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European Institute for Peptide Research (IFRMP 23), Laboratory of Cellular and Molecular Neuroendocrinology, Institut National de la Santé et de la Recherche Médicale (INSERM U413), Centre National de la Recherche Scientifique (UA CNRS), University of Rouen, 76821 Mont-Saint-Aignan, France
Address all correspondence and requests for reprints to: Dr. H. 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 widespread distribution and abundance of these proteins in neuroendocrine cells have made granins, in particular CgA, useful markers for identifying a variety of neuroendocrine neoplasms, and have stimulated studies on their potential implication in the sorting and condensation of the coresident peptides and neurotransmitters leading to secretory granule formation (3, 4). Molecular cloning of the complementary DNA (cDNA) encoding CgA from different mammalian species has revealed that the protein contains several pairs of basic amino acids (5, 6, 7, 8, 9, 10) that are potential cleavage sites for prohormone convertases, suggesting that CgA could be processed in vivo to generate bioactive peptides. In support of this hypothesis, the cloning of porcine CgA cDNA (11) has revealed that pancreastatin (Pst), a peptide originally isolated from the porcine pancreas that inhibits glucose-stimulated insulin secretion (12), is in fact a processing product of CgA. Subsequently, other biological activities have been reported for Pst such as inhibition of pancreatic exocrine secretion (13) and PTH secretion (14, 15). More recently, additional peptides that could derive from CgA processing have been shown to affect secretion from neuroendocrine cells in an autocrine or paracrine manner. These peptides fall into three categories: 1) those that have been characterized from tissue extracts such as vasostatins, two N-terminal processing products of CgA (16) that inhibit PTH secretion (17) and blood vessel contraction (18); 2) those that have been obtained by in vitro proteolytic cleavage of purified CgA, including parastatin, which also inhibits PTH secretion (19) or chromacin, a peptide that exhibits antibacterial activity (20); and 3) synthetic peptides designed from the CgA sequence, which were found to possess a biological activity, such as catestatin, a peptide that inhibits catecholamine release from chromaffin cells (21).
Because evolutionary pressure acts to conserve mainly functional regions of precursor proteins, cloning of CgA in nonmammalian vertebrates should provide crucial information on the biologically active determinants of the protein. Indeed, recent reviews (1, 2) have emphasized the necessity of characterizing CgA in lower species to decipher conserved structural domains that may be important for CgA function. We describe here for the first time the cloning of a cDNA encoding CgA in a nonmammalian species, the frog Rana ridibunda. We have compared the structure of frog CgA with its mammalian counterparts and characterized its tissue distribution in the central nervous system and in peripheral tissues. In addition, variations of CgA gene expression in the intermediate lobe of the pituitary gland were compared with those of POMC in response to the physiological process of background color adaptation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PCR cloning
Frog brain and adrenal RNA (5 µg) was reverse transcribed at
42 C for 1 h in a 20-µl reaction volume containing 50
mM Tris-HCl, pH 8.3, 75 mM KCl, 3
mM MgCl2, 10 mM dithiothreitol, 500
µM of each dNTP, 0.5 µg oligo(dT)12–18
(Life Technologies, Inc., Cergy-Pontoise, France), 20 U
RNase inhibitor (Promega Corp., Charbonnières,
France) and 200 U of Moloney murine leukemia virus RNase H-
Superscript (Life Technologies, Inc.). PCR was performed
on an aliquot of the RT reaction to amplify a portion of frog CgA cDNA
using degenerate primers deduced from the C-terminal part of mammalian
CgA protein sequences (5, 6, 7, 8, 9). The reaction was carried out in a 50-µl
volume containing 3 µl of the reverse transcribed RNA, 20
mM Tris-CH3COOH, pH 9, 10 mM
(NH4)2SO4, 75 mM
CH3COOK, 0.05% Tween-20, 1.5 mM
MgSO4, 200 µM of each dNTP, 1
µM of degenerate sense primer CG5,
5'-GA(A/G)GA(C/T)CA(A/G)GA(A/G)CTGGA-3' and antisense primer CG7,
5'-TG(C/T)AG(C/T)TG(A/G)TG(A/C/G/T)GC(A/C/G/T)AC(C/T)-TT(C/T)TC-3',
corresponding respectively to the amino acid sequences EDQELE and
EKVAHQLQ, and 2.5 U Tfl DNA polymerase (Promega Corp.).
