Maltose-Binding Protein Enhances Secretion of Recombinant Human Granzyme B Accompanied by In Vivo Processing of a Precursor MBP Fusion Protein

Background: The apoptosis-inducing serine protease granzyme B (GrB) is an important factor contributing to lysis of target cells by cytotoxic lymphocytes. Expression of enzymatically active GrB in recombinant form is a prerequisite for functional analysis and application of GrB for therapeutic purposes. Methods and Findings: We investigated the influence of bacterial maltose-binding protein (MBP) fused to GrB via a synthetic furin recognition motif on the expression of the MBP fusion protein also containing an N-terminal a-factor signal peptide in the yeast Pichia pastoris. MBP markedly enhanced the amount of GrB secreted into culture supernatant, which was not the case when GrB was fused to GST. MBP-GrB fusion protein was cleaved during secretion by an endogenous furinlike proteolytic activity in vivo, liberating enzymatically active GrB without the need of subsequent in vitro processing. Similar results were obtained upon expression of a recombinant fragment of the ErbB2/HER2 receptor protein or GST as MBP fusions. Conclusions: Our results demonstrate that combination of MBP as a solubility enhancer with specific in vivo cleavage augments secretion of processed and functionally active proteins from yeast. This strategy may be generally applicable t

Expression of the granzyme B-TGFα fusion protein GrB-T in NK cells.

<p>(A) Schematic representation of the lentiviral transfer vector pS-GrB-T-IEW that encodes under the control of the Spleen Focus Forming Virus promoter (SFFV) a fusion of human GrB with TGFα, followed by an internal ribosome entry site (IRES) and cDNA encoding enhanced green fluorescent protein (EGFP) as a marker. SP, GrB signal peptide; DP, GrB activation dipeptide; GrB_21–247, mature form of GrB; L, flexible linker; M, Myc tag; H, hexa-histidine tag. The similar transfer vector pS-GrB_S183A-T-IEW encodes enzymatically inactive mutant GrB_S183A fused to TGFα (not shown). After transduction with S-GrB-T-IEW or S-GrB_S183A-T-IEW vector particles, EGFP-expressing NKL/GrB-T and NKL/GrB_S183A-T cells were enriched by flow cytometric cell sorting, and analyzed for GrB-T expression. (B) GrB-T mRNA expression in NKL/GrB-T and NKL/GrB_S183A-T cells was verified by semi-quantitative RT-PCR. (C) Expression of GrB-T proteins in NKL/GrB-T and NKL/GrB_S183A-T cells was investigated by immunoblot analysis of cell lysates with GrB-specific antibody. γ-Tubulin was analyzed as a loading control. (D) Expression of GrB-T proteins in NKL/GrB-T and NKL/GrB_S183A-T cells was confirmed by intracellular staining with Myc-tag-specific antibody and flow cytometry (open areas). In all experiments parental NKL cells served as controls.</p

Cytotoxicity of NKL/GrB-T and NKL/GrB_S183A-T cells towards EGFR-expressing tumor cells.

<p>(A) Expression of EGFR on the surface of MDA-MB468 breast carcinoma and A431 squamous cell carcinoma cells was determined by flow cytometry with EGFR-specific antibody (open areas). Cells treated only with secondary antibody served as controls (shaded areas). (B) Cytotoxicity of NKL/GrB-T (filled squares) and NKL/GrB_S183A-T cells (open squares) towards MDA-MB468 and A431 cells was determined in FACS-based cytotoxicity assays at different E/T ratios. Parental NKL cells (filled circles) were included for comparison.</p

Selective cytotoxicity of GrB-T fusion protein.

