Repeated Domains of Leptospira Immunoglobulin-like Proteins Interact with Elastin and Tropoelastin

Leptospira spp., the causative agents of leptospirosis, adhere to components of the extracellular matrix, a pivotal role for colonization of host tissues during infection. Previously, we and others have shown that Leptospira immunoglobulin-like proteins (Lig) of Leptospira spp. bind to fibronectin, laminin, collagen, and fibrinogen. In this study, we report that Leptospira can be immobilized by human tropoelastin (HTE) or elastin from different tissues, including lung, skin, and blood vessels, and that Lig proteins can bind to HTE or elastin. Moreover, both elastin and HTE bind to the same LigB immunoglobulin-like domains, including LigBCon4, LigBCen7′–8, LigBCen9, and LigBCen12 as demonstrated by enzyme-linked immunosorbent assay (ELISA) and competition ELISAs. The LigB immunoglobulin-like domain binds to the 17th to 27th exons of HTE (17–27HTE) as determined by ELISA (LigBCon4, K_D = 0.50 μm; LigBCen7′–8, K_D = 0.82 μm; LigBCen9, K_D = 1.54 μm; and LigBCen12, K_D = 0.73 μm). The interaction of LigBCon4 and 17–27HTE was further confirmed by steady state fluorescence spectroscopy (K_D = 0.49 μm) and ITC (K_D = 0.54 μm). Furthermore, the binding was enthalpy-driven and affected by environmental pH, indicating it is a charge-charge interaction. The binding affinity of LigBCon4D341N to 17–27HTE was 4.6-fold less than that of wild type LigBCon4. In summary, we show that Lig proteins of Leptospira spp. interact with elastin and HTE, and we conclude this interaction may contribute to Leptospira adhesion to host tissues during infection.Pathogenic Leptospira spp. are spirochetes that cause leptospirosis, a serious infectious disease of people and animals (1, 2). Weil syndrome, the severe form of leptospiral infection, leads to multiorgan damage, including liver failure (jaundice), renal failure (nephritis), pulmonary hemorrhage, meningitis, abortion, and uveitis (3, 4). Furthermore, this disease is not only prevalent in many developing countries, it is reemerging in the United States (3). Although leptospirosis is a serious worldwide zoonotic disease, the pathogenic mechanisms of Leptospira infection remain enigmatic. Recent breakthroughs in applying genetic tools to Leptospira may facilitate studies on the molecular pathogenesis of leptospirosis (5–8).The attachment of pathogenic Leptospira spp. to host tissues is critical in the early phase of Leptospira infection. Leptospira spp. adhere to host tissues to overcome mechanical defense systems at tissue surfaces and to initiate colonization of specific tissues, such as the lung, kidney, and liver. Leptospira invade hosts tissues through mucous membranes or injured epidermis, coming in contact with subepithelial tissues. Here, certain bacterial outer surface proteins serve as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs)² to mediate the binding of bacteria to different extracellular matrices (ECMs) of host cells (9). Several leptospiral MSCRAMMs have been identified (10–18), and we speculate that more will be identified in the near future.Lig proteins are distributed on the outer surface of pathogenic Leptospira, and the expression of Lig protein is only found in low passage strains (14, 16, 17), probably induced by environmental cues such as osmotic or temperature changes (19). Lig proteins can bind to fibrinogen and a variety of ECMs, including fibronectin (Fn), laminin, and collagen, thereby mediating adhesion to host cells (20–23). Lig proteins also constitute good vaccine candidates (24–26).Elastin is a component of ECM critical to tissue elasticity and resilience and is abundant in skin, lung, blood vessels, placenta, uterus, and other tissues (27–29). Tropoelastin is the soluble precursor of elastin (28). During the major phase of elastogenesis, multiple tropoelastin molecules associate through coacervation (30–32). Because of the abundance of elastin or tropoelastin on the surface of host cells, several bacterial MSCRAMMs use elastin and/or tropoelastin to mediate adhesion during the infection process (33–35).Because leptospiral infection is known to cause severe pulmonary hemorrhage (36, 37) and abortion (38), we hypothesize that some leptospiral MSCRAMMs may interact with elastin and/or tropoelastin in these elastin-rich tissues. This is the first report that Lig proteins of Leptospira interact with elastin and tropoelastin, and the interactions are mediated by several specific immunoglobulin-like domains of Lig proteins, including LigBCon4, LigBCen7′–8, LigBCen9, and LigBCen12, which bind to the 17th to 27th exons of human tropoelastin (HTE).     

