Distinct Dibasic Cleavage Specificities of Neuropeptide-Producing Cathepsin L and Cathepsin V Cysteine Proteases Compared to PC1/3 and PC2 Serine Proteases

Proenkephalin (PE) is a prohormone containing dibasic sites that are cleaved by proteases to generate peptide neurotransmitters and hormones. Little is known about the conformational features of such protease cleavage sites within prohormone substrates. Therefore, the goal of this study was to investigate the relative accessibilities of multiple dibasic processing sites of PE by peptide amide hydrogen-deuterium exchange mass spectrometry (DXMS). DXMS demonstrated differences in the relative accessibilities of the KR, KK, and RR cleavage sites of PE to the aqueous environment. DXMS assesses relative rates of exchange of hydrogens of the polypeptide backbone of PE with deuterium atoms from D(2)O (heavy water) in solvent. Analyses of peptides spanning each of the 12 dibasic PE cleavage sites illustrated differences in H-D exchange rates that reflect relative solvent accessibility. The mid-domain cleavage sites (dibasic sites 4-8) exhibited greater accessibility to the aqueous solvent compared to regions of the NH(2) and COOH domains (dibasic sites 2, 3, and 9-11, respectively). The NH(2)- and COOH-terminal domains both exhibited relatively high H-D exchange rates. The hydrogen exchange rate profile of PE, as well as its circular dichroism (CD) features for secondary structure, was modified in trifluoroethanol, an organic solvent that represents a more hydrophobic environment. These findings suggest that the dibasic protease cleavage sites of the PE prohormone with differences in accessibility to the aqueous environment undergo proteolytic processing to generate active neuropeptides for cell-cell communication in neuroendocrine systems.

Mutagenesis of Conserved Residues at the Yellow Fever Virus 3/4A and 4B/5 Dibasic Cleavage Sites: Effects on Cleavage Efficiency and Polyprotein Processing

Consensus sequence for processing of peptide precursors at monobasic sites

The cleavage specificity of a monobasic processing dynorphin converting endoprotease is examined with a series of quench fluorescent peptide substrates and compared with the cleavage specificity of prohormone convertases. A dynorphin B-29-derived peptide, Abz-Arg-Arg-Gln-Phe-Lys-Val-Val-Thr-Arg-Ser-Glneddnp (where Abz is o-aminobenzoyl and eddnp is ethylenediamine 2,4-dinitrophenyl), that contains both dibasic and monobasic cleavage sites is efficiently cleaved by the dynorphin converting enzyme and not cleaved by two propeptide processing enzymes, furin and prohormone convertase 1. A shorter prorenin-related peptide, Dnp-Arg-Met-Ala-Arg-Leu-Thr-Leu-eddnp, that contains a monobasic cleavage site is cleaved by the dynorphin converting enzyme and prohormone convertase 1 and not by furin. Substitution of the P1' position by Ala moderately affects cleavage by the dynorphin-processing enzyme and prohormone convertase 1. It is interesting that this substitution results in efficient cleavage by furin. The site of cleavage, as determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry, is N-terminal to the Arg at the P1 position for the dynorphin converting enzyme and C-terminal to the Arg at the P1 position for furin and prohormone convertase 1. Peptides with additional basic residues at the P2 and at P4 positions also serve as substrates for the dynorphin converting enzyme. This enzyme cleaves shorter peptide substrates with significantly lower efficiency as compared with the longer peptide substrates, suggesting that the dynorphin converting enzyme prefers longer peptides that contain monobasic processing sites as substrates. Taken together, these results suggest that the cleavage specificity of the dynorphin converting enzyme is distinct but related to the cleavage specificity of the prohormone convertases and that multiple enzymes could be involved in the processing of peptide hormones and neuropeptides at monobasic and dibasic sites.

Caspase-3 and -7 are considered functionally redundant proteases with similar proteolytic specificities. We performed a proteome-wide screen on a mouse macrophage lysate using the N-terminal combined fractional diagonal chromatography technology and identified 46 shared, three caspase-3-specific, and six caspase-7-specific cleavage sites. Further analysis of these cleavage sites and substitution mutation experiments revealed that for certain cleavage sites a lysine at the P5 position contributes to the discrimination between caspase-7 and -3 specificity. One of the caspase-7-specific substrates, the 40 S ribosomal protein S18, was studied in detail. The RPS18-derived P6-P5' undecapeptide retained complete specificity for caspase-7. The corresponding P6-P1 hexapeptide still displayed caspase-7 preference but lost strict specificity, suggesting that P' residues are additionally required for caspase-7-specific cleavage. Analysis of truncated peptide mutants revealed that in the case of RPS18 the P4-P1 residues constitute the core cleavage site but that P6, P5, P2', and P3' residues critically contribute to caspase-7 specificity. Interestingly, specific cleavage by caspase-7 relies on excluding recognition by caspase-3 and not on increasing binding for caspase-7.

