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Harry S. Truman Memorial Veterans’ Hospital (L.R.F., S.L.E.) and the Departments of Pharmacology, Pathology and Anatomical Sciences, Physiology, and Biochemistry, School of Medicine and the Molecular Biology Program, Missouri University (L.R.F., S.L.E., X.F., R.M.L., Y.W., L.M.R., D.T.C., R.H.F., W.J.K.), Columbia, Missouri 65212
Address all correspondence and requests for reprints to: Dr. Leonard R. Forte, Department of Pharmacology, Missouri University School of Medicine, Columbia, Missouri 65212. E-mail:lrf{at}missouri.edu
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Introduction |
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Recent studies revealed that mRNA transcripts encoding guanylin and/or uroguanylin and their GC receptors are expressed in many tissues of the opossum (12, 16). A novel mRNA transcript that hybridizes with both uroguanylin and guanylin cDNA probes and is longer than either the 1.2-kb uroguanylin or the 0.8-kb guanylin mRNA was detected by Northern assays using RNA isolated from spleen and testes of the North American opossum, Didelphis virginiana. Herein, we provide new information for cDNA and deduced protein structures, the biological activity, and mRNA expression of a unique member of the guanylin peptide family. The gene and its protein product were named lymphoguanylin because the first cDNA isolated was derived from spleen mRNA, and the active peptide domain is similar in primary structure to uroguanylin and guanylin.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cloning of opossum preprolymphoguanylin cDNAs
cDNAs encoding preprolymphoguanylin were produced using RT with
RNA isolated from spleen, thymus, lymph nodes, circulating white blood
cells, bone marrow, kidney, opossum kidney (OK) cells, heart, ovary,
cerebellum, and testis and used as templates for the PCR. Total RNA was
extracted using the RNeasy kit (Qiagen, Chatsworth, CA).
Oligo(deoxythymidine)18-primed cDNAs were synthesized from
3 µg total RNA using reverse transcriptase (Superscript II,
Life Technologies, Gaithersburg, MD). The first set of PCR
primers used for PCR amplification of uroguanylin-like cDNAs
corresponded to nucleotides -21 to -2 (5'-GGAACAAGACTGGCAGACAC-3')
and nucleotides 266–247 (5'-GCTCTGAAGATGTTGGCAGC-3') of the opossum
preprouroguanylin cDNAs isolated from a colon cDNA library (12). These
primers amplified cDNA products of 287 bp that lacked the region
containing the COOH-terminal active peptide (16). A second set of
primers consisting of 5'-GGAACAAGACTGGCAGACAC-3' and
5'-GTGGGGGCACAGAGTGAGGT-3' was derived from nucleotides -21 to -2 and
nucleotides 402 to 383 within the 3'- and 5'-untranslated domains,
respectively, of opossum preprouroguanylin cDNA (12, 16). The first
primer set generated a partial cDNA identical to a corresponding domain
within the cDNAs produced by PCR using the second set of primers. The
second pair of oligonucleotide primers generated identical 385-bp cDNA
products (excluding primers) when spleen, thymus, lymph nodes,
circulating white blood cell, bone marrow, kidney, OK cells, heart,
ovary, cerebellum, and testis cDNAs were used as templates and 30
cycles of the PCR were carried out at 93 C for 1 min, at 56.5 C for 1
min, and at 72 C for 1.5 min using Taq DNA polymerase
(U.S. Biochemical Corp., Cleveland, OH). The PCR-generated
cDNA products were ligated into the plasmid vector pCR2.1 (TA cloning
kit, Invitrogen, San Diego, CA), and molecular cDNA clones
were isolated and sequenced. Automated sequencing was performed by the
DNA Core Laboratory of the Missouri University Molecular Biology
Program (Columbia, MO). Independent PCR-generated cDNA clones derived
from the spleen and other tissues of as many as eight different animals
were sequenced.
