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Original Studies |
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 (Y.A., C.D., L.Y., J.L., H.V.), 76821 Mont-Saint-Aignan, France; and the Service of Endocrinology, Department of Medicine, Faculty of Medicine, University of Sherbrooke (L.B., N.G.-P.), Sherbrooke, Quebec, Canada J1H 5N4
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 CNRS, University of Rouen, 76821 Mont-Saint-Aignan, France. E-mail: hubert.vaudry{at}univ-rouen.fr
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The cDNA encoding SgII has been recently characterized in two amphibian species, the frog Rana ridibunda (10) and the toad Xenopus laevis (11). Comparison of the amino acid sequences of human and amphibian SgII revealed that evolutionary pressure has acted to conserve particularly the structures of two putative peptides bounded by dibasic residues, whereas other domains of SgII have been poorly preserved across vertebrate species (10, 11). One of the conserved peptides, dubbed secretoneurin (SN; SgII152–184 in the human sequence), has received much attention. The peptide has been isolated and characterized in the frog brain and thus identified as an authentic processing product of SgII (12).
Subsequently, the occurrence of SN has been demonstrated in a wide
range of tissues (13, 14), and the peptide has been shown to exert
various biological activities (15, 16, 17, 18, 19). In contrast, the second
conserved region corresponding to a 66-amino acid peptide flanking SN
at its C-terminal extremity (Fig. 1
) has
not yet been investigated. This peptide, called EM66, is delimited by
conserved dibasic cleavage sites, and the structures of human and
amphibian EM66 exhibit 68–70% sequence identity (10, 11).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The cDNA sequence encoding the EM66 region in human SgII was amplified by PCR using 75 ng of the plasmid human SgII-Bluescript (22) and 50 pmol of the sense primer EM11 (5'-GAGAGGATGGATGAGGAGCAAAAAC-3') and the antisense primer EM3 (5'-CTAGAAGCTTACATCTCATCGTTGATTTGTTCAT-3'). A stop codon and a HindIII restriction site necessary for the production of the peptide and for subcloning, respectively (see below), were introduced at the 5'-end of the antisense primer. DNA amplification was carried out in a reaction mixture containing 67 mmol/L Tris-HCl (pH 8.8), 16 mmol/L (NH4)2SO4, 1.5 mmol/L MgCl2, 0.01% Tween-20, 200 µmol/L deoxy-NTP, and 2.5 U Taq DNA polymerase (Eurobio, Les Ulis, France) in the following conditions: denaturation at 94 C for 4 min followed by 30 cycles of 94 C for 1 min, 60 C for 1 min, and 72 C for 1 min before a final extension step at 72 C for 7 min. The PCR product was purified, its ends were blunted with Pfu DNA polymerase (Stratagene, La Jolla, CA), and it was digested with HindIII before ligation into the expression plasmid pMAL-c2 (New England Biolabs, Beverly, MA) previously cut with XmnI and HindIII. The construction was verified by DNA sequencing through the EM66 region. This strategy allows for the subcloning of EM66-encoding DNA in the sense orientation, in frame with and downstream of the plasmid region encoding the bacterial maltose-binding protein (MBP). The MBP-EM66 fusion protein was expressed in isopropyl D-thiogalactopyranoside (IPTG)-induced E. coli and affinity purified from bacterial soluble extracts applied to amylose resin essentially as described by the manufacturer of the Protein Fusion and Purification System (New England Biolabs).
The purified fusion protein was cleaved by factor Xa (1 µg/100 µg fusion protein) to release EM66. The recombinant peptide was submitted to partial purification on Sep-Pak C18 cartridges (Waters, St-Quentin en Yvelines, France). Bound peptide was eluted from the cartridges with acetonitrile/water/trifluoroacetic acid (TFA; 30:69.9:0.1, vol/vol/vol) and lyophilized. The Sep-Pak-prepurified recombinant peptide was redissolved in 1 mL 10% acetonitrile in water (vol/vol) and injected onto a 1 x 25-cm reverse phase Vydac C18 high performance liquid chromatography (HPLC) column (Bio-Rad, Ivry-sur-Seine, France) equilibrated with acetonitrile-water-TFA (20:79.9:0.1, vol/vol/vol) at a flow rate of 5 mL/min. The concentration of acetonitrile in the eluting solvent was held at 20% for 5 min and raised to 40% over 65 min. The purified recombinant peptide was characterized by analytical reverse phase HPLC (1 mL/min) onto a 0.45 x 25-cm Vydac C18 column (Bio-Rad) using a linear acetonitrile gradient (10–60% over 25 min) and by mass spectrometry using a Nermag R10–10U equipped with an API-ESI from Analytica-Branford (Quad Service, Poissy, France).