<p>(A) Induction of apoptosis after treatment of MDA-MB468 cells for 24 h with 100 µg/mL of total proteins from supernatants of activated NKL, NKL/GrB-T, or NKL/GrB_S183A-T cells in the presence of 25 µM chloroquine was analyzed by determining the percentage of Annexin V and propidium iodide (PI) double-positive cells by flow cytometry. (B) EGFR-positive MDA-MB468 (left) and EGFR-negative MDA-MB453 cells (right) were treated with 100 µg/mL of total proteins from supernatants of activated NKL, NKL/GrB-T, or NKL/GrB_S183A-T cells in the presence of 25 µM chloroquine as indicated. Controls cells were treated with medium containing PMA, ionomycin and chloroquine. After 24 h, the relative number of viable cells was determined in WST-1 assays. (C) To confirm specificity of cell killing, MDA-MB468 cells were pre-incubated with 50 µg/mL of EGFR-specific antibody 425 as a competitor prior to addition of proteins from NK cell supernatants and determination of cytotoxicity as described in (B). Control cells were pre-incubated with isotype-matched control antibody. (D) Dependence of cell killing on GrB activity was confirmed by pre-incubation of culture supernatants from activated NKL cells with 400 µM of GrB-specific peptide aldehyde inhibitor Ac-IETD-CHO before addition to MDA-MB468 cells and determination of cytotoxicity as described in (B). In all cases mean values ± SEM are shown; n = 3 (A); n = 6 (B–D). ***, <i>P</i><0.001; **, <i>P</i><0.01; *, <i>P</i><0.05; ns, <i>P</i>>0.05.</p

Release of GrB-T protein upon degranulation of NK cells.

<p>(A) Degranulation of NKL, NKL/GrB-T and NKL/GrB_S183A-T cells was induced by treatment with PMA and ionomycin for 5 h at 37°C, and culture supernatants were harvested. To confirm activation of cells, CD107a expression was analyzed by flow cytometry (open areas). Unstimulated cells served as controls (shaded areas). (B) To determine enzymatic activity of GrB and GrB-T proteins, GrB-specific peptide substrate Ac-IETD-pNA was incubated with 50 to 200 µg/mL of total proteins from supernatants of activated NKL (filled circles), NKL/GrB-T (filled squares) or NKL/GrB_S183A-T cells (open squares). Substrate cleavage was determined by measuring the absorbance at 405 nm. Mean values ± SEM are shown; n = 4. *, <i>P</i><0.05. (C) Binding of GrB-T (bold line) and mutant GrB_S183A-T protein (dotted line) released by activated NKL/GrB-T and NKL/GrB_S183A-T cells to EGFR-positive MDA-MB468 and EGFR-negative MDA-MB453 breast carcinoma cells was determined by flow cytometry with GrB-specific antibody. Cells treated with medium (shaded areas) or proteins released by activated parental NKL cells (regular line) served as controls.</p

Cytotoxic activity of proteins released by activated NK cells.

<p>(A) EGFR-positive MDA-MB468 cells were treated with increasing concentrations of total proteins from supernatants of activated NKL (closed circles), NKL/GrB-T (closed squares), or NKL/GrB_S183A-T cells (open squares). After 24 h, the relative number of viable cells in comparison to medium-treated controls was determined in WST-1 assays. Mean values ± SEM are shown; n = 9. (B) To investigate uptake and intracellular localization of GrB-T protein, MDA-MB468 cells were treated with GrB-T-containing supernatant from NKL/GrB-T cells for 60 min at 4°C, washed and incubated for another 90 min at 37°C, before staining with GrB-specific antibody (red) and confocal laser scanning microscopy. Control cells were incubated with supernatant from activated parental NKL cells. Nuclei were stained with DAPI (blue). Merged images are shown. (C) To facilitate release of internalized proteins from endosomes, MDA-MB468 were treated with total proteins from supernatants of activated NKL, NKL/GrB-T, or NKL/GrB_S183A-T cells in the presence of 25 µM of the endosomolytic reagent chloroquine, and the relative number of viable cells in comparison to controls treated with chloroquine-containing medium was determined in WST-1 assays. Mean values ± SEM are shown; n = 9. ***, <i>P</i><0.001; **, <i>P</i><0.01.</p

Analysis of GrB secreted into the culture medium.