Crystal Structure of Bacillus anthracis Transpeptidase Enzyme CapD

Bacillus anthracis elaborates a poly-γ-d-glutamic acid capsule that protects bacilli from phagocytic killing during infection. The enzyme CapD generates amide bonds with peptidoglycan cross-bridges to anchor capsular material within the cell wall envelope of B. anthracis. The capsular biosynthetic pathway is essential for virulence during anthrax infections and can be targeted for anti-infective inhibition with small molecules. Here, we present the crystal structures of the γ-glutamyltranspeptidase CapD with and without α-l-Glu-l-Glu dipeptide, a non-hydrolyzable analog of poly-γ-d-glutamic acid, in the active site. Purified CapD displays transpeptidation activity in vitro, and its structure reveals an active site broadly accessible for poly-γ-glutamate binding and processing. Using structural and biochemical information, we derive a mechanistic model for CapD catalysis whereby Pro⁴²⁷, Gly⁴²⁸, and Gly⁴²⁹ activate the catalytic residue of the enzyme, Thr³⁵², and stabilize an oxyanion hole via main chain amide hydrogen bonds.Spores of Bacillus anthracis are the causative agents of anthrax disease (1). Upon entry into their hosts, spores germinate and replicate as vegetative bacilli (1). The formation of a thick capsule encasing vegetative forms enables bacilli to escape granulocyte0 and macrophage-mediated phagocytosis, and the pathogen eventually disseminates throughout all tissues of an infected host (2, 3). Bacilli secrete lethal and edema toxins, which cause macrophage necrosis and precipitate anthrax death (4–7). The genes providing for toxin and capsule formation are carried on two large virulence plasmids, pXO1 and pXO2, respectively (8, 9). Loss of any one plasmid leads to virulence attenuation, a feature that has been exploited for the generation of vaccine-type strains (10–14).Unlike polysaccharide-based capsules that are commonly found in bacterial pathogens, the capsular material of B. anthracis is composed of poly-γ-d-glutamic acid (PDGA)³ (3). All the genes necessary for capsule biogenesis are located in the capBCADE gene cluster on plasmid pXO2 (15–19). CapD is the only protein of this cluster that is located on the bacterial surface (16). CapD shares sequence similarity with bacterial and mammalian γ-glutamyl transpeptidases (GGTs; EC 2.3.2.2) (17). GGTs belong to the N-terminal nucleophile hydrolases (Ntn) family (Protein Structure Classification (Class (C), Architecture (A), Topology (T) and Homologous superfamily (H)) (CATH) id 3.60.60.10) (20). These enzymes assemble as a single polypeptide chain and acquire activity by undergoing autocatalytic processing to heterodimer.Bacterial GGTs catalyze the first step in glutathione degradation. For example, Helicobacter pylori GGT removes glutamate from glutathione tripeptide via the formation of a γ-glutamyl acyl enzyme. This intermediate is resolved by the nucleophilic attack of a water molecule, causing the release of γ-glutamate (21, 22). Mammalian enzymes transfer the γ-glutamyl intermediate to the amino group of a peptide, thereby completing a transpeptidation reaction (23). The B. anthracis CapD precursor is also programmed for autocatalytic cleavage (17). Similar to mammalian GGTs, CapD also catalyzes a transpeptidation reaction; however, this reaction promotes the covalent linkage of PDGA to the bacterial envelope (16, 24). We have recently demonstrated the cell wall anchor structure of capsule filaments in the envelope of B. anthracis, identifying an amide bond between the terminal carboxyl group of PDGA and the side amino group of m-diaminopimelic acid cross-bridges within muropeptides (24). The CapD-catalyzed transpeptidation reaction could be recapitulated in vitro using purified recombinant CapD, γ-d-Glu_n peptide, and muropeptide substrates (24). In the absence of the physiological nucleophile (muropeptides), CapD acyl intermediates can be resolved by the nucleophilic attack of water to generate hydrolysis products.Here, we report the high resolution crystal structure of CapD in the absence and presence of a glutamate dipeptide and compare it with the known structures of H. pylori and Escherichia coli GGTs. By combining structural, genetic, and biochemical approaches, we identify the unique features of CapD that distinguish the protein from GGTs and detect several residues that are important for CapD autocatalytic cleavage and PDGA processing. This structural information will further the development of small molecule inhibitors that disrupt CapD activity and that may be useful as anti-infective therapies for anthrax.     

Identification of Two Genes from Streptomyces argillaceus Encoding Glycosyltransferases Involved in Transfer of a Disaccharide during Biosynthesis of the Antitumor Drug Mithramycin

Mithramycin is an antitumor polyketide drug produced by Streptomyces argillaceus that contains two deoxysugar chains, a disaccharide consisting of two d-olivoses and a trisaccharide consisting of a d-olivose, a d-oliose, and a d-mycarose. From a cosmid clone (cosAR3) which confers resistance to mithramycin in streptomycetes, a 3-kb PstI-XhoI fragment was sequenced, and two divergent genes (mtmGI and mtmGII) were identified. Comparison of the deduced products of both genes with proteins in databases showed similarities with glycosyltransferases and glucuronosyltransferases from different sources, including several glycosyltransferases involved in sugar transfer during antibiotic biosynthesis. Both genes were independently inactivated by gene replacement, and the mutants generated (M3G1 and M3G2) did not produce mithramycin. High-performance liquid chromatography analysis of ethyl acetate extracts of culture supernatants of both mutants showed the presence of several peaks with the characteristic spectra of mithramycin biosynthetic intermediates. Four compounds were isolated from both mutants by preparative high-performance liquid chromatography, and their structures were elucidated by physicochemical methods. The structures of these compounds were identical in both mutants, and the compounds are suggested to be glycosylated intermediates of mithramycin biosynthesis with different numbers of sugar moieties attached to C-12a-O of a tetracyclic mithramycin precursor and to C-2-O of mithramycinone: three tetracyclic intermediates containing one sugar (premithramycin A1), two sugars (premithramycin A2), or three sugars (premithramycin A3) and one tricyclic intermediate containing a trisaccharide chain (premithramycin A4). It is proposed that the glycosyltransferases encoded by mtmGI and mtmGII are responsible for forming and transferring the disaccharide during mithramycin biosynthesis. From the structures of the new metabolites, a new biosynthetic sequence regarding late steps of mithramycin biosynthesis can be suggested, a sequence which includes glycosyl transfer steps prior to the final shaping of the aglycone moiety of mithramycin.

