NeurocanGFP Fusion Protein : A New Approach to Detect Hyaluronan on Tissue Sections and Living Cells
Department of Experimental Pathology, Lund University, Lund, Sweden (HZ,MS,UR); Department of Pathology, AstraZeneca R&D Sodertalje, Sodertalje, Sweden (HZ); Institute of Anatomy and Cell Biology University of Bonn, Bonn, Germany (SLB); and Institute for Physiological Chemistry University of Bonn, Bonn, Germany (JK)
Correspondence to: Uwe Rauch, Dept. of Experimental Pathology, University Hospital, S221 85 Lund, Sweden. E-mail: uwe.rauch{at}pat.lu.se
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Summary |
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Key Words: hyaluronan affinity histochemistry neurocan GFP fusion protein time-lapse video microscopy
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Introduction |
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Because of its conserved structure and ubiquitous expression, histological detection of hyaluronan requires specific methods other than application of antibodies. The currently used method utilizes hyaluronan-binding proteins and protein fragments modified with biotin. To generate these probes, hyaluronan and its binding proteins are co-purified from animal tissue, and the proteins are biotinylated and separated from hyaluronan (Ripellino et al. 1988). These biotinylated probes are then visualized by avidin/strepavidin conjugated with enzymes or other detection aids.
Here we introduce a simple one-step method to detect hyaluronan with high spatial resolution. It is based on a fusion protein consisting of a GFP module and the hyaluronan-binding domain of neurocan, a PG of the lectican family (Rauch et al. 2001). The fusion protein was expressed and secreted by mammalian cells. To validate the fusion protein as a histochemical detection aid, mammalian eyes were used because their sections comprise many different types of tissues in a relatively small area. Furthermore, the distribution of hyaluronan in this organ appeared well characterized (Chan et al. 1997
; Gong et al. 1997
; Hollyfield et al. 1997
,1998
; Lerner et al. 1997
,1998
). Our analysis demonstrated the suitability of the fusion protein for affinity histochemical applications. In addition, we found that the fusion protein was also able to visualize hyaluronan on living cells using time-lapse video microscopic observation.
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Materials and Methods |
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SDS-PAGE and Blotting
SDS-PAGE was performed on slab gels using the Bio-Rad (Hercules, CA) mini gel system. Proteins in conditioned medium were precipitated with trichloroacetic acid, washed with ice-cold acetone, and dissolved in SDS-PAGE sample buffer containing 2% SDS with no reducing agents. Blots were performed in Tris/glycine buffer containing 10% methanol for 1 hr at 100 V using the Bio-Rad mini gel system. For immunodetection, proteins were transferred to PVDF membrane (Amersham; Arlington Heights, IL), which was blocked with 5% dry milk in 150 mM NaCl, 20 mM Tris/HCl, pH 8, with 0.1% Tween-20 (TBST) overnight, and incubated with an antiserum against the N-terminal neurocan domain in TBST (1:7000). This rabbit antiserum was raised against the N-terminal neurocan fragment 359H recombinantly expressed in HEK 293 cells and purified by ConA affinity and consecutive Superose 12 chromatography (Retzler et al. 1996). After a further incubation with horseradish peroxidase-linked donkey anti-rabbit antibody (Jackson; West Grove, PA) in TBST (1:20 000), the blot was developed with the ECL plus detection system (Amersham).
Tissue Preparation
Procedures for handling mice were approved by the Committee for Ethics in Animal Experiments in Malmö and Lund. Mice (129SW) were anesthetized by CO2 and decapitated. The eyes were enucleated and immersion-fixed in cold Bouin fixation solution for 1218 hr at 4C. The eyes were embedded in paraffin, sectioned at 6 µm and stored at 80C.