Antibodies against EM66 were raised by injecting New Zealand rabbits with the fusion protein MBP-EM66 emulsified with Freund’s adjuvant.
SDS-PAGE and Western blot analysis
Proteins were analyzed by PAGE under denaturing conditions (23) and either stained with Coomassie blue (Bio-Rad) or electroblotted onto a nitrocellulose sheet (Amersham, Courtaboeuf, France) and revealed with antibodies, as described previously (24), using a chemiluminescence detection kit (Amersham).
Tissue preparation
Adult human adrenal glands were obtained from patients undergoing nephrectomy for kidney cancer. For biochemical studies, the adrenal medulla was dissected from the adrenal cortex. Fetal adrenal glands were obtained from fetuses, aged 17–20 weeks (postconception), at the time of therapeutic abortion. Fetal age was estimated by foot length and time after the last menstruation, according to the method of Streeter et al. (25). After collection, glands were frozen at -80 C. The protocols of collection of the tissues were approved by the human subject review committee at our Institutions.
Immunohistochemical procedure
Frozen adrenal glands were sliced into 10-µm sections with a
cryomicrotome (Frigocut, Reicher-Jung, Germany). Sections were thaw
mounted on gelatin-coated slides, dried in a desiccator overnight at 4
C, and dipped for 3 h in McLean’s fixative [4%
paraformaldehyde, 20 mmol/L lysine, and 2.5 mmol/L sodium metaperiodate
in 0.1 mol/L phosphate buffer (PB), pH 7.4]. After several rinses in
PB, slices were processed for indirect immunofluorescence microscopy,
as previously described (26). Briefly, tissue sections were incubated
overnight at 4 C with the EM66 antiserum (code no. 736-1806) diluted
1:500 or with antibodies against human chromogranin A (CgA; Dako,
Glostrup, Denmark) diluted 1:500 in PB containing 0.3% Triton X-100
and 1% BSA. The tissues were rinsed in PB buffer for 30 min and
incubated for 1 h at room temperature with fluorescein
isothiocyanate-conjugated goat antirabbit 
-globulins (Caltag
Laboratories, San Francisco, CA) diluted 1:100. Finally, adrenal slices
were rinsed in PB, mounted with buffer/glycerol (1:1), coverslipped,
and observed on a Leitz Orthoplan microscope (Leitz, Heidelberg,
Germany) equipped with a Vario-Orthomat photographic system. To
verify the specificity of the immunoreaction, the following controls
were performed: 1) substitution of the primary antisera with PB, 2)
incubation with nonimmune rabbit serum instead of the EM66 or CgA
antisera, and 3) preincubation of the EM66 antiserum (diluted 1:500)
with purified recombinant EM66 (10-6 mol/L).
Tissue extraction
For Western blot analysis, adult adrenomedullary tissue or whole fetal adrenal glands were homogenized in 10 mmol/L Tris-HCl, pH 7.4, containing 0.05% Triton X-100 and 1 mmol/L phenylmethylsulfonylfluoride. The extracts were centrifuged at 12,000 x g for 15 min at 4 C, and the proteins were contained in the supernatants were analyzed.
For HPLC analysis, frozen fetal adrenal or adult adrenal medulla were immersed for 10 min in a cooled solution of 75% ethanol-18% hydrochloric acid. The tissue samples were homogenized, sonicated, and centrifuged (13,000 x g, 4 C) for 30 min. The supernatant was collected, dried by vacuum centrifugation (Speed-Vac Concentrator, Savant AES 2000, Hicksville, NY), and kept dry until chromatographic analysis.