By taking advantage of the recently published furin structure, whose catalytic domain shares high homology with other proprotein convertases, we designed mutations in the catalytic domain of PC2, altering residues Ser206, Thr271, Asp278, ArgGlu282, AlaSer323, Leu341, Asn365, and Ser380, which are both conserved and specific to this convertase, and substituting residues specific to PC1 and/or furin. In order to investigate the determinants of PC2 specificity, we have tested the mutated enzymes against a set of proenkephalin-derived substrates, as well as substrates representing Arg, Ala, Leu, Phe, and Glu positional scanning variants of a peptide B-derived substrate. We found that the exchange of the Ser206 residue with Arg or Lys led to a total loss of activity. Increased positive charge of the substrate generally resulted in an increased specificity constant. Most intriguingly, the RE281GR mutation, corresponding to a residue placed distantly in the S6 pocket, evoked the largest changes in the specificity pattern. The D278E and N356S mutations resulted in distinct alterations in PC2 substrate preferences. However, when other residues that distinguish PC2 from other convertases were substituted with PC1-like or furin-like equivalents, there was no significant alteration of the PC2 specificity pattern, suggesting that the overall structure of the substrate binding cleft rather than individual residues specifies substrate binding.

Haemophilia A is an X-linked bleeding disorder caused by the loss of factor VIII function. Factor VIII is a plasma glycoprotein that functions as a cofactor for the serine protease factor IXa in the proteolytic activation of factor X to Xa. A significant complication of factor VIII replacement therapy, in which purified factor VIII is administered to the patient, is the development of antibody inhibitors. Porcine factor VIII often displays limited cross-reactivity to inhibitor plasmas and is used as an alternative therapeutic agent to evade inhibition of factor VIII procoagulant function. The most frequent inhibitor incidence occurs on epitopes within the A2 and C2 domains. Inhibitors to the A3 domain and the acidic region between A1 and A2 have also been observed. Recent studies have elucidated the high-resolution three-dimensional structures of the factor VIII C2 domain and the C2 domain bound to a C2-specific inhibitor antibody, BO2C11. BO2C11 functions in vitro by inhibiting the ability of factor VIII to associate with negatively charged phospholipid surfaces and von Willebrand factor (VWF). Subsequent mutational analyses of factor VIII indicated that residues involved in the putative membrane binding surface of the C2 domain were essential for binding to both phospholipid surfaces and antibody inhibitors. A low-resolution model of the domain architecture of factor VIII associated on a negatively charged phospholipid surface has been proposed based on two-dimensional electron crystallography. The regions of factor VIII that display elevated inhibitor binding were mapped onto the factor VIII model. Using the present structure and sequence information, improvements to recombinant factor VIII molecules have been proposed that may decrease antigenicity and increase specific activity. Haemophilia A is the most common blood coagulation disorder, affecting one in 5000 males. It is an X-linked disease caused by defects in the expression or function of the plasma glycoprotein factor VIII. Factor VIII is an essential protein cofactor in the intrinsic pathway of blood coagulation (Davie et al, 1991; Davie, 1995; Mann, 1999). It functions by dramatically increasing the catalytic efficiency of the serine protease factor IXa in the proteolytic activation of factor X to Xa (van Dieijen et al, 1981). The clinical manifestations of haemophilia A are categorized with respect to the severity of factor VIII deficiency. Factor VIII activity levels of 5–25% are associated with a mild disease state, 1–5% moderate, and less than 1% as severe (Hoyer, 1994). Severe haemophiliacs may have spontaneous haemorrhages, requiring therapy two to four times monthly (Levine & Brettler, 1991). Haemorrhaging into joints and muscles is frequently observed in haemophilia A patients, which may eventually cause pain, swelling and severe limitations of motion. The most common and effective therapy for factor VIII deficiency is replacement therapy with either plasma-derived or recombinant factor VIII (Scharrer et al, 1999; Hoots, 2001; Mauser-Bunschoten et al, 2001). With the advent of rigorous and comprehensive testing of blood replacements for viral contaminants (Evatt et al, 1999; Srivastava, 1999; Philipp, 2001), the most significant complication of factor VIII replacement therapy is the development of factor VIII inhibitor antibodies (Hoyer & Scandella, 1994). These inhibitors are usually polyclonal immunoglobulin (Ig)G populations, often belonging to the IgG4 subclass (Fulcher et al, 1987). Anti-factor VIII inhibitor antibodies manifest in 3–13% of mild/moderate patients (Hay, 1998; Hay et al, 1998) and in 25–35% of severe patients (Rizza & Biggs, 1973; McMillan et al, 1988; Tuddenham et al, 1994; Antonarakis et al, 1995; Schwaab et al, 1995). The spontaneous development of factor VIII inhibitor antibodies in non-haemophilia patients also occurs rarely (approximately one per million persons) and is most often seen in postpartum women (Hoyer & Scandella, 1994; Bossi et al, 1998). The precise combination of mechanisms for the highly variable development of inhibitors in haemophilia A patients remains unknown (Hoyer, 1995). It is relatively clear that factor VIII gene deletions, truncations and inversions associated with a severe form of haemophilia A result in the failure to express normal forms of the protein. This results in the failure to establish central and peripheral tolerance, thus eliciting an immune response. In the case of mild or moderate forms of factor VIII deficiency, the nature and strength of the immune response depends on several variables, including the identity of the missense mutations that cause a bleeding tendency (Liu et al, 2000). Approximately 50% of missense mutations that cause haemophilia A occur in the protein core (surface exposure of less than 10%), most likely destabilizing the protein. These mutations are often associated with patients that are clinically severe and with the development of antibody inhibitors. Additionally, surface-exposed mutations may elicit an immune response to replacement therapy. These mutations may have implications for protein–protein interactions or interdomain connections. In particular, an increased antibody inhibitor incidence (approximately 40%) occurs in haemophilia patients with the missense mutations R593C and W2229C (Fijnvandraat et al, 1997). The infusion of therapeutic amounts of factor VIII in haemophilia patients that have some residual factor VIII activity may elicit a high-titre immune