Crystal Structures of TbCatB and Rhodesain,Potential Chemotherapeutic Targets and Major Cysteine Proteases of Trypanosoma brucei

Background

Trypanosoma brucei is the etiological agent of Human African Trypanosomiasis, an endemic parasitic disease of sub-Saharan Africa. TbCatB and rhodesain are the sole Clan CA papain-like cysteine proteases produced by the parasite during infection of the mammalian host and are implicated in the progression of disease. Of considerable interest is the exploration of these two enzymes as targets for cysteine protease inhibitors that are effective against T. brucei.

Methods and Findings

We have determined, by X-ray crystallography, the first reported structure of TbCatB in complex with the cathepsin B selective inhibitor CA074. In addition we report the structure of rhodesain in complex with the vinyl-sulfone K11002.

Conclusions

The mature domain of our TbCat•CA074 structure contains unique features for a cathepsin B-like enzyme including an elongated N-terminus extending 16 residues past the predicted maturation cleavage site. N-terminal Edman sequencing reveals an even longer extension than is observed amongst the ordered portions of the crystal structure. The TbCat•CA074 structure confirms that the occluding loop, which is an essential part of the substrate-binding site, creates a larger prime side pocket in the active site cleft than is found in mammalian cathepsin B-small molecule structures. Our data further highlight enhanced flexibility in the occluding loop main chain and structural deviations from mammalian cathepsin B enzymes that may affect activity and inhibitor design. Comparisons with the rhodesain•K11002 structure highlight key differences that may impact the design of cysteine protease inhibitors as anti-trypanosomal drugs.     

Cathepsin B- and L-like cysteine protease activities during the in vitro development of Hysterothylacium aduncum (Nematoda: Anisakidae), a worldwide fish parasite

Proteinases play an important role as virulence factors both in the life-cycle of parasites and in the pathogen–host relationship. Hysterothylacium aduncum is a worldwide fish parasite nematode which has been associated with non-invasive anisakidosis and allergic responses to fish consumption in humans. Cysteine proteinases have been associated with allergy to plant pollens, detergents and dust mites. In this study the presence of two types of cysteine proteinases (cathepsin B and cathepsin L) during in vitro development of H. aduncum is investigated. Specific fluorescent substrates were used to determine cathepsin activities. The activity detected with substrate Z-FR-AMC was identified as cathepsin L (optimum pH = 5.5; range 3.5–6.5). Cathepsin B activity was only identified with Z-RR-AMC (optimum pH = 7.0–7.5; range 5.0–8.0). The start of cultivation led to increased activity of both cathepsins (1.8-fold for cathepsin B and 6.3-fold for cathepsin L). These activities varied according to the developmental stage. Cathepsin B activity decreased after M4, returning to its initial level. Cathepsin L activity also decreased after M4, but still maintained a high level (4–6 times the initial level) in adult stages. Having considered these activity variations and the optimum pH values, we suggest that cathepsin L has a role in digestive processes while cathepsin B could be involved in cuticle renewal, among other possible functions.     

Cystatins from filarial parasites: evolution, adaptation and function in the host-parasite relationship

Cystatins, together with stefins and kininogens, are members of the cystatin superfamily of cysteine protease inhibitors (CPI) present across the animal and plant kingdoms. Their role in parasitic organisms may encompass both essential developmental processes and specific interactions with the parasite's vector and/or final host. We summarise information gathered on three cystatins from the human filarial nematode Brugia malayi (Bm-CPI-1, -2 and -3), and contrast them those expressed by other parasites and by the free-living nematode Caenorhabditis elegans. Bm-CPI-2 differs from C. elegans cystatin, having acquired the additional function of inhibiting asparaginyl endopeptidase (AEP), in a manner similar to some human cystatins. Thus, we propose that Bm-CPI-2 and orthologues from related filarial parasites represent a new subset of nematode cystatins. Bm-CPI-1 and CPI-3 share only 25% amino acid identity with Bm-CPI-2, and lack an evolutionarily conserved glycine residue in the N-terminal region. These sequences group distantly from the other nematode cystatins, and represent a second novel subset of filarial cystatin-like genes. Expression analyses also show important differences between the CPI-2 and CPI-1/-3 groups. Bm-cpi-2 is expressed at all time points of the parasite life cycle, while Bm-cpi-1 and -3 expression is confined to the late stages of development in the mosquito vector, terminating within 48h of infection of the mammalian host. Hence, we hypothesise that CPI-2 has evolved to block mammalian proteases (including the antigen-processing enzyme AEP) while CPI-1 and -3 function in the milieu of the mosquito vector necessary for transmission of the parasite.     