Many bioactive drugs contain sugars attached to their aglycones which are usually important or, in some cases, essential for bioactivity. Most of these sugars belong to the family of the 6-deoxyhexoses (6-DOH) (18, 20, 27) and are transferred to the different aglycones as late steps in biosynthesis. Genes involved in the biosynthesis of different 6-DOH have been reported elsewhere and participate in the biosynthesis of erythromycin (9, 12, 31, 38, 39), daunorubicin (13, 26, 36), mithramycin (22), granaticin (2), streptomycin (10, 28), and tylosin (14, 23). However, information about the glycosyltransferases (GTFs) responsible for the transfer of the sugars to the respective aglycones is quite scarce. So far, only two GTFs from antibiotic producers have been biochemically characterized in detail, and they are involved in macrolide inactivation: Mgt, from Streptomyces lividans, a nonmacrolide producer (7, 17); and OleD, from the oleandomycin producer Streptomyces antibioticus (15, 29), which inactivates oleandomycin by addition of glucose to the 2′-OH group of the desosamine attached to the macrolactone ring (40). In the last several years, a few genes have been proposed to encode GTFs involved in the transfer of sugars to various aglycones during biosynthesis: dnrS and dnrH, from Streptomyces peucetius, involved in daunorubicin (26) and baumycin (36) biosynthesis, respectively; gra-orf5, involved in granaticin biosynthesis (2); eryCIII and eryBV, involved in the transfer of desosamine and mycarose, respectively, in erythromycin biosynthesis (12, 32, 38); and tylM2, from Streptomyces fradiae, involved in sugar transfer during tylosin biosynthesis (14).Mithramycin (Fig. ​(Fig.1)1) is an aromatic polyketide which shows antibacterial activity against gram-positive bacteria and also antitumor activity (30, 37). Together with the chromomycins and the olivomycins, mithramycin constitutes the so-called aureolic acid group of antitumor drugs. The polyketide moiety of mithramycin is derived from the condensation of 10 acetate building blocks in a series of reactions catalyzed by a type II polyketide synthase (5, 21). The mithramycin aglycone is glycosylated at positions 6 and 2 with disaccharide (d-olivose- d-olivose) and trisaccharide (d-olivose-d-oliose-d-mycarose) moieties, respectively. All of these sugars belong to the 6-DOH family. In the mithramycin pathway, two genes (mtmD and mtmE) encoding two enzymes (glucose-1-phosphate:TTP thymidylyl transferase and dTDP-4,6-dehydratase, respectively) involved in the biosynthesis of the mithramycin 6-DOH have been cloned, and their participation in mithramycin biosynthesis has been demonstrated by insertional inactivation (22). Here we report the characterization of two Streptomyces argillaceus genes (mtmGI and mtmGII) that encode two putative GTFs responsible for the formation and transfer of the disaccharide chain. Inactivation of these genes by gene replacement showed identical accumulated compounds and allowed the isolation of four glycosylated compounds which are likely to be intermediates in mithramycin biosynthesis. Open in a separate windowFIG. 1Structures of mithramycin, premithramycinone, and the new premithramycins.     

Biochemical Properties and Substrate Specificities of a Recombinantly Produced Azotobacter vinelandii Alginate Lyase

Alginate is a polysaccharide composed of β-d-mannuronic acid (M) and α-l-guluronic acid (G). An Azotobacter vinelandii alginate lyase gene, algL, was cloned, sequenced, and expressed in Escherichia coli. The deduced molecular mass of the corresponding protein is 41.4 kDa, but a signal peptide is cleaved off, leaving a mature protein of 39 kDa. Sixty-three percent of the amino acids in this mature protein are identical to those in AlgL from Pseudomonas aeruginosa. AlgL was partially purified, and the activity was found to be optimal at a pH of 8.1 to 8.4 and at 0.35 M NaCl. Divalent cations are not necessary for activity. The pI of the enzyme is 5.1. When an alginate rich in mannuronic acid was used as the substrate, the K_m was found to be 4.6 × 10⁻⁴ M (sugar residues). AlgL was found to cleave M-M and M-G bonds but not G-M or G-G bonds. Bonds involving acetylated residues were also cleaved, but this activity may be sensitive to the extent of acetylation.

