Isoforms of Cathepsin B1 in Neurotropic Schistosomula of
Trichobilharzia regenti Differ in Substrate Preferences and a
Highly Expressed Catalytically Inactive Paralog Binds Cystatin

J 2020

Isoforms of Cathepsin B1 in Neurotropic Schistosomula of Trichobilharzia regenti Differ in Substrate Preferences and a Highly Expressed Catalytically Inactive Paralog Binds Cystatin

DVOŘÁKOVÁ, Hana, Roman LEONTOVYČ, Tomáš MACHÁČEK, Anthony J. O´DONOGHUE, Ondrej ŠEDO et. al.

Basic information

Original name

Isoforms of Cathepsin B1 in Neurotropic Schistosomula of Trichobilharzia regenti Differ in Substrate Preferences and a Highly Expressed Catalytically Inactive Paralog Binds Cystatin

Authors

DVOŘÁKOVÁ, Hana (203 Czech Republic), Roman LEONTOVYČ (203 Czech Republic), Tomáš MACHÁČEK (203 Czech Republic), Anthony J. O´DONOGHUE (840 United States of America), Ondrej ŠEDO (203 Czech Republic, belonging to the institution), Zbyněk ZDRÁHAL (203 Czech Republic, guarantor, belonging to the institution), Charles S. CRAIK (840 United States of America), Conor R. CAFFREY (840 United States of America), Petr HORÁK (203 Czech Republic) and Libor MIKEŠ (203 Czech Republic)

Edition

FRONTIERS IN CELLULAR AND INFECTION MICROBIOLOGY, 2020, 2235-2988

Other information

Language

English

Type of outcome

Článek v odborném periodiku

Field of Study

10606 Microbiology

Country of publisher

Switzerland

Confidentiality degree

není předmětem státního či obchodního tajemství

References:

fulltext

Impact factor

Impact factor: 5.293

RIV identification code

RIV/00216224:14740/20:00115570

Organization unit

Central European Institute of Technology

DOI

http://dx.doi.org/10.3389/fcimb.2020.00066

UT WoS

000524706100001

Keywords in English

peptidase; cathepsin B; processing; substrate specificity; occluding loop; cystatin; helminth; schistosome

Abstract

V originále

Schistosomula (the post-infective stages) of the neurotropic schistosome Trichobilharzia regenti possess multiple isoforms of cathepsin B1 peptidase (TrCB1.1-TrCB1.6) with involvement in nutrient digestion. The comparison of substrate preferences of TrCB1.1 and TrCB1.4 showed that TrCB1.4 had a very narrow substrate specificity and after processing it was less effective toward protein substrates when compared to TrCB1.1. Self-processing of both isoforms could be facilitated by sulfated polysaccharides due to a specific binding motif in the pro-sequence. Trans-activation by heterologous enzymes was also successfully employed. Expression profiling revealed a high level of transcription of genes encoding the enzymatically inactive paralogs TrCB1.5 and TrCB1.6. The transcription level of TrCB1.6 was comparable with that of TrCB1.1 and TrCB1.2, the most abundant active isoforms. Recombinant TrCB1.6wt, a wild type paralog with a Cys(29)-to-Gly substitution in the active site that renders the enzyme inactive, was processed by the active TrCB1 forms and by an asparaginyl endopeptidase. Although TrCB1.6wt lacked hydrolytic activity, endopeptidase, but not dipeptidase, activity could be restored by mutating Gly(29) to Cys(29). The lack of exopeptidase activity may be due to other mutations, such as His(110)-to-Asn in the occluding loop and Asp(224)-to-Gly in the main body of the mature TrCB1.6, which do not occur in the active isoforms TrCB1.1 and TrCB1.4 with exopeptidase activity. The catalytically active enzymes and the inactive TrCB1.6 paralog formed complexes with chicken cystatin, thus supporting experimentally the hypothesis that inactive paralogs could potentially regulate the activity of the active forms or protect them from being inhibited by host inhibitors. The effect on cell viability and nitric oxide production by selected immune cells observed for TrCB1.1 was not confirmed for TrCB1.6. We show here that the active isoforms of TrCB1 have different affinities for peptide substrates thereby facilitating diversity in protein-derived nutrition for the parasite. The inactive paralogs are unexpectedly highly expressed and one of them retains the ability to bind cystatins, likely due to specific mutations in the occluding loop and the enzyme body. This suggests a role in sequestration of inhibitors and protection of active cysteine peptidases.

Links

LQ1601, research and development project

Name: CEITEC 2020 (Acronym: CEITEC2020)

Investor: Ministry of Education, Youth and Sports of the CR

90043, large research infrastructures

Name: CIISB

90127, large research infrastructures

Name: CIISB II

Citovat

DVOŘÁKOVÁ, Hana, Roman LEONTOVYČ, Tomáš MACHÁČEK, Anthony J. O´DONOGHUE, Ondrej ŠEDO, Zbyněk ZDRÁHAL, Charles S. CRAIK, Conor R. CAFFREY, Petr HORÁK and Libor MIKEŠ. Isoforms of Cathepsin B1 in Neurotropic Schistosomula of Trichobilharzia regenti Differ in Substrate Preferences and a Highly Expressed Catalytically Inactive Paralog Binds Cystatin. FRONTIERS IN CELLULAR AND INFECTION MICROBIOLOGY. 2020, vol. 10, FEB, p. "66", 17 pp. ISSN 2235-2988. Available from: https://dx.doi.org/10.3389/fcimb.2020.00066.