Exoerythrocytic Plasmodium Parasites Secrete a Cysteine Protease Inhibitor Involved in Sporozoite Invasion and Capable of Blocking Cell Death of Host Hepatocytes

Plasmodium parasites must control cysteine protease activity that is critical for hepatocyte invasion by sporozoites, liver stage development, host cell survival and merozoite liberation. Here we show that exoerythrocytic P. berghei parasites express a potent cysteine protease inhibitor (PbICP, P. berghei inhibitor of cysteine proteases). We provide evidence that it has an important function in sporozoite invasion and is capable of blocking hepatocyte cell death. Pre-incubation with specific anti-PbICP antiserum significantly decreased the ability of sporozoites to infect hepatocytes and expression of PbICP in mammalian cells protects them against peroxide- and camptothecin-induced cell death. PbICP is secreted by sporozoites prior to and after hepatocyte invasion, localizes to the parasitophorous vacuole as well as to the parasite cytoplasm in the schizont stage and is released into the host cell cytoplasm at the end of the liver stage. Like its homolog falstatin/PfICP in P. falciparum, PbICP consists of a classical N-terminal signal peptide, a long N-terminal extension region and a chagasin-like C-terminal domain. In exoerythrocytic parasites, PbICP is posttranslationally processed, leading to liberation of the C-terminal chagasin-like domain. Biochemical analysis has revealed that both full-length PbICP and the truncated C-terminal domain are very potent inhibitors of cathepsin L-like host and parasite cysteine proteases. The results presented in this study suggest that the inhibitor plays an important role in sporozoite invasion of host cells and in parasite survival during liver stage development by inhibiting host cell proteases involved in programmed cell death.     

A major cathepsin B protease from the liver fluke Fasciola hepatica has atypical active site features and a potential role in the digestive tract of newly excysted juvenile parasites

The newly excysted juvenile (NEJ) stage of the Fasciola hepatica lifecycle occurs just prior to invasion into the wall of the gut of the host, rendering it an important target for drug development. The cathepsin B enzymes from NEJ flukes have recently been demonstrated to be crucial to invasion and migration by the parasite. Here we characterize one of the cathepsin B enzymes (recombinant FhcatB1) from NEJ flukes. FhcatB1 has biochemical properties distinct from mammalian cathepsin B enzymes, with an atypical preference for Ile over Leu or Arg residues at the P₂ substrate position and an inability to act as an exopeptidase. FhcatB1 was active across a broad pH range (optimal activity at pH 5.5–7.0) and resistant to inhibition by cystatin family inhibitors from sheep and humans, suggesting that this enzyme would be able to function in extracellular environments in its mammalian hosts. It appears, however, that the FhcatB1 protease functions largely as a digestive enzyme in the gut of the parasite, due to the localization of a specific, fluorescently labeled inhibitor with an Ile at the P₂ position. Molecular modelling and dynamics were used to predict the basis for the unusual substrate specificity: a P₂ Ile residue positions the substrate optimally for interaction with catalytic residues of the enzyme, and the enzyme lacks an occluding loop His residue crucial for exopeptidase activity. The unique features of the enzyme, particularly with regard to its specificity and likely importance to a vital stage of the parasite's life cycle, make it an excellent target for therapeutic inhibitors or vaccination.     

