The functional expression and characterisation of a cysteine peptidase from the invasive stage of the neuropathogenic schistosome Trichobilharzia regenti

Abstract

A transcriptional product of a gene encoding cathepsin B-like peptidase in the bird schistosome Trichobilharzia regenti was identified and cloned. The enzyme was named TrCB2 due to its 77% sequence similarity to cathepsin B2 from the important human parasite Schistosoma mansoni. The zymogen was expressed in the methylotropic yeast Pichia pastoris; procathepsin B2 underwent self-processing in yeast media. The peptidolytic activity of the recombinant enzyme was characterised using synthetic fluorogenic peptide substrates at optimal pH 6.0. Functional studies using different specific inhibitors proved the typical cathepsin B-like nature of the enzyme. The S(2) subsite specificity profile of recombinant TrCB2 was obtained. Using monospecific antibodies against the recombinant enzyme, the presence of cathepsin B2 was confirmed in extracts from cercariae (infective stage) and schistosomula (early post-cercarial stage) of T. regenti on Western blots. Also, cross-reactivity was observed between T. regenti and S. mansoni cathepsins B2 in extracts of cercariae, schistosomula or adults. In T. regenti, the antisera localised the enzyme to post-acetabular penetration glands of cercariae implying an important role in the penetration of host skin. The ability of recombinant TrCB2 to degrade skin, serum and nervous tissue proteins was evident. Elastinolytic activity suggests that the enzyme might functionally substitute the histolytic role of the serine class elastase known from S. mansoni and Schistosoma haematobium but not found in Schistosoma japonicum or in bird schistosomes.

Fig. 1

Multiple alignment of cathepsin B2 from Trichobilharzia regenti (TrCB2; GenBank Accession No. EF682129) with cathepsin B2 from Schistosoma japonicum (SjCB; GenBank Accession No. AY226984) and Schistosoma mansoni (SmCB2; GenBank Accession No. AJ312106). Above the sequences, the dotted line refers to the signal peptide, grey line to the pro-peptide and black line to the predicted mature enzyme. Below the sequences, identities across all three sequences are indicated by asterisks, one or more conservative substitutions are indicated by colons, non-conservative changes are represented by points. The conserved His, Cys and Asn residues forming the catalytic active site are shaded in black. Space symbol indicates the possible N-glycosylation site and arrow depicts the cleavage site between the pro-domain and mature peptidase as experimentally determined by N-terminal Edman degradation of expressed recombinant TrCB2. The ‘occluding loop’ unique to cathepsin B enzymes is underlined. The slightly modified ‘haemoglobinase motif’, ascribed to those cathepsins B which are able to cleave haemoglobin, is indicated in bold face.

Fig. 2

Fluorogenic peptide substrate hydrolysis and inhibition studies with de- and glycosylated forms of recombinant TrCB2 at pH optimum 6.0. (A) Peptidolytic activity compared with a spectrum of aminomethylcoumarine-labelled substrates. (B) Effect of cysteine and serine peptidase differentiating inhibitors on peptidolytic activity of TrCB2 with the cathepsin B-specific substrate RR. Composition of substrates is presented in single-letter aminoacid code. Values are means of three independent triplicate assays ± SD. RFU, relative fluorescent unit.

Fig. 3

Detection of purified recombinant TrCB2 by SDS–PAGE, ligand and Western blotting. (A) Labelling of TrCB2 by the cysteine peptidase-specific probe DCG-04 on blot. Lane 1, DCG-04 binding to the active site of the enzyme; lane 2, binding of DCG-04 inhibited by cysteine peptidase inhibitor E-64; lane 3, weak reaction of DCG-04 with heat-inactivated enzyme. (B) Shift in the molecular size in SDS–PAGE of yeast-produced TrCB2 after deglycosylation with endoglycosidase F1. Lane 4, non-treated enzyme; lane 5, endoglycosidase F1 treated enzyme; lane 6, deglycosylation performed in the presence of E-64. (C) Reaction of mouse and rabbit sera raised against TrCB2 and SmCB2, respectively, on blots of recombinant glycosylated TrCB2. Lanes 7 and 8, controls with buffer instead of primary antibodies; lanes 9 and 10, pre-immune mouse and rabbit sera; lane 11, serum against glycosylated TrCB2; lane 12, serum against deglycosylated TrCB2; lane 13, serum against SmCB2. Note the strong reaction of DCG-04 and immune sera with the major 32 kDa glycosylated band of yeast-produced TrCB2; reaction also occurs with a minor 30 kDa band of supposedly non-glycosylated TrCB2 produced by yeast, which is not visible in Coomassie stained gel (lane 4). TrCB2 and SmCB2, cathepsins B2 of Trichobilharzia regenti and Schistosoma mansoni, respectively.

