Genome-wide identification, classification and expression analysis of the serine carboxypeptidase-like protein family in poplar.

Previous studies have shown that the serine carboxypeptidase-like (SCPL) proteins in several plants play a key part in plant growth, development and stress responses. However, little is known about the functions of the SCPL genes in poplar. We identified 57 SCPL genes and divided into 3 subfamilies, which were unevenly distributed on 19 poplar chromosomes. Gene structure indicated that SCPL genes contain more introns, and motifs of each subfamily were relatively conserved. There were a total of 14 pairs of paralogs, with 6 pairs of these paralogs generated by segmental duplication and 1 generated by tandem duplication. In microsynteny analysis, large-scale duplication events played a key part in the expansion of Carboxypeptidase III genes. Expression of these genes was higher in mature leaf. Quantitative real-time PCR showed that majority of the SCPL genes were induced by methyl jasmonate (MeJA) treatment. PtSCPL27 and PtSCPL40 were located on the cytomembrane by conducting subcellular localization analysis. Our paper provides a theoretical basis for further functional research of PtSCPL genes and will benefit the molecular breeding for resistance to disease in poplar.

Genome-Wide Identification and Characterization of Carboxypeptidase Genes in Silkworm (Bombyx mori).

The silkworm (Bombyx mori) is an economically-important insect that can secrete silk. Carboxypeptidases have been found in various metazoan species and play important roles in physiological and biochemical reactions. Here, we analyzed the silkworm genome database and characterized 48 carboxypeptidases, including 34 metal carboxypeptidases (BmMCP1-BmMCP34) and 14 serine carboxypeptidases (BmSCP1-BmSCP14), to better understand their diverse functions. Compared to other insects, our results indicated that carboxypeptidases from silkworm have more family members. These silkworm carboxypeptidases could be divided into four families: Peptidase_M2 carboxypeptidases, Peptidase_M14 carboxypeptidases, Peptidase_S10 carboxypeptidases and Peptidase_S28 carboxypeptidases. Microarray analysis showed that the carboxypeptidases had distinct expression patterns, whereas quantitative real-time PCR demonstrated that the expression level of 13 carboxypeptidases significantly decreased after starvation and restored after re-feeding. Overall, our study provides new insights into the functional and evolutionary features of silkworm carboxypeptidases.

Functional segregation and emerging role of cilia-related cytosolic carboxypeptidases (CCPs).

Recent experimental data indicating axonal regeneration, axogenesis, dendritogenesis, and ciliary axoneme assembly and wellness have linked the role of cytosolic metallocarboxypeptidase 1 (CCP1/AGTPBP1/Nna1) to the microtubule network. In addition, 5 of the 6 mammalian ccp genes have been shown to participate in post-translational modifications of tubulin, which occur in the microtubules of neurons, mitotic spindles, cilia, and basal bodies. Here, we compile evidence for the idea that the occurrence of CCPs strongly correlates with the presence of cilia, suggesting that CCP functions might be primarily related to cilia and basal bodies (CBBs). In agreement with this hypothesis, CCPs were localized in centrioles, basal bodies, and mitotic spindles in HeLa cells by confocal microscopy. By reconstructing the evolutionary history of CCPs, we show their presence in the last eukaryotic common ancestor and relate each group of CCP orthologs to specific roles in CBBs. The clues presented in this study suggest that during the evolution of eukaryotes, mechanisms mediated by CCPs through tubulin post-translational modifications controlling assembly, trafficking, and signaling in the microtubules, were transferred from cilia to cell and axon microtubules.

A serine carboxypeptidase-like acyltransferase is required for synthesis of antimicrobial compounds and disease resistance in oats.

