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Endocrinology Vol. 138, No. 1 128-137
Copyright © 1997 by The Endocrine Society

ARTICLES

Characterization of a Novel {alpha}-Amidated Decapeptide Derived from Proopiomelanocortin-A in the Trout Pituitary1

H. Tollemer2, J. Leprince3, T. Bailhache, I. Chauveau4, F. Vandesande, M. C. Tonon, P. Jego and H. Vaudry

European Institute for Peptide Research (IFRMP 23), Laboratory of Cellular and Molecular Neuroendocrinology, INSERM U-413, Unité Affiliée au Centre National de la Recherche Scientifique, University of Rouen (H.T., J.L., M.C.T., H.V.), Mont-Saint-Aignan; and Laboratory of Cell Biology and Reproduction, Centre National de la Recherche Scientifique URA 256, University of Rennes I (T.B., I.C., P.J.), Rennes, France; and Laboratory of Neuroendocrinology, Zoological Institute, University of Leuven (F.V.), Leuven, Belgium

Address all correspondence and requests for reprints to: Dr. Hubert Vaudry, European Institute for Peptide Research (IFRMP 23), Laboratory of Cellular and Molecular Neuroendocrinology, INSERM U-413, UA Centre National de la Recherche Scientifique, University of Rouen, 76821 Mont-Saint-Aignan, France.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Two complementary DNAs encoding distinct forms of POMC have been characterized in the trout pituitary. One of the POMC variants (POMC-A) possesses a C-terminal extension of 25 amino acids, which has no equivalent in other POMCs described to date. This C-terminal peptide contains three pairs of basic amino acids, suggesting that it may be the precursor of multiple processed peptides. In addition, the presence of a C-terminal glycine residue suggests that some of the processing products may be {alpha}-amidated. To characterize the molecular forms of the peptides generated from the C-terminal domain of trout POMC-A, we have developed specific antibodies against the C-terminal pentapeptide YHFQG and its {alpha}-amidated derivative YHFQ-NH2. Immunocytochemical labeling of pituitary sections with antibodies against YHFQ-NH2 revealed the presence of numerous immunoreactive cells in the pars intermedia and the rostral pars distalis. In contrast, the antibodies against YHFQG produced only weak immunostaining. HPLC analysis combined with RIA detection revealed that extracts of the pars intermedia and pars distalis contain several peptides derived from the C-terminal extension of trout POMC-A, with the predominant molecular form exhibiting the same retention time as ALGERKYHFQ-NH2. Tryptic digestion of this major form produced a peptide that coeluted with YHFQ-NH2. These data indicate that the processing of the C-terminal extension of trout POMC-A generates several novel peptides including the decapeptide amide ALGERKYHFQ-NH2.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
POMC IS A multifunctional precursor protein that generates, through proteolytic processing, several biologically active peptides, including ACTH, {alpha}MSH, and ß-endorphin (1). The complementary DNAs encoding POMC have been characterized in a number of vertebrate species of different taxa, e.g. human (2), ox (3), pig (4), rat (5), mouse (6), toad (7) frog (8), salmon (9), trout (10), and lamprey (11). The toad Xenopus laevis (12), the salmon (9), and the trout (10) possess two POMC genes as a result of duplication of the entire genome in their respective ancestors.

Although the general organization of the POMC molecule has been conserved throughout the vertebrate phylum, some remarkable singularities have been noticed in fish. In particular, salmon and trout POMCs possess only two MSH peptides, i.e. {alpha}- and ßMSH (9, 10, 13), whereas in amphibia and mammals, a third MSH peptide ({gamma}MSH) is present in the N-terminal domain (3, 7, 8). In addition, one of the trout POMC molecules (POMC-A) possesses an unusual C-terminal extension (10). This 25-amino acid peptide contains three pairs of basic residues that represent potential cleavage sites (Fig. 1Go). Thus, processing of trout POMC-A may produce several novel peptides that may be of biological significance. In addition, the C-terminal pentapeptide possesses a Gly residue at the COOH-terminus, suggesting that {alpha}-amidation of the peptide may occur (Fig. 1Go). As many bioactive peptides are C-terminally {alpha}-amidated (14), this peptide represents a valuable marker to explore the processing of the C-terminal tail of trout POMC-A.