response that cross-reacts with all circulating factor VIII, causing a dramatic decrease in factor VIII levels. Desmopressin (DDAVP), a synthetic analogue of the pituitary hormone vasopressin, is often used to bypass factor VIII replacement therapy in mild/moderate haemophiliacs as a result of this complication (Santagostino et al, 1995). Maintaining effective treatment for haemophilia A patients with inhibitor responses remains a challenge, despite the relatively recent development of alternative methods of treatment (Hacker et al, 2001; Kulkarni et al, 2001; Penner, 2001; Rossi, 2001). Low-titre inhibitors [measured as titres of ≤5 Bethesda units (BU)/ml] pose a relatively tractable problem, with therapy consisting of either hormone-induced secretion of factor VIII by DDAVP or by simply overcoming the inhibitor response by overwhelming the antibodies with larger amounts of therapeutic factor VIII during infusions. The development of a high-titre inhibitor response (titres of > 5 BU/ml) causes a much greater problem for replacement therapy. These patients are often unresponsive to large infusions of human factor VIII administered at more frequent intervals. Alternative treatments for factor VIII inhibitors consist of factor VIII ‘bypass’ therapies, porcine factor VIII, immunosuppression and plasmapheresis. Factor VIII bypass products consist of recombinant factor VIIa (rVIIa), prothrombin complex concentrates (PCCs) and activated prothrombin complex concentrates (aPCCs). Recent advances in immune tolerance induction and gene therapy technology may also provide effective clinical treatments in the near future (Pasi, 2001; Saenko et al, 2002). In many cases, porcine factor VIII has been effective in the treatment of haemophilia patients with high-titre inhibitors as a result of less cross-reactivity against their specific antibody inhibitors, higher specific activity and increased stability (Hay et al, 1994; Gribble & Garvey, 2000; Garvey, 2002; Hay, 2002). It has been shown to be effective in up to 90% of bleeding episodes in selected haemophilia patients from an international survey (Hay, 2002). Occasional cross-reactivity (a median of 15%) between porcine and human factor VIII molecules can occur, and continuous infusion of porcine factor VIII can elicit an antiporcine factor VIII immune response. Side-effects to porcine factor VIII treatment may also consist of an allergic reaction, consisting of fever, chills, shortness of breath and thrombocytopenia (Hay et al, 1994). In some cases, however, porcine factor VIII can be administered in large amounts to overcome both human and porcine inhibitor antibodies, and haemostasis can be achieved in the absence of detectable levels of functional factor VIII (Gribble & Garvey, 2000; Garvey, 2002). If patients have no response to factor VIII replacement therapy, bypass therapies have been shown to be effective at inducing cessation of bleeding (Lusher, 1994; Scharrer, 1999; Kulkarni et al, 2001). Both PCCs and aPCCs are an inexpensive alternative that may prevent life-threatening bleeds. These concentrates consist of mixtures of vitamin K-dependent proteases and display long half-lives with respect to factor VIII. Recombinant factor VIIa is also an effective treatment. While rfVIIa has no known risk of viral infection, it is more expensive and displays a shorter half-life than both factor VIII and PCC/aPCC therapies. The principle disadvantages of bypass agents are the inability to assay the efficacy of each treatment and the risk of thrombosis due to the increased concentration of coagulation activators. The initiation of blood coagulation occurs via two principal pathways (Fig 1) (Davie et al, 1991; Davie, 1995; Mann, 1999). The initial (extrinsic) pathway is rapidly activated at the site of vascular damage when exposed tissue factor, an integral membrane glycoprotein, binds tightly to factor VII, a circulating serine protease proenzyme. This binding event leads to the serine protease-mediated activation of factor VII to form factor VIIa (Broze et al, 1985). The factor VIIa/ tissue factor complex proteolytically activates factor X to Xa (Di Scipio et al, 1977), which binds to factor Va to form a membrane-associated ‘prothrombinase’ complex (Tracy et al, 1981). This complex converts prothrombin to thrombin, which then acts directly on fibrinogen to initiate clot formation (Mann et al, 1990). The extrinsic pathway is a rapidly upregulated, but short-lived, response to vascular injury and is inhibited by a variety of circulating inhibitors (Broze & Miletich, 1987; Rao & Rapaport, 1987; Rapaport, 1989; Broze et al, 1990). For example, tissue-factor-pathway inhibitor (TFPI) blocks fibrin generation by forming an inactive complex with the factor VIIa/tissue factor complex (Broze & Miletich, 1987; Rao & Rapaport, 1987). Diagram of the extrinsic and intrinsic pathways for the activation of factor Xa. At the site of vascular damage, tissue factor (TF) is exposed and binds to factor VII (FVII). FVII is activated to factor VIIa (FVIIa) by trace amounts of protease in the presence of calcium. The TF/FVIIa complex activates factor X (FX) to factor Xa (FXa). FXa binds to factor Va (FVa) in the presence of calcium and negatively charged phospholipids (PS) on the surface of activated platelets. The FXa/FVa complex, also known as the ‘prothombinase’ complex, proteolytically activates prothrombin (II) to thrombin (IIa). Thrombin and FXa feedback to proteolytically activate factor VIII (FVIII) to factor VIIIa (FVIIIa). When FVIII is activated, it dissociates from von Willebrand factor (VWF) and binds to PS on activated platelet surfaces. The TF/FVIIa complex alters its specificity to activate factor IX (FIX) to factor IXa (FIXa). FIXa binds to FVIIIa in the presence of calcium and PS to form the intrinsic ‘tenase’ complex, which proteolytically activates FX to FXa. The secondary (intrinsic) pathway to blood coagulation is a more slowly upegulated, but longer-lived, response that amplifies and sustains clot formation (Komiyama et al, 1990). When thrombin is initially activated, it stimulates the intrinsic pathway by proteolytically activating several coagulation factors, including factors XI and VIII (Eaton et al, 1986; Naito & Fujikawa, 1991). Factor VIII is a central regulatory protein cofactor in this pathway (Lawson et al, 1994). It resides in the circulation bound to VWF in a tight, non-covalent complex (Kd ∼ 0·5 nmol/l) (Foster et al, 1987). Once factor VIII is activated to factor VIIIa by thrombin or factor Xa, it dissociates from VWF (Saenko & Scandella, 1995). Factor VIIIa subsequently binds to factor IXa in the presence of negatively charged phospholipids at the surface of activated platelets and calcium to form an activated ‘tenase’ complex responsible for converting factor X to Xa, increasing the Vmax by 200 000-fold (van Dieijen et al, 1981). Factor VIIIa functions as a localization and conformational cofactor