Despite its promise, biarsenical labeling of TC-tagged proteins has seen limited use thus far. One reason may be background presumably due to FlAsH binding to off-target cysteine-rich proteins (Stroffekova et al., 2001
; Berens et al., 2005
; Langhorst et al., 2006
; Hearps et al., 2007
), necessitating a postincubation step to reduce this background. Successful applications of this technology have used protocols similar to that originally reported for FlAsH (Perlman and Resh, 2006
; Arhel et al., 2006
; Roberti et al., 2007
; Tour et al., 2007
) but have not addressed the specificity issue to allow one-step labeling of any protein, especially those localized to cell surfaces. A previous attempt to label cell surface proteins required a custom-synthesized membrane-impermeable probe, which still showed some background labeling (Adams et al., 2002
). Thus, the biarsenical labeling approach has not yet realized its full potential with the inability to conveniently target cell surface proteins.
Labeling PrPres with fluorescent tags in live cells presented an exceptional challenge because of the sensitivity of its formation to steric effects by foreign tags. Being one of the smallest fluorescent labels, FlAsH/TC-motif labeling seemed a promising option so we sought to develop strategies to overcome the above-mentioned limitations. Fortunately, DTT can rapidly reduce endoplasmic reticulum proteins in live cells without affecting cell viability (Braakman et al., 1992
), suggesting it was a good candidate reducing agent to enhance biarsenical labeling efficiency while ensuring reduction of cystine residues within the TC motif of cell surface proteins. We discovered DTT worked and simplified the procedure by eliminating the preincubation and postincubation steps traditionally used to reduce background labeling. To our knowledge, the method reported here is among the most rapid to achieve specific fluorescent labeling of cell surface proteins, facilitating the time resolution of pulse-chase labeling for subsequent biochemical and/or live cell imaging experiments. Our success in fluorescent-tagging PrP and APP proves that extracellular proteins can be labeled specifically and efficiently with the commercially available FlAsH reagent.
Because of the potential power of TC-tagging, we and others have developed new biarsenical derivatives (Bhunia and Miller, 2007
; Cao et al., 2007
; Chen et al., 2007
; Liu et al., 2007
; Tour et al., 2007
). We synthesized FlAsH conjugates containing either an Alexa Fluor dye or biotin that worked as efficiently and specifically as FlAsH, suggesting that conjugation to carboxy-FlAsH would not substantially affect the affinity of the biarsenical moiety for the TC-motif. This is important because such probes are useful only when they are specific. Biarsenical scaffolds conjugated to Alexa Fluor dye (Bhunia and Miller, 2007
) or biotin (with an additional dopamine moiety) (Liu et al., 2007
) have been described by other groups, but the affinity and specificity of these reagents for the minimal CCPGCC TC motif remain to be shown in the context of the highly challenging application of labeling TC-tagged proteins expressed in mammalian cells where the ratio of TC-tagged protein to total protein is usually very low and off-target biarsenical-binding proteins are common (Griffin et al., 2000
; Stroffekova et al., 2001
). Furthermore, without a method to label cell-surface TC-tagged proteins these membrane-impermeable biarsenical compounds would be restricted to cell-free or fixed/permeabilized cell applications. With the advent of IDEAL-labeling, biarsenical labeling becomes a technique to rapidly add a functional molecule onto a designated part of a cell surface target protein in situ on live cells. The molecule may have properties other than fluorescence. Bio-FlAsH is one example, which has tremendous potential for many applications via detection with the plethora of commercially available biotin-binding conjugates. Because carboxy-FlAsH is itself a fluorophore, it serves as a scaffold for the creation of multifunctional derivatives.
The successful conversion of FlAsH-PrPs to FlAsH-PrPres demonstrates that the steric effects of the FlAsH/TC-motif are small, encouraging its application to other proteins with steric sensitivities. It is difficult to modify the PK-resistant core region of PrP without impairing its convertibility, as demonstrated in part by barriers for prion transmission between species consisting of differences in only a few amino acid residues in this region between inoculated PrPres and host-encoded PrPsen. The precise structure of PrPres has not been determined. It seems to consist of PrP oligomers with tightly packed β-sheet strands, some of which are generated from refolding of either α-helical and/or unstructured regions of PrPsen. This ordered assembly of PrP molecules during the conversion process may contribute to its sensitivity to PrP modifications. Although PrP(230TC) could convert to PrPres, a mutant PrP containing a 15 amino acid tag at the same site as PrP(230TC) did not convert to PrPres (data not shown), suggesting a limitation to tag sizes even in relatively tolerant locations. We speculate that, besides the minimal size, the hairpin structure of the TC-motif might further limit its steric effects. Whatever the explanation, the minimal steric effect of the TC-motif would extend its application to many other proteins.
