Alguns aspectos geneticos dos enzimas lisosomicos Isolamento de um ADN complementar da catepsina D

Abstract

The present work deals with some molecular and functional aspects of lysosomes. Cathepsin D, an aspartic protease serves as a model to study the biosynthetic pathway of the lysosomal enzymes, because the proteolytic modifications of this lysosomal protease have been studied in detail. Cathepsin D characteristics and physiopathological aspects are reviewed as well as the general properties of aspartic proteases, with special relevance to their evolutionary aspects. This work relates the basic, molecular aspects to the broader framework of clinical medicine and clinical genetics. The work describes the general cellular pathways of the lysosomal enzymes and the biosynthetic pathway of cathepsin D and debates some hypotheses viewing a better unterstanding of cathepsin D turnover. Physiologic as well as pathophysiologic aspects of some lysosomal enzymes are also discussed. The experimental work describes the isolation of a cathepsin D cDNA clone. The cDNA clone for cathepsin D was obtained by screening a human fibroblast library in the pCD expression vector. This library was divided in sub-libraries based on the size of the cDNA inserts. Two mixed sequence aligonucleotides, III and IV, corresponding to aminoacids 25 to 34 and 198 to 205, respectively, of the known aminoacid sequence of porcine spleen were used to screen the above mentioned sub-libraries. A plasmid pCGS 586 (14kb-15kb) containing prochymosin cDNA was also used to screen the same sub-libraries. This bovine chymosin probe was chosen, because it showed 75% of identical residues when compared to the aminoacid sequence from the carboxy terminal end of cathepsin D. The cathepsin D cDNA was subcloned into Mp13 filamentous phage vector, and the nucleotide sequence was determined. The deduced protein sequence proved to be similar to the sequence of cathepsin D determined previously, a similar reflecting the high evolutionary conservation of the protein sequence among the different mammalian species. This clone demonstrated the low allelic polymorphism in the human cathepsin D gene, in comparison to the sequence that was, in the meantime, isolated from human kidney tissue. Therefore, this work reflects..Available from Fundacao para a Ciencia e a Tecnologia, Servico de Informacao e Documentacao, Av. D. Carlos I, 126, 1200 Lisboa / FCT - Fundação para o Ciência e a TecnologiaSIGLEPTPortuga