for factor IXa, inducing an essential conformational change to co-ordinate active serine protease activity and increasing the affinity of factor IXa for activated platelet surfaces by fivefold (the Kd decreases from 2·5 to 0·56 nmol/l). Factor VIII is a large plasma glycoprotein that consists of 2332 amino acid residues (Fig 2) (Kane & Davie, 1988; Lenting et al, 1998; Fay, 1999). The complete, unprocessed protein contains six sequential domains arranged on a single polypeptide chain in the order NH3-A1-(a1)-A2-(a2)-B-(a3)-A3-C1-C2-CO2 (Toole et al, 1984; Vehar et al, 1984; Saenko & Scandella, 1995). The A domains are homologous to one another and display sequence similarity to the copper-binding protein ceruloplasmin (between 35% and 40% identity between each A domain, and approximately 34% identical to ceruloplasmin) (Pemberton et al, 1997). They are flanked by short spacer sequences that are highly acidic (a1, a2 and a3). The C domains are also homologous to each other and have a weak homology to the discoidin protein fold family (e.g. the lipid-binding domain of galactose oxidase) (Baumgartner et al, 1998; Pellequer et al, 1998). The unique B domain is heavily glycosylated, possesses variable cleavage sites and has no known haemostatic function (Lenting et al, 1998). Domain structure and interactions of factor VIII. Factor VIII is synthesized as a single polypeptide chain of 2332 amino acids. The protein consists of three A domains, two C domains, a B domain and three acidic regions (a1, a2 and a3). The single polypeptide chain is processed and cleaved at various positions within the B domain (residues 1648, 1313 and 740) to form a heterogeneous mixture of hetero-dimers. Factor VIII hetero-dimers are carried in the circulation by VWF multimers, which protect factor VIII from becoming activated and/or degraded by serine proteases. Factor VIII becomes proteolytically activated by thrombin (IIa) or factor Xa, dissociates from VWF and subsequently binds to PS on activated platelet surfaces. Factor VIIIa binds to factor IXa in the presence of PS and calcium to form the tenase complex. Factor VIII-specific inhibitor antibodies function by blocking the ability of factor VIII to bind to any or all of its procoagulant binding partners. Observed epitopes are mapped onto the factor VIII protein and labelled in grey with the specific mAb that has been isolated, as described in the text. The circulating form of the factor VIII protein is a heterogeneous combination of metal-bridged hetero-dimers produced by cleavage at the B–A3 junction and variable cleavages within the B domain. The factor VIII hetero-dimer contains a heavy chain, consisting of the A1, A2 and B domains (residues 1–336, 373–740 and 741–1648 respectively), and a light chain, consisting of the A3, C1 and C2 domains (residues 1690–2019, 2020–2172 and 2173–2332 respectively). This form of factor VIII is bound tightly to an N-terminal domain (D'D3) of VWF through binding sites in the third acidic region (a3) and the C2 domain (Foster et al, 1987; Saenko et al, 1994; Saenko & Scandella, 1997). The factor VIII/VWF complex is required for normal levels of factor VIII to persist and protects factor VIII from proteolytic degradation by activated protein C, factor IXa and factor Xa. Factor VIII is activated by specific thrombin or factor Xa cleavages to a hetero-trimeric form at residues 372, 740 and 1689. The activated form, factor VIIIa, dissociates from VWF and binds to negatively charged phospholipids on activated platelet surfaces (Saenko & Scandella, 1995). The carboxyl terminal C2 domain of factor VIII contains the primary binding site for negatively charged phospholipids (Saenko et al, 1999, 2001), which involves stereoselection for O-phospho-l-serine, the negatively charged head group of phosphatidylserine (PS) (Gilbert & Drinkwater, 1993). The binding of factor VIII or VIIIa to VWF or PS is mutually exclusive, even though the activation of factor VIII involves cleavages outside the C2 domain. This implies that the binding sites for VWF and PS overlap on the C2 domain surface. Additionally, the factor VIII C2 domain has binding sites for both factor Xa (residues 2253–2270) and thrombin (Nogami et al, 1999, 2000). The light chain of factor VIIIa also contains a high-affinity binding site for factor IXa, consisting of residues 1811–1818 of the A3 domain (Lenting et al, 1994, 1996). An additional factor IXa binding site on factor VIIIa resides in the A2 domain and consists of residues 484–509 and 558–565 (Fay et al, 1994; Fay & Koshibu, 1998; Fay & Scandella, 1999). Although the affinity of the A2 domain binding site is significantly lower than that of the A3 domain, it is essential for the amplification of the enzymatic activity of factor IXa, transforming the active site of the protease domain (Fay & Koshibu, 1998). The interaction between the A2 domain and factor IXa also functions to mediate the stability required for proper metastatic function in the intrinsic ‘tenase’ complex by stabilizing the association of the A2 domain with the A1/A3-C1-C2 factor VIIIa scaffold. The substrate of the intrinsic tenase complex, factor X, also has a binding site on factor VIIIa, which resides between residues 349–372 of the first acidic region, a1 (Lapan & Fay, 1997, 1998). The initial characterization of inhibitor binding regions on factor VIII consisted of expressing recombinant fragments of factor VIII in E. coli and conducting an immunoblotting assay with inhibitor plasma (Scandella et al, 1988, 1989, 1995). This proved to be successful for identifying some of the major epitopes that lead to inhibition, but detection was limited to antibodies that could retain binding after factor VIII denaturation. The development of human/porcine factor VIII chimaeras provided an alternative method for factor VIII inhibitor detection, based on the observation that porcine factor VIII sequences displayed little cross-reactivity with human-derived antibodies (Healey et al, 1995; Koshihara et al, 1995). The epitopes recognized by inhibitory antibodies for factor VIII that block its procoagulant function generally reside in specific regions of the molecule (Saenko et al, 2002). The most frequent site of inhibitor binding occurs within the A2 and C2 domains (Scandella et al, 2001). Inhibitors to the A3 and C1 domains, and the acidic region between domains A1 and A2 have also been observed, but epitopes in these regions have not been extensively characterized. Factor VIII antibody inhibitors can block factor VIII function in several ways: (1) Blocking the ability of factor VIIIa to bind and activate factors IXa and X; (2) Inhibiting the binding of factor VIII to VWF and/or negatively charged phospholipid surfaces; (3) Hindering the activation of factor VIII by thrombin (and/or factor Xa) or the subsequent release of factor VIII from VWF. Inhibitor antibodies localized to the A2 domain were found initially to interact