1. Introduction. T. Zavašnik-Bergant 1

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1 Application of monoclonal and ultrathin cryosections in immunogold electron microscopy: a study on recombinant human inhibitor expressed in bacterial cells T. Zavašnik-Bergant 1 1 Department of Biochemistry, Molecular and Structural Biology, Jožef Stefan Institute, Jamova c. 39, SI-1000 Ljubljana, Slovenia The preparation of ultrathin cryosections represents a method where cell proteins preserve their antigenicity well which further enables the application of monoclonal antibodies as probes for efficient immunogold labelling prior to transmission electron microscopy (TEM). As a representative application of this technique the labelling signal and the specificity of mouse monoclonal against the protein of interest have been compared to polyclonal prepared and isolated against the same recombinant protein by fluorescence (confocal) and electron microscopy, respectively. In this paper the emphasis is put on the preparation of ultrathin cryosections of bacterial cells (Escherichia coli) expressing recombinant human protein named cystatin C with an intention to find a highly specific monoclonal which would later also detect inhibitory active recombinant cystatin C used as a modulator of proteolytic activity of lysosomal enzymes in human cells. Keywords monoclonal ; immunogold labelling; ultrathin cryosection; microscopy; recombinant protein 1. Introduction Antibodies are Y-shaped glycoproteins made by humans and animals. They have the ability to recognize and discriminate among very similar proteins. Monoclonal antibodies are often used as highly specific probes for identification, evaluation and quantification of selected cellular proteins within a multitude of other cell components. Many specific monoclonal antibodies are nowadays produced in vitro by bulk tissue cultures of selected mouse hybridoma cell clones and are widely available to users. Their detection is performed using various microscopic techniques, i.e. through the measurement of fluorescence signal (fluorescence microscopy), the formation of coloured product (light microscopy) or the presence of bound colloidal gold particles (electron microscopy). When the intention within a research project or a biotechnological process is to monitor the expression of recombinant protein, fluorescence (confocal) microscopy is generally recognized as a suitable microscopic method and is more commonly used, also in combination with specific (monoclonal) labelling. On the other hand and despite of the high specificity of monoclonal antibodies, for transmission electron microscopy (TEM) rabbit polyclonal antibodies are preferentially used as probes for more precise immunolocalization of recombinant proteins. The reason lies in an inefficient application of many monoclonal antibodies used as specific probes for immunogold labelling of cells embedded into resins (i.e. plastic sections). Often and due to the noticeable loss of antigenic determinants changed antigen could not be recognized, labelled and localized with a specific monoclonal basically recognizing only one epitope on a protein (antigen) of interest. Contrary to resin embedment, the preparation of cryosections [1, 2] represents a method where cell proteins preserve their antigenicity well which further enables the application of monoclonal antibodies as probes for efficient immunogold labelling prior to TEM observation. In this paper emphasis is put on the preparation of ultrathin cryosections of bacterial cells (E. coli) expressing recombinant human protein named cystatin C. As a representative application of this technique and as shown in Fig. 1 and Fig. 2 the labelling signal and the specificity of mouse monoclonal against cystatin C have been compared to polyclonal prepared and isolated against the same protein by fluorescence (confocal) and electron microscopy, respectively. Cystatins are important natural reversible inhibitors of lysosomal proteolytic enzymes (cysteine proteases) [3]. They are wide spread in many living organisms and are involved in various biological processes where they regulate normal proteolysis and take part in disease pathology [4]. Precise understanding of their regulatory roles on proteolytic enzymes will provide a valuable insight how proteolysis could be modulated to treat a specific disease. The purpose of this study was to find a highly specific (possibly monoclonal) which would later also be appropriate for the detection of inhibitory active recombinant cystatin C used as a modulator of proteolytic activity of lysosomal enzymes in human cells. FORMATEX

