase activity is shared by a metalloprotease converting the fragments RYGGFM and KRYGGFM in Metenkephalin (YGGFM) (38). The carboxypeptidase H, also named carboxypeptidase E or enkephalin-convertase, removes the basic C-terminal residues (54) and is found as a soluble and a membrane-bound forms (54 -57). After exocytosis, a circulating form of IRCM serine protease 1 may involved in the processing of PEA. This enzyme has been identified as the plasma kallikrein, capable of cleaving several prohormones at the Cterminal side of dibasic sites or between Lys-Lys, Lys-Arg, and Arg-Arg sites (58). Table II, the cleavage at 24 sites can be explained through the activity of the different enzymes previously described to be involved in PEA maturation (Table II). Among these 24 cleavage sites, 17 are located close to dibasic sites, five are located at the proximity of arginine residue, and two result from the splitting of weak peptidic bonds (Gly-Xaa or Xaa-Gly). Because acidic residues, proline, and phosphorylated serine alter the proteolytic activities, five dibasic sites (Lys 85 -Lys 86 , Lys 180 -Arg 181 , Arg 187 -Arg 188 , and Lys 200 -Lys 201 ) and six basic residues (Lys 49 , Lys 131 , Arg 140 , Lys 176 , Arg 191 , and Lys 224 ) were not the targets to these enzymes. Furthermore, the putative cleavage sites close to the five basic residues included in the three-disulfide bridge arrangement (Arg 12 , Arg 15 , Lys 31 , Lys 36 , and Lys 42 ) were not used as such. In addition, prohormone convertases 1/3 and 2, prohormone thiolprotease, proopiomelanocortin-converting enzyme, the serine proteases, and the carboxypeptidases are predominantly involved. The peptidylglycine-␣-amidating monooxygenase activity was revealed because of the removing of the glycine 106, which precedes the C-amidation of amidorphin (80 -105).

As shown in
Several publications described a different processing in other tissues such as brain (59), breast tumor cell lines (60), adenomas (61), bone marrow, and immune cells (62)(63)(64). PEA processing was described to be dependent on the age of the patients (65) or to stress (18,66) and to be directly related to the proteolytic enzyme expressions that may depend on various environmental conditions (60).
The complete analysis of the processing of PEA in bovine adrenal medulla chromaffin granules will assist in revealing the regulation and activation of proteolytic enzymes that are critical to signal molecule expression. Thus far, only few biological effects have been attributed to PEAPs. Enkephalins are known to exhibit analgesia (67), organ development (68), positive ionotropic effects in isolated heart muscle cells (69), as well as immunomodulatory activity (5,70). Furthermore, among other PEAPs, the opioid heptapeptide (YGGFMRF) (71), BAM-12 and -22, as well as peptides E and F display analgesic effects (72). Synenkephalin-derived peptides were also recently TABLE II Proteolytic enzymes implicated in the intragranular and extracellular processing of PEA A, intragranular processing; B, extracellular processing. PTP, prohormone thiol-protease; AGE, adrenorphin-generating enzyme; ACE, angiotensin-converting enzyme; PAM, peptidylglycine-␣-amidating monooxygenase. described to be involved in human lymphocyte proliferation (64).
As shown in Fig. 6, the major identified cleavage sites exhibit a high phylogenetic conservation (73), suggesting that these new peptides described here should be able to share important biological activities among species. As an example, enkelytin and its extended form PEA209 -239, because of their relative abundance, their conservation, and their antimicrobial activity, appear to display an important role in neuroimmunity (74,75).