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  		Figure 1. Schematic representation of the structure of pre-POMC (POMC) in tetrapods and pre-POMC-A in trout. Vertical bars indicate pairs of basic amino acids. The dotted zone represents the {gamma}-MSH sequence, which is missing in trout POMC. The hatched zone represents the C-terminal extension of trout POMC-A. The sequence of this C-terminal peptide is shown, and the three pairs of basic amino-acids are underlined.

 
The aim of the present study was to determine whether processing of trout POMC-A generates novel regulatory peptides. To this end, we have raised specific antibodies against the pentapeptide YHFQG and its {alpha}-amidated derivative YHFQ-NH2. These antibodies were used for immunocytochemical localization and biochemical characterization of peptides derived from the C-terminal extension of POMC-A in the trout pituitary.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
A total of 130 adult rainbow trout, Oncorhynchus mykiss, of both sexes were used in the present study. The animals were obtained from a fish farm (Montville, France) and killed by decapitation, and the brains and pituitaries were quickly removed. For immunohistochemical studies, the tissues were fixed overnight in a solution of 4% paraformaldehyde in PBS, pH 7.4. For biochemical studies, the pars distalis and pars intermedia were separated and immediately frozen on dry ice.

Reagents and peptide synthesis
Trifluoroacetic acid (TFA), bovine thyroglobulin, glutaraldehyde, hydrogen peroxide, 3,3'-diaminobenzidine tetrahydrochloride, L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin, and Sephadex G-25 (superfine) were purchased from Sigma Chemical Co. (St. Louis, MO). Paraformaldehyde (PFA) was obtained from Prolabo (Paris, France). Goat antirabbit {gamma}-globulins were purchased from Jackson ImmunoResearch (West Grove, PA). The peroxidase-antiperoxidase (PAP) complex was obtained from Affinity Research Products (Nottingham, UK). BSA (fraction V) was purchased from Boehringer (Paris, France). Methanol, phenoxyethanol, and acetic acid were obtained from Merck (Darmstadt, Germany). Acetonitrile was purchased from Carlo Erba (Milan, Italy). F-moc-protected amino acids were obtained from Applied Biosystems (St. Quentin en Yvelines, France). Na125I was purchased from CIS Bio International (Gif-sur-Yvette, France).

The peptides ALGERKYHFQ-NH2, YHFQG, and YHFQ-NH2 were synthesized (0.1-mmol scale) by the solid phase methodology on a 433A Applied Biosystems peptide synthesizer, using the standard F-moc procedure. A Rink amide 4-methylbenzhydrylamine resin (Biochem, Meudon, France) was used for synthesis of {alpha}-amidated peptides and a F-moc-Gly-HMP resin (Applied Biosystems) was used for synthesis of YHFQG. Synthetic peptides were purified by reverse phase HPLC on a 1 x 25-cm Vydac C18 column (Touzart et Matignon, Courtaboeuf, France) using a linear gradient (10–40% over 40 min) of acetonitrile-TFA (99.9:0.1, vol/vol). Analytical HPLC was performed on a 0.45 x 25-cm Vydac C18 column, and the purity of all peptides was greater than 99%. The purified peptides were characterized by fast atom bombardmentmass spectrometry.

Antibodies against YHFQG and YHFQ-NH2
Synthetic YHFQG (3.1 mg) and synthetic YHFQ-NH2 (3.9 mg) were covalently linked to 100 mg bovine thyroglobulin with glutaraldehyde as coupling agent, and the conjugates were purified by dialysis. The coupling efficiency was more than 80% for both peptides. Rabbits were injected intradermally with the synthetic peptide-thyroglobulin conjugates (equivalent of 200 µg synthetic peptide/animal) emulsified in Freund’s adjuvant. Evaluation of the titers of the antisera was carried out by RIA as previously described (15).

Immunohistochemistry
Tissue preparation. The trout were anesthetized by immersion in 2-phenoxyethanol (0.4 ml/liter water). The animals were perfused through the ventral aorta with 50 ml 0.01 M PBS. The perifusion was continued with a solution of 4% PFA in PBS. The pituitaries were dissected and postfixed overnight in 4% PFA. The tissues were rinsed in 20% sucrose-PBS for 6 h. The pituitaries were then frozen at -70 C, and 50-µm thick sections were cut in a cryostat (Microm, Francheville, France) and processed for immunohistochemistry with the PAP technique.