with its amino-terminal 18·3 kDa fragment by immunoblotting analysis with inhibitor plasma (Scandella et al, 1989). Most of these antibodies could be competed with a recombinant A2 domain polypeptide fragment (Scandella et al, 1993). Further analysis of an inhibitor epitope for a murine monoclonal antibody (mAb 413) with human/porcine chimaeras determined that residues 484–508 were most important for inhibition (Fig 2) (Healey et al, 1995). Inhibition assays performed with this hybrid also showed significantly less cross-reactivity with inhibitors from four haemophilia patient plasmas. Interestingly, porcine and human sequences of the A2 domain differ at positions 484, 485, 487–489, 492, 495, 501 and 508. Alanine-scanning mutagenesis determined that substitutions Y487A, S488A, R489A, P492A, V495A, F501A and I508A displayed a significant reduction in inhibition by most patient plasmas (Lubin et al, 1997). Alternatively, substitutions R484A and P485A were relatively ineffective. The Y487A substitution was determined to be the most effective, displaying the greatest reduction in inhibition ranging from 10% to 20%. As discussed previously, the factor VIII A2 domain directly interacts with factor IXa through residues 484–509 (Fay & Scandella, 1999). Interestingly, significant sequence changes in this region can elude the previously developed inhibitors from patient plasmas and to interact with factor IXa to form an tenase complex. The of inhibitor antibodies that the light chain of factor VIII have previously been determined to bind the C2 domain (Fig 2) et al, 1995; et al, 1995; et al, 1998; et al, 1998). studies to the epitope region on the C2 domain with the murine in the that residues were important for inhibitor binding et al, 1993). Subsequent C2 domain truncations determined that residues a common epitope important for interaction with inhibitor antibodies from a of patient plasmas (Scandella et al, 1995). In to these with human/porcine C2 domain chimaeras determined that substitution between residues displayed significantly less inhibition with respect to the human C2 domain (Healey et al, 1998). Most C2 inhibitor antibodies block the ability of factor VIII to bind to negatively charged phospholipid surfaces and VWF et al, 1998). The results from both C2 domain truncations and human/porcine that both regions of the factor VIII C2 domain (residues and are involved in binding to both phospholipids and VWF. inhibitors of the factor VIII light chain have been localized to the (Fig A mAb residues and to block another binding site for VWF et al, other inhibitor from patient plasmas competed with the A3 et al, 1998). These inhibitors block a binding site for factors IX and IXa, and thus prevent the generation of factor Xa in a which was produced from recombinant factor VIII protein fragments bound to residues et al, 1991). When the was on human/porcine sequence the inhibitor not with the This antibody blocks the cleavage of factor VIII by An additional mAb that binds to the heavy chain of factor VIII has been described (Foster et al, 1988; et al, The epitope consists of residues and between a thrombin cleavage site and an activated protein C cleavage site thus to block the activation and/or degradation of factor VIII. In recent various structures of the factor and VIII C2 domains et al, 1999; et al, and of the factor VIII C2 domain in complex with a mAb from a haemophilia A patient with inhibitor et al, have been Additionally, an electron structure has been of the factor VIII molecule bound to a phospholipid membrane surface et al, 1999; et al, 2002). Subsequent mutational and studies based on these structures have provided for factor VIII may function or The of this on the of these various sequence between human and porcine factor VIII antibody of improvements can be to factor VIII molecules based on and future studies that may of factor VIII and the antibody response. the structure of the factor VIII C2 domain to was (Fig et al, 1999). As the fold was homologous to the lipid-binding domain of galactose and consisted of an core with three and other additional from the A for the interaction of factor VIII with negatively charged phospholipid as as for the of haemophilia A mutations localized to this domain, were proposed on the of this The putative consists of two and an that from one of the residues and These residues are proposed to interact with the of a phospholipid membrane these exposed residues a of charged residues and that may present interactions with the negatively charged head of residues within the factor VIII C2 domain are known to be sites of mutations that cause haemophilia of these mutations occur at either the protein core or at the site of interdomain thus by the stability of the protein. Interestingly, it was observed that was a of missense mutations in the putative It was that the region of factor VIII is highly and residues to to the binding missense mutations with a not the function of factor VIII. two and are localized to this region of the C2 domain and are to the association of factor VIII with negatively charged phospholipids surfaces and/or VWF. These two mutations cause a mild or moderate bleeding of the structure of the factor VIII C2 domain and residues involved in the proposed surface. The C2 domain an core fold of the discoidin protein fold The putative involves residues and and a of charged residues and the structure of the factor VIII C2 domain, mutagenesis were performed to the function of the C2 domain (Gilbert et al, 2002). Using the putative single and mutations were and for the ability to bind both and VWF. of residues and displayed specific that were > of factor VIII. Alternatively, of residues and were degraded within the and thus activity was not The and and reduction in specific activity during the activated assay and and reduction in specific activity during a factor X activation assay The also displayed and > reduction in affinity for VWF in an assay These that the four residues a significant in the ability of factor VIII to bind both negatively charged phospholipid and that these residues a large of both binding It is also that most single mutations in this region not dramatically the specific activity of factor VIII, thus the that the highly nature of the putative region is The structure of the factor C2 domain displayed a fold to the factor VIII C2 domain as et al, 1999). The factor C2 domain also residues on the of two to and by residues and Additionally, the factor C2 domain was in two forms two were observed (Fig These two are as the and forms due to a of the first which up to and the of approximately The factor C2 domain was to in the form, and to the form interaction with the negatively charged phospholipid of activated platelets and bind the head This large conformational has not been observed in the factor VIII C2 domain. It is that a the ability of the factor C2 domain to change from the to the

