Phosphorylation and activation of the intestinal guanylyl cyclase receptor for Escherichia coli heat-stable toxin by protein kinase C

The heat-stable enterotoxin STa of E. coli causes diarrhea by binding to and stimulating intestinal membrane-bound guanylyl cyclase, triggering production of cyclic GMP. Agents which stimulate protein kinase C (PKC), including phorbol esters, synergistically enhance STa effects on cGMP and secretion. We investigated whether PKC causes phosphorylation of the STa receptor in vivo and in vitro.Immunoprecipitation of the STa receptor-guanylyl cyclase was carried out from extracts of T84 colon cells metabolically labelled with [³²P]-phosphate using polyclonal anti-STa receptor antibody. The STa receptor was phosphorylated in its basal state, and ³²P content in the 150 kDa holoreceptor band increased 2-fold in cells exposed to phorbol ester for 1 h. In vitro, immunopurified STa receptor was readily phosphorylated by purified rat brain PKC. Phosphorylation was inhibited 40% by 5 M of a synthetic peptide corresponding to the sequence around Ser¹⁰²⁹ of the STa receptor, a site previously proposed as a potential PKC phosphorylation site. Treatment of the immunopurified STaR/GC with purified PKC increased STa-stimulated guanylyl cyclase activity 2-fold. We conclude that PKC phosphorylates and activates the STa receptor/guanylyl cyclase in vitro and in vivo; Ser¹⁰²⁹ of the STaR/GC remains a candidate phosphorylation site by PKC.Abbreviations STa the heat-stable enterotoxin of E. coli, which has also been called ST-I and STp. The 18 amino acid variant was used throughout - PBS phosphate-buffered saline - PDB 4--12, 13-phorbol dibutyrate - ANP atrial natriuretic peptide - STaR/GC STa receptor/guanylyl cyclase, also called GC-C - PKC protein kinase C     

Functional Properties of Pro Region of Escherichia coli Heat-Stable Enterotoxin

Escherichia coli heat-stable enterotoxin Ip (STp) is synthesized as the 72-amino-acid residue precursor consisting of three regions: pre region (amino acid residues 1 to 19), pro region (amino acid residues 20 to 54), and mature ST (mST) region (amino acid residues 55 to 72). We examined the role of the pro sequence of STp in enterotoxigenicity of a strain by deleting the gene fragment encoding amino acids 22 to 57. This deletion caused a remarkable reduction of its enterotoxic activity of culture supernatant. In order to analyze the sequence responsible for the function of the pro region, two additional deletion mutants were made. The deletion of the sequence covering amino acids 29 to 38, which is conserved in all sequences of ST reported, brought about a significant reduction of enterotoxic activity but the deletion of the non-conserved sequence (amino acids 40 to 53) did not. This result shows that conserved sequence is mainly responsible for the function. Subsequently, to examine the mechanism of action of the pro region, plasmids carrying DNA sequences of hybrid proteins consisting of pre-pro-nuclease, pre-mST-nuclease, pre-pro-mST-nuclease and pre-pro-nuclease-mST were constructed. Amino acid sequence determination and SDS-polyacrylamide gel analysis revealed that these fusion proteins were cleaved between pre sequence and pro sequence during secretion and the cleaved fusion proteins were accumulated in periplasmic space. But the amount of hybrid protein accumulated in the periplasmic space varied among the strains. That is, the amount of the pre-pro-nuclease gene product that accumulated in the periplasmic space was the highest of all fusion gene products. These results indicate that the existence of the mST region strongly interferes with the translocation of the gene product into the periplasmic space and that the pro region functions to guide the mST region into the periplasmic space.     

cDNA cloning of the hydroxysteroid sulfotransferase STa sharing a strong homology in amino acid sequence with the senescence marker protein SMP-2 in rat livers

A cDNA encoding hydroxysteroid sulfotransferase a (STa), which catalyzes activation of carcinogenic polycyclic hydroxymethyl-arenes, was isolated from a lambda gtll cDNA expression library constructed from poly(A)+RNA of a female Sprague-Dawley (SD) rat liver. The cDNA, designated as ST-40, consisted of 1,015 base pairs which had an open reading frame of 852 base pairs encoding the entire rat STa subunit of 284 amino acids. The nucleotide base sequence of the ST-40 cDNA shared a strong homology of 94.4% with that of ST-20 cDNA encoding a hydroxysteroid ST which had been reported by us. The deduced amino acid sequence of STa had a homology of 73.7% with that of an SD rat liver senescence marker protein (SMP-2) consisting of 282 amino acid residues. However, STa was found to share a much stronger homology of 92% on the average with SMP-2 in their four specific regions corresponding to about 60% of the total sequences, indicating SMP-2 to be an isozyme of hydroxysteroid ST.     

