Abstract
Neutrophils contain the antimicrobial cationic peptides, defensins in the granules. To understand the mechanism of gene expression, the cDNAs for the peptides were analyzed. From a guinea-pig bone marrow cell cDNA library, three kinds of cDNA clones encoding neutrophil cationic peptides, GNCP-1 and -2 were isolated. Two cDNAs coded for GNCP-1 (GNCP-1A and -1B), and one cDNA coded for GNCP-2. The nucleotide sequences of these cDNAs were highly homologous (>99%), and the deduced amino acid sequences showed that GNCP-1A and GNCP-1B differed by only one amino acid substitution in the propeptide region, and GNCP-1B and GNCP-2 differed by only one amino acid substitution in the mature peptide region.
Next, a genomic library was screened, and the GNCP gene clones were analyzed. Among four different GNCP gene clones isolated, two clones encoded GNCP-1A and-1B, and other two clones encoded GNCP-2. The sequence analyses revealed that the nucleotide sequences of GNCP-1 and -2 genes were highly homologous (>96%) in the region sequenced, and that the GNCP-1 and -2 genes spanned 3kb, and consisted of three exons and two introns. Exon 1 encoded the 5' untranslated region, exon 2 encoded the prepro-peptide region, and exon 3 encoded the mature peptide and 3' untranslated regions.
Finally, to evaluate the promoter activity, the 5' flanking regions (0.4 kb) of the GNCP-1 and -2 genes were subcloned into the promoterless PGV-B luciferase vector, and the plasmid constructs were transfected into guinea-pig 104C1 cells and GPC-16 cells. The GNCP-PGV-B constructs expressed> 10-fold more luciferase activity than the promoterless PGV-B vector in both cells. Interestingly, the promoter activity of GNCP-1 gene was 2-fold more than that of GNCP-2 gene. Together these observations indicate that GNCP-1 and -2 are encoded by the homologous but different genes, and the expression of GNCP -1 and -2 genes are likely different at the level of transcription.
