Presence of immunoreactive salusin-alpha in human serum and urine

Salusins, identified from a full-length enriched human cDNA library by bioinformatics analyses, show mitogenic, neuromodulatory and hemodynamic activities in rats. They are expressed in a wide variety of human tissues, but their precise structures and levels in human body fluids remain unknown. We developed a radioimmunoassay suitable for the detection of immunoreactive human salusin-alpha and characterized the molecular forms and concentrations of salusin-alpha in human serum and urine. The assay allowed for measurement of immunoreactive salusin-alpha concentrations as low as 1 fmol/tube after extraction of serum with an octyl-silica column, and the concentration required for 50% inhibition of binding was 40 fmol/tube. Cross-reactivities with salusin-beta and other bioactive peptides were negligible. Salusin-alpha-like immunoreactivity in normal human serum and urine ranged from 11.0 to 40.4 pmol/l (mean+/-S.D., 23.3+/-8.1 pmol/l, n=31) and from 18.6 to 367.3 pmol/l (mean+/-S.D., 156.8+/-95.8 pmol/l), respectively. Reverse-phase high performance liquid chromatography coupled with radioimmunoassay detection revealed a major immunoreactive component that coeluted with authentic salusin-alpha. These data indicate the presence of salusin-alpha in human serum and urine, thereby verifying the initially predicted processing sites for salusin-alpha in humans.     

Salusins promote cardiomyocyte growth but does not affect cardiac function in rats

Salusin-alpha and -beta are newly found polypeptides that stimulate proliferation, hypotension and bradycardia in vascular smooth muscle cells (VSMCs) and fibroblasts. Propresalusin mRNA is widespread, and positive stains for salusins have been observed in many human tissues such as endothelium and ventricular tissue. To investigate the bio-effect of salusins on cardiovascular function, 20 nmol/kg salusin-alpha or 2 nmol/kg salusin-beta was intravenously (i.v.) injected into rats, and isolated rat hearts were perfused with 10(-12) to 10(-7) mol/l salusin-alpha or -beta. (45)Ca(2+) uptake and (3)H-Leucine incorporation were determined in cultured neonatal rat cardiomyocytes. Neither salusin-a nor -beta affected cardiac function in vivo or in vitro but salusin-beta decreased mean arterial blood pressure (MAP). The polypeptides' stimulation of (45)Ca(2+) uptake and (3)H-Leucine incorporation was concentration-dependent, and the incorporation was inhibited by nicardipine (Nic) and FK-506 [FK; an inhibitor of calcineurin (CaN)]. PD(98059) [PD; inhibitor of mitogen-activated protein kinase (MAPK)] and chelerythrine [inhibitor of protein kinase C (PKC)] inhibited salusin-stimulated (3)H-Leucine incorporation. Endothelin-1 (ET) synergistically increased salusin-induced (45)Ca(2+) uptake. Our results suggest that salusin-alpha and -beta did not directly affect cardiac function in the rat heart but that they improved calcium uptake and protein synthesis in neonatal rat cardiomyocytes through the calcium, calcineurin, MAPK and PKC signal pathways. Salusins may be regulatory factors for myocardial growth and hypertrophy.     

Biosynthesis and secretion of salusin-alpha from human cells

Salusins originally identified using bioinformatics analyses have been shown to act on the cardiovascular and endocrine systems. Although the hypotensive activity of salusin-alpha is limited, it exerts a significant anti-atherosclerotic effect via suppression of foam cell formation in human monocyte-derived macrophages by down-regulating acyl-CoA:cholesterol acyltransferase-1. Furthermore, serum salusin-alpha levels show a close negative correlation with the severity of atherosclerotic diseases. However, biosynthesis and secretion of salusin-alpha peptide from cultured mammalian cells have not been demonstrated to date. We examined the expression, synthesis and release of salusin-alpha in human-derived cell lines. Preprosalusin mRNA and protein were detected ubiquitously in all cells tested, whereas the processing of preprosalusin into salusin-alpha peptide is dependent upon each cell type. Immunohistochemical study revealed the most abundant salusin-alpha-like immunoreactivity to be present in HeLa cells which released salusin-alpha-like immunoreactivity into the culture supernatant. Analysis of extracted conditioned media from HeLa cells by reverse-phase high performance liquid chromatography coupled with radioimmunoassay detection revealed a single immunoreactive component that co-eluted with authentic salusin-alpha. These results present the first evidence that salusin-alpha is biosynthesized and released from human-derived cells.     