The characterization and purification of a human transcription factor modulating the glutathione peroxidase gene in response to oxygen tension

Guanylyl cyclase C (GC-C) was found to function as the principal receptor for heat-stable enterotoxins (STa), major causative factors in E. coli-induced secretory diarrhea. GC-C is enriched in intestinal epithelium, but was also detected in other epithelial tissues. The enzyme belongs to the family of receptor guanylyl cyclases, and consists of an extracellular receptor domain, a single transmembrane domain, a kinase homology domain, and a catalytic domain. GC-C is modified by N-linked glycosylation and, at least in the small intestine, by proteolysis, resulting in a STa receptor that is coupled non-covalently to the intracellular domain. So far two endogenous ligands of mammalian GC-C have been identified i.e. the small cysteine-rich peptides guanylin and uroguanylin. The guanylins are released in an auto- or paracrine fashion into the intestinal lumen but may also function as endocrine hormones in gut-kidney communication and as regulators of ion transport in extra-intestinal epithelia. They are thought to activate GC-C by inducing a conformational change in the extracellular portion of the homotrimeric GC-C complex, which allows two of the three intracellular catalytic domains to dimerize and form two active catalytic clefts. In the intestine, activation of GC-C results in a dual action: stimulation of Cl and HCO₃ secretion, through the opening of apical CFTR Cl channels; and inhibition of Na absorption, through blockade of an apical Na/H exchanger. The principal effector of the GC-C effect on ion transport is cGMP dependent protein kinase type II, which together with GC-C and the ion transporters, may form a supramolecular complex at the apical border of epithelial cells.     

cDNA cloning of human calpastatin: sequence homology among human, pig, and rabbit calpastatins

cDNA of human calpastatin, an inhibitor protein specific for calpain (EC 3.4.22.17; Ca2(+)-dependent cysteine proteinase) was isolated by screening of a library prepared from human liver mRNA with pig calpastatin cDNA fragment as a probe. The primary structure of human calpastatin was deduced from the nucleotide sequence of the cDNA and compared with that of pig and rabbit calpastatins already reported. Human calpastatin consisted of 673 amino acid residues and had 78% and 77% identity to pig or rabbit calpastatins, respectively. Human calpastatin had a domain structure with four internally repetitive sequences and one N-terminal non-homologous sequence like the other calpastatins. Human calpastatin had two deletions, 22 and 13 residues long in domain L and domain 1, respectively, compared to pig or rabbit calpastatins.     

Molecular cloning of guinea pig angiotensin type 1 receptor

The primary structure of cDNA encoding of the angiotensin type 1 receptor (AT(1)R) was cloned from guinea pig liver. Guinea pig AT(1)R (GP-AT(1)R) cDNA clone contains a 1,077-bp open reading frame which encodes a protein consisting of 359 amino acid residues. GP-AT(1)R amino acid sequence showed a 92% level of identity among mammalian species. GP-AT(1)R is expressed in liver, kidney, adrenal gland, heart and colon.     

Isolation and expression of a guanylate cyclase-coupled heat stable enterotoxin receptor cDNA from a human colonic cell line.

Heat stable enterotoxins (STs) are low molecular-weight peptides secreted by enterotoxigenic bacteria. One type of these enterotoxins (STa) induces intestinal secretion leading to acute diarrhea by binding to a membrane form of guanylate cyclase. We have isolated a cDNA from a human colonic cell line, T84, encoding for a guanylate cyclase-coupled enterotoxin receptor (STaR). The predicted amino acid sequence of the human STa receptor is 81% identical with the previously cloned enterotoxin receptor (GC-C) from rat intestine. COS-7 cells transiently transfected with the cloned cDNA expressed specific concentration-dependent response to STa as measured by cyclic GMP accumulation and is about 20 times more sensitive to the stimulation by STa than has been shown for GC-C.     

