Characterization of the promoters of the guinea pig neutrophil cationic peptide-1 and -2 genes (original) (raw)
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Structure of the guinea pig neutrophil cationic peptide gene
FEBS Letters, 1992
Guinea pig neutrophils contain the antimicrobial cationic pcptides GNCP-I and GNCP-2 in the grander,, In this study, the GNCP gene was isolated, and the struclure was characterized. Using cDNA probes. one phage clone was isolated from a guinea pig genomic library. The gene spanned ~3 kb, and comprised three exons. and two introns. Sequence analysis revealed that the gene encoded GNCP-2. Exon 1 tnainly coded for the 5 untranslated region, cxon 2 coded for the prcpro-pcptide region of GNCP-2, and exon 3 coded for the mature peptide region of GNCP-2 and the 3' untrans!ated region. Primer extension analysis indicated that the transcription initiation site was located to a thymidine residue, 93 bp upstream of the ATG initiation codon of GNCP-2 mRNA. A possible TATA box was located 24 bp upstream of the transcription start site. Interestingly. the pyrimidine-rich sequences identiiied in the promoter regions of the human ncutrophil elastasc and myclaperoxidase genes were also found in the 5' flanking region of the GNCP-2 gene.
Guinea-pig neutrophil cationic peptides are encoded by at least three kinds of mRNA transcripts
Comparative biochemistry and physiology. B, Comparative biochemistry, 1993
1. cDNA clones encoding antimicrobial guinea-pig neutrophil cationic peptides, GNCP-1 and GNCP-2 were isolated from a bone marrow cell cDNA library. 2. Three kinds of cDNAs (GNCP-1A, GNCP-1B and GNCP-2 cDNAs) were obtained, and the analysis of these cDNAs indicated that GNCPs were produced as precursor proteins comprising 93 amino acid residues, which were composed of signal sequences (N-terminal 19 residues), pro-peptide sequences (43 residues) and mature GNCP sequences (31 residues). 3. The deduced amino acid sequences showed that GNCP-1A and GNCP-1B differed by only one amino acid substitution in the pro-peptide region, and GNCP-1B and GNCP-2 differed by only one amino acid substitution in the mature peptide region. The nucleotide sequences of these cDNAs were highly homologous (99%). 4. Together these observations indicate that GNCPs are encoded by at least three kinds of mRNA transcripts which are likely derived from the similar genes.
Evaluation of the expression of the cationic peptide gene in various types of leukocytes
FEBS Letters, 1992
To understand the regulation or the production of antimicrobial cationic pcptidc (CP) in Icukocytcs, cxprcssion of the CP gcnc was cvaluat& in vurious types ol' Icukocytcs usinp guinea pig ncutrophils. monocytcs. macrophapcs. cosinophils. lymphocytes and bone marrow cells. Acid-urea PAGE and SDS-PAGE!immunoblot annlyscs showed that CP was prcscnt in ncutrophils ;Ind bone marrow cells. but not in other Icukocytcs. Norihcrn blot und transcription run-off anlrlyscs rcvcalcd that only bone marrow cclla exprcsscd CP mRNA and transcribed the CP gcnc. Intcrcrtingly. in situ hybridization :malysis using bone m;u'row cells dcmonstrarcd that CP mRNA WdS cxprcsscd in the ncutrophil precursor culls. such us promyclocyl.cs ;Ind n~yclocytcs, but wus not c!rixtcd in ~hc muturc ncutrophils and other bone marrow cells. Morcovcr. immunocytochemical study inilicated ~11;~ CP WIS prcscnt in the ncutrophil precursor cells and ~hc mature ncutrophils in the bone marrow. Thus, the CPgcnc appcilrs to bc cxprcsscd during u limircd period ol'ncutrophil maturation. and CP is likely synthcsizcd by the ncutrophil precursor cells in the bone murrow.
The Journal of Immunology, 2000
The human neutrophil defensins (human neutrophil peptides (HNPs)), major components of azurophilic granules, contribute to innate and acquired host immunities through their potent antimicrobial activities and ability to activate T cells. Despite being encoded by nearly identical genes, HNP-1 is more abundant in the granules than HNP-3. We investigated the regulation of HNP-1 and HNP-3 expression at the transcriptional level using a promyelocytic HL-60 cell line. Luciferase analysis showed that transcriptional levels of HNP-1 and HNP-3 promoters were equivalent and that an ϳ200-bp region identical between promoters was sufficient for transcriptional activity. Furthermore, overlapping CCAAT/enhancer-binding protein (C/EBP) and c-Myb sites in the region were found to be required for efficient transcription. Gel mobility shift assay demonstrated that C/EBP␣ predominantly bound to the C/EBP/c-Myb sites using HL-60 nuclear extracts. No specific binding to C/EBP/c-Myb sites was observed in nuclear extracts from mature neutrophils, which expressed neither C/EBP␣ protein nor HNP mRNAs. Taken together, these findings suggest that the difference in the amounts of HNP-1 and HNP-3 peptides in neutrophils is caused by posttranscriptional regulation and that C/EBP␣ plays an important role in the transcription of HNP genes in immature myeloid cells.
