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SALIVARY (SD-TYPE) CYSTATINS: OVER ONE BILLION YEARS IN THE MAKING—BUT TO WHAT PURPOSE?

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 (40K): [in a new window]   Figure 1. Protein sequence alignment of select cystatins. The aligned sequences extend from 2-3 residues N-terminal to the conserved glycine to the known C-terminal. The alignment was generated and formated essentially as described (Dickinson, 2002). The gaps around the N39 residue involved in legumain inhibition were adjusted manually. Abbreviations used: SA S, SN, D, C, E/M, and F: the corresponding human type 2 cystatins (GenBank accession numbers NP_001313, NP_001890, NP_001889, NP_001891, NP_000090, NP_001314, and NP_003641, respectively); Rat S, rat salivary cystatin S (P19313); chicken, chicken egg white cystatin (P01038); adder, puff adder (Bitis arietans) venom cystatin (P08935); crab, horseshoe crab Tachypleus tridentatus hemocyte cystatin (JC4536); C. elegans, Caenorhabditis elegans cystatin R01B10.1 (NP_504565); and rice, oryzacystatin I (P09229). A majority consensus sequence and the conserved disulfide bonds are shown below the alignment. The positions of conserved domains are indicated above the alignment (see text). Numbers above the alignment indicate residue number (with the conserved glycine numbered position 11 [cystatin C numbering]). Secondary structure regions (based on chicken cystatin, see text) are indicated above. ß-A to ß-E denote the five ß-strands. -1 denotes -helix 1; -2/loop denotes the region that forms an -helix in the crystal, but a loop in solution (see text).

View larger version (50K): [in a new window]   Figure 2. Known and predicted structures of cystatins. The known structures of oryzacystatin (1EQK.pdb) (Panel A), chicken egg white cystatin (CEW1.pdb) (Panel C), and the predicted structures of C. elegans RO1B10.1 (Panel B) and human cystatin S (Panel D) are shown. The alignment in Fig. 1 was the basis for homology modeling by means of Deep View (SWISS-MODEL; Guex and Peitsch, 1997). The prominent -helix 1 is positioned in the center of all four structures, and the -2/loop region (absent in oryzacystatin) above. The conserved G, QXVXG, and PW regions, and the two disulfide bonds (SS1 is N-terminal, SS2 is C-terminal), are labeled on the chicken cystatin structure, and shown as space-filling atoms in all structures where present. The legumain inhibition region N39 (Leg) is also indicated on chicken cystatin.

View larger version (12K): [in a new window]   Figure 3. N-terminal extensions and cleavage sites. The N-terminal regions (excluding secretory peptide leader sequences) of the human type 2 cystatins are shown up to the conserved G11. Cleavage sites detected in saliva or the purified protein are shown by an arrow (Al-Hashimi et al., 1988; Saitoh et al., 1988; Popovic et al., 1990; Baron et al., 1999a). +None reported.

View larger version (11K): [in a new window]   Figure 4. Phylogenetic tree of selected vertebrate type 2 cystatins. The alignment shown in Fig. 1 was used to generate a tree by neighbor-joining by means of the PAUP 4.0b8a software package (Swofford, 2000). The scale shows the genetic distance along branch lengths. Bootstrap values were obtained from 100 replicates and are shown as a percentage beside the branches. The large arrow indicates a possible position for the root of the tree, with proteins on the cystatin C branch of the tree to the top. Abbreviations used: hCys, human cystatin; rat S, rat salivary cystatin S; and adder, puff adder (Bitis arietans) venom cystatin.

View larger version (19K): [in a new window]   Figure 5. Chromosomal organization of the type 2 cystatin and CRES genes at 20p11.2. Identified genes in the most recent version of the public domain human genome sequence map (http://www.ncbi.nlm.nih.gov/) are shown. Arrowheads denote the gene orientation, but are not to scale. The open box denotes a gap in the sequence. See text for details concerning CSTP1, CSTP2, CST1, 2, and 5.