Northern assays
Total RNA (20–40 µg) was subjected to electrophoresis in
formaldehyde-agarose gels and then transferred to nylon membranes
(Zeta-Probe, Bio-Rad Laboratories, Inc., Hercules, CA).
The blots were hybridized with either the spleen-derived cDNA or the
uroguanylin cDNA and a ß-actin cDNA as previously described (12, 16).
Prehybridization was performed for 2 h with QuickHyb
(Stratagene, La Jolla, CA) at 69 C, followed by
hybridization for 4 h at 65 C with each cDNA probe labeled by
random priming (Boehringer Mannheim, Indianapolis, IN). The blots were
washed twice with 2 x SSC (standard saline citrate)-0.1% SDS for
5 min at room temperature. Exposure to x-ray film was performed at -80
C with intensifying screens.
Synthesis of peptides
Opossum lymphoguanylin (QEECELCINMACTGY), opossum uroguanylin
(QEDCELCINVACTGC), and opossum guanylin (SHTCEICAYAACAGC) were
synthesized by the solid phase method on an PE Applied Biosystems model 431A peptide synthesizer (Foster City, CA) and
purified by reverse phase C18 chromatography as described
previously (1, 2).
Cell culture
T84 cells were cultured in DMEM and Ham’s F-12 medium (1:1)
supplemented with 5% FBS, 60 mg penicillin, and 100 mg streptomycin/ml
as previously described (2). Opossum kidney (OK-E) cells were cultured
in DMEM and Ham’s F-12 medium (1:1) with 5% FBS as previously
described (5).
cGMP accumulation bioassay
T84 or OK-E cells were cultured in 24-well plastic dishes, and
cGMP levels were measured in control and agonist-stimulated cells by
RIA (2, 5). Synthetic peptides were dissolved in 200 µl DMEM
containing 20 mM HEPES and 1 mM
isobutylmethylxanthine at pH 7.4. Cells were washed twice with DMEM
before the addition of 200 µl DMEM containing the agonist peptides or
vehicle to T84 or OK-E cells followed by incubation at 37 C for 40 min.
After incubation, the reaction medium was aspirated, cells were washed
with PBS, and 0.2 ml 3.3% perchloric acid was added per well to
extract cGMP. The extract was neutralized and used to measure cGMP by
RIA (2, 5).
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Results |
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). However,
the transcripts in spleen and testis that hybridize with this
uroguanylin cDNA are larger than the intestinal transcript. These
larger mRNAs also hybridized with a guanylin cDNA probe (data not
shown). Then, RNA was isolated from spleen to make cDNAs, and
oligonucleotide primers were prepared that anneal to uroguanylin cDNAs
for amplification by the PCR of a 287-bp cDNA product (16). This cDNA
was cloned, and nucleotide sequence analyses revealed that the spleen
cDNA was related to uroguanylin and guanylin cDNAs, but this mRNA in
the spleen was clearly derived from a novel guanylin-like gene. Because
this spleen cDNA did not contain the COOH-terminus of the precursor
polypeptide, which encodes the bioactive peptide domain, a different
3'-primer was prepared for PCR cloning (12, 16). This primer anneals to
a region of the 3'-untranslated domain distal to the stop codon in the
uroguanylin cDNAs. The 5'-primer used in all subsequent PCR-based
cloning experiments anneals within the 5'-untranslated region flanking
the initiation codon of the open reading frame of uroguanylin cDNAs
(12, 16). Using spleen-derived cDNA templates and these primers, a
385-bp cDNA was amplified by PCR and isolated by molecular cloning
(Fig. 2A). Sequence analyses of multiple
independent cDNA clones from several different animals revealed that
this cDNA is 92.7% identical to the corresponding nucleotide sequences
for preprouroguanylin reported previously (12). The nucleotide and
deduced amino acid sequences of the protein demonstrate that the
uroguanylin/guanylin-like mRNA transcripts in spleen are derived from a
new gene in the guanylin family. The open reading frame within the
spleen-derived cDNA encodes a 109-amino acid polypeptide that is 84%
identical to preprouroguanylin and 40% identical to preproguanylin.
