Identification of eNAP-1, an antimicrobial peptide from equine neutrophils

Abstract

Endogenous, cysteine-rich antimicrobial peptides known as defensins are prominent components of human, rabbit, and rat neutrophils, yet little is known about their occurrence in other mammalian species. Although we did not detect mature (i.e., processed) defensins in equine neutrophil granules, we found that these granules contained small amounts of other cysteine-rich peptides with antimicrobial activity. One of these, eNAP-1, was purified by a combination of gel permeation and reversed-phase high-performance liquid chromatography from acid extracts prepared from the cytoplasmic granules of equine neutrophils. The molecular mass of eNAP-1 was approximately 7.2 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Amino acid analysis revealed that eNAP-1 had an unusually high cysteine content and that it was relatively enriched in alanine, glycine, lysine, and proline residues. The partial (N-terminal) amino acid sequence of eNAP-1 was DVQCGEGHFCHDXQTCCRASQGGXACCPYSQGVCCADQRHCCPVGF. Thirtysix of these residues (78.3%) were identical to those of a recently cloned human neutrophil peptide of unknown function and belonging to the granulin family. Homologous peptides have also been noted in rat bone marrow cells and rat kidney epithelins. We tested the ability of eNAP-1 to kill several equine uterine pathogens. Streptococcus zooepidemicus was killed most effectively, sustaining a >99.8% decrease in CFU per milliliter after a 2-h exposure to 100 ,g of eNAP-1 per ml (-15 FM). Escherichia coli and Pseudomonas aeruginosa were somewhat less susceptible, manifesting 87.0 and 87.1% mean decreases in CFU per milliliter, respectively, after incubation for 2 h with 200 ,ug of eNAP-1 per ml. KlebsieUla pneumoniae numbers were not significantly reduced after exposure to eNAP-1. These antimicrobial properties suggest that eNAP-1 may contribute to phagocyte-mediated host defense against equine infections.

Antimicrobial proteins of murine macrophages

“…The amino acid composition of the purified polypeptides was determined by quantitating amino acids as phenylthiocarbamyl derivatives on RP-HPLC by using a Novapak C18 column (Millipore) as described previously (2,14). The N-terminal amino acid sequence of the purified peptides was determined on a model 475A sequencing system (Applied Biosystems Inc., Foster City, Calif.) as previously described (6,30). Sequence data were analyzed by using the Swiss-Prot 19 data base and the FASTA algorithm (36).…”

Flow cytometric analysis of defensins in blood and marrow neutrophils

“…PMN granules contain numerous proteins including the defensins. These small 3±4 kDa cysteine-rich peptides have been previously found in PMN of mammals such as rabbits (7), humans (8), guinea pigs (9) and rats (10) but not in PMN of mice (11) or horses (12). As the name indicates, defensins play a major role in host defense against bacteria, spirochetes, fungi, mycobacteria, yeast and some enveloped viruses, but they can be cytotoxic (13±15).…”

A Review of Antimicrobial Peptides: Defensins and Related Cationic Peptides

“…Among the known 46 N-terminal amino acids, there are at least 10 cysteines (Table 2). 38 Porcine neutrophils also contain 16 to 18 residue, cationic peptides with 4 cysteines and structures which are similar to the antimicrobial peptides isolated from leukocytes of other species. They have been termed protegrins and three have been identified: PG-1, 2, and 3.39p40…”