Metabolic Mapping of Protease Activity
- Metabolic Mapping of Proteinase Activity with Emphasis on In Situ Zymography of Gelatinases : Review and Protocols
Over 30 years ago, Robert E. Smith and colleagues developed synthetic substrates for specific proteinases with the leaving group 4-methoxy-2-naphthylamine (MNA; Smith et al. 1972; Smith and Van Frank 1975). The substrates consist of the MNA-leaving group attached to an amino acid sequence specific for the proteinase under study (Lojda 1984). The latter can be coupled after proteolytic removal of the amino acids either to a diazonium salt, such as Fast Blue BB, to give a colored final reaction product (Lojda 1984), to hexazotized pararosanilin or new fuchsin for electron microscopic purposes (Schroeder and Gossrau 1982), or to 5-nitrosalicylaldehyde, which results in a yellow fluorescent reaction product (Dolbeare and Smith 1977). Figure 1 shows the ultrastructural localization of cathepsin B activity in lysosomes of rat liver parenchymal cells, as demonstrated with Z-ala-arg-arg-MNA as substrate and hexazotized pararosanilin as coupling reagent. The procedure was performed as described by Schellens et al. (2003).
Diazonium salts have been applied in simultaneous coupling methods for localization of various proteinases and peptidases (Lojda 1984). However, cysteine proteinases cannot be demonstrated very well with the simultaneous coupling method because these proteinases require SH groups for their activity and SH groups destroy diazonium salts. Moreover, diazonium salts strongly inhibit the activity of proteinases noncompetitively. Therefore, either the fluorescence method with the use of 5-nitrosalicylaldehyde (Van Noorden et al. 1987) or a postcoupling method with Fast Blue BB as coupling reagent (Van Noorden et al. 1989) is the method of choice to localize activity of cysteine proteinases. The synthetic substrates for proteases with MNA as leaving group that are commercially available are listed in Table 1.
Yi et al. (2001) applied synthetic fluorogenic substrates with 7-amino-4-trifluoromethylcoumarin (AFC) as leaving group to demonstrate protease activities in the wound of swine skin, which approach was proposed by Lojda (1996). The substrate was incorporated into agarose so that the water-soluble fluorescent tag AFC could be properly localized. Substrates with AFC as leaving group are commercially available for many proteases. However, the activity of only a limited number of proteases has been localized thus far (Lojda 1996; Yi et al. 2001). It should be emphasized that small peptide substrates (with either MNA or AFC as leaving group) are not necessarily very specific. There is considerable overlapping in the affinity of different proteinases for various peptide substrates.
It is becoming increasingly recognized that enzymes may behave differently in living cells and tissues than in frozen or fixed cells or tissue sections. Therefore, techniques are being developed for the detection of protease activities, such as those of dipeptidyl peptidase IV (DPPIV) and cathepsin B in living cells (Van Noorden et al. 1997,1998). A review on fluorogenic substrates available for metabolic mapping in living cells was recently published (Boonacker and Van Noorden 2001). Rhodamine-based fluorogenic dipeptide substrates were first synthesized by Leytus et al. (1983) for aminopeptidase, DPPIV, cathepsin B, cathepsin K, and cathepsin L. Recently, a new type of fluorogenic substrate for proteases has been synthetized based on the leaving group cresyl violet (Van Noorden et al. 1997,1998; Lee et al. 2003). The methods to localize protease activity in living cells using cresyl violet-based and rhodamine-based substrates have been critically evaluated by Boonacker et al. (2003).
Fluorogenic substrates for caspases are based on peptide sequences that are 18 amino acids long containing motifs that are specifically recognized by caspases and two identical fluorophores covalently attached near their termini. In such dimers, the fluorophore fluorescence is 90% quenched and fluorescence is generated when the substrate is cleaved (Komoriya et al. 2000; Kohler et al. 2002). The same type of substrate with quenched fluorogenic properties is also available for cathepsin D and MMP-2 (Bremer et al. 2001a,b). Thus far, these methods have been applied to living cells. Whether the localization of the final fluorescent reaction product is precisely enough to localize protease activity in tissue sections has yet to be established.
Proteolysis in tumor cells has been used by Weissleder et al. (1999) to develop an in vivo imaging system for tumors. An optimally quenched near-infrared fluorescence (NIRF) probe generates a strong NIRF signal after proteolytic activation. The probe is cleaved by lysosomal cysteine and serine proteases. This approach may also provide perspectives for localization of protease activities in tissue sections.
An entirely different approach to demonstrate activated caspases in living cells is the use of Caspa Tags, which are carboxyfluorescein-labeled fluoromethylketone inhibitors (Kohler et al. 2002). These cell-permeable inhibitors bind more or less specifically and irreversibly to the active site of caspases. Because the active site is available only in mature caspases, the Caspa Tags can exclusively stain cells containing the processed caspases of interest and the probes will not accumulate in normal (non-apoptotic) cells. This principle may have applications for the demonstration of other active proteases as well.
In conclusion, reliable techniques are available for the demonstration of activities of cysteine proteinases, serine proteinases, and aspartic proteinases using artificial substrates that contain small numbers of peptides, but demonstration of activities of MMPs with these small substrates had only limited success owing to the large numbers of peptide bonds that can be cleaved by MMPs and the overlap among different MMPs.
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