title: Analysis of Fasciola cathepsin L5 by S2 subsite substitutions and determination of the P1-P4 specificity reveals an unusual preference.
abstract: Fasciola parasites (liver flukes) express numerous cathepsin L proteases that are believed to be involved in important functions related to host invasion and parasite survival. These proteases are evolutionarily divided into clades that are proposed to reflect their substrate specificity, most noticeably through the S(2) subsite. Single amino acid substitutions to residues lining this site, including amino acid residue 69 (aa69; mature cathepsin L5 numbering) can have profound influences on subsite architecture and influence enzyme specificity. Variations at aa69 among known Fasciola cathepsin L proteases include leucine, tyrosine, tryptophan, phenylalanine and glycine. Other amino acids (cysteine, serine) might have been expected at this site due to codon usage as cathepsin L isoenzymes evolved, but C69 and S69 have not been observed. The introduction of L69C and L69S substitutions into FhCatL5 resulted in low overall activity indicating their expression provides no functional advantage, thus explaining the absence of such variants in Fasciola. An FhCatL5 L69F variant showed an increase in the ability to cleave substrates with P(2) proline, indicating F69 variants expressed by the fluke would likely have this ability. An FhCatL2 Y69L variant showed a decreased acceptance of P(2) proline, further highlighting the importance of Y69 for FhCatL2 P(2) proline acceptance. Finally, the P(1)-P(4) specificity of Fasciola cathepsin L5 was determined and, unexpectedly, aspartic acid was shown to be well accepted at P(2,) which is unique amongst Fasciola cathepsins examined to date.
authors.affiliation: School of Applied Sciences, RMIT University, Bundoora, Victoria 3083, Australia.
authors.forename: Luke J
authors.initials: LJ
authors.lastname: Norbury
authors.forename: Andrew
authors.initials: A
authors.lastname: Hung
authors.forename: Simone
authors.initials: S
authors.lastname: Beckham
authors.forename: Robert N
authors.initials: RN
authors.lastname: Pike
authors.forename: Terry W
authors.initials: TW
authors.lastname: Spithill
authors.forename: Charles S
authors.initials: CS
authors.lastname: Craik
authors.forename: Youngchool
authors.initials: Y
authors.lastname: Choe
authors.forename: John V
authors.initials: JV
authors.lastname: Fecondo
authors.forename: Peter M
authors.initials: PM
authors.lastname: Smooker
mesh_terms: D000818:Animals; D002403:Cathepsins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005210:Fasciola hepatica / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017433:Protein Structure, Secondary; D013379:Substrate Specificity
chemical_list: D002403:Cathepsins; C426647:cathepsin L5