The 3D structure and function of digestive cathepsin L-like proteinases of Tenebrio molitor larval midgut |
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| Authors: | Beton Daniela Guzzo Cristiane R Ribeiro Alberto F Farah Chuck S Terra Walter R |
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| Institution: | 1. Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, C. P. 26077, 05513-970 São Paulo, Brazil;2. Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, C. P. 11461, 05422-970 São Paulo, Brazil;1. Novartis Institute for Biomedical Research, Novartis Campus, CH-4002 Basel, Switzerland;2. Novartis Institute for Biomedical Research, 6201 South Freeway, Fort Worth, TX 76134-2099, United States;3. Novartis Institute for Biomedical Research, 4560 Horton Street, Emeryville, CA 94608-2916, United States;1. Department of Nephrology, Radboud University Medical Center, Nijmegen, the Netherlands;2. Department of Pathology, Radboud University Medical Center, Nijmegen, the Netherlands;3. Department of Matrix Biochemistry, Radboud University Medical Center, Nijmegen, the Netherlands;4. Department of Pharmacology and Toxicology, Radboud University Medical Center, Nijmegen, the Netherlands;5. Department of Nephrology, Einthoven Laboratory for Vascular Medicine, Leiden University Medical Center, Leiden, the Netherlands;6. Institute of Molecular Medicine and Cell Research and BIOSS Centre for Biological Signaling Studies, Albert-Ludwigs-University, Freiburg, Germany;1. Instituto de Patobiología, Centro de Investigaciones en Ciencias Veterinarias y Agronómicas (CICVyA), INTA-Castelar, Los Reseros y Nicolas Repetto s/n, 1686, Hurlingham, Argentina;2. Laboratório de Diagnóstico Biomolecular, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa and Instituto de Biologia Experimental e Tecnologica, Av. da República, Oeiras, 2780-157, Portugal;3. Cátedra de Enfermedades Infecciosas, Facultad de Ciencias Veterinarias, Universidad de Buenos Aires, Chorroarín 280, Buenos Aires, C1427CWO, Argentina;4. CONICET, C1033AAJ, Ciudad Autónoma de Buenos Aires, Argentina;5. University of Glasgow, College of Medical, Veterinary and Life Sciences, Garscube Campus, Bearsden Road, Glasgow, G61 1QH, UK;1. Department of Biochemistry, Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), Rua 3 de Maio 100, 04044-020, São Paulo, SP, Brazil;2. Instituto de Patobiologia Veterinaria, Centro de Investigaciones en Ciencias Veterinarias y Agronómicas (CICVyA), INTA-Castelar, Los Reseros y Nicolas Repetto s/n, Hurlingham, 1686, Argentina;3. National Council of Scientific and Technological Research (CONICET), Ciudad Autónoma de Buenos Aires C1033AAj, Argentina;4. Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular (INCT-EM), RJ, Brazil |
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| Abstract: | Cathepsin L-like proteinases (CAL) are major digestive proteinases in the beetle Tenebrio molitor. Procathepsin Ls 2 (pCAL2) and 3 (pCAL3) were expressed as recombinant proteins in Escherichia coli, purified and activated under acidic conditions. Immunoblot analyses of different T. molitor larval tissues demonstrated that a polyclonal antibody to pCAL3 recognized pCAL3 and cathepsin L 3 (CAL3) only in the anterior two-thirds of midgut tissue and midgut luminal contents of T. molitor larvae. Furthermore, immunocytolocalization data indicated that pCAL3 occurs in secretory vesicles and microvilli in anterior midgut. Therefore CAL3, like cathepsin L 2 (CAL2), is a digestive enzyme secreted by T. molitor anterior midgut. CAL3 hydrolyses Z-FR-MCA and Z-RR-MCA (typical cathepsin substrates), whereas CAL2 hydrolyses only Z-FR-MCA. Active site mutants (pCAL2C25S and pCAL3C26S) were constructed by replacing the catalytic cysteine with serine to prevent autocatalytic processing. Recombinant pCAL2 and pCAL3 mutants (pCAL2C25S and pCAL3C26S) were prepared, crystallized and their 3D structures determined at 1.85 and 2.1 Å, respectively. While the overall structure of these enzymes is similar to other members of the papain superfamily, structural differences in the S2 subsite explain their substrate specificities. The data also supported models for CAL trafficking to lysosomes and to secretory vesicles to be discharged into midgut contents. |
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