Lopap, a prothrombin activator from Lonomia obliqua belonging to the lipocalin family: recombinant production, biochemical characterization and structure-function insights

• Published in: Biochemical journal 2006-09, Vol.398 (2), p.295-302
• Main Authors: Reis, Cleyson Valença, Andrade, Sonia Aparecida, Ramos, Oscar Henrique Pereira, Ramos, Celso Raul Romero, Ho, Paulo Lee, Batista, Isabel de Fátima Correia, Chudzinski-Tavassi, Ana Marisa
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Circular Dichroism; DNA, Complementary - genetics; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Factor X - metabolism; Humans; Hydrolysis; Insect Proteins - chemistry; Insect Proteins - classification; Insect Proteins - genetics; Insect Proteins - metabolism; Lactoglobulins - chemistry; Lepidoptera; Lonomia obliqua; Models, Molecular; Molecular Sequence Data; Protein Conformation; Prothrombin - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Sequence Alignment; Serine Endopeptidases - chemistry; Serine Endopeptidases - classification; Serine Endopeptidases - genetics; Serine Endopeptidases - metabolism; Structure-Activity Relationship
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/BJ20060325

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.