Fig. 4

Preference profiles of recombinant TrCB2 using a complete diverse positional scanning synthetic combinatorial peptide substrate library. S₂ specificities of the enzyme to corresponding P1–P4 peptide amino acid positions were determined with a library in which randomised positions were incorporated by addition of the isokinetic mixture of 20 amino acids. The amino acids are designated by the single-letter code on the x-axis (n, norleucine). The y-axis is a 100% scale of picomolar fluorophore produced per second.

Fig. 5

Reaction of mouse antiserum against TrCB2 and rabbit antiserum against SmCB2 with extracts from various stages of Trichobilharzia regenti and Schistosoma mansoni life cycles. (A) Cercarial extract of T. regenti, (B) cercarial extract of S. mansoni, (C) schistosomular extract of T. regenti and (D) extract from adult S. mansoni were resolved by reducing SDS–PAGE, electroblotted to polyvinylidene difluoride membrane and incubated with serum from a mouse immunised with TrCB2 (lanes 1), a rabbit immunised with SmCB2 (lanes 2), a non-immunised mouse (lanes 3) and from a non-immunised rabbit (lanes 4). Arrowheads show presumptive cathepsin B2 bands.

Fig. 6

Immunolocalisation of cathepsin B2 in longitudinal and transverse sections of cercariae of Trichobilharziaregenti by indirect immuno-fluorescence microscopy. (A) Sections incubated with mouse anti-TrCB2 and then with AlexaFluor-labelled anti-mouse IgG. Positive reaction is apparent in post-acetabular penetration glands and their ducts. (B) Section of T. regenti cercariae was stained with H & E solution. Post-acetabular glands and their ducts appear in black. Arrowheads point to the circumacetabular glands. (C) Control with pre-immune mouse serum; no reaction is visible.

Fig. 7

Digestion of selected natural substrates by recombinant cathepsin B2 from Trichobilharzia regenti (TrCB2). The substrates at a final concentration of 0.2 mg/ml were incubated in the presence (+) or absence (−) of recombinant TrCB2 (1 μg) in 50 μl of citrate–phosphate buffer pH 6 containing 2 mM DTT at 37 °C overnight. Control reactions (−) contained CPB instead of the enzyme. Aliquots (20 μl) of the resulting hydrolysates were separated by electrophoresis and stained with Coomassie brilliant blue. Substrates used: Gel (A) haemoglobin, lanes 1, from duck; lanes 2, bovine; lanes 3, turkey; Gel (B) lanes 4, collagen type IV; lanes 5, keratin; lanes 6, fibrinogen type III; lanes 7, myelin basic protein. Arrowheads indicate bands corresponding to recombinant TrCB2. Note that all substrates were efficiently degraded, except for haemoglobins hydrolysed only at a negligible rate.

Fig. 8

Digestion of elastin by recombinant cathepsin B2 from Trichobilharzia regenti (TrCB2). Insoluble (A and B) or soluble (C) elastin in citrate-phosphate buffer pH 6 was incubated with recombinant TrCB2. (A) The reaction was stopped by adition of trichloracetic acid, centrifuged and an aliquot combined with a 0.2 M sodium borate buffer, pH 8.5 and fluorescamine solution. The fluorescence of labelled peptides was measured at λ_ex = 390 nm and λ_em = 480 nm. Graph shows the difference in fluorescence of sample containing both substrate and enzyme compared with controls with either the enzyme or substrate alone, indicating the presence of new peptides originating from elastin digested by TrCB2. Values are means of triplicate assays ± SD. E, elastin; RFU, relative fluorescence unit. (B) The samples were concentrated and applied directly on nano-reverse phase-HPLC-MALDI-TOF-TOF. Bolded amino acids show partial sequences deciphered by mass spectrometry analysis as unique for sample containing both elastin and TrCB2. (C) SDS–PAGE proof of soluble elastin degradation by the 32 kDa TrCB2 (lane 1); no hydrolysis was observed in the presence of E-64 inhibitor of cysteine peptidases (lane 2); soluble elastin band (lane 3) is indicated by an arrowhead.