Research High Throughput Substrate Specificity Profiling of Serine and Cysteine Proteases Using Solution-phase Fluorogenic Peptide Microarrays* Dhaval N. Gosalia‡§, Cleo M. Salisbury¶§, Jonathan A. Ellman¶ʈ, and Scott L. Diamond‡**ʈ Proteases regulate numerous biological processes with a ptosis, and coagulation and play an important role in the degree of specificity often dictated by the amino acid pathogenicity and progression of many diseases (1). Pro- sequence of the substrate cleavage site. To map prote- teases comprise one of the largest protein families in organ- Downloaded from ase/substrate interactions, a 722-member library of flu- isms from Escherichia coli to humans (2–4). Improved under- orogenic protease substrates of the general format Ac- standing of proteases will provide insight into biological ؍ Ala-X-X-(Arg/Lys)-coumarin was synthesized (X all systems and will likely provide a number of important new natural amino acids except cysteine) and microarrayed therapeutic targets (1). with fluorescent calibration standards in glycerol nano- To properly function, proteases must preferentially cleave www.mcponline.org droplets on glass slides. Specificities of 13 serine pro- teases (activated protein C, plasma kallikrein, factor VIIa, their target substrates in the presence of other proteins. While factor IXa␤, factor XIa and factor ␣ XIIa, activated com- many factors impact protease substrate selection, one of the plement C1s, C1r, and D, tryptase, trypsin, subtilisin key aspects is the complementary nature of the enzyme- Carlsberg, and cathepsin G) and 11 papain-like cysteine active site with the residues surrounding the cleaved bond in proteases (cathepsin B, H, K, L, S, and V, rhodesain, pa- the substrate. As such, determination of the residues that pain, chymopapain, ficin, and stem bromelain) were ob- comprise the preferred cleavage site of a protease provides at Univ of Penn on November 29, 2007 tained from 103,968 separate microarray fluorogenic re- critical information regarding substrate selection. Further- ؋ ؋ actions (722 substrates 24 different proteases 6 more, determination of substrate specificity also provides a replicates). This is the first comprehensive study to report framework for the design of potent and selective inhibitors. the substrate specificity of rhodesain, a papain-like cys- Here we exploit solution-phase substrate nanodroplet mi- teine protease expressed by Trypanasoma brucei rhod- croarrays (5), in which fluorogenic substrates suspended in esiense, a parasitic protozoa responsible for causing glycerol droplets are treated with aerosolized aqueous en- sleeping sickness. Rhodesain displayed a strong P2 pref- -zyme solutions, to provide protease substrate specificity pro ؍ erence for Leu, Val, Phe, and Tyr in both the P1 Lys and Arg libraries. Solution-phase microarrays facilitate prote- files (6). These arrays allow high throughput characterization ase/substrate specificity profiling in a rapid manner with of the preferred residues on the P side (7) of the substrate in minimal peptide library or enzyme usage. Molecular & a highly parallel and miniaturized format. We report the use of Cellular Proteomics 4:626–636, 2005. these arrays here to map the substrate specificity of 24 serine and cysteine proteases in a rapid and efficient manner. EXPERIMENTAL PROCEDURES Because of their critical roles in biological pathways like 1 hormone activation, proteasomal degradation, and apoptosis, Materials—Purified human activated protein C (APC), human plasma kallikrein, human factor VIIa, human factor IXa ␤, human proteases are essential for cellular function and viability. Pro- factor XIa, and human factor ␣ XIIa were purchased from Enzyme teases regulate hormonal activation, cellular homeostasis, apo- Research Laboratories (South Bend, IN). Human two-chain activated complement C1r, human two-chain activated complement C1s, hu- man complement factor D, human tryptase (lung), bovine trypsin From the ‡Department of Bioengineering, Institute for Medicine and (pancreas, high-purity endotoxin-free), subtilisin Carlsberg (Bacillus Engineering, University of Pennsylvania, Philadelphia, Pennsylvania licheniformis), human cathepsin G (neutrophil), human cathepsin B 19104; ¶Center for New Directions in Organic Synthesis, Department (liver), human cathepsin H (liver), human cathepsin K (recombinant, of Chemistry, University of California, Berkeley, California 94720; and E. coli), human cathepsin L (liver), human cathepsin S (spleen), human **Department of Chemical and Biomolecular Engineering, Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104 1 The abbreviations used are: APC, activated protein C; ACC, 7- Received, January 5, 2005, and in revised form, February 10, 2005 amino-4-carbamoylmethylcoumarin; FRET, fluorescence resonance Published, MCP Papers in Press, February 10, 2005, DOI energy transfer; PS-SCL, positional scanning-synthetic combinatorial 10.1074/mcp.M500004-MCP200 library. 