Molecular cloning and sequence analysis of cDNA encoding human kidney D-amino acid oxidase

cDNA clones encoding D-amino acid oxidase were isolated from a human kidney cDNA library by hybridization with cDNA for the pig enzyme. The cDNA insert of 2.0 kilobase pairs long provided coding information for a protein consisting of 347 amino acids. The molecular mass of the enzyme was calculated to be 39,410 Da. The amino acid sequence similarity between the pig and human enzymes is 84.4%, and among the active site residues proposed from chemical modification studies, methionine-110 of the pig enzyme was replaced by threonine. Northern blot analysis confirmed the expression of an mRNA of 2.0 kilobases encoding the D-amino acid oxidase in human kidney.     

Demonstration of apolipoprotein CII in guinea pigs. Functional characteristics, cDNA sequence, and tissue expression

In contrast to plasma from other mammals, guinea pig plasma does not stimulate the activity of lipoprotein lipases in vitro. This had led previously to the conclusion that guinea pigs lack an analogue to apolipoprotein CII (apoCII). By adsorption of lipid-binding proteins to lipid droplets, thereby separating them from other plasma components, we could demonstrate apoCII-like activity in guinea pig plasma. On electrophoresis, the CII-like activity co-migrated with one isoform of guinea pig apolipoprotein CIII, identified by amino-terminal amino acid sequence determination (40 residues). By isoelectric focusing in a narrow pH gradient, the activating protein was separated sufficiently from the dominating apoCIII isoform to allow sequence determination of 8 residues from the amino terminus. Six of these were identical to corresponding residues in apoCII from dog and monkey. With the aid of a human apoCII cDNA probe we identified one cross-hybridizing mRNA species (approximately 600 nucleotides) on Northern blots of guinea pig liver. Three positive clones were isolated from a guinea pig liver cDNA library using the same cDNA probe. The nucleotide sequence showed extensive similarities to the previously known human, monkey, and canine sequences, but the signal peptide was 3 amino acid residues longer in the guinea pig protein, and there was a deletion of 4 residues in the putative lipid binding domain. Northern blot analyses indicated that guinea pig apoCII is mainly expressed in the liver with little or no contribution from the intestine.     

Prosequence switching: An effective strategy to produce biologically active E. coli heat-stable enterotoxin STh

Enterotoxigenic Escherichia coli (ETEC) infections account for the majority of cases of acute secretory diarrhea. The causative agents are enterotoxins secreted by ETEC, among them is the heat-stable enterotoxin, STh. STh is a 19-amino acid peptide containing three disulfide bonds that stimulates fluid secretion in the bowel by binding to the receptor domain of intestinal guanylyl cyclase C (GC-C). Since GC-C agonists have pharmacologic potential for diagnosis and treatment of disorders such as constipation-predominant irritable bowel syndrome (IBS-C), chronic constipation, and colorectal carcinoma, it is crucial to develop methods for the large-scale production of STh and related peptides. Here, we present a strategy for recombinant expression of STh that relies on the use of the prosequence of human uroguanylin to support proper folding and disulfide bond formation. The chimeric protein CysCys-STh consisting of the propeptide of uroguanylin as N-terminus and the STh peptide as C-terminus was expressed in E. coli, and an efficient purification protocol was developed. Trypsin digestion of this protein released the enterotoxin which could be obtained in high purity. NMR and mass spectrometry confirmed the identity and homogeneity of the toxin, and its biological activity was confirmed by a cell-based in vivo assay. The expression scheme introduced here represents a cost-efficient and scalable way of STh production.     

Biological activity of synthetic peptides analogous to heat-stable enterotoxin produced by Yersinia enterocolitica

Abstract 3 peptides were synthesized chemically by following the primary structure of heat-stable enterotoxin (ST) produced by Yersinia enterocolitica . A peptide 1–30, having the whole sequence of 30 amino-acid residues, showed a ST activity similar to that of analogue peptide 15–30 composed of the C-terminal 16 amino acid residues. The c-GMP levels of L cells increased through an interaction with peptide 1–30 but not with peptide 15–30, while membranes isolated from broken L cells responded to both. Peptide 1–11, composed of the N-terminal 11 amino-acid residues, showed no biological activity.     