Peptidomic identification and biological validation of neuroendocrine regulatory peptide-1 and -2

Recent advances in peptidomics have enabled the identification of previously uncharacterized peptides. However, sequence information alone does not allow us to identify candidates for bioactive peptides. To increase an opportunity to discover bioactive peptides, we have focused on C-terminal amidation, a post-translational modification shared by many bioactive peptides. We analyzed peptides secreted from human medullary thyroid carcinoma TT cells that produce amidated peptides, and we identified two novel amidated peptides, designated neuroendocrine regulatory peptide (NERP)-1 and NERP-2. NERPs are derived from distinct regions of the neurosecretory protein that was originally identified as a product of a nerve growth factor-responsive gene in PC12 cells. Mass spectrometric analysis of the immunoprecipitate using specific antibodies as well as reversed phase-high performance liquid chromatography coupled with radioimmunoassay analysis of brain extract demonstrated the endogenous presence of NERP-1 and NERP-2 in the rat. NERPs are abundant in the paraventricular and supraoptic nuclei of the rat hypothalamus and colocalized frequently with vasopressin but rarely with oxytocin. NERPs dose-dependently suppressed vasopressin release induced by intracerebroventricular injection of hypertonic NaCl or angiotensin II in vivo. NERPs also suppressed basal and angiotensin II-induced vasopressin secretion from hypothalamic explants in vitro. Bioactivity of NERPs required C-terminal amidation. Anti-NERP IgGs canceled plasma vasopressin reduction in response to water loading, indicating that NERPs could be potent endogenous suppressors of vasopressin release. These findings suggest that NERPs are novel modulators in body fluid homeostasis.     

The bioactive peptides salusins and apelin-36 are produced in human arterial and venous tissues and the changes of their levels during cardiopulmonary bypass

This study aimed to examine the effects of CPB on salusin-α, salusin-β and apelin-36 bioactive peptides in people who are planned to undergo coronary artery bypass graft (CABG) operation due to coronary artery disease and to explore whether these peptides are produced in human aortic, saphenous and arterial tissues. The study included age and BMI matched 15 patients who underwent CABG operation by CPB. In order to determine salusin-α, salusin-β and apelin-36 levels, venous blood samples were collected before induction of anesthesia (T1), before CPB (T2), 5min before the removal of cross-clamp (T3), 5min after the removal of cross-clamp (T4), upon arrival in the intensive care (T5), at postoperative 24th hour (T6) and 72nd hour (T7). Salusin and apelin expressions of the tissues were shown by immunohistochemical method. Peptide amounts of sera and tissues were measured using ELISA. Salusins production by vessels occurs in fibroblast cells of the media in the aorta and smooth muscle cells of the media in the LIMA and saphena. Apelin is produced by endothelial cells of the intima and fibroblast cells of the media in the aorta and by smooth muscle cells of the media in the LIMA and saphena. Changes in the levels of salusin-β and apelin-36 were significant during CPB. Salusin-α, salusin-β and apelin-36 are locally synthesized in the arteries and veins. Salusins and apelin-36 might be important markers in the CPB, and also that salusin-β was more specific in comparison to salusin-α.     