Fusion Protein Staining
Sections were dewaxed with xylene (three times for 5 min) and an ethanol series (100%, 90%, 70%, and 50%) 5 min for each. After washes in phosphate buffer (20 mM NaxH3-xPO4, 150 mM NaCl, pH 7.4) three times for 5 min, the sections were blocked in blocking solution (2% normal goat serum in phosphate buffer) for 1 hr at room temperature. The fusion protein was diluted 1:3 in blocking solution and briefly centrifuged at 14,000 x g before it was applied to sections. The fusion protein was incubated on sections overnight at 4C. The next day unbound fusion protein was washed away with phosphate buffer (three times for 5 min). Slides were counterstained with propidium iodide and then coverslipped with Vectashield (Vector Laboratories; Burlingame, CA). For triple staining the fusion protein was incubated together with an anti-laminin-1 antiserum (455, 1:500; kindly provided by Dr. Lydia Sorokin, Lund) in blocking solution overnight at 4C. Omitting the fusion protein, the secondary antibody (Cy3-conjugated goat anti-rabbit antibody; Jackson) was incubated at RT for 30 min in phosphate buffer. The slides were washed in phosphate buffer and counterstained with DAPI.
Hyaluronan Digestion In Situ
Deparaffinized sections were incubated for 3 hr at 37C with 100 U/ml Streptomyces hyaluronate lyase (Sigma; St Louis, MO) in 50 mM Na acetate buffer, pH 5, in the presence of 5 mM EDTA, 5 mM benzamidine/HCl, 5 mM N-ethylmaleimide, and 1 mM freshly added phenylmethylsulfonylfluoride. Control samples were incubated under the same conditions without the enzyme.
Probe Preabsorption
The fusion protein was preincubated with 1 mg/ml hyaluronic acid (Sigma H5388) for 30 min at RT and then applied to adjacent sections.
Fluorescence Microscopy
Specimens were viewed with an epifluorescent microscope (Axiophot; Zeiss, Oberkochen, Germany) and images were taken with digital cameras AxioCam (Zeiss) or C4742-95-12NRG (Hamamatsu; Tokyo, Japan) with Openlab software (Improvision; Tucson, AZ). The images were processed using Openlab and Photoshop software (Version 6.0; Adobe, Tucson, AZ).
Time-lapse Video Microscopy
Specimens were viewed every 2 min by phase-contrast and epifluorescent microscopy (Axiovert 200M; Zeiss) at 37C and 5% CO2 in a humidified chamber. Images were taken with the digital camera C4742-95-12ERG (Hamamatsu) and merged with Openlab software (Improvision). The images were combined using Photoshop software (Version 6.0; Adobe).
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Results |
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In the dog eye, hyaluronan staining was seen clearly in the vitreous (Figure 3A) , where hyaluronan was first identified and commonly is considered as reference for hyaluronan deposition in mammals. In addition, this staining, which extended in radial strands into the ganglion cell layer (Figure 3C), was no longer evident after treatment with hyaluronidase (Figures 3B and 3D).
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Because it is possible to apply the fusion protein without any secondary incubation or color reaction, it was tested for its suitability in a real-time application such as time-lapse video microscopy. The culture medium of native, untransfected HEK 293 cells was changed to medium that had been conditioned by cells secreting the fusion protein. Consecutively distinct fluorescent patches appeared on the cells. Time-lapse video microscopy revealed a dynamic redistribution of the fluorescence signal in a reproducible pattern. The staining frequently accumulated at sites where cells showed a tendency to physically segregate, but parts of the cell membrane were still attached to each other (Figure 4) .
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Discussion |
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To ensure that this fusion protein would bind to hyaluronan, proper folding of this molecule, especially of the hyaluronan-binding domain, was considered to be very important. We chose to express the fusion protein in mammalian cells, where molecules in the secretory pathway are usually subjected to an endogenous "proofreading" system that does not allow the export of misfolded proteins (Vashist et al. 2001). The hyaluronan-binding domain of neurocan alone has been previously successfully expressed in this system (Retzler et al. 1996
). An analysis of the medium conditioned by fusion protein-secreting HEK 293 cells by Western blotting confirmed that it was possible to obtain distinct monomeric neurocanGFP fusion proteins. Because the antiserum used was raised against the N-terminal neurocan domain, which represents the major part of the fusion protein, the observation of a dominant band at the expected size of the fusion protein and only a minor band of a size expected for the hyaluronan-binding domain alone indicated that no extensive degradation had occurred. Moreover, because the SDS-PAGE was performed under non-reducing conditions, this observation also excludes extensive aberrant covalent associations of the fusion proteins via disulfide bonds.
