Isolation of cDNA encoding guinea pig neutrophil cationic antibacterial polypeptide of 11 kDa (CAP11) and evaluation of CAP11 mRNA expression during neutrophil maturation.
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 G1LRKKFRKTRKRIQKLGRKIGKTGRKVWKAWREYGQIPYPCRI43 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. Together these observations indicate that CAP11 is a member of cathelicidin family and its mRNA is preferentially expressed at the later stage of neutrophil maturation (i.e. metamyelocyte stage). Furthermore, CAP11 may be stored in the granule subset, possibly the gelatinase granule.
Nagaoka
I
I
Department of Biochemistry, Juntendo University, School of Medicine, Hongo, Bunkyo-ku, Tokyo 113, Japan. nagaokai@med.juntendo.ac.jp
Tsutsumi-Ishii
Y
Y
Yomogida
S
S
Yamashita
T
T
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D001853:Bone Marrow / Q000378:metabolism; D001854:Bone Marrow Cells; D018076:DNA, Complementary; D006168:Guinea Pigs; D007150:Immunohistochemistry; D017404:In Situ Hybridization, Fluorescence; D008297:Male; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000166:cytology* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000378:metabolism; D023084:alpha-Defensins / Q000235:genetics*
D018076:DNA, Complementary; D012333:RNA, Messenger; D023084:alpha-Defensins