ENAP-1

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. Thirty-six 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 greater than 99.8% decrease in CFU per milliliter after a 2-h exposure to 100 micrograms of eNAP-1 per ml (approximately 15 microM). 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 micrograms of eNAP-1 per ml. Klebsiella 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.

Stimulation of bovine aortic endothelial cells (BAEC) with bradykinin produces cycles of tyrosine phosphorylation/dephosphorylation of a 90 kDa endothelial nitric oxide synthase (eNOS)-associated protein which we have termed ENAP-1 (for endothelial nitric oxide synthase-associated protein 1). ENAP-1 interacts specifically and tightly with eNOS in BAEC and is co-immunoprecipitated from cell lysates with anti-eNOS antibodies. In addition, anti-phosphotyrosine antibodies co-precipitate eNOS. Bradykinin-stimulated tyrosine phosphorylation of ENAP-1 is blocked by the tyrosine kinase inhibitor, tyrphostin. Dephosphorylation is blocked by the tyrosine phosphatase inhibitor, orthovanadate. Treatment of BAEC with bradykinin or the tyrosine phosphatase inhibitor, phenylarsine oxide promotes tyrosine phosphorylation of detergent-insoluble, cytoskeletal proteins accompanied by translocation of eNOS to the cytoskeletal subcellular compartment. Translocation is blocked by the tyrosine kinase inhibitor, geldanamycin and does not appear to alter enzyme catalytic activity. Tyrosine phosphorylation-dependent association of eNOS with the cytoskeleton may have a role in targeting NO production to specific subcellular locations.

Previously we described ENAP-1, a 90-kDa protein that is tyrosine-phosphorylated in endothelial cells in response to bradykinin (BK) stimulation and is associated with endothelial nitric oxide synthase (eNOS). Subsequently, other investigators demonstrated that eNOS interacts with heat shock protein 90 (Hsp90) following stimulation of endothelial cells with vascular endothelial growth factor (VEGF), histamine, or fluid shear stress. Therefore, we tested the hypotheses that ENAP-1 and Hsp90 are the same protein and that BK activation of eNOS is dependent on Hsp90. Immunoblotting of immunoprecipitated Hsp90 with anti-phosphotyrosine antibody shows that Hsp90 is tyrosine-phosphorylated in response to BK stimulation of bovine aortic endothelial cells (BAECs). Coimmunoprecipitation of Hsp90 with anti-eNOS antibody reveals a Hsp90-eNOS complex in endothelial cells under basal conditions that is increased following BK stimulation. Taken together with the tyrosine phosphorylation data, these data suggest that ENAP-1 is Hsp90. BK-stimulated nitric oxide (NO) release is completely blocked by pretreatment with geldanamycin, a specific inhibitor of Hsp90, illustrating the importance of the Hsp90-eNOS interaction. In vitro binding assays with Hsp90-glutathione-S-transferase fusion proteins show direct binding of eNOS with the middle domain (residues 259-615) of Hsp90.