Immunohistochemical procedure. The tissue sections were preincubated for 10 min with 0.6% hydrogen peroxide in methanol to inhibit endogenous peroxidases, then incubated overnight at 4 C with the antisera against either YHFQG or YHFQ-NH2 diluted in PBS supplemented with 0.1% Triton X-100. The slices were rinsed and incubated for 1 h at room temperature with goat antirabbit {gamma}-globulins diluted 1:200 and then for another hour with the PAP complex diluted 1:200. The enzymatic activity was revealed with 0.04% 3,3'-diaminobenzidine tetrahydrochloride and 0.012% hydrogen peroxide. To verify the specificity of the immunoreaction, the following controls were performed: 1) the primary antisera were replaced with nonimmune rabbit serum or PBS; and 2) the primary antisera were preabsorbed with synthetic YHFQG, YHFQ-NH2, or ALGERKYHFQ-NH2.

Tissue extraction
Trout pituitaries were immersed in 1 ml boiling 2 N acetic acid and maintained in a boiling water bath for 10 min to ensure inactivation of proteolytic enzymes (16). The tissues were homogenized by sonication, and the homogenates were centrifuged at 13,000 xg for 30 min. The pellets were used for the measurement of protein concentrations. The supernatants were evaporated in a Speed-Vac concentrator (Savant Instruments, Hicksville, NY) and kept dry until analysis. Tissue extracts were submitted to partial purification on Sep-Pak C18 cartridges (Alltech Associates, Deerfield, IL). Bound material was recovered by elution with acetonitrile-water-TFA (60:39.96:0.04, vol/vol) and evaporated in a Speed-Vac concentrator. The partially purified extracts were kept in a dry atmosphere until chromatographic analysis or direct RIA.

HPLC analysis of endogenous peptides
Pituitary extracts were redissolved in 1 ml acetonitrile-methanol-TFA-water (120:30:0.85:849.15, vol/vol) and centrifuged for 10 min. The supernatant was collected and injected onto a 0.4 x 25-cm reverse phase C18 HPLC column (Merck) equilibrated with a solution of 15% acetonitrile-methanol (80:20) and 85% water-TFA (999:1). The concentration of acetonitrile-methanol was held at 15% for 10 min, raised to 35% over 20 min, and finally raised to 67.5% over 10 min. The synthetic peptides ALGERKYHFQ-NH2, YHFQG, and YHFQ-NH2, used as standards, were chromatographed on the same gradient. Fractions (1 ml) were collected, dried by vacuum centrifugation, and radioimmunoassayed in duplicate.

Tryptic mapping
The two major immunoreactive peaks resolved by HPLC were isolated and submitted to trypsin digestion. Dried fractions were dissolved in 100 µl 0.2 M sodium bicarbonate buffer (pH 7.8) and incubated with L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin for 16 h at 37 C, as previously described (17). Synthetic ALGERKYHFQ-NH2 was digested with trypsin under the same conditions. Tryptic digests were prepurified on a Sep-Pak C18 cartridge and analyzed by HPLC with the previous elution conditions, and the retention times were compared with that of synthetic YHFQ-NH2.

Gel filtration
The apparent mol wt of the two main HPLC peaks exhibiting YHFQG- and YHFQ-NH2-like immunoreactivity (-LI) were determined by Sephadex G-25 gel filtration. The column (0.9 x 30 cm) was equilibrated with 0.1 M acetic acid supplemented with 0.1% BSA and calibrated with the following monoiodinated peptides: [125I]YHFQ-NH2 (mol wt, 718), [125I]YHFQG (mol wt, 775), iodinated prepro-TRH-(160–169) (mol wt, 1319) (18), [125I]ALGERKYHFQ-NH2 (mol wt, 1372), iodinated octadecaneuropeptide (mol wt, 2036) (16). The HPLC-purified peaks were chromatographed on the G-25 column. Fifty fractions (0.7 ml) were collected, evaporated in a Speed-Vac concentrator, and the YHFQG or YHFQ-NH2 content was measured in each fraction by RIA.