The gene and its protein product were named lymphoguanylin because the
cDNA was isolated from spleen mRNA/cDNA and the polypeptide belongs to
the guanylin peptide family.
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). Illustrated for comparison are the primary
structures of the opossum forms of lymphoguanylin, uroguanylin,
guanylin, and an Escherichia coli ST peptide. A major
difference within the structure of lymphoguanylin is the tyrosine 109
residue found at the COOH-terminus of this peptide. All of the guanylin
and uroguanylin peptides identified to date have a cysteine at this
position (1, 2, 8, 9, 10, 11, 12, 13, 21, 22). The disulfide bonds formed between the
first and third cysteines and linking the second to fourth cysteines in
the peptide chain are considered to be required for optimal biological
activity in the guanylin and uroguanylin peptides (1, 2). The
replacement of cysteine 109 with the tyrosine 109 residue represents a
unique molecular structure within the guanylin family of bioactive
peptides (1, 2). Lymphoguanylin is uroguanylin-like because it
has two glutamate residues in the NH2-terminal domain
of this peptide. Opossum uroguanylin has glutamate and aspartate
residues, and all other uroguanylin molecules in mammalian species have
acidic residues at these positions (11, 12, 13, 14, 15, 21). Shared between
lymphoguanylin and uroguanylin is an internal asparagine residue, which
is also found in the bacterial ST peptides (2, 21). Guanylin peptides
have an aromatic amino acid at this position, which makes guanylin
sensitive to hydrolysis and inactivation by chymotrypsin (23, 24). The
third difference is the methionine 104 substitution in lymphoguanylin
for the valine 104 of uroguanylin. The 23-residue signal peptide of
preprouroguanylin is 87% identical to a corresponding peptide domain
at the NH2-terminus of preprolymphoguanylin (24). Near the
NH2-termini of opossum prolymphoguanylin, prouroguanylin,
and proguanylin polypeptides are highly conserved domains consisting of
the peptide sequence L(D/E)SVKKL (12, 16). A biological function for
this portion of the prohormones has not yet been elucidated.
A lymphoguanylin peptide (Q95EECELCINMACTGY109)
was synthesized and then oxidized to form a disulfide bond between
cysteine 98 and cysteine 106. The cysteine 100 residue was protected so
that a single intramolecular disulfide would result from oxidation of
this peptide. The crude peptide was active in the T84 cell cGMP
accumulation bioassay; thus, we partially purified the synthetic
lymphoguanylin by reverse phase HPLC (RP-HPLC) using this bioassay to
detect the active peptide. Bioactive lymphoguanylin eluted at
approximately 25% acetonitrile, whereas uroguanylin and guanylin
eluted at about 20–21% acetonitrile under these RP-HPLC conditions
(Fig. 3A). About 360 µg active
lymphoguanylin were isolated from about 3 mg peptide. The
RP-HPLC-purified lymphoguanylin stimulates cGMP production in human T84
intestinal cells, but its apparent potency is less than that of either
uroguanylin or guanylin (Fig. 3B). Concentration-response curves for
these peptide agonists suggest that all three peptides are full
agonists in the stimulation of the intestinal form of GC receptors
expressed in T84 cells (1, 2). Although lymphoguanylin was less potent
than either uroguanylin or guanylin, this novel peptide stimulated cGMP
accumulation in T84 cells greater than 300-fold. When the potencies of
these peptides were examined in an OK cell line, we observed that GC
receptors in kidney cells were also activated by lymphoguanylin (Fig. 3C). The rank order of peptide agonist potencies remained similar
(uroguanylin > guanylin > lymphoguanylin), but 100
µM lymphoguanylin stimulated cGMP accumulation by only
about 5-fold over the basal level in kidney cells. In contrast, 10
µM uroguanylin increased cGMP by 206-fold, and 10
µM guanylin increased cGMP by 88-fold over the basal
level in OK cells.