The direct fusion of the fluorescent detection module to the target recognition domain should reveal the localization of hyaluronan with the highest possible resolution. To fully exploit the spatial resolution of the fusion protein as probe, we used Bouin fixation, which is considered to maintain the tissue structure very well. As presented in this study, the probe gives a detailed staining pattern, e.g., showing the localization of hyaluronan between cells within the retinal inner plexiform/ganglion cell layer. It should be noted that those signals were obtained without any amplification mediated by secondary detection aids. Those are likely to cause a broadening of the stained area, thereby obscuring the exact spatial distribution. Furthermore, compared with the conventional detection method, this probe offers the possibility to be used together with biotinylated probes, such as lectins, for double or triple staining.
The specificity of hyaluronan detection was demonstrated by abolition of the binding after preincubation of the probe with excess hyaluronan, which specifically absorbed the probe from the concentrated conditioned medium. A second demonstration of the specificity was the lack of binding of the probe after treatment of the tissue sections with Streptomyces hyaluronidase, which selectively digests hyaluronan. Both experiments indicate that the fusion protein is a specific probe for the detection of hyaluronan comparable to the established method with hyaluronan-binding proteins isolated from bovine cartilage or sheep brain (Wang et al. 1996; Victor et al. 1999
). Because the international exchange of products derived from animals is prone to restrictions, fusion proteins, which can be produced in any laboratory culturing cells, may become a reliable source for affinity histochemical probes.
Considering that hyaluronan was first identified as a component of the vitreous of the eye, a surprisingly low level of hyaluronan was evident in the mouse vitreous. To be sure that the lack of hyaluronan in the mouse vitreous is not due to the detection method, we analyzed also the vitreous of dog eyes, in which hyaluronan was clearly apparent. The low hyaluronan levels might be related to the small size of the mouse vitreous compared with that of other mammalian species, and might indicate that, for this structure, hyaluronan is not of crucial functional importance.
The observation of fluorescent patches on living cells rather than a general staining of the entire cell surface indicates a focal concentration of hyaluronan to distinct areas. This distribution was possibly mediated by hyaluronan-binding cell surface molecules. Investigations of cultured periodontal ligament fibroblasts, which were fixed with methanol, indicate indeed a localization of CD44 in focal deposits at the edges and in cytoplasmic processes of cells (Zohar et al. 2000).
GFP fusion proteins produced in mammalian cells appear to be especially suitable for the detection of carbohydrate structures. These structures are less sensitive to denaturing and crosslinking fixation procedures and are therefore structurally better conserved in histological applications, while their usually extracellular location renders them accessible to fusion proteins supplied in the culture medium in cytological and cell biological applications. On the other hand, protein domains, which recognize carbohydrates, can be quite complex and require more than a single protein module to achieve the necessary specificity (Tu et al. 1996). These domains may display complex disulfide linkage patterns or depend for their activity on typical vertebrate glycosylations (Sgroi et al. 1996
). However, this study demonstrates that it is possible to express in a preparative fashion carbohydrate-binding protein domains linked to GFP in mammalian cells.
In summary, a GFP fusion protein produced in and secreted from mammalian cells successfully detected hyaluronan in fixed and living biological material, opening up new perspectives for using GFP fusion proteins as detection tools in histological and cytological studies.
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Acknowledgments |
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We thank Drs Reinhard Fässler, Lydia Sorokin, and Ronny Fransson-Steen for support and Dr Berndt Ehinger for critical comments. The dog sections were kindly provided by Department of Pathology, AstraZeneca in Sweden.
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Footnotes |
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