Amplification was achieved by using 35 cycles of 94 C for 1 min, 48 C
for 1 min and 72 C for 1 min in a Robocycler Gradient 40
(Stratagene, La Jolla, CA). PCR products were
electrophoresed on a 2% agarose gel and a DNA fragment with the
expected size (69 bp) was purified and ligated into the pGEMT vector
(Promega Corp.). Sequencing revealed that this PCR product
was highly homologous to the corresponding C-terminal sequence of
mammalian CgA.
cDNA library screening and sequence analysis
A frog (Rana ridibunda) pituitary cDNA library made
in the CDM7/amp plasmid (22) was screened with the oligonucleotide CG8,
5'-GAGGATCAAGAACTGGAGAGCCTGGCTGCTATCGAGGCTGAGCTG-3', whose
sequence was deduced from the PCR DNA fragment. This oligonucleotide
was 3' end-labeled with [
-32P]dCTP (Amersham Pharmacia Biotech, Les Ulis, France) using Terminal
Deoxynucleotide Transferase (Promega Corp.). After
prehybridization, filters were hybridized with 32P-labeled
CG8 in 50 mM
NaH2PO4/Na2HPO4, pH
6.5, 50% formamide, 4 x SSC (1 x SSC = 0.15
M NaCl and 0.015 M sodium citrate), 1 x
Denhardt’s solution, 200 µg/ml salmon sperm DNA, 100 µg/ml yeast
transfer RNA and 0.1% SDS at 42 C, overnight. The filters were washed
four times with 2 x SSC, 0.1% SDS at room temperature and twice
with 0.2 x SSC, 0.1% SDS at 42 C. A positive clone, FPL4CGA8,
was sequenced on both strands on a LI-COR 4000L DNA Sequencer
(ScienceTec, Les Ulis, France) using the Thermosequenase Kit
(Amersham Pharmacia Biotech) and fluorescent universal
primers (MWG/ScienceTec, Les Ulis, France). Nucleotide and deduced
amino acid sequences were analyzed by the DNASIS V2.1 software
(Hitachi, Olivet, France).
Northern blot analysis
Total RNA was extracted by the acid-guanidinium
thiocyanate-phenol-chloroform method of Chomczynski and Sacchi (23)
using the Tri reagent (Sigma Chemical Co., Saint-Quentin
Fallavier, France). RNA was dissolved in denaturing buffer, heated at
65 C for 15 min, and fractionated on formaldehyde-agarose gels. After
staining with ethidium bromide, gels were blotted on Hybond NX
(Amersham Pharmacia Biotech) membranes that were
subsequently hybridized at 42 C with 32P-labeled random
primed (Prime-a-Gene Labeling System, Promega Corp.)
probes as described previously (24). Filters were analyzed by
autoradiography on Kodak X-OMAT films (Sigma Chemical Co.).
In situ hybridization histochemistry
Frogs were anesthetized and perfused transcardially with 4%
paraformaldehyde. Frontal sections (12 µm-thick) of embedded brains
were cut on a cryostat (Frigocut, Reicher-Jung, Germany) and kept at
-80 C until use. Tissue sections were hybridized at 60 C in the
presence of 107 cpm/ml 35S-labeled riboprobe as
previously described (22, 25). Sense and antisense riboprobes were
prepared by in vitro transcription of a BamHI
fragment (nucleotides 66–715 of clone FPL4CGA8) subcloned into
pBluescript II KS (Stratagene), in the presence of
[35S]UTP (Amersham Pharmacia Biotech) and T7
or T3 RNA polymerase (Promega Corp.). Tissue slices were
dehydrated and exposed onto Hyperfilm ß max (Amersham Pharmacia Biotech) for 8 days. Anatomical structures were identified by
hematoxylin and eosin staining of tissue slices.
Background color adaptation experiments
Frogs were adapted on white or black background under constant
illumination for 2 weeks at 18 C. Neurointermediate or distal lobes of
the pituitary from four to five groups of ten animals adapted to white
or black background were dissected and immediately processed for RNA
extraction. RNA samples were analyzed by Northern blot using FPL4CGA8
or POMC cDNAs as probes. Messenger RNA (mRNA) signals were quantified
using a BIO-500 image analysis system (Biocom, Les Ulis, France) and
were corrected for RNA loading variations by scanning the ethidium
bromide-stained 18S ribosomal RNA of each sample using the DensyLab
2.0.5 software (Bioprobe Systems, Montreuil, France). Statistical
analysis was performed using the Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Based on the PCR product sequence, a homologous oligonucleotide was
synthesized and used to screen a frog pituitary cDNA library. Of
105 clones probed, more than 100 were positive. Restriction
analysis of a few clones revealed that most of them contained a 1.3-kb
insert. Sequencing showed that this cDNA contained an open reading
frame of 1197 bp encoding a 399-amino acid preprotein (Fig. 1
), which shares several similarities
with mammalian CgA. The first 18 amino acids correspond to the signal
peptide and the remaining 381 amino acids represent the mature frog CgA
protein with a predicted molecular mass of 44 kDa. This protein, like
its mammalian counterparts, is highly acidic, 28% of its amino acids
being either glutamic acid (19%) or aspartic acid residues (9%). Frog
CgA contains a putative N-glycosylation site at position 216, and two
cysteine residues at positions 17 and 38 that have been shown to form a
disulfide loop in mammalian CgA (26). The amino acid sequence of frog
CgA comprises 11 pairs of basic residues.