626 Molecular & Cellular Proteomics 4.5 © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. This paper is available on line at http://www.mcponline.org Microarrays for Profiling Protease Substrate Specificity FIG.1.Overall design of fluorogenic substrates. cathepsin V (recombinant, NSO cells), papain (carica papaya), and each array, assuring that the assays were run within the linear range. stem bromelain (pineapple) were purchased from Calbiochem (La The activated slides were scanned using a cooled CCD-based imager Jolla, CA). Chymopapain (papaya latex) and ficain (fig tree latex) were (NovaRay; Alpha Innotech, San Leandro, CA), at Ex ␭ ϭ 405/40 nm purchased from Sigma Aldrich (St. Louis, MO). The rhodesain, a gift and Em ␭ ϭ 475/40 nm with integration times of 2,500 msec and from the L. S. Brinen laboratory at University of California, San Fran- 15-␮m pixel resolution. Images were acquired in a 16-bit format, and cisco, was expressed from Pichia pastoris as previously reported (8). the analysis and presentation of the data was performed using Array Lipidated recombinant human tissue factor was purchased from Vision (Imaging Research, Ontario, Canada) and Cluster and Treeview American Diagnostica (Stamford, CT). All enzymes were stored ac- (10). For the most preferred substrates, the signal/background ratio cording to manufacturers’ instructions. was typically Ͼ60 with coefficient of variance Ͻ5%. Downloaded from Methods—The fluorogenic substrate library was synthesized and printed according to protocols previously described (5, 6, 9). The RESULTS AND DISCUSSION ϭ ϭ P1 Arg and P1 Lys sublibraries, along with calibration standards (unacylated 7-amino-4-carbamoylmethylcoumarin (ACC), acetyl- A 722-member, spatially separated ACC library of the for- ␮ capped ACC, and blanks), were printed at either 50 or 100 M in a mat Ac-P4-P3-P2-P1-ACC-NH2 was prepared with Ala at the 16 ϫ 24 format equivalent to a 384-well plate on polylysine-coated P site, all combinations of proteinogenic amino acids (except ϫ 4 www.mcponline.org glass slides (Erie Scientific, Portsmouth, NH) using a 1 1 pin Cys) at the P and P sites, and a Lys or Arg residue at the P (Telechem, Sunnyvale, CA) protocol on an OmniGrid Accent (Gene 2 3 1 Machines, San Carlos, CA) microarrayer. Calibration standards were site (Fig. 1) (6). The substrate specificities of 13 serine pro- printed on each array, to enable quantification and normalization of teases and 11 cysteine proteases (Table I, Figs. 2 and 3) were fluorescence intensity between slides. profiled using these substrates in a microarray-based format. The proteases were reconstituted and diluted in buffers recom- The proteases profiled here span three evolutionarily unre- mended by the manufacturers. The enzyme solutions were delivered lated clans (PA, SB, and C1). Multiple statistically homologous at Univ of Penn on November 29, 2007 to the microarrays at the following concentrations: 10 ␮M APC in 20 mM Tris-HCl, 100 mM NaCl (pH 7.4); 1 ␮M plasma kallikrein in 4 mM proteases from the PA clan (the S1 family) and the C1 clan (the NaOAc-HCl, 150 mM NaCl (pH 5.3); 5 ␮M factor VIIa in 20 mM Tris-HCl, CA family) were chosen to show that sequence homology 100 mM NaCl, 1 mM CaCl2 (pH 7.4), and 15 nM lipidated recombinant need not correlate with substrate specificity. ␮ ␤ human tissue factor; 10 M factor IXa in 20 mM Tris-HCl, 100 mM Serine Proteases—All the serine proteases profiled are from NaCl (pH 7.4); 100 nM factor XIa in 4 mM NaOAc-HCl, 150 mM NaCl the S1 family of clan PA except subtilisin Carlsberg, which is (pH 5.3); 10 ␮M factor ␣ XIIa in 4 mM NaOAc-HCl, 150 mM NaCl (pH ␮ from the S8 family of clan SB. The two clans differ in the order 5.3); 10 M two-chain activated complement C1r in 47 mM NaH2PO4 buffer, 5 mM EDTA, 1 mM benzamidine (pH 7.4); 10 ␮M two-chain of their overall fold and catalytic residues, with clan PA having activated complement C1s in 47 mM NaH2PO4 buffer, 5 mM EDTA, 1 double ␤ barrels with His-Asp-Ser as the catalytic triad and ␮ mM benzamidine (pH 7.4); 4 M complement factor D in 15 mM clan SB having parallel ␤ sheets and Asp-His-Ser as the ␮ NaH2PO4, 135 mM NaCl (pH 7.2); 1 M tryptase in 50 mM NaOAc, 1 M ␮ ␮ catalytic triad (3, 11). NaCl, 50 M heparin, 0.01% NaN3 (pH 5.0); 1 M trypsin in a 0.9% NaCl aqueous solution; 1 ␮M subtilisin Carlsberg in water; 10 ␮M The proteases profiled from the S1 family showed a pref- ␮ ϭ cathepsin G in 50 mM NaOAc, 150 mM NaCl (pH 5.5); 10 M cathepsin erence for P1 Lys or Arg due to the presence of the highly Bin20mM NaOAc buffer, 1 mM EDTA (pH 5.0); 5 ␮M cathepsin H in conserved negatively charged Asp189 (chymotrypsinogen 100 mM Na2HPO4,2mM EDTA, 2 mM DTT (pH 6.0); 500 nM cathepsin numbering) present at the bottom of the S1 pocket (12, 13). P1 Kin50mM NaOAc, 100 mM NaCl, 2.5 mM EDTA, 2.5 mM DTT (pH 5.5); Arg residues are able to form a direct ionic bond with Asp189, 5 ␮M cathepsin L in 20 mM malonate buffer, 400 mM NaCl, 1 mM EDTA ␮ whereas the corresponding Lys interaction is water-bridged (pH 5.5); 5 M cathepsin S in 100 mM Na2HPO4,2mM EDTA, 2 mM DTT, 0.4% Triton X-100 (pH 6.0); 500 nM cathepsin V in 50 mM because of the shorter side-chain of Lys.