Bm-CPI-2, a cystatin from Brugia malayi nematode parasites, differs from Caenorhabditis elegans cystatins in a specific site mediating inhibition of the antigen-processing enzyme AEP.
The filarial parasite Brugia malayi survives for many years in the human lymphatic system. One immune evasion mechanism employed by Brugia is thought to be the release of cysteine protease inhibitors (cystatins), and we have previously shown that the recombinant cystatin Bm-CPI-2 interferes with protease-dependent antigen processing in the MHC class II antigen presentation pathway. Analogy with vertebrate cystatins suggested that Bm-CPI-2 is bi-functional, with one face of the protein blocking papain-like proteases, and the other able to inhibit legumains such as asparaginyl endopeptidase (AEP). Site-directed mutagenesis was carried out on Bm-CPI-2 at Asn-77, the residue on which AEP inhibition is dependent in vertebrate homologues. Two mutations at this site (to Asp and Lys) showed 10-fold diminished and ablated activity respectively, in assays of AEP inhibition, while blocking of papain-like proteases was reduced by only a small degree. Comparison of the B. malayi cystatins with two homologues encoded by the free-living model organism, Caenorhabditis elegans, suggested that while the papain site may be intact, the AEP site would not be functional. This supposition was tested with recombinant C. elegans proteins, Ce-CPI-1 (K08B4.6) and Ce-CPI-2 (R01B10.1), both of which block cathepsins and neither of which possess the ability to block AEP. Thus, Brugia CPI-2 may have convergently evolved to inhibit an enzyme important only in the mammalian environment.
Murray
Janice
J
Institute of Immunology and Infection Research, Ashworth Laboratories, University of Edinburgh, West Mains Road, Scotland EH9 3JT, UK.
Manoury
Bénédicte
B
Balic
Adam
A
Watts
Colin
C
Maizels
Rick M
RM
D000595:Amino Acid Sequence; D000818:Animals; D017951:Antigen Presentation / Q000187:drug effects*; D017178:Brugia malayi / Q000201:enzymology* / Q000235:genetics; D017173:Caenorhabditis elegans / Q000201:enzymology* / Q000235:genetics; D015891:Cystatins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000187:drug effects*; D019143:Evolution, Molecular; D000949:Histocompatibility Antigens Class II; D006801:Humans; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D000099240:Asparaginyl Endopeptidase
D015891:Cystatins; D003546:Cysteine Endopeptidases; D000949:Histocompatibility Antigens Class II; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D000099240:Asparaginyl Endopeptidase