The propeptide of Fasciola hepatica cathepsin L is a potent and selective inhibitor of the mature enzyme

Abstract Background The zoonotic worm parasite Fasciola hepatica secretes an abundance of cathepsin L peptidases that are associated with virulence, invasiveness, feeding and migration. The peptidases are produced as inactive zymogens that activate at low pH by autocatalytic removal of their N-terminal pro-domain or propeptide. Propeptides bind to their cognate enzyme with high specificity. Little is known, however, about the mechanism by which the propeptide of FhCL3, a cathepsin L peptidase secreted by the infective newly excysted juveniles (NEJs), regulates the inhibition and activation of the mature enzyme before it is secreted into host tissues. Results Immunolocalisation/immunoblotting studies show that the FhCL3 zymogen is produced and secreted by gastrodermal cells of the NEJs gut. A recombinant propeptide of FhCL3 (ppFhCL3) was shown to be a highly potent and selective inhibitor of native and recombinant F. hepatica FhCL3 peptidase, and other members of the cathepsin L family; inhibition constant (Ki) values obtained for FhCL1, FhCL2 and FhCL3 were 0.04 nM, 0.004 nM and < 0.002 nM, respectively. These values are at least 1000-fold lower than those Ki obtained for human cathepsin L (HsCL) and human cathepsin K (HsCK) demonstrating the selectivity of the ppFhCL3 for parasite cathepsins L. By exploiting 3-D structural data we identified key molecular interactions in the specific binding between the ppFhCL3 and FhCL3 mature domain. Using recombinant variants of ppFhCL3 we demonstrated the critical importance of a pair of propeptide residues (Tyr46Lys47) for the interaction with the propeptide binding loop (PBL) of the mature enzyme and other residues (Leu66 and Glu68) that allow the propeptide to block the active site. Conclusions The FhCL3 peptidase involved in host invasion by F. hepatica is produced as a zymogen in the NEJs gut. Regulation of its activation involves specific binding sites within the propeptide that are interdependent and act as a “clamp-like” mechanism of inhibition. These interactions are disrupted by the low pH of the NEJs gut to initiate autocatalytic activation. Our enzyme kinetics data demonstrates high potency and selectivity of the ppFhCL3 for its cognate FhCL3 enzyme, information that could be utilised to design inhibitors of parasite cathepsin L peptidases.

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Recognition Pattern of the Fasciola hepatica Excretome/Secretome during the Course of an Experimental Infection in Sheep by 2D Immunoproteomics

Pathogens ◽

10.3390/pathogens10060725 ◽

2021 ◽

Vol 10 (6) ◽

pp. 725

Author(s):

David Becerro-Recio ◽

Javier González-Miguel ◽

Alberto Ucero ◽

Javier Sotillo ◽

Álvaro Martínez-Moreno ◽

...

Keyword(s):

Experimental Infection ◽

Fasciola Hepatica ◽

Cathepsin L ◽

Helminth Parasites ◽

Glutathione S Transferase ◽

Serum Samples ◽

Secretory Products ◽

Immunogenic Proteins ◽

Host Parasite ◽

First Time

Excretory/secretory products released by helminth parasites have been widely studied for their diagnostic utility, immunomodulatory properties, as well as for their use as vaccines. Due to their location at the host/parasite interface, the characterization of parasite secretions is important to unravel the molecular interactions governing the relationships between helminth parasites and their hosts. In this study, the excretory/secretory products from adult worms of the trematode Fasciola hepatica (FhES) were employed in a combination of two-dimensional electrophoresis, immunoblot and mass spectrometry, to analyze the immune response elicited in sheep during the course of an experimental infection. Ten different immunogenic proteins from FhES recognized by serum samples from infected sheep at 4, 8, and/or 12 weeks post-infection were identified. Among these, different isoforms of cathepsin L and B, peroxiredoxin, calmodulin, or glutathione S-transferase were recognized from the beginning to the end of the experimental infection, suggesting their potential role as immunomodulatory antigens. Furthermore, four FhES proteins (C2H2-type domain-containing protein, ferritin, superoxide dismutase, and globin-3) were identified for the first time as non-immunogenic proteins. These results may help to further understand host/parasite relationships in fasciolosis, and to identify potential diagnostic molecules and drug target candidates of F. hepatica.