Porcine pyridoxal kinase: c-DNA cloning, expression and confirmation of its primary sequence

Porcine brain pyridoxal kinase has been cloned. A 1.2 kilo-based cDNA with a 966-base pair open reading frame was determined from a porcine brain cortex cDNA library using PCR technique. The DNA sequence was shown to encode a protein of 322 amino acid residues with a molecular mass of 35.4 kDa. The amino acid sequence deduced from the nucleotide sequence of the cDNA was shown to match the partial primary sequence of pyridoxal kinase. Expression of the cloned cDNA in E. coli has produced a protein which displays both pyridoxal kinase activity and immunoreactivity with monoclonal antibodies raised against natural enzyme from porcine brain. With respect to the physical properties, it is shown that the recombinant protein exhibits identical kinetic parameters with the pure enzyme from porcine brain. Although the primary sequence of porcine pyridoxal kinase has been shown to share 87% homology with the human enzyme, we have shown that the porcine enzyme carries an extra peptide of ten amino acid residues at the N-terminal domain.     

Molecular Cloning and Chromosomal Localization of Human Salt-Tolerant Protein

We have isolated a novel human cDNA coding for human salt-tolerant protein (HSTP), that is a homologue of the rat salt-tolerant protein (STP) and may contribute to salt-induced hypertension by modulating renal cation transport. The nucleotide sequence (1988 bp) of the HSTP cDNA contains an open reading frame encoding a polypeptide comprising 545 amino acids, two residues fewer than the rat STP cDNA. The predicted amino acid sequence exhibits 92% identity to that of the rat protein. HSTP contains predicted coiled-coil domains and Src Homology 3 domain, and shows a high degree of identity to CIP4 (Cdc42 target protein) and human Trip 10 (thyroid-hormone receptor interacting protein). We have mapped the HSTPgene to human chromosome 19 by fluorescence in situhybridization. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cloning and Sequencing of the xynA Gene Encoding Xylanase A of Aspergillus kawachii

We have cloned the xynA gene coding for xylanase A, a major component of the xylanase family, from Aspergillus kawachii. The cDNA was isolated from an A. kawachii cDNA library by immunoscreening using antibody raised against the purified xylanase A protein. Nucleotide sequence analysis of the cDNA showed a 981-bp open reading frame that encoded a protein of 327 amino acid residues. The signal peptide was composed of 25 amino acid residues and the N-terminus of the mature protein was pyroglutamic acid. The transformed yeast with a cloned cDNA produced xylanase. The genomic DNA was arranged as ten exons and nine introns.     

The structure and expression of the guinea pig Fc receptor for IgG1 and IgG2 (Fc gamma 1/gamma 2R).

A cDNA clone encoding the receptor for guinea pig immunoglobulin G was isolated from a guinea pig peritoneal macrophage cDNA library. The cloned cDNA encoded 271 amino acids containing an N-terminal signal sequence. The deduced amino acid sequence is most homologous to murine Fc gamma RII beta 2. The receptor protein could be expressed in COS-7 and L cells transfected with the cDNA, suggesting that the expression of this receptor does not require the co-expression of a second chain such as gamma chain of Fc epsilon RI or CD3 zeta chain. The transformant L cells showed the binding to both the guinea pig IgG1 and IgG2 antibodies complexed with antigen, indicating that the cDNA we cloned was the one for guinea pig Fc gamma 1/gamma 2R.     

