Isolation, biochemical characterization, and molecular modeling of American lobster digestive cathepsin D1 Liliana Rojo a,1 , Rogerio Sotelo-Mundo b , Fernando García-Carreño a,1 , László Gráf c, ⁎ a Centro de Investigaciones Biológicas del Noroeste (CIBNOR), Mar Bermejo 195, Col. Playa Palo de Santa Rita, La Paz, B.C.S. 23096, Mexico b Centro de Investigación en Alimentación y Desarrollo (CIAD), Apdo. Postal 1735, Hermosillo, Sonora 83000, Mexico c Department of Biochemistry, Eötvös Loránd University, Budapest, Pázmány Péter st. 1/C, H-1117 Budapest, Hungary abstract article info Article history: Received 10 June 2010 Received in revised form 30 August 2010 Accepted 30 August 2010 Available online 15 September 2010 Keywords: Aspartic proteinase Cathepsin D Structure modeling Digestive An aspartic proteinase was isolated from American lobster gastric fluid. The purified cathepsin D runs as a single band on native-PAGE displaying proteolytic activity on a zymogram at pH 3.0, with an isoelectric point of 4.7. Appearance of the protein in SDS-PAGE, depended on the conditions of the gel electrophoresis. SDS treatment by itself was not able to fully unfold the protein. Thus, in SDS-PAGE the protein appeared to be heterogeneous. A few minute of boiling the sample in the presence of SDS was necessary to fully denature the protein that then run in the gel as a single band of ~50 kDa. The protein sequence of lobster cathepsin D1, as deduced from its mRNA sequence, lacks a ‘polyproline loop’ and β-hairpin, which are characteristic of some of its structural homologues. A comparison of amino acid sequences of digestive and non-digestive cathepsin D-like enzymes from invertebrates showed that most cathepsin D enzymes involved in food digestion, lack the polyproline loop, whereas all non-digestive cathepsin Ds, including the American lobster cathepsin D2 paralog, contain the polyproline loop. We propose that the absence or presence of this loop may be characteristic of digestive and non-digestive aspartic proteinases, respectively. © 2010 Elsevier Inc. All rights reserved. 1. Introduction Aspartic proteinases comprise a protein superfamily present across different taxa including bacteria (Hill and Phylip, 1997), yeast (Tang and Wong, 1987), plants (Runeberg-Roos et al., 1991), viruses (Wlodawer and Gustchina, 2000) and vertebrates (Fruton, 2002). Based on their characteristics and tissue/cellular locality, aspartic proteinases of vertebrates have been classified as cathepsin D, cathepsin E, chymosin, pepsin and renin (Barrett et al., 1998). Eukaryotic aspartic proteainases exhibit some key features, including the well conserved triad Asp-Thr-Gly around the two aspartic residues of the active site (D 32 and D 215 , from the human pepsin A numbering) (Fusek and Větvička, 2005). Although similarities surpass this characteristic, aspartic proteinases are also diverse in a number of ways, including the shape of their binding sites and cellular localization. These features point to their particular physiological functions (Davies, 1990) including extracellular protein hydrolysis at acid pH, digestion of intracellular proteins and activation of proenzymes (Benes et al., 2008) to name a few. After pepsin, cathepsin D is the second most studied aspartic proteinase (Benes et al., 2008). The physiological functions of cathepsin D that have been explored include intracellular protein hydrolysis (Metcalf and Fusek, 1993), activation of some proenzymes (Khan et al., 1999), regulation of programmed cell death (Gui et al., 2006) and digestion of food proteins in invertebrates (Blanco-Labra et al., 1996; Williamson et al., 2002; Padilha et al., 2009; Rojo et al., 2010). Cathepsin D has been known to function at acid pH (Takahashi and Tang, 1981). Its structure may contain high mannose oligosaccharides linked at the so-called N-sites to the polypeptide chain (Takahashi et al., 1983). Cathepsin D pre-pro-enzyme is synthesized in the rough endoplasmic reticulum. Some posttranslational modifications must occur before its conversion to a fully active form, first the removal of a signal sequence of ~20 amino acid occurs within the rough endoplasmatic reticulum (Richter et al., 1998) to form a zymogen. Then, a 44 amino acid propetide is split-off the zymogen forming a single-chain intermediate form that is subsequently further processed in the so-called processing region (Yonezawa et al., 1988) and then converted to a fully activated two- chain linked proteinase (Richo and Conner, 1994). In cathepsin D enzymes, amino acid sequences around the activation peptides and the position of cysteine residues are highly conserved (Fusek et al., 1992; Richo and Conner, 1994). Two loops surround the active site and they are probably involved in substrate binding. One of these, the polyproline loop contains three consecutive prolines, the other loop, the “Y 75 flap” (Tyr 75 from porcine pepsin numbering) is flexible and partly covers the active site (Metcalf and Fusek, 1993). Aspartic proteinases along with cysteine proteinases are the main contributors to the proteolytic activity found in the gastric fluid in American and European lobster (Laycock et al., 1989; Navarrete del Comparative Biochemistry and Physiology, Part B 157 (2010) 394–400 ⁎ Corresponding author. E-mail addresses: fgarcia@cibnor.mx (F. García-Carreño), graf@elte.hu (L. Gráf). 1 Tel.: +52 612 123 8484; fax: +52 612 125 3625. 1096-4959/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2010.08.009 Contents lists available at ScienceDirect Comparative Biochemistry and Physiology, Part B journal homepage: www.elsevier.com/locate/cbpb