Pilosulin 5, a novel histamine-releasing peptide of the Australian ant, Myrmecia pilosula (Jack Jumper Ant)

Venom of the Australian ant species Myrmecia pilosula contains a number of allergenic peptides including pilosulins. To obtain novel cDNA clones that encode the pilosulin-related bioactive peptides, mRNA of M. pilosula species complex was subjected to RT-PCR in which the forward primer corresponds to a nucleotide sequence in the leader sequences of pilosulins. We isolated a cDNA clone encoding the novel bioactive peptide pilosulin 5. Tandem mass analysis was entirely consistent with the cDNA derived sequence, and indicated that pilosulin 5 is connected by a single disulfide bridge to create a dimmer peptide of 8546 Da. Synthetic pilosulin 5 peptide caused a significant histamine release in a dose-dependent manner, and the mastoparan homologous region of pilosulin 5 was responsible for the activity.     

The cDNA sequence coding for prepro-PGS (prepro-magainins) and aspects of the processing of this prepro-polypeptide

Amphibian skin is well known as a source of peptides homologous to bioactive peptides found in mammalian gut and brain. A systematic investigation of the skin secretions from Xenopus laevis revealed several peptides not derivable from known precursors. The sequence elucidation, utilizing fast atom bombardment/mass spectrometry, of two peptides, PGS and PGS Gly-10;Lys-22, is reported. These have been independently characterized and named magainins and found to have antimicrobial activity. A mixed sequence oligonucleotide probe complementary to the mRNA sequence coding for PGS was synthesized and used to screen a Xenopus skin cDNA library. A full length cDNA species encoding prepro-PGS was isolated and characterized, and its sequence is reported. The deduced precursor sequence was found to contain one copy of PGS Gly-10;Lys-22 and five copies of PGS. The proteolytic processing of this prepro-polypeptide is discussed.     

cDNA sequence of neuroendocrine protein 7B2 expressed in beta cell tumors of transgenic mice

The cDNA for a widely distributed neuroendocrine protein called 7B2 has been cloned from beta cell tumors of transgenic mice and sequenced. As deduced from the cDNA sequence, 7B2 is a secretory protein of 186 amino acids, nearly identical to its human and porcine homologs. The presence of several pairs of basic residues in the carboxyl terminal portion of the protein suggests that 7B2 can undergo proteolytic maturation in secretory granules and thus generate potential bioactive peptides. 7B2 mRNA is about 1.5 kilobase long and is apparently transcribed from a single gene per haploid genome. The use of tissue-specific promoters to express oncogenes in rare cell types of transgenic mice is a powerful tool for immortalization and expansion of these cells, and it facilitates the isolation and the study of rare proteins such as 7B2.     

Characterization of three constituent chains of collagen type VI by peptide sequences and cDNA clones

Pepsin-solubilized collagen VI was prepared from human placenta and used to separate three constituent chains for determining partial amino acid sequences. Antibodies raised against the chains assisted in the identification and purification of several cDNA clones from three expression lambda gt11 libraries. Most of the clones hybridized to either a 3.5-kb or 4.2-kb mRNA species which by matching peptide and nucleotide sequences could be identified as coding for the alpha 2(VI) or alpha 1(VI) chain, respectively. Other clones hybridized to either an 8.5-kb mRNA which very likely encoded the alpha 3(VI) chain or to an unknown 2.0-kb mRNA. Northern blots revealed a considerable variation in the mRNA levels for each collagen VI chain in both skin and cornea fibroblasts and in several tumor cell lines. Limited sequence data generated from peptides and cDNA clones demonstrated a characteristic cysteine pattern at the junction between N-terminal globular domain and triple helix in all three chains. In addition, the data showed occasional interruptions of triplet sequences within the triple-helical domain and the presence of two Arg-Gly-Asp sequences which are potential cell-binding structures.     