Hamster D-amino-acid oxidase cDNA: rodents lack the 27th amino acid residue in D-amino-acid oxidase

Summary.  The nucleotide sequence of cDNA that encodes hamster d-amino-acid oxidase (DAO) was determined. The cDNA consisted of 1,590 nucleotides and a poly(A) tail. It had an open reading frame for a protein consisting of 346 amino acid residues. The number of the amino acid residues is the same as that of the rat DAO. However, the hamster DAO has one residue more than mouse DAO and one residue less than human, pig, rabbit, and guinea pig DAOs. Amino acid sequence of the hamster DAO was highly similar to those of mouse and rat DAOs: 89% and 88% of the amino acid residues were identical between the hamster and mouse DAOs and between the hamster and rat DAOs, respectively. The homology was slightly less between the hamster DAO and the human (81%), pig (78%), rabbit (78%), or guinea pig DAO (82%). It has been proposed that the mouse and rat DAOs lack an amino acid residue corresponding to the 25th residue of the DAOs of other mammals. However, a detailed comparison of the amino acid sequences as well as the underlying nucleotide sequences by inclusion of the hamster ones revealed that the rodent DAOs does not lack the 25th, but the 27th residue. Received January 16, 2002 Accepted June 20, 2002 Published online November 14, 2002 Authors' address: Dr. Ryuichi Konno, Department of Microbiology, Dokkyo University School of Medicine, Mibu, Tochigi 321-0293, Japan, Fax: +81-282-86-5616     

Cloning and tissue distribution of Leptin mRNA in the pig∗

Abstract

The sequence of the pig ob cDNA, which codes for the protein leptin, has been determined by screening a pig adipose cDNA library with an RT‐PCR amplified cDNA fragment of this gene. The 501 bp ob cDNA has 89% identity to the human ob cDNA, 92% identity to the bovine ob cDNA, 84% identity to the mouse ob cDNA and 84% identity to the rat ob cDNA. At the amino acid level, pig leptin which codes for a protein with a predicted molecular weight of 18,661‐dalton, has 86% identity to human leptin, 93% identity to bovine leptin, 84% identity to rat leptin and 84% identity to mouse leptin. RT‐PCR screening of RNA isolated from pig adipose, skeletal muscle, cardiac muscle, pancreas, stomach, kidney, spleen and jejunum detected ob mRNA only in adipose tissue; Northern blots with an ob cDNA probe identified a 4.0 kb species in adipose tissue. The conservation of sequence and expression pattern of leptin in the pig reported here indicates that as in other species, this protein likely plays an important role in controlling food intake and fat deposition in the pig.     

Gene expression of D-amino acid oxidase in rabbit kidney

Although D-amino acid oxidase (DAO) [EC 1.4.3.3] activity in rabbit kidney extract was undetectable, protein immunoreactive toward rabbit anti-pig kidney DAO antiserum and RNAs that hybridized with fragments of human and pig DAO cDNAs were detected distinctly in the rabbit kidney. A cDNA clone, RD22, was isolated from the rabbit kidney cDNA library by hybridization with a fragment of human DAO cDNA. Analysis of the nucleotide sequence revealed a 2,018 nucleotide sequence encoding a protein consisted of 347 amino acids. The number of amino acid residues was identical with those of human and pig DAOs, and the amino acid sequence showed 80 and 83% identity with pig and human DAOs, respectively. RNAs that hybridized with RD22 DNA fragment also existed in rabbit kidney, and their sizes were the same as those of the RNAs detected with the human and pig DAO cDNA fragments. RD22-derived protein was hardly synthesized by an in vitro expression system. However, a cDNA fragment lacking most of the 5'-untranslated region and its mutants containing base changes around the initiation codon did direct protein synthesis. Moreover, the protein derived from the partial cDNA fragment containing a large part of the coding region sequence showed immunoreactivity toward anti-pig DAO antiserum. The results suggest that one of the causes of the very poor synthesis of DAO protein in rabbit kidney is translational suppression in the synthetic process.     

Cloning and sequence analysis of a cDNA encoding human non-selective type of endothelin receptor.

A cDNA encoding non-selective type (ETB) of endothelin receptor was isolated from a human liver cDNA library. The cDNA had an open reading frame encoding a protein of 442 amino acid residues with a relative Mr of 49,643. The deduced amino acid sequence of human ETB receptor was 88% and 64% identical to those of rat lung ETB receptor and bovine lung ET-1-specific (ETA) receptor, respectively, and contained a relatively long and proline-rich extracellular N-terminal region in addition to a significant similarity with the G protein-coupled receptor super-family with seven transmembrane segments.