Dual function of the propeptide of prouroguanylin in the folding of the mature peptide: disulfide-coupled folding and dimerization.

Guanylyl cyclase activating peptide II (GCAP-II), an endogenous ligand of guanylyl cyclase C, is produced via the processing of the precursor protein (prepro-GCAP-II). We have previously shown that the propeptide in pro-GCAP-II functions as an intramolecular chaperone in the proper folding of the mature peptide, GCAP-II (Hidaka, Y., Ohno, M., Hemmasi, B., Hill, O., Forssmann, W.-G., and Shimonishi, Y. (1998) Biochemistry 37, 8498-8507). Here, we report an essential region in pro-GCAP-II for the correct disulfide pairing of the mature peptide, GCAP-II. Five mutant proteins, in which amino acid residues were sequentially deleted from the N terminus, and three mutant proteins of pro-GCAP-II, in which N-terminal 6, 11, or 17 amino acid residues were deleted, were overproduced using Escherichia coli or human kidney 293T cells, respectively. Detailed analysis of in vivo or in vitro folding of these mutant proteins revealed that one or two amino acid residues at the N terminus of pro-GCAP-II are critical, not only for the chaperone function in the folding but also for the net stabilization of pro-GCAP-II. In addition, size exclusion chromatography revealed that pro-GCAP-II exists as a dimer in solution. These data indicate that the propeptide has two roles in proper folding: the disulfide-coupled folding of the mature region and the dimerization of pro-GCAP-II.

HCC-1, a novel chemokine from human plasma.

A novel CC chemokine, HCC-1, was isolated from the hemofiltrate of patients with chronic renal failure. HCC-1 has a relative molecular mass of 8,673 and consists of 74 amino acids including four cysteines linked to disulfide bonds. HCC-1 cDNA was cloned from human bone marrow and shown to code for the mature protein plus a putative 19-residue leader sequence. Mature HCC-1 has sequence identity of 46% with macrophage inflammatory protein (MIP)-1 alpha and MIP-1 beta, and 29-37% with the other human CC chemokines. Unlike MIP-1 alpha and the other CC chemokines, HCC-1 is expressed constitutively in several normal tissues (spleen, liver, skeletal and heart muscle, gut, and bone marrow), and is present at high concentrations (1-80 nM) in plasma. HCC-1 has weak activities on human monocytes and acts via receptors that also recognize MIP-1 alpha. It induced intracellular Ca2+ changes and enzyme release, but no chemotaxis, at concentrations of 100-1,000 nM, and was inactive on T lymphocytes, neutrophils, and eosinophil leukocytes. In addition, HCC-1 enhanced the proliferation of CD34+ myeloid progenitor cells. It was as effective as MIP-1 alpha, but about 100-fold less potent.

A new human guanylate cyclase-activating peptide (GCAP-II, uroguanylin): precursor cDNA and colonic expression.

We have amplified, cloned, and sequenced 583 bp GCAP-II/uroguanylin-specific cDNA from human colon cDNA first strand. The cDNA codes for a putative 112 amino-acid precursor protein including the sequence of uroguanylin and GCAP-II. Northern blot hybridization revealed a high level expression of the GCAP-II gene in human colon, but not in the kidney. This expression of GCAP-II indicates a pivotal role in cGMP-mediated functions of the colon.

hBD-1: a novel beta-defensin from human plasma.

We report the isolation and characterization of a novel peptide with significant sequence homology to beta-defensins from human blood filtrate. The human beta-defensin-1 (hBD-1) is a short basic peptide of 36 amino acid residues. It contains six cysteines forming three intramolecular disulfide bonds. The molecular mass of hBD-1 is 3928.6 Da. Cloning of the specific cDNA confirmed the amino acid sequence of the native peptide. hBD-1 shares the nine conserved amino acids characteristic for beta-defensins from respiratory epithelial cells and neutrophils of cattle and chicken leukocytes. hBD-1 is present in nanomolar concentration in human plasma.

Coexistence of atrial natriuretic factor (ANF) and oxytocin in neurons of the rat hypothalamus.

Sections of rat hypothalamus were examined immunocytochemically for coexistence of atrial natriuretic factor (cardiodilatin) and oxytocin. These biosynthetically unrelated neuropeptides were demonstrated using the peroxidase-antiperoxidase method followed by immunofluorescence. Neurons immunoreactive for atrial natriuretic factor were found in the supraoptic, periventricular and paraventricular nuclei. A sub-population of these neurons contained oxytocin-like immunoreactivity. They constitute only a small portion of the total number of oxytocinergic neurons.

Cardiodilatin-immunoreactive neurons in the hypothalamus of Tupaia.

Using various region specific antibodies raised against partial sequences of synthetic cardiodilatin (CDD) we detected immunoreactive neurons with their perikarya in the nucleus periventricularis of Tupaia belangeri. The fibers could be traced laterally directed towards the amygdaloid complex and some varicosities were also observed in the lateral parts of the nucleus periventricularis. It is postulated that brain CDD represents a new neuropeptide and that these CDD-IR neurons are involved in specific functions related to the modulation of the cardiovascular centers.

A new hypothalamic substance, and not luteinizing hormone-releasing hormone, is detected immunocytochemically by antibody to luteinizing hormone-releasing hormone.

Adjacent paraffin sections of rat hypothalami fixed in Bouin's fluid were treated either with buffer or with luteinizing hormone-releasing hormone (LHRH) before immunocytochemical staining with anti-LHRH. Upon buffer pretreatment, pituitary gonadotrophs were unstained and hypothalamic fibers were stained. Upon LHRH pretreatment, pituitary gonadotrophs were stained (receptor reaction) and hypothalamic fibers were unstained. Extension of washes and use of series of neutralizing antisera between LHRH application and immunocytochemical staining, as well as the absence of inhibiting concentrations of LHRH in the later washes and neutralizing antisera removed from the sections, excluded the possibility that the disappearance of visualization of hypothalamic fibers was due to blockage of anti-LHRH in immunocytochemical staining. The results suggested that LHRH removed from the sections an immunocytochemically stainable but as yet unknown analog of LHRH and replaced it with LHRH, which in turn became lost during subsequent immunocytochemical processing. This idea was confirmed by the isolation by high-pressure liquid chromatography of a peak, distinct from LHRH, upon treatment of hypothalami with LHRH. It is suggested that the new substance may be carrier-held and that this substance, rather than LHRH, is normally detected by immunocytochemistry with anti-LHRH. Added LHRH binds not only to high-affinity pituitary receptors but also to low-affinity hypothalamic carriers.