(Received for publication, May 5, 1995)
From the
Antisecretory factor (AF) is a protein known to inhibit
intestinal fluid secretion induced by cholera toxin. cDNA clones,
expressing immunoreactivity to AF were isolated from a human pituitary
gland library and sequenced. The sequence contained 1309 base pairs
plus a poly(A) tail; Northern blot analysis of pituitary RNA confirmed
this size. One large open reading frame was found to code for 382 amino
acids. The protein was expressed in pGEX-1T/Escherichia coli and purified. The recombinant AF was extremely potent, 9 ng
(2
10
mol), giving a significant antisecretory
activity against cholera toxin-induced fluid secretion in rat.
Antiserum against recombinant AF was used in immunohistochemical and
Western blot analysis. Sections from human pituitary glands manifested
specific intracellular staining in cells exclusively located in the
anterior part. Both recombinant AF and AF extracted from pituitary
gland appeared in SDS-polyacrylamide to have a molecular mass of 60
kDa, although the real value was 41 kDa. The protein sequence
manifested homology (29% identity) with one protein, a putative Saccharomyces cerevisiae 30-kDa protein of unknown function.
A large number of regulatory peptides occur both in the gastrointestinal tract and in the central nervous system where they are predominantly confined to the nerves themselves or to endocrine cells (1, 2) . Some of these peptides affect the transport of water and electrolytes across the intestinal mucosa. Thus, vasoactive intestinal polypeptide and substance P stimulate ion secretion from the serosal to the mucosal side of the epithelium(3) . Somatostatin, on the other hand, enhances absorbtion of ion and water in the gut, a property which has been used therapeutically for reducing the stool output during acute diarrhea(4, 5) .
During cholera diarrhea, a
pronounced fluid and electrolyte output takes place, due to the action
of the potent cholera toxin on the permeability of the intestinal
mucosa(4) . As a result, a protein is formed that counteracts
the permeability changes by the toxin. This protein, named
antisecretory factor (AF), ()seems mainly to be formed in
the pituitary gland and transported with the blood to the
gut(6) . AF appear to have a molecular mass about 60 kDa and an
isoelectric point of 5.0; 10
mol of AF is
sufficient to abolish intestinal secretion in a rat exposed to cholera
toxin, and 10
mol reverses intestinal secretion in
pigs exposed to cholera toxin or Escherichia coli heat-labile
toxin(7) . The action is not species specific, as AF from man
or pig exerts activity in rat. Also, other enterotoxins than cholera
toxin, such as those from Campylobacter jejuni, Clostridium
difficile, E. coli, and Dinophysis, are inhibited by
AF(7) . Moreover, the protein is a potent blocker of chloride
ion transport across nerve cell membranes, a property which might be
involved in its inhibitory effects on intestinal secretion(8) .
In the present work, we describe the cloning and sequencing of cDNA coding for a protein with the same immunoreactivity as AF. The coding sequence is expressed in E. coli, and the resulting fusion protein is isolated and characterized. Antibodies against the recombinant AF were produced in rabbits and used in immunohistochemical studies.
Figure 1: Nuclear acid sequence and deduced amino acid sequence of the new human protein. The confirmed amino acid sequence is underlined. Bottom, horizontal map showing cloned cDNA and oligonucleotide primers.
Protein expression was induced
by 0.1 mM isopropyl--D-thiogalactopyranoside,
and after a further 4 h of growth at 30 °C, the cells were pelleted
and resuspended in PBS. Cells were lysed by sonication, treated with 1%
Triton X-100, and centrifuged at 12,000
g for 10 min;
the supernatant containing the expressed fusion proteins was purified
by passing the lysates through glutathione-agarose (Pharmacia). The
fusion proteins were either eluted by competition with free glutathione
or were cleaved overnight with 10 units of bovine thrombin to remove
the AF protein from the GST affinity tail.
The AF clones 1, 2, and 3 were ligated into the pGEX
1T plasmid vector so that the ORF was in frame with the GST
protein. The constructs were transformed into E. coli, and
expression of fusion proteins was induced with
isopropyl-
-D-thiogalactopyranoside. The purified fusion
proteins and the thrombin-cleaved AF protein were subjected to SDS-PAGE
and Western blotting using antiserum against porcine antisecretory
factor (Fig. 2). Coomassie Brilliant Blue staining of the
proteins revealed discrete bands for each protein except for the
GST-AF-1 protein, which manifested degradation into smaller components.
However, in the Western blot analyses, the full-length protein gave a
much stronger signal than the degradated products (Fig. 2B). The strong reaction with the antiserum
against porcine AF indicated that the recombinant proteins indeed have
the same immunoreactivity as AF. The molecular mass of the full-length
protein appeared to be about 60 kDa, which is higher than the true
molecular mass of 41,139 Da estimated from the amino acid composition.
Furthermore, the proteins were also immunoblotted and probed with
antiserum raised against GST-AF-2, which bound to the thrombin-cleaved
proteins (Fig. 3).
Figure 2: Coomassie Brilliant Blue-stained SDS-polyacrylamide mini-gel (A) and immunoblot probed with antisera against porcine AF (B). Lanes with unprimednumbers contain glutathione-agarose-purified GST-AF fusion proteins AF-1, AF-2, and AF-3, whereas lanes with primednumbers contain the fusion proteins cleaved with thrombin. Molecular weight references (R) are indicated on the left. The GST-AF-1 fusion protein is highly degraded, but the immunoblot analysis shows only the detection of a full-length protein and spontaneous thrombin cleavage product. There is a 26-kDa product in the GST-AF-3 protein, probably the glutathione S-transferase tail that has been independently expressed.
