A novel domain in adenovirus L4-100K is required for stable binding and efficient inhibition of human granzyme B: possible interaction with a species-specific exosite

Abstract

Lymphocyte granule serine proteases (granzymes) play a critical role in protecting higher organisms against intracellular infections and cellular transformation. The proteases have also been implicated in the generation of tissue damage in a variety of chronic human conditions, including autoimmunity and transplant rejection. Granzyme B (GrB), one cytotoxic member of this family, achieves its effect through cleavage and activation of caspases as well as through caspase-independent proteolysis of cellular substrates. The 100,000-molecular-weight (100K) assembly protein of human adenovirus type 5 (Ad5-100K) was previously defined as a potent and specific inhibitor of human GrB. We now show that although human, mouse, and rat GrB proteases are well conserved in terms of structure, substrate specificity, and function, Ad5-100K inhibitory activity is directed exclusively against the human protease. Biochemical analysis demonstrates that the specificity of the 100K protein for human GrB resides in two distinct interactions with the protease: (i) a unique sequence within the reactive site loop (P(1))Asp(48)-(P(1'))Pro(49) in Ad5-100K which interacts with the active site and (ii) the presence of an additional inhibitor-enzyme interaction likely outside the enzyme catalytic site (i.e., an exosite). We have located this extended macromolecular interaction site in Ad5-100K within amino acids 688 to 781, and we have demonstrated that this region is essential for stable inhibitor-enzyme complex formation as well as efficient inhibition of human GrB. This novel component of the inhibitory mechanism of the 100K protein identifies a distinct target for selective inhibitor design, a finding which may be of benefit for diseases in which GrB plays a pathogenic role.

FIG. 1.

Ad5-100K inhibits human but not mouse or rat GrB. (A) Cell lysates from mock-infected (lanes 1 to 4) or Ad5-100K plasmid-transfected (lanes 5 to 8) 293T cells were incubated in the absence or presence of 100 nM human (Hu), 160 nM mouse (M), or 130 nM rat (R) GrB. Ad5-100K, NuMA, and PARP were detected by immunoblotting. Filled arrows denote intact proteins, and unfilled arrows denote GrB-specific fragments. (B) GrB (25 nM) was preincubated in the presence or absence of rAd5-100K (at the indicated enzyme/inhibitor molar ratios) at 37°C for 10 min, and residual GrB proteolytic activity was determined by cleavage of IVTT-generated radiolabeled Mi-2. As a control, the radiolabeled substrate was incubated with buffer alone (lane 1). Filled and unfilled arrows denote intact Mi-2 and its fragments, respectively. The experiments were performed on at least two separate occasions, with similar results. Numbers at left of panels indicate molecular weights.

FIG. 2.

GrB-mediated cleavage and inhibition of 100K. (A) Human GrB, mouse GrB, and rat GrB cleave Ad5-100K at different sites. [³⁵S]methionine-labeled His-Ad5-100K generated by IVTT was incubated in the absence (lanes 1, 5, and 9) or presence of mouse (lanes 2 to 4), rat (lanes 6 to 8), or human (lanes 10 and 11) GrB at the indicated concentrations. Filled and unfilled arrows denote intact Ad5-100K and the 95-kDa fragment generated by human GrB cleavage at Asp⁴⁸, respectively. (B) P₁′ Pro⁴⁹ in Ad5-100K is essential for human GrB inhibition. Cell lysates from mock-infected (lanes 1 and 2) or Ad5-100K wild-type (wt) plasmid-transfected (lanes 3 and 4) or Ad5-100K-P⁴⁹A plasmid-transfected (lanes 5 and 6) 293T cells were incubated in the absence or presence of 100 nM human GrB. Ad5-100K, NuMA, and PARP were detected by immunoblotting. Filled arrows denote intact proteins, and unfilled arrows denote their GrB-induced fragments. The experiments were repeated on two to four separate occasions, with similar results. Numbers at left of panels indicate molecular weights.

FIG. 3.

Species effects on 100K cleavage by GrB. (A) Analysis of 100K assembly protein sequences of members of the Adenoviridae family. Based on sequence homology among several adenovirus subgenera, the 100K protein can be divided into three regions, an NTR of ∼0 to 170 amino acids (shown in gray), a CCR of ∼600 to 700 amino acids (shown in white), and a CTR of ∼20 to 100 amino acids (shown in black). The percentage of homology between the various adenovirus CCRs and the Ad5-100K CCR is shown inside each CCR. The adenovirus subgenera analyzed included the following: Ad5, canine adenovirus type 2 (CAdV-2), porcine adenovirus type 3 (PAdV-3), MAV-1, duck adenovirus type 1 (DAdV-1), bovine adenovirus type 4 (BAdV-4), frog adenovirus type 1 (FrAdV-1), and fowl adenovirus type 1 (CELO). (B) Cleavage of the 100K assembly protein from MAV-1 by mouse GrB and human GrB. [³⁵S]methionine-labeled MAV1-100K generated by IVTT was incubated in the absence or presence of increasing concentrations of mouse or human GrB. The filled arrow denotes intact MAV1-100K, and unfilled arrows denote GrB cleavage fragments. The experiments were performed twice on separate occasions, with identical results. Numbers at left of panel B indicate molecular weights.

FIG. 4.

