Summary for peptidase C01.075: cruzipain

Summary Alignment Sequences Sequence features Distribution Structure Literature Substrates Inhibitors Pharma

 

Names
MEROPS Namecruzipain
Other namescongopain (Trypanosoma congolense), cruzain, evansain (Trypanosoma evansi), GP57/51
Name and HistoryThe name cruzipain was proposed (Cazzulo et al., 1990) to denote that the enzyme is the major cysteine proteinase of Trypanosoma cruzi, the causative agent of the American trypanosomiasis, Chagas disease, and that it belongs to the papain family. The name cruzain was suggested afterwards by Eakin et al., 1992. Antigen GP57/51 (Murta et al., 1990) is also synonymous. Cruzipain was discovered in cell-free extracts by Itow et al., 1977, and purified to homogeneity by Bontempi %et al, 1984; the cysteine proteinase purified by Rangel et al., 1981 is likely to be the same enzyme, despite some discrepancies, as is the 50 kDa proteinase described by Greig et al., 1990.
Domain architecture
MEROPS Classification
Classification Clan CA >> Subclan (none) >> Family C1 >> Subfamily A >> C01.075
Holotypecruzipain (Trypanosoma cruzi), Uniprot accession P25779 (peptidase unit: 122-337), MERNUM MER0001691
History Identifier created: Handbook of Proteolytic Enzymes (1998) Academic Press, London.
Activity
Catalytic typeCysteine
PeplistIncluded in the Peplist with identifier PL00071
NC-IUBMBSubclass 3.4 (Peptidases) >> Sub-subclass 3.4.22 (Cysteine endopeptidases) >> Peptidase 3.4.22.51
EnzymologyBRENDA database
Proteolytic eventsCutDB database (4 cleavages)
PreparationCruzipain (3-4 % of the total soluble protein of the cell in axenically cultured epimastigotes of T. cruzi, Tul 2 strain) can readily be purified by affinity chromatography of a cell-free extract obtained by freezing and thawing of the epimastigotes on ConA-Sepharose, followed by Mono Q ion-exchange chromatography (Parussini et al., 2003.
SpecificityWith small molecule substrates, it prefers Arg or Lys at the P1 position, and Arg, Leu, Phe or Val at P2 (Cazzulo et al., 1990). Several studies (Cazzulo et al., 1996; Serveau et al., 1996; Del Nery et al., 1997) indicated that specificity towards cruzipain was highest with Pro at P2'. Other data on subsite specificity are shown under the paragraph Substrates.
pH optimumCruzipain is an endopeptidase able to digest proteins such as casein, bovine albumin and denatured hemoglobin (optimal pH 3-5) (Bontempi et al., 1984), and synthetic, blocked, chromogenic and fluorogenic substrates, with optimal pH values 7–9 (Cazzulo et al., 1990).
StructureThe X-ray crystallographic structure of a recombinant truncated form of the enzyme (cruzain-delta c) in complex with several synthetic inhibitors has been determined, at resolutions ranging from 1.6 to 2.35 Å (McGrath et al., 1995; Gillmor et al., 1997; Brinen et al., 2000). The overall folding pattern and the arrangement of the active-site residues are similar to those in papain; however, Ser205 of papain is replaced by Glu, which is responsible for the binding of Arg at S2, giving cruzipain a cathepsin B-like specificity. Glu205 can adjust the position of its side chain, allowing the binding of positively charged or hydrophobic amino acid residues (Gillmor et al., 1997).
LocationVariable in different stages of the parasite"s biological cycle. In the epimastigote, cruzipain is mostly present in the reservosome, which contains protein that is digested during differentiation to metacyclic trypomastigotes (Souto-Padrón et al., 1990; Soares et al., 1992); inhibition of the enzyme blocks metacyclogenesis (Franke de Cazzulo et al., 1994). In addition, there are minor isoforms bound to the cell membrane, presumably through a GPI anchor (Parussini et al., 1998). The membrane localization of cruzipain is important in amastigotes (Souto-Padron et al.,1990, Tomas et al., 1997). Trypomastigotes are able to excrete cysteine proteinases, including cruzipain, into the medium (Yokoyama-Yasunaka et al., 1994; Aparicio et al., 2004).
PhysiologyNutrition of parasite by digestion of host proteins. Immune avoidance. Antigen.
Biological aspectsFunctions: 1) parasite nutrition; 2) invasion of mammalian cells by the parasite, which is essential for the development of Chagas disease. Cruzipain liberates Lys-bradykinin, directly by proteolysis of kininogen, or by activation of plasmatic pre-kallikrein (Del Nery et al., 1997); the kinin interacts with B(2) receptors eliciting an intracellular free calcium transient (Scharfstein et al., 2000). A second, bradykinin-independent increase in parasite invasion is elicited by secreted cruzipain, presumably by activating an unidentified protein in the parasite cell membrane (Aparicio et al., 2004); 3) probably provides a mechanism for the parasite to escape the immune response of the host, by digesting immunoglobulins at the 'hinge' region (Bontempi & Cazzulo, 1990); 4) it has also been proposed to participate in the differentiation steps of the parasite´s life cycle and at the last phase of autophagy (Alvarez et al., 2012).
RNA splicingWith only one exception described so far and that not related to peptidases, there are no introns in the genes of trypanosomatids.
KnockoutCruzipain is expressed from a number of genes, and no knock-out has been obtained so far.
Pharmaceutical relevancePotential drug target for Chagas disease.
Distinguishing featuresThe most important distinctive feature of cruzipain is the presence of the C-terminal domain, which, in contrast to the similar cysteine proteinases from Leishmania mexicana and Trypanosoma brucei, is retained in the natural mature form of the enzyme (Cazzulo et al., 1992).
Pathways KEGGChagas disease (American trypanosomiasis)
Contributing authorsJuan José Cazzulo, Instituto de Investigaciones Biotecnológicas "Dr. Rodolfo A. Ugalde", Universidad Nacional de San Martin (IIB-INTECH, UNSAM-CONICET), Campus Miguelete, Av. 25 de Mayo y Francia, 1650 San Martin, Buenos Aires, Argentina.
Other databases WIKIPEDIAhttp://en.wikipedia.org/wiki/Cruzipain
Cleavage site specificity Explanations of how to interpret the following cleavage site sequence logo and specificity matrix can be found here.
Cleavage patternK/K/L/krScissile bondA/ks/K/rq (based on 65 cleavages)
weblogo
Specificity matrix
 