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). However, strong amino acid sequence
conservation was found in the N- and C-terminal parts of the protein as
well as in a short segment preceding the C-terminus. These conserved
domains exhibit 70–80% amino acid identity between frog and human
sequences whereas the central part of the molecule shows a weak degree
of similarity with only about 14% amino acid identity (Fig. 2B). The
alignment revealed also that, among the eleven pairs of basic amino
acids found in the frog sequence, six pairs (amino acids 77–78,
271–272, 286–287, 342–343, 351–352 and 379–380) are located in
analogous positions in the mammalian CgA sequences, and one pair (amino
acids 119–120) is located three amino acids upstream in the mammalian
sequences (Fig. 2, A and B). Interestingly, these conserved potential
cleavage sites are located at the extremities of the three highly
conserved regions of CgA.
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). An intense
hybridization signal was observed in the pituitary, indicating a
particularly high rate of CgA gene expression in this gland. CgA mRNA
was also detected in the whole brain, the hypothalamus, the spinal
cord, and the adrenal gland, albeit at a much lower level than in the
pituitary. In contrast, CgA mRNA could not be detected in the
intestine.
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). In the diencephalon,
intense hybridization signals were detected in the dorsal part of the
ventrolateral thalamic nucleus, the anterior thalamic and ventral
hypothalamic nuclei, and the suprachiasmatic nucleus, whereas moderate
signals were observed in the ventral part of the ventrolateral thalamic
nucleus, the ventromedial and central thalamic nuclei, as well as the
magnocellular preoptic nucleus (Fig. 4, B and C). In the mesencephalon,
high CgA mRNA levels were found in the optic tectum, and lower
concentrations were observed in the anterodorsal and anteroventral
tegmental nuclei (Fig. 4D). In the pituitary, a robust signal was
detected in the distal lobe and a less intense signal was found in the
intermediate lobe (Fig. 4D). Finally, in the spinal cord, both the
dorsal and ventral horns were strongly labeled (Fig. 4E). When a sense
probe was used, no hybridization signal was observed (Fig. 4F).
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). Under the same conditions,
POMC mRNA levels were also increased in the pars intermedia from
black-adapted animals (159% ± 11 vs. white-adapted
animals, P < 0.01, Fig. 5B). In contrast, no
significant changes of either CgA or POMC mRNA levels were observed in
the distal lobe of the pituitary from black-adapted vs.
white-adapted animals (Fig. 5, A and B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We could amplify by PCR a DNA fragment encoding a C-terminal peptide
from frog CgA that allowed to isolate a full-length cDNA clone encoding
frog CgA. The predicted protein is approximately 50 amino acids shorter
than mammalian CgA, but it shares several features with the latter such
as the presence of an 18-amino acid signal peptide, several pairs of
basic residues, and a high content (28%) of acidic amino acids.
Alignment of the frog CgA protein with its mammalian homologues
revealed a rather low overall amino acid sequence identity (40–44%).