Compatibility of FlAsH with fluorescent gel analysis allows observation of the same FlAsH-labeled molecules both microscopically and biochemically as effectively demonstrated in D (lysate) and E. Whereas immunoblotting shows the steady-state of protein molecules collectively irrespective of their age, pulse-chase analysis can provide dynamic time-resolved analysis. Unlike conventional pulse-chase using radioisotopes, lack of immunoprecipitation in FlAsH-pulse chase minimizes loss of labeled proteins, and the fixed stoichiometry between FlAsH and fragments regardless of their sizes achieves more accurate quantification of labeled fragments. Altered behavior of the labeled proteins due to the tag and/or labeling procedures was a concern, but the results of FlAsH-pulse-chase experiments were consistent with reports for wild-type PrP and APP, confirming a minimal impact on the behavior of these proteins. Moreover, there were several novel results related to PrP biology.
First, C3 was identified as a new metabolic fragment of PrP. To date, PrP shedding possibly by metalloprotease-directed cleavage near the C terminus has been described (Parkin et al., 2004
), although the short C-terminal by-product has never been noted. E64-sensitive proteolysis responsible for C3 formation (B) might represent another shedding mechanism of PrP. Further experiments with E64 established that it is active against multiple strains of mouse-adapted prions but led to the surprising finding that the anti-prion activity of this compound in infected N2a cells is not mediated through inhibition of new PrPres biosynthesis. The anti-prion activity of cysteine protease inhibitors is somewhat controversial. Depending upon the host cell line, cysteine protease inhibitors can either reduce (Doh-Ura et al., 2000
;Yadavalli et al., 2004
) or increase (Luhr et al., 2004
) steady-state PrPres levels. According to Yadavalli et al. (2004)
, N-terminal truncation of PrPres by calpain-mediated cleavage assists PrPres propagation in ScN2a and SMB models of prion infection. In the present study, PrPres biosynthesis was not inhibited by E64 even at concentrations that blocked complete N-terminal truncation of newly formed PrPres, suggesting that the anti-prion effects of E64 are independent of its effects on PrPres proteolysis. Interestingly, we did observe evidence of a very small N-terminal truncation of FlAsH-PrPres formed in the presence of cysteine protease inhibitors (Supplemental Figure 3), but the protease(s) involved remains to be identified. Our data are consistent with a time dependence to the inhibitory effect of E64, raising the possibility that cysteine proteases may indirectly contribute to PrPres propagation via the modification of other cellular factors analogous to the effects of a particular tyrosine kinase (Ertmer et al., 2004
). This could explain the differential effects observed between different prion- infected cell lines. Alternatively, N-terminal proteolysis of PrPres may facilitate faithful transmission of PrPres aggregates between cells either during mitosis or by extracellular mechanisms (e.g., exosomes; Fevrier et al., 2004
), which would be important for maintenance of PrPres formation in the context of rapidly dividing cell models of infection.
Next, based on quantification of the 21-kDa protease-resistant core of PrPres with and without exogenous protease digestion, we found chymotrypsin digestion allows a more accurate evaluation of total FlAsH-PrPres. Although PK resistance has been regarded as a hallmark of disease-associated PrP, PK is not necessarily the ideal protease to assess protease resistance of PrPres. Evidence for the existence of PK-sensitive forms of disease-associated PrP has come from experiments using mild PK digestion at low concentrations or low temperatures (Tremblay et al., 2004
) or immunological assays relying on differential epitope exposure between PrPres and PrPsen (Safar et al., 1998
). Therefore, defining PrPres based specifically on PK digestion as opposed to other proteases is arbitrary. The Chy-res might represent “PK-sensitive PrPres,” and this moderately protease-sensitive fragment might aid structural studies of PrPres. Although a previous report comparing PK versus chymotrypsin digestion of brain homogenates failed to detect a difference in protease sensitivity of PrPres, their work differs significantly from ours with respect to the method of analysis, the source material for digestion, and the use of very high enzyme concentrations (Pan et al., 2005
Finally, the lack of direct labeling of TC-PrPres fortuitously enabled measurement of the “conversion rate” of FlAsH-PrPsen rather than the change in total TC-PrPres. This allowed rapid analysis of the effects of many compounds specifically on PrPres synthesis, permitting detection of early events not readily observed in analysis of steady-state PrPres levels (data not shown). This could be developed as a high-throughput screen for anti-prion therapeutics. Likewise, FlAsH-labeled TC-tagged APP constructs might assist the search for new Alzheimer's drugs by allowing rapid screening for modulators of APP processing.
In conclusion, we developed a specific method for fluorescent labeling of extracellular TC-tagged proteins, PrP and APP, and demonstrated its usefulness in imaging and protein metabolism studies. This sets the stage for important experiments in prion biology such as imaging of the formation and spread of PrPres. Such imaging experiments will themselves be challenging due to the fact only a small percentage of PrP-sen is converted to PrP-res, but our system provides valuable tools to begin these investigations. We also created powerful new biarsenical derivatives. The present report establishes the versatility of biarsenical compounds in imaging and pulse-chase studies and illustrates the potential of this technique to attach functional molecules to specific target proteins on live cells, encouraging the development of more biarsenical compounds.