RIA procedure
The concentrations of YHFQG- and YHFQ-NH2-LI in crude tissue extracts or in HPLC fractions were measured in duplicate by a double antibody RIA procedure as previously described (15). The synthetic peptides (1 µg) were iodinated by the chloramine-T method and separated from free iodine on Sep-Pak C18 cartridges with a gradient of acetonitrile (6–36%) in 0.1% TFA. Radioiodinated peptides were eluted at 14% acetonitrile and kept at -20 C in glycerol (1:1, vol/vol).

The RIAs were performed as previously described (15). The final dilutions of the antisera against YHFQG and YHFQ-NH2 (code numbers 643-1506 and 644-1506) were 1:10,000 and 1:12,000, respectively, and the total amount of tracer was 6,000 cpm/tube.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Immunocytochemical localization of YHFQG and YHFQ-NH2
Immunolabeling of pituitary sections with antibodies against YHFQ-NH2 produced intense staining of the pars intermedia and the rostral pars distalis (Fig. 2Go, A and B). A similar pattern of labeling was obtained with the antibodies against YHFQG, except that staining was less intense in the pars intermedia and very weak in the rostral pars distalis (Fig. 2CGo).



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  		Figure 2. Immunohistochemical staining of sagittal sections of rainbow trout pituitary. A, Low magnification microphotograph of a pituitary section labeled with the YHFQ-NH2 antiserum. B and C, Schematic representation of the distribution of YHFQ-NH2-immunoreactive (B) and YHFQG-immunoreactive cells (C) in the pars intermedia (PI) and the rostral pars distalis (RPD). PPD, Proximal pars distalis; IR, infundibular recess. The densely solid dotted areas indicate intense staining; the sparsely solid dotted areas indicate moderate staining; the open dotted area indicates weak staining. Scale bar = 500 µm.

 
At a higher magnification, numerous YHFQ-NH2-immunoreactive cells were observed in the pars intermedia (Fig. 3AGo). Intensely labeled YHFQ-NH2-positive cells were also visualized in the rostral pars distalis (Fig. 3BGo). A few YHFQ-NH2-immunoreactive cells were found in PRL follicles (Fig. 3BGo). Preincubation of the YHFQ-NH2 antiserum with synthetic YHFQ-NH2 (10-5 M; data not shown) or synthetic ALGERKYHFQ-NH2 (10-5 M) caused disappearance of the immunoreaction in the pars intermedia (Fig. 3CGo) and the rostral pars distalis (Fig. 3DGo). In contrast, preincubation of the YHFQ-NH2 antiserum with 10-5 M YHFQG did not cause any change in the intensity of the staining (data not shown).



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  		Figure 3. Microphotographs showing the distribution of YHFQ-NH2-immunoreactive cells in the pars intermedia (A) and the rostral pars distalis (B). Some of the immunoreactive cells in the rostral pars distalis were observed in the PRL follicles (PF). Preincubation of the antiserum with synthetic ALGERKYHFQ-NH2 (10-5 M) resulted in complete extinction of the immunoreaction in the pars intermedia (C) and rostral pars distalis (D). Scale bars = 10 µm.

 
The YHFQG antiserum produced moderate staining of pars intermedia cells (Fig. 4AGo) and a weak staining of a cell subset in the rostral pars distalis (Fig. 4BGo). Preincubation of the YHFQG antiserum with synthetic YHFQG (10-5 M) resulted in complete loss of the immunoreaction in the pars intermedia (Fig. 4CGo) and the rostral pars distalis (Fig. 4DGo). In contrast, preincubation of the YHFQG antiserum with 10-5 M YHFQ-NH2 or 10-5 M ALGERKYHFQ-NH2 did not modify the intensity of the labeling (data not shown).



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  		Figure 4. Microphotographs showing the distribution of YHFQG-immunoreactive cells in the pars intermedia (A) and the rostral pars distalis (B). Preincubation of the antiserum with synthetic YHFQG (10-5 M) resulted in complete extinction of the immunoreaction in the pars intermedia (C) and rostral pars distalis (D). Scale bars = 10 µm.