Alginate is a family of 1-4-linked copolymers of β-d-mannuronic acid (M) and α-l-guluronic acid (G). It is produced by brown algae and by some bacteria belonging to the genera Azotobacter and Pseudomonas (8, 17, 18, 31). The polymer is widely used in industry and biotechnology (36, 44), and the genetics of its biosynthesis in Pseudomonas aeruginosa has been extensively studied due to its role in the disease cystic fibrosis (33). In bacterial alginates, some of the M residues may be O-2- and/or O-3-acetylated (42). The polymer is initially synthesized as mannuronan, and the G residues are introduced at the polymer level by mannuronan C-5-epimerases (13, 22, 23). The epimerized alginates contain a mixture of blocks of consecutive G residues (G blocks), consecutive M residues (M blocks), and alternating M and G residues (MG blocks). Alginates from Pseudomonas sp. do not contain G blocks (42).Alginate lyases catalyze the depolymerization of alginates by β-elimination, generating a molecule containing 4-deoxy-l-erythro-hex-4-enepyranosyluronate at the nonreducing end. Such lyases have been found in organisms using alginate as a carbon source, in bacteriophages specific for alginate-producing organisms, and in alginate-producing bacteria (45). An alginate molecule may contain four different glycosidic bonds, M-M, G-M, M-G, or G-G, and the relative rates at which each of these bonds are cleaved vary among different lyases (36a). The lyases also differ in the extent to which they are affected by acetylation (35, 43, 46).Davidson et al. (10) described an Azotobacter vinelandii lyase which preferred M blocks as a substrate. Kennedy et al. (28) later reported the purification of periplasmic alginate lyases from A. vinelandii and from Azotobacter chroococcum which also seemed to prefer deacetylated, M-rich alginate. The activities of these enzymes were found to be optimal at pH 6.8 and 7.2, respectively, while the enzyme reported by Davidson et al. (10) was found to display optimal activity at pH 7.8.A gene, algL, encoding an alginate lyase has been cloned from P. aeruginosa (2, 41). The gene was found to be located in a cluster containing most of the genes necessary for the biosynthesis of alginate. A homologous gene cluster has recently been identified in A. vinelandii (38) and shown to encode an alginate lyase (32). In our previous report, we showed that plasmid pHE102, which contains a part of this gene cluster, contains a DNA sequence sharing homology with algL from P. aeruginosa (38). We have now subcloned, sequenced, and expressed this gene in Escherichia coli. The lyase was shown to preferentially cleave deacetylated M-M and M-G bonds, but acetylated substrates were also cleaved.     

Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.     

Phosphoproteomics of Klebsiella pneumoniae NTUH-K2044 Reveals a Tight Link between Tyrosine Phosphorylation and Virulence

Encapsulated Klebsiella pneumoniae is the predominant causative agent of pyogenic liver abscess, an emerging infectious disease that often complicates metastatic meningitis or endophthalmitis. The capsular polysaccharide on K. pneumoniae surface was determined as the key to virulence. Although the regulation of capsular polysaccharide biosynthesis is largely unclear, it was found that protein-tyrosine kinases and phosphatases are involved. Therefore, the identification and characterization of such kinases, phosphatases, and their substrates would advance our knowledge of the underlying mechanism in capsule formation and could contribute to the development of new therapeutic strategies. Here, we analyzed the phosphoproteome of K. pneumoniae NTUH-K2044 with a shotgun approach and identified 117 unique phosphopeptides along with 93 in vivo phosphorylated sites corresponding to 81 proteins. Interestingly, three of the identified tyrosine phosphorylated proteins, namely protein-tyrosine kinase (Wzc), phosphomannomutase (ManB), and undecaprenyl-phosphate glycosyltransferase (WcaJ), were found to be distributed in the cps locus and thus were speculated to be involved in the converging signal transduction of capsule biosynthesis. Consequently, we decided to focus on the lesser studied ManB and WcaJ for mutation analysis. The capsular polysaccharides of WcaJ mutant (WcaJY5F) were dramatically reduced quantitatively, and the LD₅₀ increased by 200-fold in a mouse peritonitis model compared with the wild-type strain. However, the capsular polysaccharides of ManB mutant (ManBY26F) showed no difference in quantity, and the LD₅₀ increased by merely 6-fold in mice test. Our study provided a clear trend that WcaJ tyrosine phosphorylation can regulate the biosynthesis of capsular polysaccharides and result in the pathogenicity of K. pneumoniae NTUH-K2044.Protein phosphorylation is one of the most biologically relevant and ubiquitous post-translational modifications in both eukaryotic and prokaryotic organisms. It is best known that protein phosphorylation is a reversible enzyme-catalyzed process that is controlled by various kinases and phosphatases. The aberrant functions often result in irregular protein phosphorylation and ultimately lead to serious disease states such as malignant transformation, immune disorders, and pathogenic infections in mammals (1, 2). Recently, accumulating evidences suggest that Ser/Thr/Tyr phosphorylations also contribute to regulate a diverse range of cellular responses and physiological processes in prokaryotes (1). Among them, tyrosine phosphorylation in encapsulated bacteria has been discovered to play key roles in capsular polysaccharide (CPS1; K antigen) biosynthesis, which leads to virulence (3, 4). This thick layer of exopolysaccharide on many pathogenic bacteria can act as a physical boundary to evade phagocytosis and complement-mediated killing and further inhibit complement activation of the host (1, 5, 6).In 1996, Acinetobacter johnsonii protein-tyrosine kinase (Ptk) was first discovered and categorized under the bacterial protein-tyrosine kinase (BY-kinase) family (1, 7, 8). Shortly after, its function in bacterial exopolysaccharide production and transport was characterized (1, 7, 8). From then on, many more bacterial tyrosine kinases such as Wzc of Escherichia coli (1, 9) and EpsB of Pseudomonas solanacearum (10, 11) were found to possess this conserved property; deletion of such tyrosine kinases will result in the loss of exopolysaccharide production (12). Therefore, several experiments were conducted to investigate the role of the downstream substrates of the tyrosine kinases in different strains of bacteria, and some targeted proteins were found to participate in the exopolysaccharide anabolism (13, 14). These findings demonstrated a direct relationship between bacterial tyrosine phosphorylation and exopolysaccharide biosynthesis that was directly reflected in the strain virulence.In the past, the functional roles of the critical components involved in protein phosphorylation were defined by basic biochemical and genetic approaches (1). However, there exists a salient gap between the growing number of identified protein-tyrosine kinases/phosphatases and the relative paucity of protein substrates characterized to date. Genomic sequence analyses and advanced high resolution/high accuracy MS systems with vastly improved phosphopeptide enrichment strategies are among the two key enabling technologies that allow a high efficiency identification of the scarcely detectable site-specific phosphorylations in bacterial systems (15). Mann et al. (16) were the first to initiate a systematic study of the phosphoproteome of B. subtilis in 2007 followed by similar site-specific phosphoproteomics analyses of E. coli (17), Lactococcus lactis (18), and Halobacterium salinarum (19). These pioneering works have since set the foundation in bacterial phosphoproteomics but have not been specifically carried out to address a particular biological issue of causal relevance to virulence or pathogenesis.Klebsiella pneumoniae is a Gram-negative, non-motile, facultative anaerobic, and rod-shaped bacterium. It is commonly found in water and soil (20) as well as on plants (21) and mucosal surfaces of mammals, such as human, horse, and swine (22, 23). It was demonstrated that CPS on the surface of K. pneumoniae is the prime factor of virulence and toxicity in causing pyogenic liver abscess (PLA), a common intra-abdominal infection with a high 10–30% mortality rate worldwide (24–29). There are also variations in virulence in regard to different capsular serotypes; K1 and K2 were found to be especially pathogenic in causing PLA in a mouse model (30) compared with other serotypes, which show little or no effect (31–34). The K. pneumoniae NTUH-K2044 (K2044) strain, encapsulated with K1 antigen (35), was isolated from clinical K. pneumoniae liver abscess patients. It has become an important emerging pathogen (36) because it usually complicates metastatic septic endophthalmitis and irreversible central nervous system infections independent of host underlying diseases (30, 34). The transmission rate is high (37), and it often rapidly leads to outbreaks of community-acquired infections, such as bacteremia, nosocomial pneumonia, and sepsis, common in immunocompromised individuals (38).In this study, we wanted to prove that the biosynthesis of CPS is mediated through tyrosine phosphorylation of a subset of proteins. An MS-based systematic phosphoproteomics analysis was conducted on K2044 to identify tyrosine phosphorylated proteins that are also associated with CPS biosynthesis. We further validated the relationship between tyrosine phosphorylation on those proteins and virulence of K2044 by site-directed mutagenesis, CPS quantification, serum killing, and mouse lethality assay.     