Serine carboxypeptidase-like (SCPL) proteins have recently emerged as a new group of plant acyltransferases. These enzymes share homology with peptidases but lack protease activity and instead are able to acylate natural products. Several SCPL acyltransferases have been characterized to date from dicots, including an enzyme required for the synthesis of glucose polyesters that may contribute to insect resistance in wild tomato (Solanum pennellii) and enzymes required for the synthesis of sinapate esters associated with UV protection in Arabidopsis thaliana. In our earlier genetic analysis, we identified the Saponin-deficient 7 (Sad7) locus as being required for the synthesis of antimicrobial triterpene glycosides (avenacins) and for broad-spectrum disease resistance in diploid oat (Avena strigosa). Here, we report on the cloning of Sad7 and show that this gene encodes a functional SCPL acyltransferase, SCPL1, that is able to catalyze the synthesis of both N-methyl anthraniloyl- and benzoyl-derivatized forms of avenacin. Sad7 forms part of an operon-like gene cluster for avenacin synthesis. Oat SCPL1 (SAD7) is the founder member of a subfamily of monocot-specific SCPL proteins that includes predicted proteins from rice (Oryza sativa) and other grasses with potential roles in secondary metabolism and plant defense.

Structural evidence that scytalidolisin (formerly scytalidopepsin A) is a serine-carboxyl peptidase of the sedolisin family.

Scytalidopepsin A, a pepstatin-insensitive acid endopeptidase from the fungus Scytalidium lignicolum was found to be a member of the sedolisin family of serine-carboxyl peptidases through analyses of the amino acid sequences of peptides derived from the reduced, pyridylethylated enzyme by enzymatic digestion. Hence it should be renamed scytalidolisin (or Scytalidium sedolisin).

Angiotensin-converting enzyme-2 (ACE2): comparative modeling of the active site, specificity requirements, and chloride dependence.

Angiotensin-converting enzyme 2 (ACE2), a homologue of ACE, represents a new and potentially important target in cardio-renal disease. A model of the active site of ACE2, based on the crystal structure of testicular ACE, has been developed and indicates that the catalytic mechanism of ACE2 resembles that of ACE. Structural differences exist between the active site of ACE (dipeptidyl carboxypeptidase) and ACE2 (carboxypeptidase) that are responsible for the differences in specificity. The main differences occur in the ligand-binding pockets, particularly at the S2' subsite and in the binding of the peptide carboxy-terminus. The model explains why the classical ACE inhibitor lisinopril is unable to bind to ACE2. On the basis of the ability of ACE2 to cleave a variety of biologically active peptides, a consensus sequence of Pro-X-Pro-hydrophobic/basic for the protease specificity of ACE2 has been defined that is supported by the ACE2 model. The dipeptide, Pro-Phe, completely inhibits ACE2 activity at 180 microM with angiotensin II as the substrate. As with ACE, the chloride dependence of ACE2 is substrate-specific such that the hydrolysis of angiotensin I and the synthetic peptide substrate, Mca-APK(Dnp), are activated in the presence of chloride ions, whereas the cleavage of angiotensin II is inhibited. The ACE2 model is also suggestive of a possible mechanism for chloride activation. The structural insights provided by these analyses for the differences in inhibition pattern and substrate specificity among ACE and its homologue ACE2 and for the chloride dependence of ACE/ACE2 activity are valuable in understanding the function and regulation of ACE2.

3D structure of Sulfolobus solfataricus carboxypeptidase developed by molecular modeling is confirmed by site-directed mutagenesis and small angle X-ray scattering.

Sulfolobus solfataricus carboxypeptidase (CPSso) is a thermostable zinc-metalloenzyme with a M(r) of 43,000. Taking into account the experimentally determined zinc content of one ion per subunit, we developed two alternative 3D models, starting from the available structures of Thermoactinomyces vulgaris carboxypeptidase (Model A) and Pseudomonas carboxypeptidase G2 (Model B). The former enzyme is monomeric and has one metal ion in the active site, while the latter is dimeric and has two bound zinc ions. The two models were computed by exploiting the structural alignment of the one zinc- with the two zinc-containing active sites of the two templates, and with a threading procedure. Both computed structures resembled the respective template, with only one bound zinc with tetrahedric coordination in the active site. With these models, two different quaternary structures can be modeled: one using Model A with a hexameric symmetry, the other from Model B with a tetrameric symmetry. Mutagenesis experiments directed toward the residues putatively involved in metal chelation in either of the models disproved Model A and supported Model B, in which the metal-binding site comprises His(108), Asp(109), and His(168). We also identified Glu(142) as the acidic residue interacting with the water molecule occupying the fourth chelation site. Furthermore, the overall fold and the oligomeric structure of the molecule was validated by small angle x-ray scattering (SAXS). An ab initio original approach was used to reconstruct the shape of the CPSso in solution from the experimental curves. The results clearly support a tetrameric structure. The Monte Carlo method was then used to compare the crystallographic coordinates of the possible quaternary structures for CPSso with the SAXS profiles. The fitting procedure showed that only the model built using the Pseudomonas carboxypeptidase G2 structure as a template fitted the experimental data.