2 Microscopy: Science, Technology, Applications and Education A. Méndez-Vilas and J. Díaz (Eds.) Monoclonal Primary mouse monoclonal Primary rabbit polyclonal Secondary polyclonal Polyclonal Fluorophore Fluorophore conjugated to secondary Optical section of cell with denoted target protein Fig. 1 Immunolabelling for fluorescence (confocal) microscopy. Target protein is first labelled with primary mouse (above) or with primary rabbit (below) and followed by secondary anti-mouse (above) or secondary anti-rabbit (below), conjugated to selected fluorophore. Primary polyclonal recognizes different parts of the same target protein (its different epitopes) while primary monoclonal recognizes only one epitope on the target antigen (i.e. protein of interest). Monoclonal Primary mouse monoclonal Primary rabbit polyclonal Secondary rabbit (anti-mouse) polyclonal Polyclonal Protein A Colloidal gold Protein A - gold Ultrathin section of cell with denoted target protein Fig. 2 Immunogold labelling for transmission electron microscopy (TEM). Target protein is first labelled with primary rabbit (below) or with primary mouse followed by secondary rabbit anti-mouse (above), and Protein A-gold. Polyclonal recognizes different parts of the same target protein (its different epitopes) while primary monoclonal recognizes only one epitope on the target antigen (i.e. protein of interest). 242 FORMATEX 2010

3 2. Material and methods 2.1 Specimen preparation and immunolabelling for fluorescence (confocal) microscopy Bacterial cells (E. coli) expressing recombinant human protein (protease inhibitor cystatin C) were prepared according to the described procedure [5]. Aliquots of bacterial cells were cytocentrifuged at 265 g (StatSpin Cytofuge) directly onto poly-l-lysine (Sigma)-coated glass slides. Cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) in 0.1 M phosphate buffer for 1 hour at 37 ºC and permeabilized with 0.1% Triton X-100 for additional 5 min. After this and all subsequent steps, cells were rinsed in PBS. ph of all fixatives, immunoreagents, buffers and rinsing solutions was adjusted to ph 7.4. Non-specific binding of immunoreagents (primary and secondary antibodies) was blocked with 3% BSA (Sigma) and 10% goat serum (Sigma) in PBS. Cells were labelled with primary monoclonal or polyclonal (10 µg/ml) against human cystatin C [6]. Primary was added for 1 hour at 37 ºC. Another incubation for 45 min at 37 ºC followed with secondary Alexa Fluor 488-labelled goat anti-mouse IgG and secondary Alexa Fluor 488-labelled goat anti-rabbit IgG (Invitrogen Molecular Probes), respectively. Controls omitting the primary and/or secondary were also performed. Labelled bacterial cells were mounted with ProLong AntiFade Kit (Invitrogen Molecular Probes). Fluorescence microscopy and optical slicing were performed using a confocal laser scanning microscope LSM 510, objective 60, N.A. = 1.4 (Carl Zeiss). Fluorophore Alexa Fluor 488 was excited with argon laser at 488 nm. Emission signal was filtered using BP nm filter and 400 nm-optical sections in z-dimension were studied. Carl Zeiss LSM image software was used to evaluate the localization of labelled recombinant protein inside bacterial cells. 2.2 Specimen preparation, preparation of ultrathin cryosections and immunogold labelling for transmission electron microscopy (TEM) Bacterial cells (E. coli) expressing recombinant human protein (protease inhibitor cystatin C) were prepared according to the described procedure [5]. Cells were fixed with paraformaldehyde (Sigma-Aldrich) added directly into the medium up to the final concentration of 4%. Fixation took 1 hour at 37 ºC. Mixture of growth medium and fixative was removed with a series of centrifugation steps at 400 g. After final centrifugation step a loose pellet of bacterial cells was embedded in 10% gelatine (Merck) in 0.1 M phosphate buffer. Gelatine was solidified on ice for 30 min and the pellet with cells excised from the tube and cut into small blocks (1 mm 3 cubes). Blocks with bacterial cells were