Antigen three-dimensional structure potentially limits antigen processing and presentation to helper T-cell epitopes. The association of helper T-cell epitopes with the mobile loop in Hsp10s from mycobacteria and bacteriophage T4 suggests that the mobile loop facilitates proteolytic processing and presentation of adjacent sequences. Sites of initial proteolytic cleavage were mapped in divergent Hsp10s after treatment with a variety of proteases including cathepsin S. Each protease preferentially cleaved the Hsp10s in the mobile loop. Flexibility in the 22-residue mobile loop most probably allows it to conform to protease active sites. Three variants of the bacteriophage T4 Hsp10 were constructed with deletions in the mobile loop to test the hypothesis that shorter loops would be less sensitive to proteolysis. The two largest deletions effectively inhibited proteolysis by several proteases. Circular dichroism spectra and chemical cross-linking of the deletion variants indicate that the secondary and quaternary structures of the variants are native-like, and all three variants were more thermostable than the wild-type Hsp10. Local structural flexibility appears to be a general requirement for proteolytic sensitivity, and thus, it could be an important factor in antigen processing and helper T-cell epitope immunogenicity.

Determination of protease specificity is of crucial importance for understanding protease function. We have developed the first gel-based label-free proteomic approach (DIPPS-direct in-gel profiling of protease specificity) that enables quick and reliable determination of protease cleavage specificities under large variety of experimental conditions. The methodology is based on in-gel digestion of the gel-separated proteome with the studied protease, enrichment of cleaved peptides by gel extraction, and subsequent mass spectrometry analysis combined with a length-limited unspecific database search. We applied the methodology to profile ten proteases ranging from highly specific (trypsin, endoproteinase GluC, caspase-7, and legumain) to broadly specific (matrix-metalloproteinase-3, thermolysin, and cathepsins K, L, S, and V). Using DIPPS, we were able to perform specificity profiling of thermolysin at its optimal temperature of 75°C, which confirmed the applicability of the method to extreme experimental conditions. Moreover, DIPPS enabled the first global specificity profiling of legumain at pH as low as 4.0, which revealed a pH-dependent change in the specificity of this protease, further supporting its broad applicability.

