Figure 1.
Schematic representation of His6-proHNP1.
Top Panel. The 36 amino acid residue N-terminal extension showing the His6-tag from the pET28a+ vector followed by the enterokinase site and proHNP1 sequence in the His6-proHNP1 construct are shown. The numbers above indicate the number of amino acid residues in each segment. Bottom Panel. The proHNP1 sequence showing the mature sequence (underlined), the disulfide linkages between Cys residues, and the Met residue (indicated with an asterisk) at the junction of the pro- and mature sequence. The numbers indicate the residue number in the preproHNP1 precursor.
Figure 2.
Purification of His6-proHNP1 foldamers.
A) Ni-column eluant was analyzed by SDS-tricine PAGE and Coomassie stained; arrowhead indicates the major ∼12 kDa recombinant protein in the eluant. B) HPLC chromatogram of Ni-eluant resolved by semi-preparative C18 RP-HPLC generates two major peaks, A and B. C) HPLC fractions (numbers indicated) shown in panel B were resolved on SDS-tricine gel and stained with Coomassie blue, (D) analyzed by western blotting using anti-His6-tag antibody, and by (E) Coomassie staining following AU-PAGE. F) His6-proHNP1 containing fractions 36–38 (His6-proHNP1A) and fractions 41–42 (His6-proHNP1B) were pooled and further purified by RP-HPLC on C18 column and analyzed by Coomassie staining samples resolved on SDS-tricine gel (lane 1 - His6-proHNP1A, lane 2 - His6-proHNP1B).
Figure 3.
HNP1 forms released by CNBr cleavage of His6-proHNP1.
HNP1 peptides were generated by CNBr cleavage of folded His6-proHNP1A and misfolded His6-proHNP1B. A) HNP1 generated by CNBr cleavage of His6-proHNP1B (upper tracing) and His6-proHNP1A (lower tracing) were resolved by C18 RP-HPLC. Bar indicates fractions containing peptides with molecular weight consistent with that of folded/oxidized HNP1 and peaks marked with arrows were further purified by HPLC. B) MALDI-TOF MS of CNBr-cleaved HNP1 derived from His6-proHNP1B (upper panel) and His6-proHNP1A (lower panel). C) Analytical C18 RP-HPLC using the Alliance system of HNP1 generated by CNBr-cleavage of His6-proHNP1B and His6-proHNP1A compared with neutrophil derived native HNP1. D) AU-PAGE analysis followed by Coomassie staining of 2 µg of each sample shown in panel C: peak B-derived HNP1 (lane 1), peak A-derived HNP1 (lane 2), native HNP1 (lane 3).
Figure 4.
Staphylocidal activity of HNP1 generated by CNBr cleavage of His6-proHNP1.
Microbicidal activity of native HNP1 (•) or HNP1-peptides generated by CNBr-cleavage of His6-proHNP1A (▴) and His6-proHNP1B (♦) was determined in a standard bactericidal assay against S. aureus 502a.
Figure 5.
Proteolytic processing of His6-proHNP1 by azurophil granule serine proteases.
A) His6-proHNP1A was digested with NE, PR3 and CG as described in Methods and resolved by C18 RP-HPLC. Peptides with masses matching that of HNP1 (asterisks) were identified by MALDI-TOF-MS. B) Analytical C18 RP-HPLC using the Alliance system of HNP1 isoforms derived from NE, PR3, and CG compared with native HNP1. C) AU-PAGE followed by Coomassie staining of native HNP1 (lanes 1 and 5) and HNP1 generated by processing with NE (lane 2), PR3 (lane 3) and CG (lane 4). D) The sequences of mature HNP1 and RMAD4 (underlined) and residues at the junction of their corresponding pro-segment are shown. ProHNP1 and proRMAD4 processing sites mediated by NE, PR3 and CG are indicated.
Table 1.
Mass spectroscopic analysis of folded and reduced/alkylated HNP1a.
Figure 6.
Microbicidal activity of HNP-peptides generated by NE, PR3 and CG processing of pro-HNP1.
Bactericidal activity of HNP1 isoforms generated by processing with NE (▪), PR3 (▴), and CG (♦), with native HNP1 (•) was determined using S. aureus 502a as described in Methods.