Gene Cloning,Nucleotide Sequencing,and Purification and Characterization of the Low-Specificity l-Threonine Aldolase from Pseudomonas sp. Strain NCIMB 10558

A low-specificity l-threonine aldolase (l-TA) gene from Pseudomonas sp. strain NCIMB 10558 was cloned and sequenced. The gene contains an open reading frame consisting of 1,041 nucleotides corresponding to 346 amino acid residues. The gene was overexpressed in Escherichia coli cells, and the recombinant enzyme was purified and characterized. The enzyme, requiring pyridoxal 5′-phosphate as a coenzyme, is strictly l specific at the α position, whereas it cannot distinguish between threo and erythro forms at the β position. In addition to threonine, the enzyme also acts on various other l-β-hydroxy-α-amino acids, including l-β-3,4-dihydroxyphenylserine, l-β-3,4-methylenedioxyphenylserine, and l-β-phenylserine. The predicted amino acid sequence displayed less than 20% identity with those of low-specificity l-TA from Saccharomyces cerevisiae, l-allo-threonine aldolase from Aeromonas jandaei, and four relevant hypothetical proteins from other microorganisms. However, lysine 207 of low-specificity l-TA from Pseudomonas sp. strain NCIMB 10558 was found to be completely conserved in these proteins. Site-directed mutagenesis experiments showed that substitution of Lys207 with Ala or Arg resulted in a significant loss of enzyme activity, with the corresponding disappearance of the absorption maximum at 420 nm. Thus, Lys207 of the l-TA probably functions as an essential catalytic residue, forming an internal Schiff base with the pyridoxal 5′-phosphate of the enzyme to catalyze the reversible aldol reaction.β-Hydroxy-α-amino acids constitute an important class of compounds. They are natural products in their own right and are components of a range of antibiotics, for example, cyclosporin A, lysobactin, and vancomycin (30) and bouvardin and deoxybouvardin (6). 4-Hydroxy-l-threonine is a precursor of rizobitoxine, a potent inhibitor of pyridoxal 5′-phosphate (PLP)-dependent enzymes (32). 3,4,5-Trihydroxyl-l-aminopentanoic acid is a key component of polyoxins (32). l-threo-3,4-Dihydroxyphenylserine is a new drug for Parkinson’s disease therapy (13). However, the industrial production of β-hydroxy-α-amino acids has been limited to chemical synthesis processes, which need multiple steps to isolate the four isomers (l-threo form, d-threo form, l-erythro form, and d-erythro form). Threonine aldolase (EC 4.1.2.5), which stereospecifically catalyzes the retro-aldol cleavage of threonine, is a potentially useful catalyst for the synthesis of substituted amino acids from aldehyde and glycine (27, 31, 32).Two different types of threonine aldolases are known so far. l-allo-Threonine aldolase (l-allo-TA), isolated and purified from Aeromonas jandaei DK-39 (8), stereospecifically catalyzes the reversible interconversion of l-allo-threonine and glycine. Low-specificity l-threonine aldolase (l-TA) catalyzes the cleavage of both l-threonine and l-allo-threonine to glycine and acetaldehyde, as well as the reverse reaction, aldol condensation. The enzymes have been purified and characterized from Candida humicola (9, 34) and Saccharomyces cerevisiae (12). Low-specificity l-TA activity has also been shown to exist in mammals (7, 23, 26) and a variety of other microbial species (2, 4, 17, 35). The enzyme is physiologically important for the synthesis of cellular glycine in yeast (12, 15, 16). Threonine aldolases with distinct stereospecificities are ideal targets for enzymology studies on structural and functional relationships. However, information on the primary structures of threonine aldolases was limited to our recent studies (11, 12). The construction of an overproduction system for threonine aldolase will be indispensable for the industrial biosyntheses of β-hydroxy-α-amino acids.The present work focuses on the cloning, sequencing, and overexpression in Escherichia coli cells of the low-specificity l-TA gene from Pseudomonas sp. strain NCIMB 10558, the purification and characterization of the recombinant enzyme, and the identification of the active-site lysine residue of the enzyme by site-directed mutagenesis. Evidence is presented that Lys207 of low-specificity l-TA probably functions as a catalytic residue, forming an internal Schiff base with the PLP of the enzyme to catalyze the reversible aldol reaction. This is the first report showing a purified enzyme with l-β-3,4-dihydroxyphenylserine aldolase and l-β-3,4-methylenedioxyphenylserine aldolase activities, providing a new route for the industrial production of these important unnatural amino acids.     