The multicatalytic proteinase complex (MPC, proteasome) is assembled from 14 nonidentical protein subunits. It expresses five distinct proteolytic activities, including a chymotrypsin-like activity, cleaving after hydrophobic residues, and a branched chain amino acid-preferring component (BrAAP), cleaving preferentially after branched chain residues. Exposure of cells to interferons leads to replacement of the X, Y, and Z subunits by the LMP2, LMP7, and MECL1 subunits. This "immunoproteasome" is critical to processing of certain antigens. The enzymatic basis for enhanced antigen processing has not been determined. To gain insight into this question, we examined sites and relative rates of cleavage of bonds in denatured, reduced, carboxyamidomethylated lysozyme, a 129-amino acid protein, by MPC from bovine spleen, in which the X, Y, and Z subunits are replaced by LMP2, LMP7, and MECL1. We compared cleavages to those catalyzed by MPC from bovine pituitary, which contains only the X, Y, and Z subunits. We found marked increases in the rates and number of cleavages after branched chain residues in reduced, carboxyamidomethylated lysozyme by the spleen MPC. This was largely due to accelerated cleavages of bonds after a Phi-X-Br motif, where Phi is a hydrophobic residue, X is a small neutral or polar residue, and Br is a branched chain residue. Inhibitors with these structural properties were selective and potent inhibitors of the BrAAP activity of the spleen MPC. The above findings indicate that alterations in activity and substrate specificity of the BrAAP activity are important factors underlying the altered cleavages after hydrophobic residues associated with incorporation of interferon-inducible subunits. The potential relevance of the findings to antigen processing functions of MPC is discussed.