immersed with 2.6 M sucrose in 0.1 M phosphate buffer and infiltrated overnight at 4 ºC. Finally, cryoprotected cells were mounted on special aluminium pins (specimen carriers) and put in liquid nitrogen where they were stored [7, 8]. Trimming and sectioning were performed on Leica EM FC6 ultramicrotome for cryosectioning. Frozen specimen blocks were trimmed at 90 C using diamond trimming knife (Diatome). Ultrathin cryosections (60 70 nm) were cut at 120 C using diamond cryo immuno knife (35 angle, Diatome). Sections were collected on droplets of pick-up solution, i.e. 1:1 mixture of 2.3 M sucrose (Merck, in phosphate buffer) and 2% methyl cellulose (Sigma-Aldrich, in water). Retrieved ultrathin sections of bacterial cells were put on a thin layer of carbon-coated Formvar films (TAAB Lab.) spread over hexagonal Cu/Pd grids (Agar) and stored at 4 C [7]. Sections on grids were put on melted 2% gelatine in 0.1 M phosphate buffer for 20 minutes at 37 C (to remove residual gelatine used as supporting matrix around bacterial cells). Grids were transferred to droplets of buffer where grids were floating on top of the droplets with sections facing the droplets. Sections were rinsed with 20 mm Gly in PBS (to block residual aldehyde groups) and 1% BSA in PBS (to diminish non-specific binding of antibodies and Protein A-gold, respectively) [8]. For immunogold labelling sections were incubated with mouse anti-cystatin C monoclonal and rabbit anticystatin C polyclonal in PBS, respectively, using the same concentrations and the same primary antibodies as for fluorescence microscopy. Described immunolabelling procedure was performed by series of incubation/rinsing steps. Sections were incubated with primary (on 20 µl droplets) for 30 min. Secondary (bridging) (rabbit anti-mouse polyclonal, MP Biomedicals - Cappel) was used after primary mouse monoclonal (see Fig. 2 above), but not after incubation with primary rabbit polyclonal (see Fig. 2 below). PBS was used for extensive rinsing after each incubation step. After the removal of an excess of antibodies sections were incubated with Protein A gold conjugate with 10-nm gold particles for 20 min and according to the producer s instructions (Cell Microscopy Centre, University Medical Centre Utrecth). Controls omitting primary and/or secondary (bridging) were also performed. Additional extensive rinsing of grids on droplets of distilled water was performed prior to uranyl acetate staining (to remove traces of salts and thereby prevent precipitation of uranyl acetate). Sections (cell proteins) were contrasted with 0.4% uranyl acetate (SPI-Chem TM uranyl acetate, Spi Supplies), ph 4, in solution with 1.8% methyl cellulose (Sigma). FORMATEX

4 A. Méndez-Vilas and J. Díaz (Eds.) Immunogold-labelled sections of bacterial cells were observed with Philips CM120 BioTWIN transmission electron microscope at 100 kv. Micrographs were taken using KeenView digital camera and item software. For evaluation of labelled recombinant protein cystatin C two hundred gold particles on three different grids were counted for each sample [9]. 3. Results and discussion E. coli has been used for production of various recombinant proteins. However, overexpressed proteins are often inactive and accumulated in the form of inclusion bodies from which biologically active proteins can only be recovered by a refolding process after complex isolation and separation from other bacterial cell components. Human protease inhibitor cystatin C is a single-chain protein consisting of 120 amino acids with a molecular weight of 13 kda and two characteristic disulfide bonds located in the carboxy-terminal region. In presented experiment cystatin C was expressed and targeted for secretion into the periplasmic space where oxidative environment favours the formation of disulfide bonds. Furthermore, cystatin C was successfully isolated from periplasmic lysate in inhibitory active form [5]. Nevertheless, in the same population of bacterial cells accumulation of inclusion bodies was also observed. Formation of inclusion bodies did not interfere with the isolation of inhibitory active recombinant protein from periplasm. But, it gave us a useful cell model for testing various anti-cystatin C antibodies of which two (one monoclonal and one polyclonal) are presented here. The idea was to use bacterial