However, three regions of the frog protein exhibited high amino acid
sequence identity (70–80%) with their human counterparts, whereas the
other regions of the molecule showed only 14–30% identity. Similar
observations have been made with the sequences of frog and mammalian
SgII, which also show high regional conservation (22, 27), suggesting
that this property represents a common feature of members of the granin
family. As in SgII, the highly conserved segments of frog CgA are
delimited by preserved pairs of basic residues where cleavage by
prohormone convertases generally occurs. In fact, two of these peptides
have been previously isolated. One of them corresponds to the
N-terminal conserved domain (Fig. 6),
which has been identified in bovine adrenal medulla (28), the ostrich
pituitary (29), and rat insulinoma cells (30). Two molecular forms of
this peptide with 76 or 113 amino acids have been characterized and
named vasostatins I and II, respectively, in consideration of their
vasoconstriction inhibitory effects on human blood vessels (18, 28)
(Fig. 6). It has been also shown that a peptide containing only amino
acids 1–40 of vasostatins stimulates the secretion of CGRP (31) and
inhibits the secretion of PTH, PTH-related peptide, and calcitonin
in vitro (31, 32, 33). It is worth noting that the two cysteine
residues present in this peptide, which form a disulfide loop important
for sorting of CgA to secretory granules (26) as well as for the
biological activity of vasostatins (17), are also present in the frog
sequence. The second highly conserved segment is a tetradecapeptide,
named WE-14 (Fig. 6), which has been purified from a human ileal
carcinoid tumor (34) and from pheochromocytoma tissue (35). It has been
recently shown that WE-14 potentiates the stimulatory effect of IgE on
histamine release from mast cells (36). The frog counterpart of WE-14
is one amino acid shorter than the mammalian peptide; however, the
conservation of the overall structure of this peptide across the
vertebrate phylum and the fact that it exerts a biological activity
strongly suggest that WE-14 may be an important regulatory peptide. The
third conserved segment of CgA corresponds to a 35-amino acid peptide
that has not been described to date and may represent a novel
regulatory peptide. This putative peptide includes in its sequence a
conserved pair of basic amino acids and may thus give rise to two
peptides. Further studies are required to determine whether these
peptides occur in neuroendocrine or nerve cells and to define their
potential functions. In this respect, we have recently shown that a
segment of SgII whose sequence has been highly preserved in
vertebrates, does occur as a free peptide in the fetal and adult human
adrenal gland (37).
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Chromacin and catestatin have been shown to exert antibacterial
activity (20) and to inhibit nicotinic-stimulated catecholamine release
from adrenomedullary chromaffin cells (21), respectively. However, we
found that there was only a modest sequence homology for these two
peptides between mammals and frog (5 and 24%, respectively). On the
other hand, the sequence of parastatin, a 73-amino acid proteolytic
fragment of CgA that has been reported to inhibit PTH secretion (19),
comprises most of the sequence of the highly conserved C-terminal
peptide (Fig. 6
). Paradoxically, it has been found that the biological
activity of parastatin resides in its N-terminal domain that does not
overlap with the conserved region and that exhibits rather low sequence
homology between frog and mammals. It should be mentioned that these
three peptides, unlike Pst and vasostatins, have not been isolated and
therefore their existence in vivo remains to be
established.
In mammals, it has been shown that CgA is widely expressed in the nervous system and in most endocrine cells (39, 40). Similarly, frog CgA mRNA exhibited a widespread distribution in the brain and endocrine glands. A high concentration of CgA mRNA was found in the frog pituitary and particularly in the distal lobe as revealed by in situ hybridization, whereas other endocrine or neuroendocrine tissues contained lower levels of CgA mRNA. It has previously been shown that CgA mRNA and protein levels in the rat pituitary are depressed by estrogens (41) and increased by glucocorticoids (42). The physiological significance of these observations is not known yet, but given the tight regulation of CgA gene expression in the pituitary, it can be hypothesized that CgA plays an important role in the secretory activity of this gland.
CgA mRNA was also detected by in situ hybridization in the intermediate lobe of the frog pituitary. In amphibians, the pars intermedia plays a pivotal role in the process of skin color adaptation. Thus, melanotrope cells of the pituitary produce the prohormone POMC, which is processed to form melanotropic peptides that induce skin darkening (43). The POMC gene activity is therefore directly linked to background color adaptation with a high transcription level in animals adapted to a dark environment and, reciprocally, a low transcription level in animals adapted to a white background (44). The present study reveals that, in the intermediate lobe of the frog pituitary, the expression of the CgA gene parallels the variations of POMC mRNA during background adaptation, i.e. CgA mRNA levels are augmented in black- vs. white-adapted animals. The fact that POMC and CgA are coordinately regulated in melanotrope cells suggests that CgA may participate in the physiological events allowing amphibia to adapt to their environment. In particular, CgA could be involved in the intracellular mechanisms leading to POMC processing and secretion as has been shown for CgB (45). Overexpression of the latter protein in AtT-20 cells enhances the sorting of a POMC-processing product to secretory granules, suggesting that members of the granin family may influence the intracellular trafficking events leading to coresident hormone secretion. In that case, the conserved regions of CgA could prove to be important for the sorting of peptide hormone and neuropeptide precursors.
In conclusion, the present study has revealed that CgA encompasses three peptides whose amino acid sequences have been remarkably preserved from amphibians to mammals. These peptide regions are likely to be important for the physiological function of CgA. The highly specialized frog melanotrope cells, in which the expression of the CgA gene can be easily manipulated in concert with POMC, by changes of the background color, should be a very appropriate model system to investigate the function of CgA-derived peptides.
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Acknowledgments |
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Footnotes |
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2 Recipient of a postdoctoral fellowship from the Conseil
Régional de Haute-Normandie.
Received March 2, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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