Journal of Biological Chemistry, 1997
Neutrophils contain various antibacterial polypeptides and proteins in the granules that contribute to the killing of microorganisms. Recently, we have purified a cationic antibacterial polypeptide of 11 kDa (CAP11) from guinea pig neutrophil granules. CAP11 is a homodimer of G 1 LRKKFRKTRKRIQKLGRKIGKTGRKV-WKAWREYGQIPYPCRI 43 joined with one disulfide bond. In this study, to understand the regulation of CAP11 expression, we isolated and analyzed cDNA encoding CAP11. Furthermore, we investigated the expression of CAP11 mRNA during neutrophil maturation and localization of CAP11 among neutrophil granule subsets. Sequence analysis of CAP11 cDNA isolated from guinea pig bone marrow cells using rapid amplification of cDNA ends technique indicated that CAP11 is synthesized as a precursor comprising 178 amino acid residues, which is composed of a signal peptide (N-terminal 29 residues), a propeptide (106 residues), and a C-terminal mature peptide (43 residues). Interestingly, the predicted CAP11 precursor displayed the characteristic features of cathelicidins, a novel protein family of antibacterial polypeptides with a conserved cathelin-like pro-region and a variable C-terminal antibacterial domain. Northern blot and Western blot analyses using neutrophils, macrophages, eosinophils, mononuclear cells, and bone marrow cells revealed that only neutrophils and bone marrow cells expressed CAP11 mRNA and contained CAP11, suggesting that expression of CAP11 is neutrophil lineage-specific. Furthermore, Northern blot analysis using bone marrow cells separated according to their maturation stages showed that CAP11 mRNA was predominantly expressed in the cells at later stages of neutrophil maturation. Consistent with this, in situ hybridization using CAP11-specific cRNA probe demonstrated that CAP11 mRNA was primarily expressed at metamyelocyte stage. In addition, extracellular release assay revealed that CAP11 was readily released from neutrophils accompanied with gelatinase by low concentrations of N-formyl-Met-Leu-Phe without release of specific and azurophil granule components, and CAP11 was found to be exclusively present in the fraction containing gelatinase granules, prepared by Percoll density gradient centrifugation.
Computational promoter analysis of mouse, rat and human antimicrobial peptide-coding genes-0
2011
<b>Copyright information:</b>Taken from "Computational promoter analysis of mouse, rat and human antimicrobial peptide-coding genes"BMC Bioinformatics 2006;7(Suppl 5):S8-S8.Published online 18 Dec 2006PMCID:PMC1764486.BLASTN against FANTOM3 cDNA sequences applying a cut-off of equal or greater than 60% identity. The promoter regions [-1000, +200 nt] of mouse AMPcg, human and rat orthologs were extracted and submitted to Dragon Motif Builder (DMB) for motif searching. The resulting consensus motifs were passed to TRANSFAC and compared with known TFBSs using the PATCH program.
Neutrophil antibacterial peptides, multifunctional effector molecules in the mammalian immune system
Journal of Immunological Methods, 1999
The bactericidal machinery of mammalian neutrophils is built up of many components with different chemical properties, involving proteins, peptides and oxygen-dependent radicals. All these components work in synergy, leading to destruction and elimination of ingested microbes. During the eighties, it gradually became clear, that cationic peptides are a part of the oxygen-independent bactericidal effectors in phagocytic cells. In mammals, these antimicrobial peptides are represented by two families, the defensins and the cathelicidins. These potent broad spectra peptides are included as immediate effector molecules in innate immunity. The detailed killing mechanism for these effectors is partly known, but nearly all of them have membrane affinity, and permeate bacterial membranes, resulting in lysis of the bacteria. This peptide-membrane interaction includes also eukaryotic membranes, that implicates cytotoxic effects on host cells. Studies in vitro have established that the microenvironment is critical for their activities. In connection to cystic fibrosis, the effects of microenvironment changes are apparent, causing inactivation of peptide defences and leading to repeated serious bacterial infections. Thus, the importance of the microenvironment is also supported in vivo. Additional functions of these peptides such as chemotactic, mitogenic and stimulatory in the wound healing process suggest further important roles for these peptides.