A statistical investigation of amphiphilic properties of C-terminally anchored peptidases.

A number of DD-peptidases have been reported to interact with the membrane via C-terminal amphiphilic alpha-helices, but experimental support for this rests with a few well-characterized cases. These show the C-terminal interactions of DD-carboxypeptidases to involve high levels of membrane penetration, DD-endopeptidases to involve membrane surface binding and class C penicillin-binding proteins to involve membrane binding with intermediate properties. Here, we have characterized C-terminal alpha-helices from each of these peptidase groups according to their amphiphilicity, as measured by mean , and the corresponding mean hydrophobicity, . Regression and statistical analyses showed these properties to exhibit parallel negative linear relationships, which resulted from the spatial ordering of alpha-helix amino acid residues. Taken with the results of compositional and graphical analyses, our results suggest that the use of C-terminal alpha-helices may be a universal feature of the membrane anchoring for each of these groups of DD-peptidases. Moreover, to accommodate differences between these mechanisms, each group of C-terminal alpha-helices optimizes its structural amphiphilicity and hydrophobicity to fulfil its individual membrane-anchoring function. Our results also show that each anchor type analysed requires a similar overall balance between amphiphilicity for membrane interaction, which we propose is necessary to stabilize their initial membrane associations. In addition, we present a methodology for the prediction of C-terminal alpha-helical anchors from the classes of DD-peptidases analysed, based on a parallel linear model.

Carboxypeptidases from A to z: implications in embryonic development and Wnt binding.

Carboxypeptidases perform many diverse functions in the body. The well-studied pancreatic enzymes (carboxypeptidases A1, A2 and B) are involved in the digestion of food, whereas a related enzyme (mast-cell carboxypeptidase A) functions in the degradation of other proteins. Several members of the metallocarboxypeptidase gene family (carboxypeptidases D, E, M and N) are more selective enzymes and are thought to play a role in the processing of intercellular peptide messengers. Three other members of the metallocarboxypeptidase gene family do not appear to encode active enzymes; these members have been designated CPX-1, CPX-2 and AEBP1/ACLP. In this review, we focus on the recently discovered carboxypeptidase Z (CPZ). This enzyme removes C-terminal Arg residues from synthetic substrates, as do many of the other members of the gene family. However, CPZ differs from the other enzymes in that CPZ is enriched in the extracellular matrix and is broadly distributed during early embryogenesis. In addition to containing a metallocarboxypeptidase domain, CPZ also contains a Cys-rich domain that has homology to Wnt-binding proteins; Wnts are important signaling molecules during development. Although the exact function of CPZ is not yet known, it is likely that this protein plays a role in development by one of several possible mechanisms.

Families and clans of serine peptidases.

It has become clear that the proteolytic enzymes that depend upon a serine residue for their catalytic activity belong to many different families of proteins. We have attempted to group these families in "clans." A clan is defined as a group of families the members of which have a common ancestor. That is to say, they are homologous, although some of the similarities are in the "twilight zone" and the relationships cannot be proven by rigorous statistical methods. We believe we can recognize five, or perhaps six, clans of serine peptidases. In view of the separate evolutionary origins of the serine peptidases in different clans, it is not surprising to find that they may differ greatly and that indeed there are few generalizations that can be applied to all serine peptidases. In contrast, the members of a clan commonly have marked similarities.