Absence of Nitric-oxide Synthase in Sequentially Purified Rat Liver Mitochondria

Data, both for and against the presence of a mitochondrial nitric-oxide synthase (NOS) isoform, is in the refereed literature. However, irrefutable evidence has not been forthcoming. In light of this controversy, we designed studies to investigate the existence of the putative mitochondrial NOS. Using repeated differential centrifugation followed by Percoll gradient fractionation, ultrapure, never frozen rat liver mitochondria and submitochondrial particles were obtained. Following trypsin digestion and desalting, the mitochondrial samples were analyzed by nano-HPLC-coupled linear ion trap-mass spectrometry. Linear ion trap-mass spectrometry analyses of rat liver mitochondria as well as submitochondrial particles were negative for any peptide from any NOS isoform. However, recombinant neuronal NOS-derived peptides from spiked mitochondrial samples were easily detected, down to 50 fmol on column. The protein calmodulin (CaM), absolutely required for NOS activity, was absent, whereas peptides from CaM-spiked samples were detected. Also, l-[¹⁴C]arginine to l-[¹⁴C]citrulline conversion assays were negative for NOS activity. Finally, Western blot analyses of rat liver mitochondria, using NOS (neuronal or endothelial) and CaM antibodies, were negative for any NOS isoform or CaM. In conclusion, and in light of our present limits of detection, data from carefully conducted, properly controlled experiments for NOS detection, utilizing three independent yet complementary methodologies, independently as well as collectively, refute the claim that a NOS isoform exists within rat liver mitochondria.Nitric oxide (NO^·)² is a highly diffusible, hydrophobic, and gaseous free radical (1) that is responsible for autocrine and paracrine signaling activities (2). NO^· can readily partition into and through membranes (3–5) to influence biological functions such as blood pressure regulation, platelet aggregation and adhesion, neurotransmission, and cellular defense (4, 6–11). The mechanism by which NO^· influences biological functions is by binding to target proteins that contain heme and/or thiol(s). Alternatively, NO^· can combine with to produce the highly reactive species peroxynitrite.Mitochondria are highly compartmentalized, membranous organelles that contain abundant amounts of reactive hemoproteins and thiols (12, 13), to which NO^· may bind reversibly (14, 15) or irreversibly (16–18). Mitochondria also generate various amounts of during the process of cellular respiration (19, 20). Studies conducted during the past decade have suggested that NO^· can diffuse into mitochondria and cause mitochondrial dysfunction by reversibly inhibiting cytochrome c oxidase (14, 21, 22) and NADH dehydrogenase (23).In the mid-90s, a putative variant of NOS was proposed to reside within mitochondria. Initially, Kobzik et al. (24) and Hellsten and co-workers (25) observed an apparent endothelial NOS (eNOS) immunoreactivity in skeletal muscle mitochondria. Simultaneously, Bates et al. (26, 27) observed an apparent eNOS histochemical reactivity in inner mitochondrial membrane preparations, isolated from rat liver, brain, heart, skeletal muscle, and kidney. Tatoyan and Giulivi (28), acting on these initial observations, performed experiments in an attempt to confirm the identity of this putative mtNOS. Relying on immunochemical analysis, Tatoyan and Giulivi (28) claimed that inducible NOS (iNOS) was the NOS isoform present in rat liver mitochondria. This same group using mass spectrometry later presented data in support of the putative mtNOS being a variant of nNOS (29). Ghafourifar and Richter (30) had reported previously that the putative mtNOS was calcium-sensitive and constitutive in nature. Since these reports, different groups have reported the presence of each of the three main isoforms of NOS within mitochondria (29, 31, 32). Also, biochemical characterization of the putative mtNOS performed by Giulivi and co-workers (29) revealed certain post-translational modifications (myristoylation and phosphorylation of the protein) that are thought to be unique to eNOS. During the last decade, various reports have supported the presence of at least one of the three main isoforms of NOS residing in mitochondria. However, the more recent reports tend to question this claim (33–36). Because of the contradictory reports regarding the existence of a putative mtNOS, Brookes (33) compiled a critical and thorough review of the literature published up to 2003 dealing with the putative mtNOS. This review brought to light the diverse technical issues involved in the aforementioned studies. Major issues were the degree of purity of mitochondrial preparations (37, 38), shortcomings of measurement methodology (29, 39–41), use of inappropriate, or total lack of, experimental controls and confusing technical practices. Lacza et al. (42) has reviewed the more recent developments in the area of mitochondrial NO^· production and discussed some of the shortcomings of certain techniques still being used.In light of this ongoing controversy regarding the presence or absence of a mtNOS, we designed and carefully conducted properly controlled studies to either confirm or refute the existence of any NOS isoform within mitochondria. Ultrapure rat liver mitochondria were isolated using repeated differential centrifugation followed by Percoll gradient purification. Proteomic analyses were then performed using a nano-HPLC-coupled nanospray LTQ-MS. To avoid the interfering factors that are rampant in NO^· trapping assays (43), the NOS-catalyzed conversion of l-[¹⁴C]arginine to l-[¹⁴C]citrulline was used to probe for NOS activity in mitochondria. Appropriate controls were employed and, for inhibition studies, high concentrations of l-thiocitrulline (TC) (44) were used. Additionally, immunochemical analyses were performed with ultrapure mitochondria using nNOS, eNOS, and CaM antibodies. The problems faced with the commonly used techniques in mtNOS studies are discussed.     

Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Lysophosphatidic Acid Enhances Pulmonary Epithelial Barrier Integrity and Protects Endotoxin-induced Epithelial Barrier Disruption and Lung Injury

Lysophosphatidic acid (LPA), a bioactive phospholipid, induces a wide range of cellular effects, including gene expression, cytoskeletal rearrangement, and cell survival. We have previously shown that LPA stimulates secretion of pro- and anti-inflammatory cytokines in bronchial epithelial cells. This study provides evidence that LPA enhances pulmonary epithelial barrier integrity through protein kinase C (PKC) δ- and ζ-mediated E-cadherin accumulation at cell-cell junctions. Treatment of human bronchial epithelial cells (HBEpCs) with LPA increased transepithelial electrical resistance (TER) by ∼2.0-fold and enhanced accumulation of E-cadherin to the cell-cell junctions through Gα_i-coupled LPA receptors. Knockdown of E-cadherin with E-cadherin small interfering RNA or pretreatment with EGTA (0.1 mm) prior to LPA (1 μm) treatment attenuated LPA-induced increases in TER in HBEpCs. Furthermore, LPA induced tyrosine phosphorylation of focal adhesion kinase (FAK) and overexpression of the FAK inhibitor, and FAK-related non-kinase-attenuated LPA induced increases in TER and E-cadherin accumulation at cell-cell junctions. Overexpression of dominant negative protein kinase δ and ζ attenuated LPA-induced phosphorylation of FAK, accumulation of E-cadherin at cell-cell junctions, and an increase in TER. Additionally, lipopolysaccharide decreased TER and induced E-cadherin relocalization from cell-cell junctions to cytoplasm in a dose-dependent fashion, which was restored by LPA post-treatment in HBEpCs. Intratracheal post-treatment with LPA (5 μm) reduced LPS-induced neutrophil influx, protein leak, and E-cadherin shedding in bronchoalveolar lavage fluids in a murine model of acute lung injury. These data suggest a protective role of LPA in airway inflammation and remodeling.The airway epithelium is the site of first contact for inhaled environmental stimuli, functions as a physical barrier to environmental insult, and is an essential part of innate immunity. Epithelial barrier disruption is caused by inhaled allergens, dust, and irritants, resulting in inflammation, bronchoconstriction, and edema as seen in asthma and other respiratory diseases (1–4). Furthermore, increased epithelial permeability also results in para-cellular leakage of large proteins, such as albumin, immunoglobulin G, and polymeric immunoglobulin A, into the airway lumen (5, 6). The epithelial cell-cell junctional complex is composed of tight junctions, adherens junctions, and desmosomes. These adherens junctions play a pivotal role in regulating the activity of the entire junctional complex because the formation of adherens junctions subsequently leads to the formation of other cell-cell junctions (7–9). The major adhesion molecules in the adherens junctions are the cadherins. E-cadherin is a member of the cadherin family that mediates calcium-dependent cell-cell adhesion. The N-terminal ectodomain of E-cadherin contains homophilic interaction specificity, and the cytoplasmic domain binds to catenins, which interact with actin (10–13). Plasma membrane localization of E-cadherin is critical for the maintenance of epithelial cell-cell junctions and airway epithelium integrity (7, 10, 14). A decrease of adhesive properties of E-cadherin is related to the loss of differentiation and the subsequent acquisition of a higher motility and invasiveness of epithelial cells (10, 14, 15). Dislocation or shedding of E-cadherin in the airway epithelium induces epithelial shedding and increases airway permeability in lung airway diseases (10, 14, 16). In an ovalbumin-challenged guinea pig model of asthma, it has been demonstrated that E-cadherin is dislocated from the lateral margins of epithelial cells (10). Histamine increases airway para-cellular permeability and results in an increased susceptibility of airway epithelial cells to adenovirus infection by interrupting E-cadherin adhesion (14). Serine phosphorylation of E-cadherin by casein kinase II, GSK-3β, and PKD1/PKC² μ enhanced E-cadherin-mediated cell-cell adhesion in NIH3T3 fibroblasts and LNCaP prostate cancer cells (11, 17). However, the regulation and mechanism by which E-cadherin is localized within the pulmonary epithelium is not fully known, particularly during airway remodeling.LPA, a naturally occurring bioactive lipid, is present in body fluids, such as plasma, saliva, follicular fluid, malignant effusions, and bronchoalveolar lavage (BAL) fluids (18–20). Six distinct high affinity cell-surface LPA receptors, LPA-R_1–6, have been cloned and described in mammals (21–26). Extracellular activities of LPA include cell proliferation, motility, and cell survival (27–30). LPA exhibits a wide range of effects on differing cell types, including pulmonary epithelial, smooth muscle, fibroblasts, and T cells (31–35). LPA augments migration and cytokine synthesis in lymphocytes and induces chemotaxis of Jurkat T cells through Matrigel membranes (34). LPA induces airway smooth muscle cell contractility, proliferation, and airway repair and remodeling (35, 36). LPA also potently stimulates IL-8 (31, 37–39), IL-13 receptor α2 (IL-13Rα2) (40), and COX-2 gene expression and prostaglandin E₂ release (41) in HBEpCs. Prostaglandin E₂ and IL-13Rα2 have anti-inflammatory properties in pulmonary inflammation (42, 43). These results suggest that LPA may play a protective role in lung disease by stimulating an innate immune response while simultaneously attenuating the adaptive immune response. Furthermore, intravenous injection with LPA attenuated bacterial endotoxin-induced plasma tumor necrosis factor-α production and myeloperoxidase activity in the lungs of mice (44), suggesting an anti-inflammatory role for LPA in a murine model of sepsis.We have reported that LPA induces E-cadherin/c-Met accumulation in cell-cell contacts and increases TER in HBEpCs (45). Here, for the first time, we report that LPA-induced increases in TER are dependent on PKCδ, PKCζ, and FAK-mediated E-cadherin accumulation at cell-cell junctions. Furthermore, we demonstrate that post-treatment of LPA rescues LPS-induced airway epithelial disruption in vitro and reduces E-cadherin shedding in a murine model of ALI. This study identifies the molecular mechanisms linking the LPA and LPA receptors to maintaining normal pulmonary epithelium barrier function, which is critical in developing novel therapies directed at ameliorating pulmonary diseases.     