Crystal structure of the parasite protease inhibitor chagasin in complex with a host target cysteine protease

Chagasin is a protein produced by Trypanosoma cruzi, the parasite that causes Chagas' disease. This small protein belongs to a recently defined family of cysteine protease inhibitors. Although resembling well-known inhibitors like the cystatins in size (110 amino acid residues) and function (they all inhibit papain-like (C1 family) proteases), it has a unique amino acid sequence and structure. We have crystallized and solved the structure of chagasin in complex with the host cysteine protease, cathepsin L, at 1.75 A resolution. An inhibitory wedge composed of three loops (L2, L4, and L6) forms a number of contacts responsible for high-affinity binding (K(i), 39 pM) to the enzyme. All three loops interact with the catalytic groove, with the central loop L2 inserted directly into the catalytic center. Loops L4 and L6 embrace the enzyme molecule from both sides and exhibit distinctly different patterns of protein-protein recognition. Comparison with a 1.7 A structure of uncomplexed chagasin, also determined in this study, demonstrates that a conformational change of the first binding loop (L4) allows extended binding to the non-primed substrate pockets of the enzyme active site cleft, thereby providing a substantial part of the inhibitory surface. The mode of chagasin binding is generally similar, albeit distinctly different in detail, when compared to those displayed by cystatins and the cysteine protease inhibitory p41 fragment of the invariant chain. The chagasin-cathepsin L complex structure provides details of how the parasite protein inhibits a host enzyme of possible importance in host defense. The high level of structural and functional similarity between cathepsin L and the T. cruzi enzyme cruzipain gives clues to how the cysteine protease activity of the parasite can be targeted. This information will aid in the development of synthetic inhibitors for use as potential drugs for the treatment of Chagas disease.     

Evolution of conflict and cooperation of nematodes associated with solitary and social sweat bees

Parasites and mutualists can wield great influence on the fitness of social organisms, yet the effect that the host’s social structure has on the evolution of parasites, commensals, and mutualists (collectively referred to here as symbionts) is poorly known. Evolutionary theory suggests that host social structure may select for more cooperative symbiont strains in comparison to symbionts of solitary hosts. We compared the productivity of one social and one solitary bee species (Halictus ligatus and Augochlora pura) in the family Halictidae with and without the presence of their nematode symbionts (Acrostichus halicti and Acrostichus puri, respectively). We measured the number of offspring produced, the number of cells provisioned, and nesting activity (for Au. pura) to test the hypothesis that symbionts specific to a social host exhibit greater cooperation than symbionts specific to a solitary host. Infected and uninfected nests of both species did not differ in any fitness estimates indicating that: (1) Acrostichus species are commensals, or at least lack large fitness effects on their hosts, and (2) the transition from association with a solitary host to association with a social host that lives in small colonies does not have detectable effects on the evolution of conflict and cooperation in this system. This is the first comparative study to test the idea that host social structure may influence the evolution of symbionts; future work should compare closely related mutualists and parasites of more advanced eusocial insects to mutualists and parasites of solitary insects.     

Toxoplasma gondii Cathepsin L Is the Primary Target of the Invasion-inhibitory Compound Morpholinurea-leucyl-homophenyl-vinyl Sulfone Phenyl

