title: Purification and characterization of human neutrophil peptide 4, a novel member of the defensin family.
abstract: Primary (azurophil) granules of neutrophils contain proteins which play a major role in the killing and digestion of bacteria in the phagolysosome. We have isolated and characterized a novel antimicrobial peptide from the azurophil granule fraction of discontinuous Percoll gradients. We have named this peptide human neutrophil peptide 4 (HNP-4) based on its structural similarity to a group of antimicrobial polypeptides known as defensins (HNP 1-3). Using size exclusion and reverse-phase high performance liquid chromatography, HNP-4 was purified to homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino-terminal sequence analysis. The amino acid sequence determined from isolated HNP-4 and from tryptic fragments of reduced and alkylated peptide is: NH2-Val-Cys-Ser-Cys-Arg-Leu-Val-Phe-Cys-Arg-Arg-Thr-Glu- Leu-Arg-Val-Gly-Asn-Cys-Leu-Ile-Gly-Gly-Val-Ser-Phe-Thr-Tyr-Cys-Cys-Thr- Arg-Val - COOH. Based on this sequence, HNP-4 has a calculated molecular weight of 3715 and a theoretical pI of 8.61. HNP-4 shows structural similarity to the family of three human defensins. HNP-4 and the defensins have identical cysteine backbones and, like the defensins, HNP-4 is rich in arginine (15.2 mol %). However, the amino acids at 22 of the 33 positions differ between HNP-4 and human defensins. Further, HNP-4 is significantly more hydrophobic than the defensins, as determined by its retention time on reverse-phase high performance liquid chromatography. In vitro, purified HNP-4 was shown to kill Escherichia coli, Streptococcus faecalis, and Candida albicans. Compared to a mixture of the other human defensins, HNP-4 was found to be approximately 100 times more potent against E. coli and four times more potent against both S. faecalis and C. albicans.
authors.affiliation: Invitron Corporation, Redwood City, California 94063.
authors.forename: C G
authors.initials: CG
authors.lastname: Wilde
authors.forename: J E
authors.initials: JE
authors.lastname: Griffith
authors.forename: M N
authors.initials: MN
authors.lastname: Marra
authors.forename: J L
authors.initials: JL
authors.lastname: Snable
authors.forename: R W
authors.initials: RW
authors.lastname: Scott
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001770:Blood Bactericidal Activity; D001798:Blood Proteins / Q000302:isolation & purification* / Q000502:physiology; D002176:Candida albicans; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003594:Cytoplasmic Granules / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D013293:Enterococcus faecalis; D004926:Escherichia coli; D006801:Humans; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009504:Neutrophils / Q000032:analysis* / Q000648:ultrastructure; D023084:alpha-Defensins
chemical_list: D000596:Amino Acids; D001798:Blood Proteins; D023084:alpha-Defensins; C406428:human neutrophil peptide 4