HPLC analysis
Dried samples were reconstituted in 2 mL 0.1% TFA in water (solution A) and loaded onto three Sep-Pak C18 cartridges. The cartridges were rinsed with 20 mL solution A, and the bound material was eluted with 5 mL of a solution of acetonitrile-water-TFA (49.9:50:0.1, vol/vol/vol). The solvent was evaporated in a Speed-Vac Concentrator. The samples were reconstituted in 500 µL solution A and centrifuged (14,000 x g), and the supernatant was injected onto a 0.45 x 25-cm Vydac C18 column equilibrated with a solution of acetonitrile-water-TFA (9.9:90:0.1, vol/vol/vol) at a flow rate of 1 mL/min. The concentration of acetonitrile in the eluting solvent was raised to 60% over 25 min using a linear gradient. HPLC standard consisted of 1 µg purified recombinant EM66. Fractions of 0.5 mL were collected, evaporated, and kept dry until RIA.
RIA
EM66 RIA was performed with the antiserum EM66-736-1806. Purified recombinant EM66 was iodinated by the chloramine-T method and separated from free iodine on Sep-Pak C18 cartridges using a gradient of acetonitrile (0–100%) in 0.1% TFA. The radioiodinated peptide eluted at 32% acetonitrile.
The assay was performed in standard diluent consisting of 60 mmol/L
Na2HPO4, 10 mmol/L ethylenediamine
tetraacetate, 0.5% NaN3, and 0.1% Triton X-100 (pH 7.4).
The antiserum, used at a final dilution of 1:60,000, was incubated with
6,000 cpm of tracer/tube for 48 h at 4 C in the absence or
presence of standard (purified EM66) or tissue extracts. Separation of
antibody-bound from free peptide was achieved by adding 100 µL
-globulins (10 mg/mL) and 2 mL of a 20% polyethylene glycol 6000
solution in standard diluent without Triton X-100. After a 20-min
incubation at room temperature, the samples were centrifuged
(5,000 x g, 20 min, 4 C), and the precipitates were
counted in an LKB scintillation spectrometer (LKB, Rockville, MD).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The EM66 peptide was produced in E. coli as a fusion
protein downstream of the bacterial MBP. The sequence encoding EM66 in
the human SgII cDNA was amplified by PCR with specific primers (data
not shown) and inserted immediately downstream of the MBP sequences so
that the cleavage of the fusion protein with factor Xa would release
EM66 with the native amino acid sequence. The different steps of fusion
protein production and purification were monitored by SDS-PAGE (Fig. 2). The IPTG-induced fusion protein (Fig. 2, lanes 1 and 2) appeared at a relative mass of approximately 60K.
This soluble protein was enriched by centrifugation (Fig. 2, lanes 3
and 4) and purified by affinity chromatography (Fig. 2, lane 5). The
apparent mass of the recombinant protein was higher than would be
expected for a fusion protein comprising the bacterial MBP (43K; Fig. 2, lane 6) and the 66-amino acid peptide (
8K). However, it is well
known that the SgII molecule has an anomalous migration in SDS-PAGE due
to its high content in acidic amino acids (3). It can thus be assumed
that the high proportion of acidic residues in the EM66 peptide also
influenced the fusion protein behavior in SDS-PAGE. Cleavage of this
fusion protein by factor Xa yielded the 43K-MBP and a major peptide
with an apparent molecular mass of about 13K (Fig. 3, lanes 1 and 2). Unexpectedly, a second
minor peptide with a slightly lower apparent mass was also observed
(Fig. 3, lane 2). To ascertain that the fusion protein actually
contained the EM66 peptide, Western blot analysis was performed using a
polyclonal antibody raised against a large portion of human SgII (22),
including the EM66 region. This antibody recognized the 60K fusion
protein (Fig. 3, lane 3). As the antibody did not show any nonspecific
staining of the MBP after factor Xa cleavage of the fusion protein
(Fig. 3, lane 4), it was concluded that EM66 was included within the
fusion protein. It was not possible to detect the free EM66 peptide
itself with the human SgII antibody in this analysis, probably because
it was not retained during the electrotransfer. The occurrence of a
shorter peptide, in addition to EM66 after factor Xa cleavage, as
observed on SDS-PAGE, could thus be ascribed to a secondary cleavage
site within EM66 in addition to the site present at the boundary of MBP
and EM66 in the fusion protein.
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). The major
recombinant peptide was then chromatographed to apparent homogeneity
onto a semipreparative Vydac C18 column (data not shown).
The purified peptide eluted as a sharp peak on an analytical Vydac
C18 HPLC column (Fig. 4B). Mass spectrometric analysis
assigned a molecular mass of 7884.08 to this purified peptide (data not
shown) compared to a calculated mass of 7884 for the full-length EM66.