The protozoan parasite Toxoplasma gondii relies on post-translational modification, including proteolysis, of proteins required for recognition and invasion of host cells. We have characterized the T. gondii cysteine protease cathepsin L (TgCPL), one of five cathepsins found in the T. gondii genome. We show that TgCPL is the primary target of the compound morpholinurea-leucyl-homophenyl-vinyl sulfone phenyl (LHVS), which was previously shown to inhibit parasite invasion by blocking the release of invasion proteins from microneme secretory organelles. As shown by fluorescently labeled LHVS and TgCPL-specific antibodies, TgCPL is associated with a discrete vesicular structure in the apical region of extracellular parasites but is found in multiple puncta throughout the cytoplasm of intracellular replicating parasites. LHVS fails to label cells lacking TgCPL due to targeted disruption of the TgCPL gene in two different parasite strains. We present a structural model for the inhibition of TgCPL by LHVS based on a 2.0 Å resolution crystal structure of TgCPL in complex with its propeptide. We discuss possible roles for TgCPL as a protease involved in the degradation or limited proteolysis of parasite proteins involved in invasion.The recent completion of many genome-sequencing projects has allowed an unprecedented view of the complete set of proteases in biologically or medically important organisms (1). Of the five mechanistically distinct catalytic types (serine, cysteine, aspartyl, metallo, and threonine), cysteine proteases are the second largest group. In particular, cysteine proteases of the C1 papain family of “lysosomal” cathepsins have garnered intense scrutiny because of their key roles in cancer, embryogenesis, heart disease, osteoporosis, immunity, and infectious diseases. Microbial cathepsins, particularly those expressed by parasites, have also attracted attention recently because of their potential as targets for treatment of helminthic and protozoal infections (2, 3).The protozoan parasite Toxoplasma gondii infects virtually all warm-blooded animals and approximately one-third of the human population worldwide. Although most Toxoplasma infections are benign, severe opportunistic disease is seen in immunodeficient or immunosuppressed individuals or congenitally infected babies. T. gondii is an obligate intracellular organism that uses an actin-myosin-based motility system to actively invade nucleated host cells (4, 5). The parasite secretes a variety of proteins during and after cell invasion that contribute to recognition of the host cell, formation of an adhesive “moving” junction, modulation of host signaling pathways and gene expression, and remodeling of the parasitophorous vacuole in preparation for parasite growth (6, 7). Although it has been known for some time that many Toxoplasma secretory proteins are post-translationally modified by proteolysis before and/or after secretion, in most cases, the consequences of proteolysis or the specific protease involved are unclear.Analysis of the T. gondii genome indicates the existence of five genes encoding cathepsin proteases of the papain family, including three cathepsin C proteases (TgCPC1, TgCPC2, and TgCPC3), one cathepsin B (Toxopain-1 or TgCPB), and one cathepsin L (TgCPL). TgCPC1 and TgCPC2 are secreted into the parasitophorous vacuole after parasite invasion and are proposed to function in nutrient acquisition (8). TgCPC3 is not expressed in tachyzoites, a rapidly dividing form of the parasite that is most commonly studied in the laboratory. TgCPB is localized in club-shaped invasion organelles called rhoptries, where it may act as a maturase for rhoptry proteins involved in modulation of the host cell (9). TgCPL is predicted to be a type II membrane protein, and a recent report by Reed and co-workers (10) showed that it has enzymatic activity with a low pH optimum and that it occupies a membrane-bound structure in the apical region of extracellular parasites. This same study revealed that T. gondii expresses two endogenous inhibitors of cysteine proteases (TgICP1 and TgICP2), but their role in regulating parasite or host cysteine proteases remains to be determined. Similar inhibitors are expressed by other parasites, including Trypanosoma cruzi, that act on host proteases, and the crystal structure of an inhibitor (chagasin)-enzyme (human cathepsin L) complex was recently reported (11).In a recent study, we screened a small library of cathepsin and proteasome inhibitors and identified two compounds that substantially impair Toxoplasma cell invasion (12). The most effective of these compounds, morpholinurea-leucyl-homophenyl-vinyl sulfone phenyl (LHVS),² inhibited invasion with a 50% inhibitory concentration (IC₅₀) of ∼10 μm. Further analysis revealed that LHVS blocks parasite attachment and gliding motility by impairing the release of proteins from a distinct set of apical secretory organelles called micronemes. Here we definitively show, using a variety of biochemical, genetic, and structural approaches, that TgCPL is the primary target of LHVS in the parasite.     

Displacement of the occluding loop by the parasite protein, chagasin, results in efficient inhibition of human cathepsin B