<p>(A) Supernatants of yeast cultures harboring pPIC9-GrB, pPIC9-GST-furS-GrB, or pPIC9-MBP-furS-GrB were collected at the indicated time points after induction with methanol, and analyzed by immunoblotting with GrB-specific antibody. Band intensities were quantified by determining mean gray values (MGV) relative to the highest value obtained. (B) Expression of an ErbB2 protein fragment. Yeast cells carrying pPIC9-ErbB2₂₂₂ or pPIC9-MBP-furS-ErbB2₂₂₂ were induced with methanol, and ErbB2 protein in culture supernatants was analyzed by immunoblotting with ErbB2-specific antibody.</p

Expression of GrB in the yeast <i>Pichia pastoris</i>.

<p>(A) Constructs for expression of human granzyme B, and MBP-GrB and GST-GrB fusion proteins. <i>AOX1</i>, methanol-inducible alcohol oxidase I promoter; SP, α-factor signal peptide; furS, furin recognition motif; M, Myc tag; H, polyhistidine tag. (B) Culture supernatants of yeast clones carrying pPIC9-GrB, pPIC9-GST-furS-GrB, or pPIC9-MBP-furS-GrB were collected after induction with methanol for 3 days, and expression of GrB was analyzed by immunoblotting with GrB-specific antibody. Each lane represents an individual clone. Clones marked with an asterisk were used in subsequent experiments. (C) For comparison, culture supernatants of clones carrying the different expression constructs were analyzed together in a single immunoblot experiment as indicated. Supernatant of a yeast clone carrying empty pPIC9 served as a control.</p

Secretion of GST and MBP into the culture medium.

<p>The presence of GST and MBP in yeast culture supernatants after induction for the indicated time periods was analyzed by immunoblotting with GST-specific (A) or MBP-specific antibodies (B). (C) Intracellular accumulation of GrB and GrB fusion proteins was investigated by analysis of cell lysates with GrB-specific antibody. The positions of the different GrB proteins are indicated by arrows. Samples analyzed were from the same cultures examined for secretion of GrB as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014404#pone-0014404-g002" target="_blank">Fig. 2A</a>.</p

Processing of MBP-GrB fusion proteins.

<p>(A) Variants of the furin recognition motif either containing (furS) or lacking (fur) an additional C-terminal serine residue. (B) Culture supernatants of yeast clones carrying pPIC9-MBP-fur-GrB were collected after induction with methanol for 3 days, and expression and processing of the GrB fusion protein was analyzed by immunoblotting with GrB-specific antibody. (C) Samples enriched for processed (100 kDa filtrate) or unprocessed MBP-fur-GrB protein (100 kDa retentate) were prepared by filtration of culture supernatant of a representative MBP-fur-GrB clone through Amicon filters with 100 kDa membranes, and analyzed by immunoblotting with GrB-specific antibody. Initial MBP-fur-GrB containing supernatant and supernatant from an MBP-furS-GrB clone were included for comparison. Supernatant of yeast cells carrying empty pPIC9 served as a control. The lower apparent molecular weight of processed GrB in (C) when compared to (B) is most likely due to a lower degree of glycosylation (calculated molecular weight for processed GrB in non-glycosylated form: 28.4 kDa). (D) Enzymatic activity of GrB proteins from culture supernatants and kinetics of substrate cleavage were analyzed in a colorimetric peptide cleavage assay. The MBP-fur-GrB or MBP-furS-GrB containing supernatants, and the MBP-fur-GrB samples enriched for processed (100 kDa filtrate) or unprocessed protein (100 kDa retentate) analyzed in (C) were tested as indicated. A purified recombinant GrB derivative (GrB) was included as a positive control. Supernatant of yeast cells carrying empty pPIC9 displayed no GrB activity (data not shown). Means of triplicate samples are shown. Error bars indicate SEM.</p