A single amino acid substitution affects substrate specificity in cysteine proteinases from Fasciola hepatica.
The trematode Fasciola hepatica secretes a number of cathepsin L-like proteases that are proposed to be involved in feeding, migration, and immune evasion by the parasite. To date, six full cDNA sequences encoding cathepsin L preproproteins have been identified. Previous studies have demonstrated that one of these cathepsins (L2) is unusual in that it is able to cleave substrates with a proline in the P2 position, translating into an unusual ability (for a cysteine proteinase) to clot fibrinogen. In this study, we report the sequence of a novel cathepsin (L5) and compare the substrate specificity of a recombinant enzyme with that of recombinant cathepsin L2. Despite sharing 80% sequence identity with cathepsin L2, cathepsin L5 does not exhibit substantial catalytic activity against substrates containing proline in the P2 position. Molecular modeling studies suggested that a single amino acid change (L69Y) in the mature proteinases may account for the difference in specificity at the S2 subsite. Recombinant cathepsin L5/L69Y was expressed in yeast and a substantial increase in the ability of this variant to accommodate substrates with a proline residue in the P2 position was observed. Thus, we have identified a single amino acid substitution that can substantially influence the architecture of the S2 subsite of F. hepatica cathepsin L proteases.
Smooker
P M
PM
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.
Whisstock
J C
JC
Irving
J A
JA
Siyaguna
S
S
Spithill
T W
TW
Pike
R N
RN
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002403:Cathepsins / Q000235:genetics* / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000235:genetics* / Q000378:metabolism; D005210:Fasciola hepatica / Q000201:enzymology*; D015801:Helminth Proteins / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity / Q000235:genetics*
D015801:Helminth Proteins; D011994:Recombinant Proteins; D002403:Cathepsins; D003546:Cysteine Endopeptidases; C426647:cathepsin L5