Cathepsin B is a papain-like cysteine protease showing both endo- and exopeptidase activity, the latter due to a unique occluding loop that restricts access to the active site cleft. To clarify the mode by which natural protein inhibitors manage to overcome this obstacle, we have analyzed the structure and function of cathepsin B in complexes with the Trypanosoma cruzi inhibitor, chagasin. Kinetic analysis revealed that substitution of His-110e, which anchors the loop in occluding position, results in 3-fold increased chagasin affinity (Ki for H110A cathepsin B, 0.35 nm) due to an improved association rate (kon, 5 x 10(5) m(-1)s(-1)). The structure of chagasin in complex with cathepsin B was solved in two crystal forms (1.8 and 2.67 angstroms resolution), demonstrating that the occluding loop is displaced to allow chagasin binding with its three loops, L4, L2, and L6, spanning the entire active site cleft. The occluding loop is differently displaced in the two structures, indicating a large range of movement and adoption of conformations forced by the inhibitor. The area of contact is slightly larger than in chagasin complexes with the endopeptidase, cathepsin L. However, residues important for high affinity to both enzymes are mainly found in the outer loops L4 and L6 of chagasin. The chagasin-cathepsin B complex provides a structural framework for modeling and design of inhibitors for cruzipain, the parasite cysteine protease and a virulence factor in Chagas disease.     

SmCL3, a Gastrodermal Cysteine Protease of the Human Blood Fluke Schistosoma mansoni

Background

Blood flukes of the genus Schistosoma are platyhelminth parasites that infect 200 million people worldwide. Digestion of nutrients from the host bloodstream is essential for parasite development and reproduction. A network of proteolytic enzymes (proteases) facilitates hydrolysis of host hemoglobin and serum proteins.

Methodology/Principal Findings

We identified a new cathepsin L termed SmCL3 using PCR strategies based on S. mansoni EST sequence data. An ortholog is present in Schistosoma japonicum. SmCL3 was heterologously expressed as an active enzyme in the yeast, Pichia pastoris. Recombinant SmCL3 has a broad pH activity range against peptidyl substrates and is inhibited by Clan CA protease inhibitors. Consistent with a function in degrading host proteins, SmCL3 hydrolyzes serum albumin and hemoglobin, is localized to the adult gastrodermis, and is expressed mainly in those life stages infecting the mammalian host. The predominant form of SmCL3 in the parasite exists as a zymogen, which is unusual for proteases. This zymogen includes an unusually long prodomain with alpha helical secondary structure motifs. The striking specificity of SmCL3 for amino acids with large aromatic side chains (Trp and Tyr) at the P2 substrate position, as determined with positional scanning-synthetic combinatorial library, is consistent with a molecular model that shows a large and deep S2 pocket. A sequence similarity network (SSN) view clusters SmCL3 and other cathepsins L in accordance with previous large-scale phylogenetic analyses that identify six super kingdoms.

Conclusions/Significance

SmCL3 is a gut-associated cathepsin L that may contribute to the network of proteases involved in degrading host blood proteins as nutrients. Furthermore, this enzyme exhibits some unusual sequence and biophysical features that may result in additional functions. The visualization of network inter-relationships among cathepsins L suggests that these enzymes are suitable ‘marker sequences’ for inclusion in future phylogenetic analyses.     

Pearl millet cysteine protease inhibitor. Evidence for the presence of two distinct sites responsible for anti-fungal and anti-feedent activities.

