Active-site mutagenesis of a Lys49-phospholipase A2: biological and membrane-disrupting activities in the absence of catalysis.
Bothropstoxin-I (BthTx-I) is a myotoxic phospholipase A(2) variant present in the venom of Bothrops jararacussu, in which the Asp(49) residue is replaced with a lysine, which damages artificial membranes by a Ca(2+)-independent mechanism. Wild-type BthTx-I and the mutants Lys(49)-->Asp, His(48)-->Gln and Lys(122)-->Ala were expressed in Escherichia coli BL21(DE3) cells, and the hydrolytic, myotoxic and membrane-damaging activities of the recombinant proteins were compared with native BthTx-I purified from whole venom. The Ca(2+)-independent membrane-damaging and myotoxic activities of the native and wild-type recombinant BthTx-I, His(48)Gln and Lys(49)Asp mutants were similar; however, the Lys(122)Ala mutant demonstrated reduced levels of both activities. Although a low hydrolytic activity against a mixed phospholipid substrate was observed with native BthTx-I, no substrate hydrolysis was detected with the wild-type recombinant enzyme or any of the mutants. In the case of the Lys(49)Asp mutant, this demonstrates that the absence of catalytic activity in Lys(49)-PLA(2) is not a consequence of the single Asp(49)-->Lys replacement. Furthermore, these results provide unambiguous evidence that the Ca(2+)-independent membrane-damaging and myotoxic activities are maintained in the absence of hydrolysis. The evidence favours a model for a hydrolysis-independent, membrane-damaging mechanism involving an interaction of the C-terminal region of BthTx-I with the target membrane.
Ward
Richard J
RJ
Departamento de Química, FFCLRP-USP, Universidade de São Paulo, Brazil. rjward@fmrp.usp.br
Chioato
Lucimara
L
de Oliveira
Arthur H C
AH
Ruller
Roberto
R
Sá
Juliana M
JM
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D002462:Cell Membrane / Q000187:drug effects; D002942:Circular Dichroism; D003435:Crotalid Venoms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D017931:DNA Primers; D008239:Lysine / Q000737:chemistry*; D008297:Male; D051379:Mice; D008958:Models, Molecular; D018482:Muscle, Skeletal / Q000187:drug effects; D016297:Mutagenesis, Site-Directed; D010741:Phospholipases A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D013056:Spectrophotometry, Ultraviolet
D003435:Crotalid Venoms; D017931:DNA Primers; D011994:Recombinant Proteins; C078097:bothropstoxin; D010741:Phospholipases A; D008239:Lysine