 
No immunostaining was detected when the primary antisera were replaced by nonimmune rabbit serum or PBS (data not shown).

Peptide concentrations in tissue extracts
The detection limits of the YHFQG and YHFQ-NH2 RIAs were 3.5 and 72 pg/tube, respectively. The IC50 values of the assays were 61 and 2310 pg, respectively.

The cross-reactivities of the YHFQG antibodies with synthetic YHFQ-NH2 and ALGERKYHFQ-NH2 were 0.2% and less than 0.01%, respectively (Fig. 5AGo). The cross-reactivities of the YHFQ-NH2 antibodies with synthetic YHFQG and synthetic ALGERKYHFQ-NH2 were 0.8% and 11.3%, respectively (Fig. 5BGo). The antibodies against YHFQG or YHFQ-NH2 did not exhibit any cross-reactivity with ACTH, {alpha}MSH, or ß-endorphin (Fig. 5Go, A and B).



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  		Figure 5. Semilogarithmic curves comparing competitive inhibition of antibody-bound 125I-labeled YHFQG (A) and YHFQ-NH2 (B) by increasing concentrations of synthetic YHFQG, YHFQ-NH2, or ALGERKYHFQ-NH2 and by serial dilutions of pituitary intermediate lobe (IL) or distal lobe (DL) extracts. The cross-reactivities of three other POMC-derived peptides (ACTH, {alpha}MSH, and ß-endorphin) are also indicated.

 
Serial dilutions (1:2) of trout pituitary extracts produced displacement curves that were parallel to the standard curve obtained with synthetic YHFQG (Fig. 5AGo). In contrast, the displacement curves obtained with pituitary extracts were not parallel to that obtained with synthetic YHFQ-NH2 (Fig. 5BGo). The total content and relative concentrations of YHFQG- and YHFQ-NH2-LI in Sep-Pak-prepurified pars distalis and pars intermedia extracts are reported in Table 1Go. The amounts of YHFQG- and YHFQ-NH2-LI were 5.5 and 3.4 times higher in the pars distalis than in the pars intermedia, respectively. The concentration of YHFQ-NH2-LI was 11 and 6.7 times higher than that of YHFQG-LI in the pars intermedia and pars distalis, respectively.


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  		Table 1. Content and concentration of immunoreactive YHFQG and YHFQ-NH2 in the trout pituitary

 
Reverse phase HPLC analysis
Characterization of the YHFQG- and YHFQ-NH2-LI detected in the tissue extracts was performed by combining reverse phase HPLC analysis and RIA detection. On the acetonitrile-methanol gradient shown in Fig. 6Go, the synthetic peptides YHFQG, YHFQ-NH2, and ALGERKYHFQ-NH2 could be resolved. In both pars intermedia (Fig. 6AGo) and pars distalis extracts (Fig. 6BGo), the YHFQG RIA detected a predominant peak with a retention time (26 min) higher than that of synthetic YHFQG (21.2 min). A minor peak corresponding to approximately 10% of the YHFQG immunoreactivity coeluted with the synthetic peptide (Fig. 6Go, A and B). The YHFQ-NH2 RIA detected a major form that coeluted with the decapeptide ALGERKYHFQ-NH2 (25.4 min) in the pars intermedia (Fig. 6CGo) and pars distalis extracts (Fig. 6DGo). A minor component coeluting with synthetic YHFQ-NH2 was also identified (Fig. 6Go, C and D).



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  		Figure 6. Reverse phase HPLC analysis of YHFQG- and YHFQ-NH2-LI in the trout pituitary. Tissue extracts consisting of five intermediate lobes or five distal lobes were prepurified on Sep-Pak C18 cartridges and chromatographed on a LiChrosorb C18 column. Fractions (1 ml each) were collected, dried, and assayed for YHFQG (A and B) and YHFQ-NH2 (C and D) contents. The arrows indicate the elution time of the synthetic standards. The recoveries of YHFQG and YHFQ-NH2 were 70% and 84%, respectively. The dashed lines show the concentration of acetonitrile-methanol (80:20) used to elute the column.