The discrepancy between this value and the relative mass observed by
SDS-PAGE analysis (13K) confirmed the anomalous migration of EM66 on
SDS-PAGE.
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Polyclonal antibodies were raised in rabbits against the fusion
protein MBP-EM66. The sensitivity and specificity of the EM66
antibodies generated were determined by RIA (Fig. 5). The sensitivity threshold of the
assay was between 25–100 pg (3.2–12.8 fmol)/tube. The antiserum did
not exhibit any significant cross-reactivity with pituitary adenylate
cyclase-activating polypeptide (PACAP), calcitonin gene-related peptide
(CGRP), galanin, or the other human SgII-derived peptide, SN.
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). The band at 13K may correspond to the
EM66 peptide.
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Immunofluorescence labeling of adult human adrenal slices with the
antiserum against EM66 produced intense staining of medullary cells,
whereas adrenocortical cells were totally devoid of immunoreactivity
(Fig. 7A). Labeling of consecutive
sections with an antiserum against CgA (a selective marker of
chromaffin cells) confirmed that the EM66-like immunoreactivity was
restricted to the medulla (Fig. 7B).
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). Incubation of consecutive
sections with antibodies against human CgA labeled the same nests of
cells (data not shown), indicating that the EM66-immunopositive cells
actually correspond to chromaffin progenitor cells. Steroid-producing
cells of the outer neocortex and the large fetal zone were not
immunolabeled (Fig. 7C). Preabsorption of the EM66 antiserum with
10-6 mol/L EM66 totally abolished the immunoreaction
(Fig. 7D). When the antiserum was substituted with either
nonimmune rabbit serum or PB, no immunofluorescence was observed. Characterization of EM66-like material in human adrenal extracts
Adult and fetal human adrenal gland extracts were analyzed by
HPLC, and the EM66-like immunoreactivity contained in the eluted
fractions was quantified by RIA (Fig. 8).
The chromatograms revealed the existence of a major peak of
EM66-immunoreactive material exhibiting the same retention time as
recombinant EM66 in both adult (Fig. 8A) and fetal (Fig. 8B) adrenal
gland extracts. However, although the adult adrenal gland extract
contained a single immunoreactive peak, several minor components
exhibiting EM66-like immunoreactivity were resolved in the fetal
adrenal gland extract.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The recent characterization of the cDNA encoding SgII in the frog Rana ridibunda has revealed that the sequence of four peptides, delimited by pairs of basic amino acids, has been remarkably preserved across vertebrates (10). Selective regional conservation of the sequence of SgII has also been reported in the toad Xenopus laevis, particularly for two regions that are also highly preserved in frog, i.e. the neuropeptide SN and the peptide EM66, which has not been investigated previously (11).
As the large size of EM66 precluded its chemical synthesis, the peptide was produced in E. coli. The identity of the recombinant peptide was verified by Western blot and mass spectrometric analysis, thus validating the molecular strategy employed. Recombinant EM66 exhibited abnormal migration on SDS-PAGE with an apparent mass of approximately 13K compared to a molecular mass of 7884.08 as determined by mass spectrometry. This anomalous behavior on SDS-PAGE, which has been described for the whole SgII protein as well as for other members of the granin family, can be ascribed to the acidic nature of these proteins (1, 2, 3).
A sensitive and specific RIA has been developed using antibodies raised against a fusion protein comprising EM66 and a bacterial protein. These antibodies did not show any cross-reactivity with various peptides present in the adrenal medulla such as galanin, PACAP, CGRP, or SN (27, 28, 29). Analysis of human adrenal extracts by Western blotting revealed that the EM66 antibodies recognize to some extent human SgII and intermediate processing products. Combination of HPLC analysis and RIA detection was used to characterize EM66 in adult and fetal human adrenal extracts. An immunoreactive peak exhibiting the same retention time as recombinant EM66 was found in both adult and fetal gland extracts. These data indicate that in the human adrenal gland, SgII serves as a precursor to generate the peptide EM66. Within SgII, the sequence of EM66 is flanked by Lys-Arg dibasic sites, which are efficient cleavage sites for PCs in other hormone and neuropeptide precursors (30). The processing of EM66 at the N-terminal Lys-Arg was predictable because the latter is shared with SN, which has been shown to occur in the human adrenal (29). The fact that EM66 is formed in the human adrenal shows that the C-terminal Lys-Arg site of this peptide is also processed. This finding contributes to the identification of the pairs of basic amino acids of SgII that are actually cleaved, as it has been shown that not all of these sites are processed (13, 31). Complete characterization of the sites of SgII that are cleaved in vivo will undoubtedly facilitate the study of the function of SgII and its derived peptides.
