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CYSTEINE PEPTIDASES OF MAMMALS: THEIR BIOLOGICAL ROLES AND POTENTIAL EFFECTS IN THE ORAL CAVITY AND OTHER TISSUES IN HEALTH AND DISEASE

Medical College of Georgia, School of Dentistry, Department of Oral Biology and Maxillofacial Pathology, 1120 15th Street, Augusta, GA 30912; ddickins{at}mail.mcg.edu

View larger version (147K): [in a new window]   Figure 1. Protein sequence alignments of mature regions of known human cathepsins, other rodent cathepsins and related proteins, and plant papain and aleurain. Although only human and selected sequences are shown, the original dataset used for generating the alignments contained 43 vertebrate and two plant sequences, identified by a BLAST search of the GenBank non-redundant database (Altschul et al., 1990). The sequence abbreviations and GenBank entries for the sequences shown are: hDPP I, human dipeptidyl peptidase I, P53634; hB, human cathepsin B, NP_001899; hV, human cathepsin V, AAC23593; hL, human cathepsin L, NP_001903; rtestin, rat testin, P15242; mP, mouse cathepsin P, NP_036137; rCLRP, rat cathepsin L-related protein, I58002; rQ, rat cathepsin Q, AAF01247; mM, mouse cathepsin M, AAF68224; hK, human cathepsin K, P43235; hS, human cathepsin S, P25774; hH, human cathepsin H, P09668; Aleurain, plant, P05167; Papain, plant, P00784; hF, human cathepsin F, NP_003784; hX, human cathepsin X, NP_001327; hLym, human lymphopain, P56202; and hO, human cathepsin O, NP_00135. Propeptide cleavage sites were obtained from the GenBank entries, or from preliminary alignments. Alignments of the predicted mature proteins were generated based on the default settings of ClustalX (Thompson et al., 1997). Introduced gaps are shown as "-". The alignment is in good agreement with that of other published ones, including those based on structure. To derive consensus sequences, we processed alignment files using the public domain software BOXSHADE (written by K. Hofmann and M. Baron, www.ch.embnet.org/software/BOX_form.html). Predominant identical residues (> 50%) at a position are shown with a black background, predominant similar residues on a grey background. The majority consensus residue is shown under each alignment. An uppercase letter shows a residue conserved in all aligned sequences. An * under the consensus sequence denotes the active-site cysteine and histidine residues conserved in all functional peptidases.

View larger version (24K): [in a new window]   Figure 2. Phylogenetic tree of vertebrate and select plant CPs. The full alignment of CPs described in the legend to Fig. 1 was used, except that bovine cathepsin L was excluded, due to the incomplete C-terminal sequence. The PAUP 4b4a software package (Swofford, 2000) was used to search for trees by means of the distance optimality criterion and default parameters, and starting trees obtained by neighbor-joining. The tree shown is an unrooted 50% majority rule consensus tree obtained by the bootstrap method (500 replicates) with heuristic search. Values shown adjacent to branches are percentage support for a branch. The large arrow indicates the position of the presumptive root of the tree: CPs on the group of branches to the right of this point comprise Branch A, those to the left Branch B (see text for explanation of Branches A and B). The abbreviations used for species are: bo, bovine; ch, chicken; h, human; m, mouse; pig, pig; r, rat; ra, rabbit; rh, rhesus; sh, sheep; and zf, zebrafish.

View larger version (67K): [in a new window]   Figure 3. Proregion alignments of known human cathepsins and selected rodent and plant proteins. Proregion alignments (excluding signal peptides and the N-terminal extension of cathepsin F) are shown in Panel A (Branch B proteins), Panel B (Branch A cathepsins B and X) and Panel C (DPP I). The same dataset described in Fig. 1 was used to generate the alignment, with the inclusion of rat CTLA-2ß (CTLA-2b, GenBank P12400). Signal peptide and propeptide cleavage sites were obtained from the GenBank entries, or from preliminary alignments. Many of the proregions are relatively short, and at least some are potentially unrelated to each other. To avoid spurious alignments, we constructed a preliminary tree of the mature protein dataset using ClustalX. This was used to guide the construction of sets of profile alignments of proregions of most closely related proteins (e.g., cathepsin L proregions, cathepsins F, lymphopain and O). These profiles were then aligned to each other, beginning with the cathepsin L profile, and progressively adding more distantly related Branch B vertebrate cathepsins. After aligning cathepsins V, we added the placental cathepsins (M, P, and Q), K, S, and H, papain, and then aleurain, followed by the F, lymphopain, and O profiles, then testin and finally CTLA-2ß, to produce a Branch B proregion alignment. A similar strategy was used to align the cathepsin B profile with the cathepsin X profile. Efforts to align cathepsin B and DPP I profiles with the Branch B alignment produced a large number of gaps, suggesting spurious alignments. The cathepsin X profile produced an alignment with the Branch B proregion showing modest similarity and a limited number of gaps. This could have been due to chance, or it could reflect a transitional form between a Branch A and Branch B type proregion. Predominant identical residues (> 50%) at a position are shown with a black background, predominant similar residues on a grey background. The majority consensus residue is shown under each alignment. An uppercase letter shows a residue conserved in all aligned sequences (excluding cathepsin O, which was not included due to its extensive divergence). Consensus motifs implicated in Branch B proregion function are shown above the alignment in Panel A; residues implicated in cathepsin B proregion function are denoted by an * under the consensus sequence in Panel B.

View larger version (82K): [in a new window]   Figure 4. Cathepsin F-like and chicken cystatin C protein alignment. Consensus residues are indicated as described in Fig. 1. Residues identical in all six sequences are denoted by an uppercase letter in the consensus sequence. Presumptive N-terminal residues produced by cleavage of the leader peptide and the proregion are denoted by an * under the consensus sequence. The GenBank entries for the sequences shown are: hF, human cathepsin F, NP_003784; mF, mouse cathepsin F, AAF13147; flF, Japanese flounder Paralichthys olivaceus, AU050404 (partial peptide sequence derived from +1 frame of mRNA sequence); DrF, Drosophila melanogaster CG12163 gene product, AAF52055; CeF, Caenorhabditis elegans cathepsin F-like hypothetical protein F41E6.6, AAB65956; and chC, chicken egg white cystatin, P01038. The alignment was produced by separately aligning chicken cystatin to flounder cathepsin F, and human, mouse, Drosophila, and C. elegans cathepsin F proteins to each other by means of ClustalX. These two profiles were then aligned, and this alignment was manually adjusted by means of an alignment of all five CPs. The alignment of chicken cystatin with the cathepsin proregions agrees quite well with an alignment based on threading reported by Nagler et al. (1999a).