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). Tissues with the most abundant
lymphoguanylin mRNAs of about 1.6 kb are atria and ventricles of heart
and kidney cortex. The lymphoguanylin cDNA also hybridizes with an
approximately 1.2-kb uroguanylin mRNA in mucosae from the duodenum,
jejunum, and ileum of small intestine and the cecum and colon of large
intestine and in esophagus (Fig. 4B). We have also detected about
1.6-kb mRNAs for lymphoguanylin using Northern assays in RNA from
spleen, thymus, and testis (Fig. 1 and data not shown). When total RNA
from either lymphoid tissues or brain were tested in Northern assays,
we often observed that lymphoguanylin mRNAs are either undetectable or
marginally detectable using this relatively insensitive method. Spleen
appears to have the most abundant levels of lymphoguanylin mRNA within
tissues of the lymphoid/immune system when examined by Northern assays
(data not shown). Because of the lower expression levels of
lymphoguanylin mRNA transcripts in these tissues, RT-PCR was used to
amplify the mRNA-cDNAs prepared from spleen, thymus, lymph nodes,
circulating white blood cells, bone marrow, cerebellum, and testis.
Lymphoguanylin cDNAs of the anticipated size were detected by ethidium
bromide and confirmed by Southern blots using a
[32P]lymphoguanylin cDNA (Fig. 4C). These cDNAs were then
isolated by molecular cloning, and nucleotide sequence analyses
revealed that the cDNA clones derived from RNA in the thymus, lymph
nodes, circulating white blood cells, bone marrow, spleen, cerebellum,
kidney, OK cells, testis, ovary, and heart are identical to the 385-bp
lymphoguanylin cDNAs that were first isolated from the spleen and shown
in Fig. 2A. In addition to these lymphoguanylin cDNAs, we also isolated
and sequenced uroguanylin cDNA clones when RNA from the kidney and
heart was reverse transcribed and used as template in the PCR (data not
shown).
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Discussion |
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E. coli ST peptides have six cysteines and three intramolecular disulfide bonds that are important determinants of biological potency (26). These disulfide pairings in STs provide a peptide conformation with the greatest potency. Isolation of guanylin and uroguanylin containing four cysteines and two disulfides identified ST-like endogenous mammalian peptides (1, 2). Two disulfides in guanylin and uroguanylin produce molecular conformations that elicit optimal bioactivity (1, 2, 25). Thus, it was surprising to find that synthetic lymphoguanylin containing a single disulfide also activates intestinal and renal GC receptors. The molecular conformation elicited by a single disulfide may contribute to the reduced potency of lymphoguanylin. However, this form of lymphoguanylin may not accurately reflect the bioactive lymphoguanylin in vivo because this peptide was identified by cDNA cloning, whereas guanylin and uroguanylin were isolated as bioactive peptides from intestinal mucosa and urine (1, 2).
In the intestine, uroguanylin and guanylin stimulate chloride and bicarbonate secretion via local release of these enteric peptides into the intestinal lumen (1, 2, 17, 18). Through this paracrine mechanism, guanylin and uroguanylin regulate intestinal fluid secretion and also neutralize strong acids within the intestinal lumen by controlling bicarbonate secretion during digestion. Another physiological role for uroguanylin occurs in an endocrine link between the intestine and kidney via circulating uroguanylin and prouroguanylin to regulate urinary NaCl excretion (12, 27). A natriuretic factor such as uroguanylin was predicted to exist in the digestive system as a mechanism to regulate salt balance (28). Consistent with this endocrine axis is the finding that high salt diets stimulate uroguanylin and sodium excretion in the urine (29). Plasma uroguanylin is delivered in the glomerular filtrate to GC receptors located on apical surfaces of renal tubular cells (5, 30). Uroguanylin, guanylin, or E. coli ST are potent natriuretic, kaliuretic, and diuretic peptides in both the perfused rat kidney and in mice in vivo, indicating that renal tubular GC receptors are accessible to circulating peptides (19, 20, 31). An intrarenal paracrine mechanism for uroguanylin may also exist, because uroguanylin mRNAs are expressed in the kidney (8, 13, 22). A different physiological role for lymphoguanylin in the kidney is implied because of the high renal mRNA expression of lymphoguanylin coupled with a relatively low potency and efficacy of this peptide as an agonist at renal GC receptors. Accordingly, a novel receptor for lymphoguanylin could be present in the kidney and/or other tissues that produce lymphoguanylin mRNA transcripts.