Recently, pearl millet cysteine protease inhibitor (CPI) was, for the first time, shown to possess anti-fungal activity in addition to its anti-feedent (protease inhibitory) activity [Joshi, B.N. et al. (1998) Biochem. Biophys. Res. Commun. 246, 382-387]. Characterization of CPI revealed that it has a reversible mode of action for protease inhibition. The CD spectrum exhibited a 35% alpha helix and 65% random coil structure. The intrinsic fluorescence spectrum was typical of a protein devoid of tryptophan residues. Demetallation of Zn2+ resulted in a substantial change in the secondary and tertiary structure of CPI accompanied by the complete loss of anti-fungal and inhibitory activity indicating that Zn2+ plays an important role in maintaining both structural integrity and biological function. The differential response of anti-fungal and inhibitory activities to specific modifiers showed that there are two different reactive sites associated with anti-fungal and anti-feedent activity in CPI located on a single protein as revealed from its N-terminal sequence data (AGVCYGVLGNNLP). Modification of cysteine, glutamic/aspartic acid or argnine resulted in abolition of the anti-fungal activity of CPI, whereas modification of arginine led to an enhancement of the inhibitory activity in solution. Modification of histidine resulted in a twofold increase in the protease inhibitory activity without affecting the anti-fungal activity, whereas modification of serine led to selective inhibition of the protease inhibitory activity. The differential nature of the two activities was further supported by differences in the temperature stabilities of the anti-fungal (60 degrees C) and inhibitory (40 degrees C) activities. Binding of papain to CPI did not abolish the anti-fungal activity of CPI, supporting the presence of two active sites on CPI. The differential behavior of CPI towards anti-fungal and anti-feedent activity cannot be attributed to changes in conformation, as assessed by their CD and fluorescence spectra. The interaction of CPI modified for arginine or histidine with papain resulted in an enhancement of CPI activity accompanied by a slight decrease in fluorescence intensity of 15-20% at 343 nm. In contrast, modification of serine resulted in inhibition of CPI activity with a concomitant increase of 20% in the fluorescence intensity when complexed by the enzyme. This implies the involvement of enzyme-based tryptophan in the formation of a biologically active enzyme-inhibitor complex. The presence of anti-fungal and anti-feedent activity on a single protein, as evidenced in pearl millet CPI, opens up a new possibility of raising a transgenic plant resistant to pathogens, as well as pests, by transfer of a single CPI gene.     

Genomic organization of the Schistosoma mansoni aspartic protease gene, a platyhelminth orthologue of mammalian lysosomal cathepsin D

Schistosomes are considered the most important of the helminth parasites of humans in terms of morbidity and mortality. Schistosomes employ proteolytic enzymes to digest host hemoglobin from ingested human blood, including a cathepsin D-like, aspartic protease that is overexpressed in the gut of the adult female schistosome. Because of its key role in parasite nutrition, this enzyme represents a potential intervention target. To continue exploration of this potential, here we have determined the sequence, structure and genomic organization of the cathepsin D gene locus of Schistosoma mansoni. Using the cDNA encoding S. mansoni cathepsin D as a probe, we isolated several positive bacterial artificial chromosomes (BAC) from a BAC library that represents an approximately 8-fold coverage of the schistosome genome. Sequencing of BAC clone 25-J-24 revealed that the cathepsin D gene locus was approximately 13 kb in length, and included seven exons interrupted by six introns. The exons ranged in length from 49 to 294 bp, and the introns from 30 to 5025 bp. The genomic organization of schistosome cathepsin D was similar in sequence, structure and complexity to human cathepsin D, including to a greater or lesser extent the conservation of all six exon/intron boundaries of the schistosome gene. It was less similar to aspartic protease genes of the nematodes Caenorhabditis elegans and Haemonchus contortus, and dissimilar to those of plasmepsins from malarial parasites. Examination of the introns revealed the presence of endogenous mobile genetic elements including SR2, the ASL-associated retrotransposon, and the SINE-like element, SMalpha. Phylogenetically, schistosome cathepsin D appeared to be more closely related to mammalian cathepsin D than to other sub-families of eukaryotic aspartic proteases known from mammals. Taken together, these features indicated that schistosome cathepsin D is a platyhelminth orthologue of mammalian lysosomal cathepsin D.     

Cathepsin B Gene Disruption Induced Leishmania donovani Proteome Remodeling Implies Cathepsin B Role in Secretome Regulation

Leishmania cysteine proteases are potential vaccine candidates and drug targets. To study the role of cathepsin B cysteine protease, we have generated and characterized cathepsin B null mutant L. donovani parasites. L. donovani cathepsin B null mutants grow normally in culture, but they show significantly attenuated virulence inside macrophages. Quantitative proteome profiling of wild type and null mutant parasites indicates cathepsin B disruption induced remodeling of L. donovani proteome. We identified 83 modulated proteins, of which 65 are decreased and 18 are increased in the null mutant parasites, and 66% (55/83) of the modulated proteins are L. donovani secreted proteins. Proteins involved in oxidation-reduction (trypanothione reductase, peroxidoxins, tryparedoxin, cytochromes) and translation (ribosomal proteins) are among those decreased in the null mutant parasites, and most of these proteins belong to the same complex network of proteins. Our results imply virulence role of cathepsin B via regulation of Leishmania secreted proteins.     

