Type-II cystatin gene cluster region overview.

<p>Organization of the <i>CST1</i>-<i>5</i> genes in human (<i>Homo sapiens</i>), chimpanzee (<i>Pan troglodytes</i>), orangutan (<i>Pongo abelii</i>), gorilla (<i>Gorilla gorilla</i>), rhesus monkey (<i>Macaca mulatta</i>), marmoset (<i>Callithrix jacchus</i>), rat (<i>Rattus norvegicus</i>), mouse (<i>Mus musculus</i>), pig (<i>Sus scrofa</i>), cattle (<i>Bos taurus</i>) and dog (<i>Canis lupus familiaris</i>). The transcriptional orientation of the genes is shown; the pseudogenes are highlighted in yellow (data from ENSEMBL and NCBI databases). The genes used in the subsequent analysis are highlighted in blue.</p

Phylogenetic tree inferred by using Maximum Likelihood (ML) and Bayesian inference (BI).

<p>TPM3+I+G was the best fitting mutation model. For ML 1000 bootstrap replicates were considered and for BI posterior probabilities were calculated; posterior probabilities (<b>bold</b>) over 0.95 and bootstrap confidence (<i>italic</i>) over 90% are considered valid support and are shown in the tree.</p

Evolution of C, D and S-Type Cystatins in Mammals: An Extensive Gene Duplication in Primates

<div><p>Cystatins are a family of inhibitors of cysteine peptidases that comprises the salivary cystatins (D and S-type cystatins) and cystatin C. These cystatins are encoded by a multigene family (<i>CST3</i>, <i>CST5</i>, <i>CST4</i>, <i>CST1</i> and <i>CST2</i>) organized in tandem in the human genome. Their presence and functional importance in human saliva has been reported, however the distribution of these proteins in other mammals is still unclear. Here, we performed a proteomic analysis of the saliva of several mammals and studied the evolution of this multigene family. The proteomic analysis detected S-type cystatins (S, SA, and SN) in human saliva and cystatin D in rat saliva. The evolutionary analysis showed that the cystatin C encoding gene is present in species of the most representative mammalian groups, i.e. Artiodactyla, Rodentia, Lagomorpha, Carnivora and Primates. On the other hand, D and S-type cystatins are mainly retrieved from Primates, and especially the evolution of S-type cystatins seems to be a dynamic process as seen in <i>Pongo abelii</i> genome where several copies of <i>CST1-like</i> gene (cystatin SN) were found. In Rodents, a group of cystatins previously identified as D and S has also evolved. Despite the high divergence of the amino acid sequence, their position in the phylogenetic tree and their genome organization suggests a common origin with those of the Primates. These results suggest that the D and S type cystatins have emerged before the mammalian radiation and were retained only in Primates and Rodents. Although the mechanisms driving the evolution of cystatins are unknown, it seems to be a dynamic process with several gene duplications evolving according to the birth-and-death model of evolution. The factors that led to the appearance of a group of saliva-specific cystatins in Primates and its rapid evolution remain undetermined, but may be associated with an adaptive advantage.</p></div

Cystatins found by LC-MS/MS from human, dog, sheep, cattle, horse, rabbit and rat saliva.

<p>Cystatins found by LC-MS/MS from human, dog, sheep, cattle, horse, rabbit and rat saliva.</p

Diagram of salivary cystatin evolution.

<p>Cystatin genes estimated emergence is shown (divergence times were based on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109050#pone.0109050-Hedges1" target="_blank">[45]</a>).</p

Amino acid composition of cystatins.