Figure 3: Western blot using antiserum against recombinant protein AF-2. To the left, porcine (P) and three human (H1, H2, H3) pituitary glands were applied; to the right, the three recombinant proteins AF-1, AF-2, and AF-3 (see Fig. 2) were applied; in the center is the molecular weight standard (R).
Protein sequence analysis was performed to further validate the identified ORF. Attempts to obtain the N-terminal protein sequence of clone 1 were unsuccessful, probably because of chemical blocking of the N-terminal end. However, the N-terminal sequences of clone 2 and clone 3 were determined and were shown to perfectly match amino acids 63-75 and 130-140, respectively, of the predicted sequence (Fig. 1).
Figure 4:
Autoradiogram of Northern blots of RNA
from a human and porcine pituitary gland. 5 µg of purified mRNA was
applied in each basin; 3`-end-labeled P oligonucleotide
probes were used, and the autoradiogram was developed after 7
days.
Antiserum against recombinant GST-AF-2 reacted with the naturally occurring AF protein of an apparent molecular mass of 60 kDa and with some smaller components, probably enzymatic degradation products (Fig. 3).
The distribution of AF in sections of human pituitary glands was studied with immunohistochemical techniques (Fig. 5). In all specimens investigated, a moderate number of cells in the adenohypophysis were stained; the immunostained material appeared to be located in granules in the cytoplasm; preabsorption of the immune serum with an excess of GST-AF-2 protein abolished the signal. No staining was observed in the posterior part (neurohypophysis).
Figure 5: Cryosections of adenohypophysis stained with antiserum against recombinant protein GST-AF-2. A, sections incubated with immune serum showing scattered cells with varying degrees of positive immunoreactivity (solidarrows). Many cells completely lack staining (openarrows). B, serial sections to A incubated with immune serum preabsorbed with excess of recombinant protein GST-AF-2. There is no specific staining of the cells. C and D, larger magnification of immunopositive cells demonstrating cytoplasmatic staining of the endocrine cells. n, nucleus; c, cytoplasma. Bars in A and B = 100 µm, bars in C and D = 10 µm.
Figure 6: Biological activity of recombinant protein AF-1 testing inhibition of cholera toxin-induced fluid secretion. Graded doses of the protein were injected intravenously in rat; 3 µg of cholera toxin was injected into an intestinal loop; after 5 h, the accumulated fluid (mg/cm intestine) in the loop was measured. Each value represents the mean ± S.E. of a group of six animals.
The nucleotide sequence of the cloned human pituitary cDNA contained a large open reading frame, which coded for a 382-residue protein. The recombinant protein manifested similar immunoreactivity, localization, migration in SDS-PAGE, isoelectric point, and biological activity as the previously characterized AF(7) . Accordingly, the sequenced protein is in all probability identical with the previously characterized AF.
The sequenced AF resembles no other mammalian protein, but portions of the coding DNA sequence were almost identical to some expressed sequence tags originating from human tissues. The observed lack of homology in the 3`-end of eight of them is probably due to errors arising from automatic sequence determination, and all of them might originate from the AF transcript. The homology of the yeast cDNA is noteworthy since it begins in the start codon of the open reading frame coding for a hypothetical 30-kDa protein(21) . Hopefully, it will be possible to express this protein and compare its biological effect with that of AF.
The terminal parts of the AF protein are basic, the calculated pI of the 100 first and 50 last residues being 9.2 and 12.2, respectively (calculated with the MacVector 4.1 software). The middle part of AF is predominantly acidic, resulting in a pI of the entire protein of 4.9. All of the recombinant proteins migrated abnormally slowly in SDS-PAGE, resulting in an overestimation of their molecular mass. This phenomenon may have been due to the extremely positive charge of the C-terminal combined with a large number of proline residues, which might obstruct the loading with negative SDS molecules. The first 10 residues of the protein appear to be relatively hydrophobic when analyzed according to Kyte-Doolittle (22) and might constitute a signal peptide, which is cleaved out prior to exocytosis of the protein. This interpretation is supported by the Western blot analyses (Fig. 3) in which the recombinant protein appeared to have a slightly higher molecular mass than the protein extracted from pituitary gland. Some of this difference, however, might also be due to the additional five amino acids in the recombinant protein constituting the thrombin cleavage site of the fusion protein.
The biological activity of recombinant AF was similar to naturally occurring AF, and the potency is greater than that of any enterotoxin known to us and greater than that of any intestinal hormone or neuropeptide modifying water and electrolyte transport. Moreover, the level of activity of human AF in rat is surprisingly high, which probably reflects a ubiquitous structure conserved in AF molecules from different species. This hypothesis is supported by the cross-reactivity between human and porcine material obtained in the Western blot and Northern blot analyses. It remains to be seen whether the 30-kDa protein from yeast also show cross-reactivity.
The distribution of immunoreactive material in the pituitary gland demonstrated solely intracellular distribution of AF in secreting cells of the anterior lobe (adenohypophysis). The proteins emanating from this lobe include growth hormone, thyrotropin, corticotropin, prolactin, and luteinizing hormone. The passage of these hormones from intracellular localization to the vascular system is triggered by releasing factors produced by neuroendocrinic cells in the hypothalamus. The secretion of AF into the blood is stress-sensitive (7) , indicating an involvement of hypothalamus and regulation by releasing factors. As protein kinases probably mediate this signal-transmission, it is noteworthy that residues 321-325, 370-373, and 364-367 of the protein (Fig. 1) constitute possible sites for phosphorylation by tyrosine, cyclic AMP-dependent, and calcium-dependent protein kinases, respectively(23- 25). Intestinal challenge with cholera toxin enhances the level of active AF in the pituitary gland(7) . The signal for this induction is probably triggered in the intestinal mucosa by a nerve reflex, which is transmitted to the central nervous system and the pituitary gland via the vagus nerve(7, 18) .
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank[GenBank].