Proteases of the nematode Caenorhabditis elegans

Crude homogenates of the soil nematode Caenorhabditis elegans exhibit strong proteolytic activity at acid pH. Several kinds of enzyme account for much of this activity: cathepsin D, a carboxyl protease which is inhibited by pepstatin and optimally active toward hemoglobin at pH 3; at least two isoelectrically distinct thiol proteases (cathepsins Ce1 and Ce2) which are inhibited by leupeptin and optimally active toward Z-Phe-Arg-7-amino-4-methylcoumarin amide at pH 5; and a thiol-independent leupeptin-insensitive protease (cathepsin Ce3) with optimal activity toward casein at pH 5.5. Cathepsin D is quantitatively most significant for digestion of macromolecular substrates in vitro, since proteolysis is inhibited greater than 95% by pepstatin. Cathepsin D and the leupeptin-sensitive proteases act synergistically, but the relative contribution of the leupeptin-sensitive proteases depends upon the protein substrate.     

Cathepsin B of Cynoglossus semilaevis: identification, expression, and activity analysis

Cathepsin B (EC 3.4.22.1) is a member of the papain family cysteine protease and in mammals is known to be involved in protein degradation and other biological functions. However, very little is known about the function of cathepsin B in fish. In this study, we identified and analyzed a cathepsin B homologue (CsCatB) from tongue sole (Cynoglossus semilaevis, Pleuronectiformes), an economic fish species cultured in China. CsCatB is composed of 322 amino acid residues and shares 70-81.3% overall sequence identities with its counterpart in teleosts and humans. CsCatB possesses typical cathepsin B structural features including the propeptide region and the papain family cysteine protease domain, the latter containing the four catalytic residues (Q101, C107, H277, and N297) that are conserved in lower and higher vertebrates. Quantitative real time RT-PCR analysis showed that CsCatB expression occurred in multiple tissues and was positively regulated by bacterial infection and by immunization with a subunit vaccine. Recombinant CsCatB purified from Escherichia coli exhibited apparent protease activity, which was optimal at 35 °C and pH 5.5. In contrast, a mutant CsCatB bearing glutamic acid substitution at H277 was dramatically reduced in proteolytic activity. These results indicate that CsCatB is a biologically active protease that is likely to be involved in host immune response during bacterial infection and vaccination.     

Structural basis for dual-inhibition mechanism of a non-classical Kazal-type serine protease inhibitor from horseshoe crab in complex with subtilisin

Serine proteases play a crucial role in host-pathogen interactions. In the innate immune system of invertebrates, multi-domain protease inhibitors are important for the regulation of host-pathogen interactions and antimicrobial activities. Serine protease inhibitors, 9.3-kDa CrSPI isoforms 1 and 2, have been identified from the hepatopancreas of the horseshoe crab, Carcinoscorpius rotundicauda. The CrSPIs were biochemically active, especially CrSPI-1, which potently inhibited subtilisin (Ki = 1.43 nM). CrSPI has been grouped with the non-classical Kazal-type inhibitors due to its unusual cysteine distribution. Here we report the crystal structure of CrSPI-1 in complex with subtilisin at 2.6 Å resolution and the results of biophysical interaction studies. The CrSPI-1 molecule has two domains arranged in an extended conformation. These two domains act as heads that independently interact with two separate subtilisin molecules, resulting in the inhibition of subtilisin activity at a ratio of 1:2 (inhibitor to protease). Each subtilisin molecule interacts with the reactive site loop from each domain of CrSPI-1 through a standard canonical binding mode and forms a single ternary complex. In addition, we propose the substrate preferences of each domain of CrSPI-1. Domain 2 is specific towards the bacterial protease subtilisin, while domain 1 is likely to interact with the host protease, Furin. Elucidation of the structure of the CrSPI-1: subtilisin (1∶2) ternary complex increases our understanding of host-pathogen interactions in the innate immune system at the molecular level and provides new strategies for immunomodulation.