<p><i>Homo sapiens</i> cystatin C (P01034); <i>Bos taurus</i> cystatin C (P01035); <i>Rattus norvegicus</i> cystatin C (P14841); <i>Canis lupus familiaris</i> cystatin C (J9NS29); <i>Homo sapiens</i> cystatin D (P28325); <i>Callithrix jacchus</i> cystatin D (ENSCJAP00000001156); <i>Macaca mulatta</i> cystatin D (G7N352); <i>Homo sapiens</i> cystatin SN (P01037); <i>Pan troglodytes</i> cystatin SN (H2QK35); <i>Homo sapiens</i> cystatin S (P01036); <i>Pan troglodytes</i> cystatin S (H2QK34); <i>Homo sapiens</i> cystatin SA (P09228); <i>Pan troglodytes</i> cystatin SA (H2QK36) and <i>Rattus norvegicus</i> cystatin S (P19313). Filled grey boxes indicate conserved amino acid motifs; empty boxes indicate conserved amino acids characteristic of each cystatin; asterisks (*) mark the codons on <i>CST3</i> under negative selection.</p

Phylogenetic tree of European rabbit 15 IgA’s.

<p>The ML phylogenetic tree of European rabbit 15 IgA’s is shown. Sequences used include IgA1 to IgA13 (GenBank accession numbers X51647 and X82108 to X82119), the expressed IgA14 and the new IgA15 (GenBank accession numbers MH120867 and MH120868).</p

Amino acids sequence alignment of European rabbit 15 IgA’s.

<p>The entire hinge region sequences are depicted. For the remaining constant region domains, only the positions for which IgA15 and IgA14 Cα chains have unique amino acid residues are shown. Human IgA1 numbering for these positions is shown above. Dots (.) represent identity with the uppermost sequence, dashes (-) stand for gaps in the alignment. IgA6 is presumably an allele to IgA1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0201567#pone.0201567.ref029" target="_blank">29</a>]. GenBank accession numbers for IgA1 to IgA14 are X51647, X82108 to X82119, and AF314407. Accession number for the new IgA15 and expressed IgA14 are MH120868 and MH120867.</p

(A) Distribution of <i>rFut1</i> polymorphisms along the coding region.

<p>The protein domains are represented by black boxes (tm, transmembrane domain; S, stem region). Nucleotide variations are shown above boxes (R = A or G; Y = C or T; K = G or T; S = G or C; M = A or C; D = A, G or T). Amino acid variations are shown below boxes. Positions numbering begins at the start codon and first methionine respectively. The positions in bold refer to the polymorphic positions shared by Sec1, Fut2 and Fut1 (positions 780, 852, 862, 867 and 885) and to the polymorphic positions shared by Fut2 and Fut1 (positions 483 and 954). Asterisks represent non-synonymous substitutions. <b>(B; C) Flow cytometry analysis of wild rabbit <i>rFut1</i> variants</b>. Enzymes were produced by transfection in CHO cells as N-term GFP fusion proteins. Synthesis of H type 2 and H type 3 was determined using the UEA-I lectin (<b>B</b>) and the Mbr1 mAb (<b>C</b>), respectively. Transfection of a human null allele (hFUT2 KO) fused to GFP provided a negative control and transfection of the rFut2 allele variant E corresponding to the rFut2 reference sequence provided a positive control. The upper panels show examples of results obtained with two selected rFut1 variants. The lower panels show the ratios between the percentages of UEA-I (H type 2) or Mbr1 (H type 3) positive cells and the percentage of GFP positive cells. In each case, the activity of rFut1 variants is compared to that of the reference rFut2 (variant E).</p

Relationship between expression of <i>rSec1</i> RNA and recognition of individual rabbits by RHDV strains.

<p>Binding to individual duodenum extracts of six strains of RHDV representative of the virus diversity was determined as described in the materials and methods section. <i>rSec1</i> RNA expression in the duodenum of the same rabbits corresponds to the values shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004759#ppat.1004759.g003" target="_blank">Fig 3</a>. Plain lines separate high and low RHDV binders defined as above or below median values. Dashed lines separate high and low expressors of rSec1 mRNA defined as animals being above or below the <i>Fut1</i> and <i>Fut2</i> range of expression. For each RHDV strain P values were obtained from analysis of these categories by Fisher’s exact test. Treating values as continuous variable and testing correlations between rSec1 RNA expression and RHDV binding using the Spearman correlation test also yielded significant relationships (p<0.05) for G2, G3, G4 and G6.</p