Although a single immunoreactive form coeluting with EM66 was found in the adult adrenal medulla extract, several minor peaks exhibiting shorter retention times than EM66 were also detected in the fetal adrenal extract. The early elution position of these immunoreactive peptides suggests that they may correspond to truncated forms of EM66.
Immunohistochemical staining of human adrenal slices with the EM66 antiserum revealed intense labeling of the adrenomedullary cells in the adult gland and nests of cells in the fetal zone of the developing adrenal. Labeling of consecutive sections with an antibody against CgA confirmed that in both the adult and fetal adrenal glands, the EM66-immunoreactive material was contained in adrenochromaffin cells. These observations along with the HPLC data indicate that EM66 can be generated early during development of the human adrenal gland. Consistent with this finding, it has been recently reported that SgII is processed to the free peptide SN at all developmental stages of the rat brain (32).
The existence of EM66 strengthens the concept that SgII is processed in vivo to give rise to several peptides that might play a role in the nervous, neuroendocrine, and endocrine systems. In fact, SgII exhibits most of the characteristics of neuropeptide precursors. First, SgII is exclusively expressed in endocrine and neuronal cells and is stored in secretory vesicles from which it is released upon stimulation (1, 2, 3). Second, the synthesis of SgII is finely regulated by mechanisms generally operating on other hormones and neuropeptides (7, 11, 33, 34). Third, like neuropeptide precursors, SgII contains several pairs of basic residues that represent potential cleavage sites by the subtilisin/kexin-like PCs (35, 36). The conservation of particular regions of SgII throughout vertebrates, the occurrence of free peptides corresponding precisely to these conserved regions in fetal and adult tissues (Ref. 32 and this study), and the fact that one of them (SN) exerts biological activities (15, 16, 17, 18, 19) argue for a role of SgII as a precursor to several bioactive peptides. Further studies are clearly required to determine the possible activities of EM66.
However, SgII may exert functions other than that of a peptide precursor. It has been suggested that SgII and other granins play also a role in prohormone packaging and secretory granule formation in neuroendocrine and neuronal cells. This assumption is based on the selective ability of granins to aggregate in high calcium and acidic pH conditions (6, 37). Such conditions are found in the trans-Golgi network, where biogenesis of secretory granules begins. The aggregation of granins in this compartment could assist the sorting of other secretory granule constituents and/or contribute to the biogenesis of secretory granules (3, 6, 37). Thus, an alternative function of some of the conserved domains of SgII such as EM66 would be to participate in these intracellular trafficking events. The processing of such particular domains could be a prerequisite for their intracellular function. Consistent with this hypothesis is the finding that the neuroendocrine polypeptide 7B2, an acidic polypeptide that is considered a member of the granin family (1, 2), acts as a chaperone for the prohormone convertase PC2 and as an inhibitor of the activity of this enzyme through two separate and conserved regions in its sequence that are processed in vivo (38, 39). In addition, it has been recently shown that overexpression of CgB in AtT20 cells resulted in increased storage and secretion of some of the POMC-processing products (40). Together, these observations suggest that specific domains of granins, in particular those with conserved sequences across different species, bind to certain secretory products to facilitate their sorting, secretion, and/or activity.
In conclusion, the present study has revealed the existence of a novel SgII-derived peptide in the human adrenal gland. The conservation of the sequence of EM66 in vertebrates and its occurrence early during ontogenesis of the human adrenal gland strongly suggest that this peptide could exert physiological activities. The production of the recombinant EM66 peptide and the development of specific antibodies described in this report will be instrumental to define the function of this novel SgII-derived peptide.
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Acknowledgments |
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Footnotes |
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2 Recipient of a fellowship from the Conseil Régional de
Haute-Normandie.
3 Recipient of a fellowship from ORIL-SERVIER Laboratories and the
Conseil Régional de Haute-Normandie.
4 Recipient of a fellowship from the Fonds de la Recherche en
Santé du Québec and the Fonds pour la Formation de
Chercheurs et l’Aide à la Recherche.
Received February 11, 1988.
Revised April 13, 1998.
Accepted April 28, 1998.
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