@article{1649841,
   author = {Dvořáková, Hana and Leontovyč, Roman and Macháček, Tomáš and O´Donoghue, Anthony J. and Šedo, Ondrej and Zdráhal, Zbyněk and Craik, Charles S. and Caffrey, Conor R. and Horák, Petr and Mikeš, Libor},
   article_number = {FEB},
   doi = {http://dx.doi.org/10.3389/fcimb.2020.00066},
   keywords = {peptidase; cathepsin B; processing; substrate specificity; occluding loop; cystatin; helminth; schistosome},
   language = {eng},
   issn = {2235-2988},
   journal = {FRONTIERS IN CELLULAR AND INFECTION MICROBIOLOGY},
   title = {Isoforms of Cathepsin B1 in Neurotropic Schistosomula of Trichobilharzia regenti Differ in Substrate Preferences and a Highly Expressed Catalytically Inactive Paralog Binds Cystatin},
   url = {https://www.frontiersin.org/articles/10.3389/fcimb.2020.00066/full},
   volume = {10},
   year = {2020}
}

TY  - JOUR
ID  - 1649841
AU  - Dvořáková, Hana - Leontovyč, Roman - Macháček, Tomáš - O´Donoghue, Anthony J. - Šedo, Ondrej - Zdráhal, Zbyněk - Craik, Charles S. - Caffrey, Conor R. - Horák, Petr - Mikeš, Libor
PY  - 2020
TI  - Isoforms of Cathepsin B1 in Neurotropic Schistosomula of Trichobilharzia regenti Differ in Substrate Preferences and a Highly Expressed Catalytically Inactive Paralog Binds Cystatin
JF  - FRONTIERS IN CELLULAR AND INFECTION MICROBIOLOGY
VL  - 10
IS  - FEB
SP  - "66"
EP  - "66"
SN  - 22352988
KW  - peptidase
KW  - cathepsin B
KW  - processing
KW  - substrate specificity
KW  - occluding loop
KW  - cystatin
KW  - helminth
KW  - schistosome
UR  - https://www.frontiersin.org/articles/10.3389/fcimb.2020.00066/full
L2  - https://www.frontiersin.org/articles/10.3389/fcimb.2020.00066/full
N2  - Schistosomula (the post-infective stages) of the neurotropic schistosome Trichobilharzia regenti possess multiple isoforms of cathepsin B1 peptidase (TrCB1.1-TrCB1.6) with involvement in nutrient digestion. The comparison of substrate preferences of TrCB1.1 and TrCB1.4 showed that TrCB1.4 had a very narrow substrate specificity and after processing it was less effective toward protein substrates when compared to TrCB1.1. Self-processing of both isoforms could be facilitated by sulfated polysaccharides due to a specific binding motif in the pro-sequence. Trans-activation by heterologous enzymes was also successfully employed. Expression profiling revealed a high level of transcription of genes encoding the enzymatically inactive paralogs TrCB1.5 and TrCB1.6. The transcription level of TrCB1.6 was comparable with that of TrCB1.1 and TrCB1.2, the most abundant active isoforms. Recombinant TrCB1.6wt, a wild type paralog with a Cys(29)-to-Gly substitution in the active site that renders the enzyme inactive, was processed by the active TrCB1 forms and by an asparaginyl endopeptidase. Although TrCB1.6wt lacked hydrolytic activity, endopeptidase, but not dipeptidase, activity could be restored by mutating Gly(29) to Cys(29). The lack of exopeptidase activity may be due to other mutations, such as His(110)-to-Asn in the occluding loop and Asp(224)-to-Gly in the main body of the mature TrCB1.6, which do not occur in the active isoforms TrCB1.1 and TrCB1.4 with exopeptidase activity. The catalytically active enzymes and the inactive TrCB1.6 paralog formed complexes with chicken cystatin, thus supporting experimentally the hypothesis that inactive paralogs could potentially regulate the activity of the active forms or protect them from being inhibited by host inhibitors. The effect on cell viability and nitric oxide production by selected immune cells observed for TrCB1.1 was not confirmed for TrCB1.6. We show here that the active isoforms of TrCB1 have different affinities for peptide substrates thereby facilitating diversity in protein-derived nutrition for the parasite. The inactive paralogs are unexpectedly highly expressed and one of them retains the ability to bind cystatins, likely due to specific mutations in the occluding loop and the enzyme body. This suggests a role in sequestration of inhibitors and protection of active cysteine peptidases.
ER  -

DVOŘÁKOVÁ, Hana, Roman LEONTOVYČ, Tomáš MACHÁČEK, Anthony J. O´DONOGHUE, Ondrej ŠEDO, Zbyněk ZDRÁHAL, Charles S. CRAIK, Conor R. CAFFREY, Petr HORÁK and Libor MIKEŠ. Isoforms of Cathepsin B1 in Neurotropic Schistosomula of Trichobilharzia regenti Differ in Substrate Preferences and a Highly Expressed Catalytically Inactive Paralog Binds Cystatin. \textit{FRONTIERS IN CELLULAR AND INFECTION MICROBIOLOGY}. 2020, vol.~10, FEB, p.~''66'', 17 pp. ISSN~2235-2988. Available from: https://dx.doi.org/10.3389/fcimb.2020.00066.

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