 
Tryptic mapping
The major immunoreactive peptides detected with the YHFQG (Fig. 6AGo; peak I) and YHFQ-NH2 RIAs (Fig. 6CGo; peak II) were collected and subjected to tryptic digestion. The elution profiles of the tryptic digests are shown in Fig. 7Go. Peak I generated an immunoreactive peptide that exhibited the same retention time as synthetic YHFQG (Fig. 7AGo). Peak II generated an immunoreactive peptide that exhibited the same retention time as YHFQ-NH2 (Fig. 7BGo). Tryptic digestion of ALGERKYHFQ-NH2 also generated an immunoreactive peptide that coeluted with synthetic YHFQ-NH2 (data not shown).



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  		Figure 7. Reverse phase HPLC analysis of the tryptic digest of peak I collected from Fig. 6AGo (A) and peak II collected from Fig. 6CGo (B). The arrows indicate the elution times of synthetic standards or purified peak I. Purified peak II coeluted with ALGERKYHFQ-NH2. The peptide ALGER was generated by tryptic digestion of ALGERKYHFQ-NH2. The recoveries of YHFQG and YHFQ-NH2 were 45% and 35%, respectively. The dashed lines show the concentration of acetonitrile-methanol (80:20) used to elute the column.

 
Apparent mol wt of peaks I and II
The predominant peptides detected with the YHFQG and YHFQ-NH2 RIAs (peaks I and II, respectively) were analyzed by Sephadex G-25 gel filtration. A series of radioiodinated peptides of different mol wt were used to calibrate the column. Peaks I and II had the same elution volume as [125I]ALGERKYHFQ-NH2 (Fig. 8Go, A and B).



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  		Figure 8. Sephadex G-25 gel filtration chromatography of peak I (A) and peak II (B). Fractions (0.7 ml each) were collected, dried, and assayed for YHFQG-LI (A) or YHFQ-NH2-LI (B). The recoveries of YHFQG and YHFQ-NH2 were 63% and 61%, respectively. Horizontal bars indicate the elution volumes of [125I]octadecaneuropeptide ([125I]ODN; recovery, 65%), [125I]ALGERKYHFQ-NH2 (recovery, 63%), [125I]YHFQG (recovery, 80%), and [125I]YHFQ-NH2 (recovery, 75%).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The structure of trout POMC-A deduced from the complementary DNA sequence indicates that the protein possesses an unusual extension at the C-terminal end of the ß-endorphin sequence (10), which has never been described in any POMC characterized to date. To determine whether trout POMC-A can generate novel peptides, we raised antibodies against the C-terminal pentapeptide YHFQG and its {alpha}-amidated variant YHFQ-NH2. The antibodies thus obtained fulfilled the criteria required for unambiguous characterization of each peptide. In particular, the YHFQG antibodies exhibited only 0.2% cross-reactivity with YHFQ-NH2 and did not cross-react with ALGERKYHFQ-NH2. The YHFQ-NH2 antibodies exhibited less than 1% cross-reactivity with YHFQG, but cross-reacted appreciably (11%) with ALGERKYHFQ-NH2, so that these latter antibodies could be used to detect both the tetrapeptide amide YHFQ-NH2 and the decapeptide ALGERKYHFQ-NH2.

Immunocytochemical studies revealed the presence of a peptide(s) immunologically related to YHFQ-NH2 in most cells of the pars intermedia and in a subpopulation of cells located in the rostral aspect of the pars distalis. The distribution of YHFQ-NH2-immunoreactive cells corroborates in situ hybridization studies performed with a POMC-A riboprobe (10) and immunocytochemical data obtained with antibodies against the NH2-terminal peptide of POMC (20) or with ACTH and {alpha}MSH antibodies (21). In contrast, the YHFQG antibodies produced weaker staining in the pars intermedia and only faint labeling in the rostral pars distalis, suggesting that trout POMC-A and its processing products are C-terminally {alpha}-amidated. Measurement of the concentrations of YHFQG- and YHFQ-NH2-LI in crude pituitary extracts confirmed that a large proportion of the peptide(s) was C-terminally {alpha}-amidated in both the pars intermedia and pars distalis. In mammals, two POMC-derived peptides possess a C-terminally {alpha}-amidated residue: {alpha}MSH, which is produced exclusively in melanotrope cells (22), and joining peptide, which is produced in both melanotrope and corticotrope cells (23, 24). In fish, however, {alpha}MSH is the sole POMC-derived {alpha}-amidated peptide that has been characterized to date (25, 26, 27, 28, 29, 30, 31, 32). The present data reveal that the trout pituitary can generate a family of novel C-terminally {alpha}-amidated peptides. Our data also imply that peptidylglycine {alpha}-amidating monooxygenase (33) is expressed in both melanotrope and corticotrope cells of the trout pituitary.

