ABSTRACT
Xylella fastidiosa is responsible for a wide range of economically important plant diseases. We report here the crystal structure and kinetic data of Xylellain, the first cysteine protease characterized from the genome of the pathogenic X. fastidiosa strain 9a5c. Xylellain has a papain-family fold, and part of the N-terminal sequence blocks the enzyme active site, thereby mediating protein activity. One novel feature identified in the structure is the presence of a ribonucleotide bound outside the active site. We show that this ribonucleotide plays an important regulatory role in Xylellain enzyme kinetics, possibly functioning as a physiological mediator.
Subject(s)
Bacterial Proteins/chemistry , Cysteine Proteases/chemistry , Models, Molecular , Xylella/enzymology , Amino Acid Substitution , Bacterial Proteins/agonists , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biocatalysis , Catalytic Domain , Crystallography, X-Ray , Cysteine Proteases/genetics , Cysteine Proteases/metabolism , Cysteine Proteinase Inhibitors/pharmacology , Enzyme Activation , Kinetics , Mutagenesis, Site-Directed , Mutant Proteins/agonists , Mutant Proteins/antagonists & inhibitors , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Point Mutation , Protein Folding , Protein Structure, Quaternary , Recombinant Proteins/agonists , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Uridine Diphosphate/chemistry , Uridine Diphosphate/metabolismABSTRACT
The lysosomal cysteine proteases cathepsin B and L play important roles in tumor cell invasion. An imbalance between these cathepsins and their endogenous inhibitors, the cystatins, has been associated with development of the metastatic phenotype. Accordingly, many studies have indicated potential use of cystatins in therapeutic approaches. We report a novel cystatin from sugarcane (Saccharum officinarum), CaneCPI-4, with strong inhibitory activity against cathepsins B (K(i) = 0.83 nM) and L (K(i) = 0.021 nM). The invasive ability of MDA-MB-231 human breast cancer cells expressing CaneCPI-4 was only slightly decreased. In contrast, addition of low, non-toxic concentrations of recombinant His-tagged CaneCPI-4 significantly reduced invasion through a Matrigel matrix. Immunoblot analyses failed to detect the recombinant protein inside cells, indicating that the cystatin was not internalized by endocytosis, but exerted its anti-invasive effect mainly through inhibition of extracellular cathepsins. Our findings open the possibility of considering phytocystatins for anti-cancer strategies.
Subject(s)
Cathepsin B/metabolism , Cathepsins/metabolism , Cystatins/metabolism , Cystatins/pharmacology , Saccharum/metabolism , Amino Acid Sequence , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cathepsin B/antagonists & inhibitors , Cathepsins/antagonists & inhibitors , Cell Line, Tumor , Cystatins/genetics , Humans , Immunoblotting , Molecular Sequence Data , Neoplasm Invasiveness/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino AcidABSTRACT
Xylella fastidiosa is a xylem-limited, Gram-negative bacterium responsible for citrus variegated chlorosis (CVC) in sweet oranges. In the present study, we present the recombinant expression, purification and characterization of an X. fastidiosa cysteine protease (dubbed Xylellain). The recombinant Xylellain ((HIS)Xylellain) was able to hydrolyze carbobenzoxy-Phe-Arg-7-amido-4-methylcoumarin (Z-FR-MCA) and carbobenzoxy-Arg-Arg-7-amido-4-methylcoumarin (Z-RR-MCA) with similar catalytic efficiencies, suggesting that this enzyme presents substrate specificity requirements similar to cathepsin B. The immunization of mice with (HIS)Xylellain provided us with antibodies, which recognized a protein of c. 31 kDa in the X. fastidiosa pathogenic strains 9a5c, and X. fastidiosa isolated from coffee plants. However, these antibodies recognized no protein in the nonpathogenic X. fastidiosa J1a12, suggesting the absence or low expression of this protein in the strain. These findings enabled us to identify Xylellain as a putative target for combating CVC and other diseases caused by X. fastidiosa strains.
Subject(s)
Cysteine Endopeptidases/genetics , Cysteine Endopeptidases/isolation & purification , Plant Diseases , Xylella/enzymology , Xylella/pathogenicity , Amino Acid Sequence , Blotting, Western , Citrus sinensis/microbiology , Cysteine Endopeptidases/chemistry , Cysteine Endopeptidases/metabolism , Molecular Sequence Data , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Alignment , Xylella/geneticsABSTRACT
Phytocystatins are cysteine proteinase inhibitors from plants implicated in the endogenous regulation of protein turnover, programmed cell death, and in defense mechanisms against pathogens. To date, only few cystatin genes have been characterized in most plant species. We have previously characterized the protein Canecystatin, the first cystatin described in sugarcane. In an attempt to study novel Canecystatins, we identified two ORFs encoding cystatins (referred as CaneCPI-2 and CaneCPI-3) using the data from the Sugarcane EST genome project. These ORFs were then subcloned and expressed in Escherichia coli using pET28 expression vector. High amounts (approximately 20 mg/L) of pure recombinant proteins were obtained by affinity chromatography in a single step of purification. Polyclonal antibodies against the recombinant Canecystatins were raised, allowing the immunodetection of the endogenous proteins in the plant tissues. Moreover, the proteins were able to inhibit papain in a fluorometric assay with K(i) values of 0.2 and 0.25 microM for CaneCPI-2 and CaneCPI-3, respectively. These findings contribute to a better understanding of the activity of sugarcane cystatins and encourage future activity and structural studies of these proteins.