Characterization of the sporulation-related gamma-D-glutamyl-(L)meso-diaminopimelic-acid-hydrolysing peptidase I of Bacillus sphaericus NCTC 9602 as a member of the metallo(zinc) carboxypeptidase A family. Modular design of the protein.

The sporulation-related gamma-D-glutamyl-(L)meso-diaminopimelic-acid-hydrolysing peptidase I of Bacillus sphaericus NCTC 9602 has been analysed by proton-induced X-ray emission. It contains 1 equivalent Zn2+ per mol of protein. As derived from gene cloning and sequencing, the B. sphaericus Zn peptidase I is a two-module protein. A 100-amino-acid-residue N-terminal domain consisting of two tandem segments of similar sequences, is fused to a 296-amino-acid-residue C-terminal catalytic domain. The catalytic domain belongs to the Zn carboxypeptidase A family, the closest match being observed with the Streptomyces griseus carboxypeptidase [Narahashi (1990) J. Biochem. 107, 879-886] and with the family prototype, bovine carboxypeptidase A. The catalytic domain of the B. sphaericus peptidase I possesses, distributed along the amino-acid sequence, peptide segments, a triad His162-Glu165-His307 and a dyad Tyr347-Glu366 that are equivalent to secondary structures, the zinc-binding triad His69-Glu72-His196 and the catalytic dyad Tyr248-Glu270 of bovine carboxypeptidase A respectively. The N-terminal repeats of the B. sphaericus peptidase I have similarity with the C-terminal repeats of the Enterococcus hirae muramidase 2, the Streptococcus (now Enterococcus) faecalis autolysin and the Bacillus phi PZA and phi 29 lysozymes, to which a role in the recognition of a particular moiety of the bacterial cell envelope has been tentatively assigned. Detergents enhance considerably the specific activity of the B. sphaericus peptidase I.

[Cellular and plasma carboxypeptidases]. / Karboksypeptydazy komórkowe i osoczowe.

The literature on carboxypeptidases of cells and plasma is reviewed. Subcellular localization, physico-chemical properties, chemical structure and biological significance of these enzymes are discussed.

Purification and properties of five different forms of human procarboxypeptidases.

Three different procarboxypeptidases A and two different procarboxypeptidases B have been isolated for the first time, in a pure and native state, from human pancreatic extracts. These proteins were purified in one or two quick steps by anion-exchange HPLC. All these forms have been biochemically characterized. Two of the procarboxypeptidases A, the A1 and A2 forms, are obtained in a monomeric state while the other, the A3 form, is obtained as a binary complex of a procarboxypeptidase A with a proproteinase E. This complex is stable in aqueous buffers at various ionic strengths and develops carboxypeptidase A and proteinase E activities in the presence of trypsin. The A1 and A2 forms show clear differences in electrophoretic mobility in SDS/polyacrylamide gels, isoelectric point, proteolytic activation process with trypsin and susceptibility to thermal denaturation. In contrast, these properties are similar in the A1 and A3 (binary complex) forms. On the other hand, with respect to the properties listed above, the B1 and B2 forms differ from each other mainly in isoelectric point. An overall comparison of the above properties reveals the unusual character of the A2 form, midway between the other A and B forms. N-terminal extended sequence analysis carried out on these proenzymes confirm that they constitute different isologous forms.

Considerations of the concept of structural homology as applied to bovine carboxypeptidases A and B.

An argument is presented in favor of the hypothesis that, bovine carboxypeptidases A and B fit by and large, the concept of homology on the levels of both amino acid sequence and three-dimensional structure. Two peptide sequences from carboxypeptidases A and B, comprising 46 amino acid residues, display structural homology to the extent of 51 per cent. One of the two peptides contains two of the amino acid residues which have been identified by X-ray crystallography as zinc ligands in carboxypeptidase A. The same residues are conserved in carboxypeptidase B in a loop of comparable dimensions. The other homologous pair of amino acid sequences appears to occur in different regions of the two enzymes which include in carboxypeptidase B the cysteinyl residue proposed to function as the third zinc ligand but not the amino acid residue believed to be the analogous ligand in carboxypeptidase A. The uncertainties of this prediction are considered in the light of existing chemical and structural evidence.