A communication pathway for lymphoguanylin may also exist between cells within the lymphoid/immune system. It was previously reported that a rat basophilic leukemia cell responds to E. coli ST with increases in cGMP and histamine release (32, 33). Moreover, administration of E. coli ST to mice modulates the immune response to treatment with sheep red blood cells (34). In recent studies, we isolated a cDNA that encodes the catalytic domain of a GC receptor for guanylin peptides, and the mRNA encoding this GC receptor is expressed in lymphoid as well as other tissues (16). This GC receptor in cells within the lymphoid/immune system may serve as a target for regulation of intracellular cGMP by either lymphoguanylin or other guanylin peptides that are produced locally in lymphoid tissues.
Isolation by molecular cloning of lymphoguanylin cDNAs from mRNA transcripts expressed in tissues within the lymphoid/immune, cardiovascular/renal, reproductive, and central nervous organ systems reveals that three different genes encode lymphoguanylin, guanylin, and uroguanylin (2, 12, 16). It is possible that a homologous lymphoguanylin gene is present in the genomes of all mammals. On the other hand, the lymphoguanylin gene could be unique to Marsupialia (metatherian mammals) and may not have evolved in Eutheria (placental mammals). Guanylin and uroguanylin genes were derived from a common ancestral gene early in mammalian evolution because both genes are present in eutherian and metatherian mammals (1, 2, 8, 9, 10, 11, 12, 13, 14, 15, 16). Guanylin and uroguanylin genes are located close together on mouse chromosome 4 and human chromosome 1, suggesting their formation by gene duplication (35, 36). The primary structure of opossum preproguanylin shares only 40% identity with preprouroguanylin (12, 16). Preprouroguanylin and preprolymphoguanylin are 92.7% and 84% identical at nucleotide and amino acid sequence levels, respectively, which is consistent with a more recent gene duplication event. Whether the lymphoguanylin gene was present in mammalian ancestors of extant marsupial and placental mammals is an important question. Vertebrate ancestors of Eutheria and Marsupialia lived more than 130 million yr ago; thus, the lymphoguanylin gene could have arisen after the evolutionary divergence of placental and marsupial mammals (37). However, a long form of an uroguanylin-like mRNA transcript found in the testis of rats is an intriguing candidate mRNA encoding a putative eutherian homolog of lymphoguanylin (13).
In conclusion, three different guanylin regulatory peptides together with their GC receptors provide a novel signal transduction pathway for cGMP-mediated regulation of cellular function. Discovery of lymphoguanylin coupled with recent evidence that additional subtypes of membrane GC receptors occur in the intestine suggest that signaling mechanisms for guanylin peptides is complex (14, 16, 38). Novel biological actions of these peptides are emerging with the demonstration that uroguanylin modulates pulmonary function in guinea pigs and that human airway cells have guanylin-regulated chloride conductances (39, 40). Identification of lymphoguanylin offers the intriguing possibility of finding a fourth guanylin-like gene and bioactive peptide in opossums. Uroguanylin and lymphoguanylin were both identified in experiments with opossums (2, 5). It is likely that additional insights into the cell and molecular biology of guanylin regulatory peptides will be provided using this novel experimental animal.
Received September 29, 1998.
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