Fig 1.
Arrows indicate major sites of proteolytic cleavage. Positively and negatively charged amino acids are indicated in red and green, respectively. Lines indicate the disulphide linkage of cysteines (C; orange). HNP-3 is identical to HNP-1 except for having substituted alanine (A) at position 65 for aspartic acids (D).
Fig 2.
Subcellular localization of proHNP processing.
(A) PLB-985 cells were pelleted and disrupted by nitrogen cavitation. After low speed centrifugation, the cavitate was divided in a postnuclear pellet (P1) consisting of nuclei and unbroken cells and a post-nuclear supernatant (S1) containing cytosol, organelles (including granules), and cell membranes. S1 was underlaid with a two-layer 1.05/1.07 PBS/Percoll density gradient and centrifuged at 37.000g for 30 minutes. Fractions were collected from the bottom of the gradient. Percoll was removed from fractions by ultracentrifugation. (B) Fractions were subjected to Western blotting for HNP, proHNPs, the endoplasmic reticulum (ER) marker calnexin, and the Golgi marker RCAS1. (C) PLB-985 cells were pulsed overnight in medium containing 100 μCi/mL 35S-methionine/cysteine. Cells were pelleted and the supernatant used for isolation of 35S-labelled proHNP by affinity chromatography with an antibody specific for proHNP. Radioactive fractions were pooled, dialyzed against PBS, and tested for proHNP by 16% SDS-Tricine-PAGE and fluorography. (D) 35S-proHNP was incubated with subcellular fractions of PLB-985 for 15 hours at 37°C. Processing was tested by 16% SDS-Tricine-PAGE and fluorography.
Fig 3.
35S-proHNP processing assay using promyelocytic PLB-985 proteases.
(A) PLB-985 cells was subjected to nitrogen cavitation followed by pelleting of the postnuclear supernatant containing cytosol, organelles (including granules), and cell membranes. This pellet (P2) was permeabilized in PBS/1% Triton X-100. Preservation of serine protease activity was verified by measuring elastase/proteinase 3 activity by spectrophotometry following degradation rate of methoxysuccinyl-Ala-Ala-Pro-Val-P-nitroanilide. (B) 35S-proHNP was incubated with P2 from PLB-985 cells, incubated at 37°C for 3 hours, and processing tested by SDS-Tricine-PAGE followed by fluorography. (C) P2 was mixed with the serine proteinase inhibitors DFP, PMSF, or elastase inhibitor IV, mixed with 35S-proHNP, and incubated for 6 hours. Processing was analyzed by SDS-Tricine-PAGE followed by fluorography. Complete inhibition of serine proteases by DFP was verified by protease inhibition assays (data not shown).
Fig 4.
Biosynthesis of proHNP and HNP in human bone marrow.
(A) Human bone marrow cells were sedimented with dextran. Supernatant was laid on Lymphoprep and centrifuged at 400g for 30 minutes. Interphase cells were depleted of nongranulocytic cells by immunomagnetic sorting, spun onto slides and May-Grünwald Giemsa stained. Bar represents 20 μm. (B) Purified granulocytic precursors were electroporated with 3x siRNA (against neutrophil elastase (NE; ELANE), cathepsin G (CG; CTSG), and proteinase 3 (PR3; PRTN3)), control siRNA, or without siRNA and incubated for 24h in a humidified incubator with 5% CO2 at 37°C. Comparative quantification mRNA for DEFA1, ELANE, CTSG, and PRTN3 was performed by real-time PCR. Figure depicts expression levels relative to cells electroporated without siRNA. Bars represent means and lines represent standard deviation. (C) Transfected granulocyte precursors were pulsed with 35S-methionine/cysteine for 2 hours and chased overnight. Cell lysates and medium were immunoprecipitated with antibodies in the following order: anti-proHNP, anti-HNP, anti-CG, anti-NE, and anti-PR3. CG (top) and NE (mid) immunoprecipitates were analyzed by 12% SDS-PAGE and fluorography. Immunoprecipitation with anti-PR3 did not yield a specific PR3 band (data not shown). ProHNP and HNP immunoprecipitates were pooled and analyzed by 16% SDS-Tricine-PAGE and fluorography (bottom).
Fig 5.
Processing of proHNP-1 by MPRO cells transfected with human serine proteases.
(A) MPRO cells and MPRO cells transfected with HNP-1 were spun onto slides, fixed, permeabilized, and immunocytochemically stained for IgG (top), HNPs (middle), and proHNPs (bottom). Bars represent 20 μm. (B) MPRO-HNP-1 cells were further transfected with the human neutrophil serine proteases neutrophil elastase (ELANE), cathepsin G (CTSG), and proteinase 3 (PRTN3). Cells were pulsed with 35S-methionine/cysteine for 1 hour and chased overnight. Cell lysates and medium were immunoprecipitated with antibodies in the following order: anti-proHNP, and anti-HNP. Immunoprecipitates were pooled and analyzed by 16% SDS-Tricine-PAGE and fluorography using 35S-proHNP and 35S-HNP from the proHNP processing assay as controls. (C) Comparative quantification mRNA for ELANE, CTSG, and PRTN3 was performed by real-time PCR. Figure depicts expression levels relative to cells electroporated human serine proteases. Bars represent means and lines represent standard deviation. (D) Activity of transfected human neutrophil elastase and proteinase 3 was asserted by lysis of transfected MPRO cells followed by spectrophotometry following degradation rate of methoxysuccinyl-Ala-Ala-Pro-Val-P-nitroanilide.
Fig 6.
Serine protease activity is not required for HNP processing in vivo.
Peripheral blood neutrophils from two patients suffering from Papillon–Lefèvre syndrome (PLS). (A) Neutrophil elastase (NE) and proteinase 3 (PR3) activity was measured as absorbance (Abs) following degradation of methoxysuc-AAPV-p-nitroanilide. Assay from patient 1 is shown and is representative of both patients. (B) Cathepsin G (CG) activity was measured as absorbance following degradation of N-suc-AAPF-p-nitroanilide. Assay from patient 1 is shown and is representative of both patients. (C) Western blotting of NE, CG, and PR3 in two PLS patients and controls. (D) Western blotting of HNP and β-actin in two PLS patients and controls.
Table 1.
Papillon-Lefévre patients.