Molecular cloning and nucleotide sequence of cDNA encoding human muscle glycogen debranching enzyme.

cDNA comprising the entire length of the human muscle glycogen debranching enzyme was cloned and its nucleotide sequence determined. The debrancher mRNA includes a 4545-base pair coding region and a 2371-base pair 3'-nontranslated region. The calculated molecular mass of the debrancher protein derived from cDNA sequence is 172,614 daltons, consistent with the estimated size of purified protein (Mr 165,000 +/- 500). A partial amino acid sequence (13 internal tryptic peptides with a total of 213 residues) determined on peptides derived from purified porcine muscle debrancher protein confirmed the identity of the cDNA clone. Comparison of the amino acid sequence predicted from the human glycogen debrancher cDNA with the partial protein sequence of the porcine debrancher revealed a high degree (88%) of interspecies sequence identity. RNA blot analysis showed that debrancher mRNA in human muscle, lymphoblastoid cells, and in porcine muscle are all similar in size (approximately 7 kilobases). Two patients with inherited debrancher deficiency had a reduced level of debrancher mRNA, whereas two other patients had no detectable abnormality in RNA blots. The isolation of the debrancher cDNA and determination of its primary structure is an important step toward defining the structure-function relationship of this multifunctional enzyme and in understanding the molecular basis of the type III glycogen storage disease.     

Cloning, expression and processing of the CP2 neuropeptide precursor of Aplysia

The cDNA sequence encoding the CP2 neuropeptide precursor is identified and encodes a single copy of the neuropeptide that is flanked by appropriate processing sites. The distribution of the CP2 precursor mRNA is described and matches the CP2-like immunoreactivity described previously. Single cell RT-PCR independently confirms the presence of CP2 precursor mRNA in selected neurons. MALDI-TOF MS is used to identify additional peptides derived from the CP2 precursor in neuronal somata and nerves, suggesting that the CP2 precursor may give rise to additional bioactive neuropeptides.     

Cloning, expression and processing of the CP2 neuropeptide precursor of Aplysia.

The cDNA sequence encoding the CP2 neuropeptide precursor is identified and encodes a single copy of the neuropeptide that is flanked by appropriate processing sites. The distribution of the CP2 precursor mRNA is described and matches the CP2-like immunoreactivity described previously. Single cell RT-PCR independently confirms the presence of CP2 precursor mRNA in selected neurons. MALDI-TOF MS is used to identify additional peptides derived from the CP2 precursor in neuronal somata and nerves, suggesting that the CP2 precursor may give rise to additional bioactive neuropeptides.     

Cloning of a cDNA encoding porcine brain natriuretic peptide

Complimentary DNA (cDNA) clones encoding porcine brain natriuretic peptide (BNP) were isolated from a porcine atrial cDNA library. The longest of the cDNA clones (1507 nucleotides) apparently originated from an unprocessed messenger RNA, since the nucleotide sequence encoding BNP-26 was interrupted by an intron of 554 nucleotides. A partial cDNA clone representing processed BNP mRNA was prepared by polymerase chain reaction. A comparison of the sequence of these two cDNAs reveals the presence of an additional intron within the sequence encoding the BNP precursor. The identification of these introns suggests that the BNP gene structure differs from the atrial natriuretic peptide gene in the location of intron 2. BNP mRNA encodes a propeptide of 131 amino acids, including a signal peptide domain (25 amino acids) and a prohormone domain (106 amino acids). Like atrial natriuretic peptide, the bioactive BNP sequence is localized at the carboxyl terminus of the prohormone. Although the carboxyl-terminal peptide sequences of porcine atrial natriuretic peptide and BNP are well conserved, there is relatively little homology within their propeptide regions.     

Molecular cloning and sequencing of the human erythrocyte 2,3-bisphosphoglycerate mutase cDNA: revised amino acid sequence.