Amino acid P4 P3 P2 P1 P1' P2' P3' P4'
Gly 0 1 0 5 1 0 2 1
Pro 0 4 1 0 0 4 6 3
Ala 3 2 2 2 30 1 3 2
Val 1 3 5 1 1 2 1 0
Leu 0 3 39 3 5 3 1 4
Ile 3 0 0 1 0 2 0 2
Met 0 0 0 4 2 0 0 0
Phe 0 1 9 4 7 0 0 0
Tyr 0 0 3 3 2 1 1 1
Trp 0 0 1 1 0 1 0 0
Ser 5 4 0 1 4 9 2 1
Thr 0 0 0 1 0 1 0 0
Cys 1 1 2 0 0 1 2 2
Asn 0 0 0 1 0 0 1 1
Gln 1 0 0 1 2 0 1 8
Asp 0 0 0 0 0 0 0 0
Glu 2 1 0 1 0 1 1 1
Lys 25 30 2 17 3 26 29 3
Arg 1 0 1 12 0 3 4 25
His 1 0 0 1 0 1 0 0
Specificity from combinatorial peptides
 
Organism comment P4 P3 P2 P1 P1' P2' P3' P4' optimal substrate fluorophore or acceptor-donor pair Reference
Trypanosoma cruzi recombinant H/K R F R - - - - HRFR ACC Choe et al., 2006
Trypanosoma cruzi recombinant - - - Amf - - - - xxxAmf+xxxx Abz-EDDnp Alves et al., 2001