Cathepsin L is a member of the papain superfamily of lysosomal cysteine proteases and is one of the most powerful endopeptidases. Its usual function is regulating cellular protein turnover in lysosome (Kirschke et al., 1995; Kazunobu et al., 2004; Kramer et al., 2007). It plays an important role with cathepsin B and H in the degradation of both endogenous and exogenous proteins. Cathepsin L, initially translated as preprocathepsin L, is then transferred through the Golgi as procathepsin L and stored in lysosomes as mature cathepsin L. (Chauhan et al., 1993). Over expression of procathepsin L in human melanoma cells increases their tumorigenicity and switches their phenotype from non-metastatic to highly metastatic (Nathalie et al., 2004). Therefore the enforced expression and secretion of procathepsin L by human melanoma cells arms them with the ability to inactivate complement-mediated cell lyses and contributes to tumor growth and metastasis (Frader et al., 1998). Cathepsin L is found to be upregulated in rat kidney during aging (Debata et al., 2007). Cathepsin-L influences the expression of extracellular matrix in lymphoid organs and plays a role in the regulation of thymic output and of peripheral T cell number (Lombardi et al., 2005). It was reported that in human SARS-CoV or SARS-CoV spike protein- pseudotyped retroviruses utilize the enzymatic activity of endosomal cathepsin L protease for viral entry (Huang et al., 2006; Li et al., 2006). Cathepsin L is a lysosomal cysteine protease that digests proteins of both intracellular and extracellular origin. It is translated as a precursor protein pre-procathepsin L, transferred through the Golgi apparatus as procathepsin L and then stored in lysosomes as mature cathepsin L (Ishidoh et al., 1998). It plays a diverse role in different organs and tissues such as maintenance of heart structure and function (Stypmann et al., 2002), epidermal differentiation, hair follicle morphogenesis and cycling (Benavides et al., 2002), development of type 1 diabetes in NOD mouse (Maehr et al., 2005), tumor metastasis (La et al., 1998), thyroid function (Friedrichs et al., 2003), a modifier of extra cellular matrices in precomovulatory follicles (Salustri et al., 1999), degradation of basement membrane in kidney (Baricos et al., 1998), podocyte migration in nephrotic syndrome (Reiser et al., 2004), regulation of thymic output and peripheral T cell number (Lombardi et al., 2005) and generation of MHC class I I-bound peptide ligands presented by cortical thymic epithelial cells (Honey et al., 2002). Our previous study has documented that the expression of cathepsin L gene is significantly up-regulated in rat kidney during aging (Debata et al., 2007). A similar result was also demonstrated at protein level (Kim et al., 2004). The upregulation of cathepsin L is also found in skeletal muscle wasting in septic muscle (Deval et al., 2001) and in scrapie-infected Neuro2a cells (Zhang et al., 2003).

In this communication, an effort was made to generate a three-dimensional (3D) model of cathepsin L protein based on the available template crystal structure of procathepsin L from protein data bank (PDB code: 1CS8) (Berman et al., 2000). The structural information of cathepsin L could prove useful to further characterizing the protein.

BLAST (Altschul et al., 1990) search was performed against Brookhaven Protein Data Bank (PDB) with the default parameters to find suitable templates for homology modeling. Based on the maximum identity with high score and lower e-value in the BLAST search, 1CS8 (PDB code) is used as the structural template for modeling the rat cathepsin L protein. The sequence – structure alignment used for model building shown in Figure 1. The alignment is characterized by some insertions and deletions in the loop regions. Since the first 17 residues from the N-terminal end did not have corresponding equivalent regions in 1CS8, the modeling was carried out from the 18th to the 317th residue, followed by a rigorous refinement of the model by means of EM and the final stable structure of the rat cathepsin L obtained is shown in Figure 2. The model has 89% of the residues in the most favored regions of the Ramachandran Map Figure 3 with a PROCHECK G-score value of 0.03 and a satisfactory VERIFY-3D profile. The predicted 3-D model of rat cathepsin L protein will be very useful while studying the real structure of the protein.

Validation of the model was carried out after the refinement process using Ramachandran Map calculations computed with the PROCHECK program. The ψ and φ distributions of the Ramachandran Map of non-glycine, non-proline residues are summarized in Figure 3 and Table 1. The model has 89% of the residues in the most favored regions of the Ramachandran Map with a PROCHECK G-score value of 0.03 and a satisfactory VERIFY3D profile.

The structural superimposition of C trace of template and rat cathepsin L is shown in Figure 4. The weighted root mean square deviation of C trace between the template and final refined model was 0.43 Å which suggest that the model is reliable.

The amino acid sequences of template and final structure are generated using JOY server (protein sequencestructure representation and analysis (Mizuguchi et al., 1998), were aligned using CLUSTAL W. Given their PDB files, secondary structures were also analyzed and compared by the JOY program. The secondary structures of template and final model of rat cathepsin L are highly conserved which showed that final model is highly reliable as shown in Figure 5.

In this report, a molecular model of rat cathepsin L protein has been constructed through homology modeling which could be used for further characterization.