Consensus sequence for precursor processing at mono-arginyl sites. Evidence for the involvement of a Kex2-like endoprotease in precursor cleavages at both dibasic and mono-arginyl sites.

Regulation of Cathpsin L by the Serpin Protein C Inhibitor.

West Nile Virus is becoming a widespread pathogen, infecting people on at least four continents with no effective treatment for these infections or many of their associated pathologies. A key enzyme that is essential for viral replication is the viral protease NS2B-NS3, which is highly conserved among all flaviviruses. Using a combination of molecular fitting of substrates to the active site of the crystal structure of NS3, site-directed enzyme and cofactor mutagenesis, and kinetic studies on proteolytic processing of panels of short peptide substrates, we have identified important enzyme-substrate interactions that define substrate specificity for NS3 protease. In addition to better understanding the involvement of S2, S3, and S4 enzyme residues in substrate binding, a residue within cofactor NS2B has been found to strongly influence the preference of flavivirus proteases for lysine or arginine at P2 in substrates. Optimization of tetrapeptide substrates for enhanced protease affinity and processing efficiency has also provided important clues for developing inhibitors of West Nile Virus infection.

Cellular synthesis of peptide hormones requires PCs (prohormone convertases) for the endoproteolysis of prohormones. Antral G-cells synthesize the most gastrin and express PC1/3, 2 and 5/6 in the rat and human. But the cleavage sites in progastrin for each PC have not been determined. Therefore, in the present study, we measured the concentrations of progastrin, processing intermediates and alpha-amidated gastrins in antral extracts from PC1/3-null mice and compared the results with those in mice lacking PC2 and wild-type controls. The expression of PCs was examined by immunocytochemistry and in situ hybridization of mouse G-cells. Finally, the in vitro effect of recombinant PC5/6 on progastrin and progastrin fragments containing the relevant dibasic cleavage sites was also examined. The results showed that mouse G-cells express PC1/3, 2 and 5/6. The concentration of progastrin in PC1/3-null mice was elevated 3-fold. Chromatography showed that cleavage of the Arg(36)Arg(37) and Arg(73)Arg(74) sites were grossly decreased. Accordingly, the concentrations of progastrin products were markedly reduced, alpha-amidated gastrins (-34 and -17) being 25% of normal. Lack of PC1/3 was without effect on the third dibasic site (Lys(53)Lys(54)), which is the only processing site for PC2. Recombinant PC5/6 did not cleave any of the dibasic processing sites in progastrin and fragments containing the relevant dibasic processing sites. The complementary cleavages of PC1/3 and 2, however, suffice to explain most of the normal endoproteolysis of progastrin. Moreover, the results show that PCs react differently to the same dibasic sequences, suggesting that additional structural factors modulate the substrate specificity.

The replicase of equine arteritis virus, an arterivirus, is processed by at least three viral proteases. Comparative sequence analysis suggested that nonstructural protein 4 (Nsp4) is a serine protease (SP) that shares properties with chymotrypsin-like enzymes belonging to two different groups. The SP was predicted to utilize the canonical His-Asp-Ser catalytic triad found in classical chymotrypsin-like proteases. On the other hand, its putative substrate-binding region contains Thr and His residues, which are conserved in viral 3C-like cysteine proteases and determine their specificity for (Gln/Glu) downward arrow(Gly/Ala/Ser) cleavage sites. The replacement of the members of the predicted catalytic triad (His-1103, Asp-1129, and Ser-1184) confirmed their indispensability. The putative role of Thr-1179 and His-1199 in substrate recognition was also supported by the results of mutagenesis. A set of conserved candidate cleavage sites, strikingly similar to junctions cleaved by 3C-like cysteine proteases, was identified. These were tested by mutagenesis and expression of truncated replicase proteins. The results support a replicase processing model in which the SP cleaves multiple Glu downward arrow(Gly/Ser/Ala) sites. Collectively, our data characterize the arterivirus SP as a representative of a novel group of chymotrypsin-like enzymes, the 3C-like serine proteases.