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Figure 1. A history of gene duplication is shown by protein sequence similarities, gene synteny, and structural conservation within the M14 family of metallocarboxypeptidases. (a) A phylogenetic tree showing the relationships of the core catalytic domains of all human M14 proteins, clearly indicating the three major subfamilies (A/B, N/E, and CCP). Outgroups used for this tree were the aminoacylases, ASPA and ACY3, considered by some to be a fourth, although quite distant, subfamily of MCPs. Bootstrap values are shown at key nodes. Scale bar indicates substitutions per site. The CPD protein contains three tandem carboxypeptidase domains, hence this protein is represented by three branches. (b) The chromosomal location of each metallocarboxypeptidase gene. Those showing a tandem arrangement, suggestive of recent duplications, are shown in red with arrows below indicating their precise synteny. CPXM1 and CPD are duplicated in the zebrafish genome, thus two chromosomes are indicated. (c) Representative structures are shown for each of the three subfamilies of metallocarboxypeptidases, illustrating their homologous CP domains, yet different N- and C-terminal domains. Human CPA2 (1AYE) from the CPA/B subfamily, duck CPD2 (1QMU) from the CPN/E subfamily, and Pseudomonas aeruginosa cytosolic carboxypeptidase (4A37) representing the CCP subfamily.
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Figure 2. Several genes within the M14 family are further duplicated within many species. All orthologs of human M14 metallocarboxypeptidases of the (a) A/B subfamily, (b) N/E subfamily, and (c) CCP subfamily were identified in Ensembl Release 98. These were manually validated using information on gene synteny and completeness and updated with information from Ensembl Release 100.
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Figure 3. Gene synteny suggests that tandem CPO duplicates were formed through unequal crossing over. Gene synteny information was collected from Ensembl. (a) Aebp1, CPZ, and cpxm1 gene paralogs were found on different chromosomes within large blocks of common genes, suggesting a largescale duplication event. (b) CPO gene paralogs were always found in tandem, suggesting unequal crossing-over errors. (c) CPO gene paralogs in Xenopus tropicalis were found within a gamma crystallin gene cluster and just upstream of a transposase-like gene.
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Figure 4. Gene size may contribute to rate of gene duplication or duplicate maintenance. The sizes of the indicated genes from 188 species were obtained from Ensembl (Release 98). An independent sample t test was performed comparing all frequently duplicated genes (red) to all others (white; p = 1.59E−37).
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Figure 5. Indicators of gene function suggest changes in metallocarboxypeptidase paralog activity and specificity, yet purifying selection to maintain function. Predicted cDNA and amino acid sequences were curated for all duplicated members of the A/B subfamily of metallocarboxypeptidases found in Ensembl. (a, b) Predicted proteins were classified as active enzymes, pseudoenzymes (containing substitutions at active site residues), or pseudogenes (containing large deletions or other deleterious structural mutations). (c, d) Substrate specificity for each predicted protein was predicted based on the identity of the bovine CPA1 residue 255 equivalent. Hydrophobic = hydrophobic residue 255; acidic = basic residue 255; basic = acidic residue 255; polar = polar residue 255. (e, f) The predicted coding sequences for each paralog pair were used in a codon-based test of purifying selection, where greater Ds–dN indicates greater probability of purifying selection. The variance of the difference of synonymous and nonsynonymous substitutions per site was computed using the analytical method. Analyses were conducted using the Nei–Gojobori method in MEGA7. (g) The probability of rejecting the null hypothesis of strict-neutrality (dN = dS) in favor of the alternative hypothesis (dN < dS, purifying selection) is shown. A t-test was used to compare CPO paralogs with all others.
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Figure 6. Xenopus tropicalis CPO orthologs are expressed and processed by an endopeptidase. (a) HEK293T cells were transfected with plasmids encoding the four HA-tagged X.t. CPO orthologs, or an empty plasmid (−). Cell lysates (equal amounts of protein) were resolved by SDS-PAGE and western blotted with an anti-HA antibody. The nitrocellulose membrane was also stained with Ponceau S as a loading control. (b) The distribution of HA-tagged X.t. CPO orthologs (green, HA antibody) was analyzed in transfected HEK293T cells by immunocytochemistry and compared with the distribution of transfected human CPO (red, CPO antibody). (c) The four X.t. CPO orthologs were expressed in Sf9 cells using recombinant baculoviruses. Cells were infected with wild-type virus (wtv) as a control. One percent of each cell lysate, or 0.06% of each collected medium, was resolved by SDS-PAGE and western blotted using an HA antibody. (d) Sf9 conditioned media (containing cpo.1, 2, 3, or 4, or wild-type virus (W)) and the same media incubated with 2.5 µg/ml trypsin (T) for 5 min at room temperature were resolved by SDS-PAGE and western blotted with an HA antibody. The membrane was stained with Ponceau S as a loading control.
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Figure 7. Xenopus tropicalis CPO orthologs exhibit different substrate preferences. (a) One hundred microliters of each trypsinized media was incubated with 900 µl of 0.5 mM substrate (FA-EE, FA-FA, FA-FF, pH 7.5) at room temperature. Change in absorbance at 340 nm was measured over time and the rate of reaction shown as the change in absorbance (milli-absorbance-units) per minute. n = 3–6. Error bars indicate standard deviation. *p < 0.05, comparing to the corresponding WTV dataset, as determined by ANOVA and Tukey–Kramer post-hoc analysis. (b) Each Xenopus tropicalis CPO paralog was modeled with AlphaFold2 and aligned in Pymol with X-ray crystal structures for Bos taurus CPA (3CPA) and Homo sapiens CPO (5MRV, chain a). All images show the zinc cofactor from Hs CPO as a gray sphere, the zinc cofactor from Bt CPA as a yellow sphere (largely superimposed by the gray sphere) and the Gly-Tyr dipeptide bound to the active site of Bt CPA as a yellow stick model. Key active site residues from each structure are indicated. Cavity surfaces, as viewed from the inside of the protein and with the prodomains removed, are shown in dark grays, while the protein outer surface is shown with white carbons, red oxygens, and blue nitrogens. No substrate binding pocket is shown for Bt CPA, as the pocket is filled with the Gly-Tyr and so not rendered as a surface.
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