cells having recombinant human cystatin C in periplasm as well as in inclusion bodies to select an appropriate and specific anti-human cystatin C. Possibly a monoclonal which would be applicable with microscopic techniques, such as fluorescence microscopy and TEM, and be able to specifically recognize inhibitory active cystatin C (mostly present in bacterial periplasm) but not cystatin C inside inclusion bodies (misfolded and not active protein). By using immunofluorescence microscopy (see Fig. 3) no significant differences in recombinant cystatin C localization were observed after labelling with monoclonal (Fig. 3a, 3c) and polyclonal (Fig. 3b, 3d), respectively. Distinct fluorescently labelled structures were observed after labelling with anti-cystatin C antibodies, but it was difficult to decide whether it is the part of bacterial periplasm that is being extensively labelled due to high concentration of active cystatin C in periplasmic space. Furthermore, with both antibodies some bacteria in population were strongly labelled throughout the cell indicating strong expression of cystatin C (Fig. 3). Furthermore, in cells distinguished fluorescently labelled spherical structures (presumably inclusion bodies, Fig. 3b, 3d) and ring-like structures (presumably periplasm, Fig. 3a) were observed. But the lack of morphological information behind the fluorescence labelling prevented precise confirmation whether tested antibodies really specifically labelled (and distinguished) between properly folded and active cystatin C (in periplasm) and misfolded cystatin C (in inclusion bodies). Therefore, to overcome the limitation of light microscopy, ultrathin cryosections were prepared and immunogold labelled with the same antibodies as they were used for fluorescence microscopy. The method itself was quick; sections of bacterial cells were cut and labelled within one day. Ultracryotomy of fixed specimens in combination with immunogold labelling has been used for ultrastructural localization of many interesting molecules. Since the introduction of this technique, vast improvements in techniques and machinery have been established and the entire process has been made easier and more accessible [7, 8]. Here it is shown how this method was successfully used for studying recombinant proteins in bacterial cells. By using TEM (Fig. 4, Fig. 5, Tab. 1) it was clearly demonstrated that polyclonal recognized both, recombinant cystatin C in inclusion bodies (insoluble aggregates of recombinant cystatin C, inactive and misfolded) and recombinant cystatin C in periplasmic space (soluble, properly folded recombinant protein) (Fig. 4b, 4d, Fig. 5b). On the other hand, selected monoclonal recognized predominantly recombinant protein in cell periplasm (72% of a total signal (Fig. 4c, Fig. 5a, Tab. 1) which was later also isolated as properly folded inhibitor cystatin C [5]. The rest of the gold particles were distributed in cytoplasm (23%) and only a minority was bound to inclusion bodies (5%) further indicating the monoclonal recognized cystatin C in periplasm but not cystatin C in inclusion bodies (Fig. 4a, 4c, Fig. 5a, Tab. 1). Contrarily, after immunogold labelling with rabbit polyclonal 45% of gold particles were counted in inclusion bodies, 47% in periplasm and 8% in cytoplasm (Tab. 1) indicating that polyclonal anti-cystatin C recognised also cystatin C in inclusion bodies, i.e. misfolded and non-active inhibitor. That was not a surprise since polyclonal (which is actually always a mixture of different monoclonal antibodies) recognizes different epitopes on a protein (antigen) of interest (Fig. 1, Fig. 2). Control omitting primary mouse anti-cystatin C monoclonal (Fig. 4e) and control omitting primary rabbit anti-cystatin C polyclonal (Fig. 4f) were performed. Only negligible background signal was observed conforming the immunogold labelling of cystatin C (Fig. 4a 4d) did not result neither as a consequence of unspecific binding of bridging rabbit anti-mouse (Fig. 4e) nor Protein A-gold (Fig. 4f), but it was a result of anti-cystatin C binding to cystatin C. 244 FORMATEX 2010

5 Microscopy: Science, Technology, Applications and Education a) b) c) d) Fig. 3 Fluorescence microscopy of labelled recombinant human cystatin C in E. coli. Fixed bacteria were labelled with mouse anticystatin C monoclonal (a, c) or rabbit anti-cystatin C polyclonal (b, d), followed by secondary goat anti-mouse (a, c) and goat anti-rabbit (b, d), respectively, both conjugated to Alexa Fluor 488 fluorophore. Bars: 5 µm. FORMATEX