The human erythrocyte 2,3-bisphosphoglycerate mutase (BPGM) is a multifunctional enzyme which controls the metabolism of 2,3-diphosphoglycerate, the main allosteric effector of haemoglobin. Several cDNA banks were constructed from reticulocyte mRNA, either by conventional cloning methods in pBR322 and screening with specific mixed oligonucleotide probes, or in the expression vector lambda gt 11. The largest cDNA isolated contained 1673 bases [plus the poly(A) tail], which is slightly smaller than the size of the intact mRNA as estimated by Northern blot analysis (approximately 1800 bases). This cDNA encodes for a protein of 258 residues; the protein yielded 34 tryptic peptides which were subsequently isolated by h.p.l.c. Our nucleotide sequence data were entirely confirmed by the amino acid composition of these tryptic peptides and reveal several major differences from the published sequence; the revised amino acid sequence of human BPGM is presented. These findings represent the first step in the study of the expression and regulation of this enzyme as a specific marker of the erythroid cell line.     

Structure of the precursor to an enzyme mediating COOH-terminal amidation in peptide biosynthesis

Many bioactive peptides terminate with an amino acid alpha-amide at their COOH terminus. The enzyme responsible for this essential posttranslational modification is known as peptidyl-glycine alpha-amidating monooxygenase or PAM. We identified cDNAs encoding the enzyme by using antibodies to screen a bovine intermediate pituitary lambda gt11 expression library. Antibodies to a beta-galactosidase/PAM fusion protein removed PAM activity from bovine pituitary homogenates. The 108,207 dalton protein predicted by the complete cDNA is approximately twice the size of purified PAM. An NH2-terminal signal sequence and short propeptide precede the NH2 terminus of purified PAM. The sequences of several PAM cyanogen bromide peptides were localized in the NH2-terminal half of the predicted protein. The cDNA encodes an additional 430 amino acid intragranular domain followed by a putative membrane spanning domain and a hydrophilic cytoplasmic domain. The forms of PAM purified from bovine neurointermediate pituitary may be generated by endoproteolytic cleavage at a subset of the 10 pairs of basic amino acids in the precursor. High levels of PAM mRNA were found in bovine pituitary and cerebral cortex. In corticotropic tumor cells, levels of PAM mRNA and pro-ACTH/endorphin mRNA were regulated in parallel by glucocorticoids and CRF.     

Molecular cloning and biological characterization of novel antimicrobial peptides, pilosulin 3 and pilosulin 4, from a species of the Australian ant genus Myrmecia

Venom of an Australian ant species of the Myrmecia pilosula species complex (mss. name Myrmecia banksi Taylor) contains two major allergenic peptides, pilosulin 1 and pilosulin 2. To obtain novel cDNA clones that encode the pilosulin-related bioactive peptides, mRNA of another Myrmecia species was subjected to RT-PCR in which the forward primer corresponds to a nucleotide sequence in the leader sequences of pilosulin 1 and pilosulin 2. As a result, we isolated cDNA clones encoding the novel antimicrobial peptides pilosulin 3 and pilosulin 4. The nucleotide and the amino acid sequences of all four pilosulins have high homology except for the mature peptide coding regions. Synthetic pilosulin 3 and pilosulin 4 peptides displayed antimicrobial activity with histamine-releasing and low hemolytic activities.     

cDNA encoding type B subunit of rat phosphoglycerate mutase: its isolation and nucleotide sequence.

A cDNA encoding the nonmuscle-specific (type B) subunit of phosphoglycerate mutase (PGAM-B) was isolated and characterized. A cDNA probe, synthesized by the polymerase chain reaction (PCR) from rat liver cell mRNA using mixed primers specific to the amino acid sequence of human PGAM-B, was used to screen a rat liver cell cDNA library. The identity of the cDNA was confirmed by amino acid sequence data for 24 peptides obtained by digesting the purified protein with three different endopeptidases. The coding region encoded a polypeptide composed of 253 amino acid (plus the initiator Met). RNA blot analysis showed a single mRNA species of 1.7 kilobases in rat liver cell. The deduced amino acid sequence of rat PGAM-B was identical to that of human PGAM-B except for only one substitution at position 251 near the carboxyl terminus (valine for the rat and alanine for the human).