Immunohistochemical staining revealed extensive labeling of the pars intermedia and more restricted labeling of the rostral pars distalis. In contrast, measurement of peptide contents by RIA showed that the actual amounts of peptides were higher in the pars distalis than in the pars intermedia, suggesting that POMC-A is processed more actively in the rostral pars distalis than in the pars intermedia. Alternatively, the mature peptides may be released more rapidly by melanotrope than by corticotrope cells.

Characterization of the molecular forms of the peptides by HPLC analysis and Sephadex G-25 gel filtration combined with RIA detection revealed that ALGERKYHFQ-NH2 constitutes the major YHFQ-NH2-immunoreactive peptide. Tryptic digestion of the endogenous immunoreactive peptides confirmed that the predominant components were precursor forms of YHFQG and YHFQ-NH2. The apparent ratio of YHFQ-NH2 to ALGERKYHFQ-NH2 was 1:4.3 in the pars intermedia and 1:3.4 in the pars distalis. However, inasmuch as the cross-reactivity of ALGERKYHFQ-NH2 in the YHFQ-NH2 RIA was only 11%, the actual ratios of YHFQ-NH2 to ALGERKYHFQ-NH2 were approximately 1:40 in the pars intermedia and 1:30 in the pars distalis. Similarly, only a small proportion of YHFQG-immunoreactive material contained in pars intermedia and pars distalis extracts coeluted with synthetic YHFQG. Taken together, these data indicate that the Lys-Arg pair preceding the ALGERKYHFQ-NH2 and ALGERKYHFQG sequences is an efficient cleavage site, whereas the Arg-Lys pair contained within these sequences is poorly cleaved. Consistent with this observation, it has been demonstrated that most prohormone convertases are far more efficient on the Lys-Arg than on the Arg-Lys cleavage site (for review, see Ref.34).

It has been previously shown that trout neurointermediate lobe extracts contain two forms of ß-endorphin that exhibit the same retention times as the salmon endorphin I and II variants (35). This finding indicates that the Lys-Arg pair located between the ß-endorphin sequence and the C-terminal extension of trout POMC-A is efficiently cleaved, at least in the intermediate lobe. This observation together with the present data indicate that posttranslational processing of trout POMC-A generates two peptides: EQWGREEGEE and ALGERKYHFQ-NH2. Further studies should be conducted to determine the possible actions of these novel decapeptides.


   Acknowledgments
 
The authors thank D. Tranchand Bunel for generating the antibodies, and L. Galas for his help in tissue collection.


   Footnotes
 
1 This work was supported by grants from INSERM (U-413), CNRS (URA 256), EU Human Capital and Mobility (Grant ERBCHRXCT 92–0017), and the Conseil Régional de Haute-Normandie Back

2 Recipient of a fellowship from the EU Human Capital and Mobility Program. Back

3 Recipient of a fellowship from ORIL Laboratories and the Conseil Régional de Haute-Normandie. Back

4 Recipient of a fellowship from the Ministère de l’Enseignement Supérieur et de la Recherche. Back

Received July 25, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Eipper BA, Mains RE 1980 Structure and biosynthesis of proadrenocorticotropin/endorphin and related peptides. Endocr Rev 1:1–27[Abstract/Free Full Text]
  2. Whitfeld PL, Seeburg PH, Shine J 1982 The human pro-opiomelanocortin gene: organization, sequence and interspersion with repetitive DNA. DNA 1:133–143[Medline]
  3. Nakanishi S, Inoue A, Kita T, Nakamura M, Chang ACY, Cohen SN, Numa S 1979 Nucleotide sequence of cloned cDNA for bovine corticotropin-ß-lipotropin precursor. Nature 278:423–427[CrossRef][Medline]
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