Lopap, a prothrombin activator from Lonomia obliqua belonging to the lipocalin family: recombinant production, biochemical characterization and structure-function insights.
Using a cDNA library made from Lonomia obliqua caterpillar bristles, we identified a transcript with a 603 bp open reading frame. The deduced protein corresponds to Lopap, a prothrombin activator previously isolated by our group from the bristles of this species. The mature protein is composed by 185 amino acids and shares similarity with members of the lipocalin family. The cDNA encoding the mature form was amplified by PCR, subcloned into pAE vector and used to transform Escherichia coli BL21(DE3) cells. As for the native Lopap, the recombinant fusion protein shows enzymatic activity, promotes prothrombin hydrolysis, generates fragments similar to prethrombin-2 and fragment 1.2 as intermediates, and generates thrombin as the final product. In addition, structural bioinformatics studies indicated several interesting molecular features, including the residues that could be responsible for Lopap's serine protease-like activity and the role of calcium binding in this context. Such catalytic activity has never been found in other members of the lipocalin family. This is the first report describing the recombinant production and biochemical characterization of a Lonomia obliqua lipocalin, as well as the structural features that could be responsible for its serine protease-like catalytic activity.
Reis
Cleyson Valença
CV
Laboratório de Bioquímica e Biofísica, Instituto Butantan, 1500 Av. Vital Brazil, CEP 05503-900, São Paulo, SP, Brazil.
Andrade
Sonia Aparecida
SA
Ramos
Oscar Henrique Pereira
OH
Ramos
Celso Raul Romero
CR
Ho
Paulo Lee
PL
Batista
Isabel de Fátima Correia
IF
Chudzinski-Tavassi
Ana Marisa
AM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002942:Circular Dichroism; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005170:Factor X / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D019476:Insect Proteins / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D007782:Lactoglobulins / Q000737:chemistry; D007915:Lepidoptera; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011516:Prothrombin / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship
D018076:DNA, Complementary; D019476:Insect Proteins; D007782:Lactoglobulins; D011994:Recombinant Proteins; D011516:Prothrombin; D005170:Factor X; C119835:Lonomia obliqua prothrombin activator protease; D012697:Serine Endopeptidases