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Early immunodiagnosis of fasciolosis in ruminants using recombinant Fasciola hepatica cathepsin L-like protease

International Journal for Parasitology ◽

10.1016/s0020-7519(01)00175-8 ◽

2001 ◽

Vol 31 (7) ◽

pp. 728-737 ◽

Cited By ~ 80

Author(s):

Jan B.W.J Cornelissen ◽

Cor P.H Gaasenbeek ◽

Fred H.M Borgsteede ◽

Wicher G Holland ◽

Michiel M Harmsen ◽

...

Keyword(s):

Fasciola Hepatica ◽

Cathepsin L

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Hurpin Is a Selective Inhibitor of Lysosomal Cathepsin L and Protects Keratinocytes from Ultraviolet-Induced Apoptosis†

Biochemistry ◽

10.1021/bi027307q ◽

2003 ◽

Vol 42 (24) ◽

pp. 7381-7389 ◽

Cited By ~ 49

Author(s):

Thomas Welss ◽

Jiuru Sun ◽

James A. Irving ◽

Rainer Blum ◽

A. Ian Smith ◽

...

Keyword(s):

Cathepsin L ◽

Selective Inhibitor ◽

Induced Apoptosis

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Partial Characterization of a Novel Cathepsin L-like Protease from Fasciola hepatica

Biochemical and Biophysical Research Communications ◽

10.1006/bbrc.2000.3633 ◽

2000 ◽

Vol 277 (1) ◽

pp. 79-82 ◽

Cited By ~ 4

Author(s):

Susan J. Hawthorne ◽

Maurice Pagano ◽

David W. Halton ◽

Brian Walker

Keyword(s):

Fasciola Hepatica ◽

Cathepsin L ◽

Partial Characterization

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Purification and characterisation of a second cathepsin L proteinase secreted by the parasitic trematode Fasciola hepatica

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1994.tb18969.x ◽

1994 ◽

Vol 223 (1) ◽

pp. 91-98 ◽

Cited By ~ 68

Author(s):

Andrew J. DOWD ◽

Angela M. SMITH ◽

Sharon McGONIGLE ◽

John P. DALTON

Keyword(s):

Fasciola Hepatica ◽

Cathepsin L

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Synthesis and processing of cathepsin L, an elastase, by human alveolar macrophages

Biochemical Journal ◽

10.1042/bj2570493 ◽

1989 ◽

Vol 257 (2) ◽

pp. 493-498 ◽

Cited By ~ 54

Author(s):

J J Reilly ◽

R W Mason ◽

P Chen ◽

L J Joseph ◽

V P Sukhatme ◽

...

Keyword(s):

Culture Medium ◽

Cathepsin L ◽

Intermediate Form ◽

Human Macrophages ◽

Lung Macrophages ◽

Synthesis And Processing ◽

Mature Enzyme ◽

Cultivation In Vitro ◽

Apparently Healthy

Cathepsin L was partially purified from lysates of freshly isolated macrophages lavaged from lungs of apparently healthy adults and found to be chromatographically and catalytically identical with liver cathepsin L. Western-blotting analysis showed that lung macrophages contain significant levels of a precursor of cathepsin L (43 kDa) in addition to mature enzyme (25 kDa). After culturing for a further 24 h, the precursor disappeared and a new band, corresponding to 34 kDa, appeared, suggesting that the precursor had been processed to an intermediate form of cathepsin L. Biosynthetic labelling of macrophages in vitro with [35S]methionine followed by immunoprecipitation with the cathepsin L antibody confirmed that the cells synthesize cathepsin L as a 43 kDa precursor that is then processed to the mature form (25 kDa) via a 34 kDa intermediate. The precursor, but not the processed forms, was released into the culture medium. During culture in vitro the 34 kDa intermediate accumulated, and little enzyme was processed to the 24 kDa form, consistent with the immunoblot data. Human lung macrophages contain a 1.5 kb transcript of cathepsin L mRNA, whereas none is detectable in human monocytes. These results establish that differentiation of human macrophages within the lung is accompanied by synthesis and expression of an elastinolytic enzyme, cathepsin L. The altered processing of cathepsin L observed during cultivation in vitro suggests caution in the assessment of the elastinolytic potential of human macrophages based on assay in vitro.

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