Neither MAV-1 nor its assembly protein has inhibitory activity against mouse and rat GrB. (A) MAV-1 does not encode inhibitors for human, mouse, or rat GrB. Cell lysates from mock-infected (lanes 1 to 4) or MAV-1-infected (lanes 5 to 8) NIH 3T3 cells were incubated in the absence or presence of 100 nM human (Hu), 160 nM mouse (M), or 130 nM rat (R) GrB. MAV1-100K, NuMA, and Mi-2 were detected by immunoblotting. Filled arrows denote intact proteins, and unfilled arrows denote prominent GrB-specific fragments. (B) Purified rMAV1-100K does not inhibit the proteolytic activities of mouse GrB and rat GrB. Mouse or rat GrB (25 nM) was preincubated in the absence or presence of rMAV1-100K (enzyme/inhibitor molar ratio, 1:20) at 37°C for 10 min, and the residual GrB proteolytic activity was determined by cleavage of IVTT-generated radiolabeled Mi-2. As a control, the radiolabeled substrate was incubated with buffer alone (lane 9). Filled and unfilled arrows denote intact Mi-2 and its fragments, respectively. The experiments were performed on two separate occasions, with identical results. Numbers at left of panels indicate molecular weights.

FIG. 5.

The chimera Ad5-MAV100K is a potent inhibitor of human GrB. (A) Schematic representation of Ad5-100K and the chimeras containing the NTR from Ad5-100K (Ad5-MAV100K and Ad5-GFP). The Ad5-100K NTR (amino acids 1 to 93) is depicted in gray, the CCR is depicted in white, the CTR is depicted in black, and full-length GFP is depicted by stippling. (B) Cleavage of Ad5-100K, Ad5-MAV100K, and Ad5-GFP by human GrB. [³⁵S]methionine-labeled substrates generated by IVTT were incubated in the presence of 0, 30, or 80 nM human GrB. Filled arrows denote intact molecules. (C) The CCR and the Ad5-100K NTR are important in human GrB inhibition. Increasing amounts of rAd5-100K, rMAV1-100K, recombinant GFP (rGFP), or the chimeras rAd5-MAV100K and rAd5-GFP were preincubated with 25 nM human GrB (at the indicated enzyme/inhibitor molar ratios) as described in the legend to Fig. 4, and the residual GrB proteolytic activity was determined by cleavage of IVTT-generated radiolabeled Mi-2. As a control, the radiolabeled substrate was incubated with buffer alone (lane 7). Filled and unfilled arrows denote intact Mi-2 and its fragments, respectively. All experiments were performed on at least two separate occasions, with similar results. Numbers at left of panels B and C indicate molecular weights.

FIG. 6.

Analysis of human GrB inhibition by wild-type Ad5-100K and its deletion mutants. Schematic representation of wild-type Ad5-100K (100K-wt) and its deletion mutants. The Ad5-100K NTR is depicted in gray, the CCR is depicted in white, and the CTR is depicted in black. Human GrB inhibition was determined by preincubating increasing amounts of purified recombinant proteins with a fixed amount (25 nM) of human GrB (enzyme/inhibitor molar ratios of 1:0, 1:1, 1:3, 1:5, 1:10, and 1:20), and the residual GrB activity was determined by cleavage of IVTT-generated radiolabeled substrate Mi-2 (data not shown). Maximum GrB inhibition was defined as the residual GrB activity obtained at a 1:1 ratio of wild-type Ad5-100K and human GrB. Inhibition is represented as follows: +++++, 1:1 ratio; ++++, 1:3 ratio; +++, 1:5 ratio; ++, 1:10 ratio; +, 1:20 ratio; −, no inhibition at any ratio (from 1:1 to 1:20). Results are representative of at least two separate experiments.

FIG. 7.

Amino acids 688 to 781 (CT688-781) in Ad5-100K are required for efficient cleavage and/or recognition, inhibition, and stable complex formation between Ad5-100K and human GrB. (A) Wild-type Ad5-100K (100Kwt) and deletion mutant 100KΔ5 inhibit human GrB with similar efficiencies. Increasing amounts of purified recombinant proteins were preincubated with 25 nM human GrB, and the residual GrB activity was determined by cleavage of IVTT-generated radiolabeled Mi-2. Filled and unfilled arrows denote intact Mi-2 and its fragments, respectively. (B) The CT688-781 domain is required for efficient cleavage of wild-type 100K protein and its deletion mutants. [³⁵S]methionine-labeled substrates generated by IVTT were incubated in the presence of 0, 20, 60, or 200 nM human GrB for 30 min at 37°C. After the reactions were terminated, samples were electrophoresed. The intact polypeptide and the cleavage products were detected by fluorography. WT, wild type. (C) The CT688-781 domain is required for stable Ad5-100K/human GrB complex formation. Equimolar amounts (300 nM) of human GrB and recombinant wild-type protein (r100K-wt) (lane 2), recombinant 100K-NT-245 (r100K-NT-245) (lane 3), or recombinant 100KΔ5 (r100KΔ5) (lane 4) were coincubated for 1 h at 4°C. As a control, GrB was incubated with buffer alone (lane 1). After incubation, an aliquot of each sample was boiled in SDS sample buffer and analyzed by immunoblotting with a monoclonal antibody against human GrB (bottom panel). The rest of the samples were immunoprecipitated with a polyclonal antibody against the 100K protein. After electrophoresis, r100K-wt, r100K-NT-245, and r100KΔ5 were visualized by immunoblotting with a monoclonal antibody against epitope tag T7 (which is present at the N terminus of all recombinant 100K proteins), and coimmunoprecipitated GrB was detected with the monoclonal antibody against GrB. The unfilled arrow denotes human GrB before immunoprecipitation (pre-IP). Filled arrows denote r100K-wt, r100K-NT-245, r100Δ5, and GrB after immunoprecipitation. Experiments were performed on two separate occasions, with similar results. Numbers at left of panels indicate molecular weights.