6 Microscopy: Science, Technology, Applications and Education A. Méndez-Vilas and J. Díaz (Eds.) a) b) c) d) e) f) Fig. 4 Transmission electron microscopy of immunogold labelled recombinant human cystatin C expressed in E. coli. Fixed bacteria were cryosectioned and their ultrathin sections (70 nm) retrieved and labelled with mouse anti-cystatin C monoclonal (a, c) or with rabbit anti-cystatin C polyclonal (b, d). Control omitting mouse anti-cystatin C monoclonal but not secondary rabbit anti-mouse polyclonal (bridging ) (e) and control omitting rabbit anti-cystatin C polyclonal (f) are shown. Protein A-gold (with 10 nm gold particles) was added in all experiments (a f). Gold particles indicate the position of labelled cystatin C. Uranyl acetate stained bacterial proteins whereas membranes remained unstained and appear white on presented micrographs. Big inclusion bodies are also noticeable (a, b, d, e, f). Bars: 500 nm (a, b, e) and 200 nm (c, d, f). 246 FORMATEX 2010

7 cytoplasm cytoplasm inclusion bodies inclusion bodies periplasm periplasm gold particles a) gold particles Fig. 5 Comparison of immunogold labelling of recombinant human inhibitor cystatin C expressed in E. coli cells labelled with mouse anti-cystatin C monoclonal (a) and with rabbit anti-cystatin C polyclonal (b). Gold particles were counted on three grids for each samples. Distributions of 200 gold particles (± SD) are shown for cytoplasm, inclusion bodies and periplasm. Table 1 Distribution of gold particles in immunogold-labelled E. coli expressing recombinant human inhibitor cystatin C (shown as % of the total count for each labelling). b) Anti-cystatin C Cytoplasm Inclusion bodies Periplasm monoclonal 23% 5% 72% polyclonal 8% 45% 47% 4. Conclusions Fixed cells have preserved their cellular proteins well with their antigenic determinants unchanged in a sufficient number of copies so that selected recombinant protein of interest (human protease inhibitor cystatin C) was successfully and specifically labelled with monoclonal recognising properly folded recombinant protein in bacterial periplasm but not misfolded recombinant protein in inclusion bodies. These results show that described technique of simple and rapid generation of ultrathin cryosections together with immunogold electron microscopy and monoclonal application offers a useful alternative method to fluorescence and confocal microscopy, also for the detection of recombinant proteins. Acknowledgements Valuable help of M. Prebanda-Trstenjak (Jožef Stefan Institute) with the cultivation of bacterial cells for recombinant protein expression is acknowledged. Microscopy was performed at EMBL Heidelberg and granted by EMBO short-term fellowship (ASTF ) to T.Z.B. References [1] Tokuyasu KT. A study of positive staining of ultrathin frozen sections. Journal of Ultrastructure Research. 1978;63: [2] Tokuyasu KT. Immunocytochemistry of ultrathin frozen sections. Histochemistry Journal. 1980;12: [3] Zavašnik-Bergant T. Cystatin protease inhibitors and immune functions. Frontiers in Bioscience. 2008;13: [4] Zavašnik-Bergant T, Turk B. Cysteine proteases: destruction ability versus immunomodulation capacity in immune cells. Biological Chemistry. 2007;388: [5] Cimerman N, Trstenjak-Prebanda M, Turk B, Popovič T, Dolenc I, Turk V. Interaction of cystatin C variants with papain and human cathepsins B, H and L. Journal of Enzyme Inhibition. 1999;14: [6] Zavašnik-Bergant T, Repnik U, Schweiger A, Romih R, Jeras M, Turk V, Kos J. Differentiation- and maturation-dependent content, localization and secretion of cystatin C in human dendritic cells. Journal of Leukocyte Biology. 2005;78: [7] Griffith JM, Posthuma G. A reliable and convenient method to store ultrathin thawed cryosections prior to immunolabelling. The Journal of Histochemistry & Cytochemistry. 2002;50: [8] Slot JW, Geuze HJ. Cryosectioning and immunolabelling. Nature Protocols. 2007;2: [9] Mayhew TM, Griffiths G, Lucocq JM. Applications of an efficient method for comparing immunogold labelling patterns in the same sets of compartments in different groups of cells. Histochemistry and Cell Biology. 2004;122: FORMATEX