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LOPAP, proteína isolada da toxina de lagartas Lonomia obliqua, possui ação ativadora de protrombina, efeito pró-coagulante e ação citoprotetora em células do endotélio humano, em cultura. Tem cadeia única com 181 resíduos de aminoácidos e 21 kDa. Sua estrutura terciária é formada por oito folhas-b fechadas em uma extremidade, mantidas juntas por pontes de hidrogênio, em formato de barril. Está classificada como pertencente ao grupo das Lipocalinas (proteínas de transporte). Neste trabalho estudou-se o LOPAP, que foi produzido recombinante em cultivo de Pichia pastoris em biorreator e purificado. Avaliou-se sua estabilidade quanto às atividades enzimática e citoprotetora, e sua estrutura secundária. Não foi detectada ativação de protrombina para o r-LOPAP obtido, mas foi observada ação citoprotetora. Considerando estes resultados e a análise de sua estrutura secundária por dicroísmo circular, concluiu-se que a proteína foi expressa com tamanho e sequência corretos, mas sem uma estrutura terciária correta, o que é determinante para a atividade enzimática. / LOPAP, a protein isolated from the toxin of Lonomia obliqua caterpillars, has prothrombin activation action, procoagulant effect and cytoprotection action in human endothelium cells culture. It has only chain with 181 amino acid residues and 21 kDa of size. Its tertiary structure is made by eight b-sheets closed at one end, hold together by hydrogen bonds, barrel-shaped. It is classified as belonging to the Lipocalin group (proteins of transport). This work studied the LOPAP, which was produced recombinant in Pichia pastoris culture in bioreactor, was purified, and it was evaluated its stability related to enzymatic and cytoprotection activities, and its secondary structure. It was not detected prothrombin activation for the r-LOPAP obtained, but it was observed a cytoprotective effect. Regarding these results and the analysis of its secondary structure, by circular dichroism, it was concluded that the protein was expressed with correct size and sequence, but without a correct tertiary structure, which is determinant for the enzymatic activity.

Humans have two plasminogen activators (PAs), tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), which generate plasmin to breakdown fibrin and other barriers to cell migration. Both PAs are used as pharmaceuticals but their efficacies are limited by their rapid clearance from the circulation, predominantly by parenchymal cells of the liver. At the commencement of the work presented here, the hepatic receptors responsible for mediating the catabolism of the PAs were little understood. tPA degradation by hepatic cell lines was known to depend on the formation of binary complexes with the major PA inhibitor, plasminogen activator inhibitor type-1 (PAI-1). Initial studies presented here established that uPA was catabolised in a fashion similar to tPA by the hepatoma cell line, HepG2. Other laboratories around this time found that the major receptor mediating the binding and endocytosis of the PAs is Low Density Lipoprotein Receptor-related Protein (LRP1). LRP1 is a giant 600 kDa protein that binds a range of structurally and functionally diverse ligands including, activated α2 macroglobulin, apolipoproteins, β amyloid precursor protein, and a number of serpin-enzymes complexes, including PA??PAI-1 complexes. Further studies for the work presented here centred on this receptor. By using radiolabelled binding assays, ligand blots, and Western blots on cultured cells, the major findings are that: (1) basal LRP1 expression on HepG2 is low compared to a clone termed, HepG2a16, but appears to increase in long term culture; (2) a soluble form of LRP1, which retains ligand-binding capacity, is present in human circulation; (3) soluble LRP1 is also present in cerebral spinal fluid where its role in neurological disorders such as Alzheimer??s disease is a developing area of interest; and (4) the release of LRP1 is a mechanism conserved in evolution, possibly as distantly as molluscs. The discovery, identification, and characterisation of soluble LRP1 introduces this protein in the human circulation, and presents a possible further level of regulation for its associated receptor system.