Printor, a Novel TorsinA-interacting Protein Implicated in Dystonia Pathogenesis

Early onset generalized dystonia (DYT1) is an autosomal dominant neurological disorder caused by deletion of a single glutamate residue (torsinA ΔE) in the C-terminal region of the AAA⁺ (ATPases associated with a variety of cellular activities) protein torsinA. The pathogenic mechanism by which torsinA ΔE mutation leads to dystonia remains unknown. Here we report the identification and characterization of a 628-amino acid novel protein, printor, that interacts with torsinA. Printor co-distributes with torsinA in multiple brain regions and co-localizes with torsinA in the endoplasmic reticulum. Interestingly, printor selectively binds to the ATP-free form but not to the ATP-bound form of torsinA, supporting a role for printor as a cofactor rather than a substrate of torsinA. The interaction of printor with torsinA is completely abolished by the dystonia-associated torsinA ΔE mutation. Our findings suggest that printor is a new component of the DYT1 pathogenic pathway and provide a potential molecular target for therapeutic intervention in dystonia.Early onset generalized torsion dystonia (DYT1) is the most common and severe form of hereditary dystonia, a movement disorder characterized by involuntary movements and sustained muscle spasms (1). This autosomal dominant disease has childhood onset and its dystonic symptoms are thought to result from neuronal dysfunction rather than neurodegeneration (2, 3). Most DYT1 cases are caused by deletion of a single glutamate residue at positions 302 or 303 (torsinA ΔE) of the 332-amino acid protein torsinA (4). In addition, a different torsinA mutation that deletes amino acids Phe³²³–Tyr³²⁸ (torsinA Δ323–328) was identified in a single family with dystonia (5), although the pathogenic significance of this torsinA mutation is unclear because these patients contain a concomitant mutation in another dystonia-related protein, ϵ-sarcoglycan (6). Recently, genetic association studies have implicated polymorphisms in the torsinA gene as a genetic risk factor in the development of adult-onset idiopathic dystonia (7, 8).TorsinA contains an N-terminal endoplasmic reticulum (ER)³ signal sequence and a 20-amino acid hydrophobic region followed by a conserved AAA⁺ (ATPases associated with a variety of cellular activities) domain (9, 10). Because members of the AAA⁺ family are known to facilitate conformational changes in target proteins (11, 12), it has been proposed that torsinA may function as a molecular chaperone (13, 14). TorsinA is widely expressed in brain and multiple other tissues (15) and is primarily associated with the ER and nuclear envelope (NE) compartments in cells (16–20). TorsinA is believed to mainly reside in the lumen of the ER and NE (17–19) and has been shown to bind lamina-associated polypeptide 1 (LAP1) (21), lumenal domain-like LAP1 (LULL1) (21), and nesprins (22). In addition, recent evidence indicates that a significant pool of torsinA exhibits a topology in which the AAA⁺ domain faces the cytoplasm (20). In support of this topology, torsinA is found in the cytoplasm, neuronal processes, and synaptic terminals (2, 3, 15, 23–26) and has been shown to bind cytosolic proteins snapin (27) and kinesin light chain 1 (20). TorsinA has been proposed to play a role in several cellular processes, including dopaminergic neurotransmission (28–31), NE organization and dynamics (17, 22, 32), and protein trafficking (27, 33). However, the precise biological function of torsinA and its regulation remain unknown.To gain insights into torsinA function, we performed yeast two-hybrid screens to search for torsinA-interacting proteins in the brain. We report here the isolation and characterization of a novel protein named printor (protein interactor of torsinA) that interacts selectively with wild-type (WT) torsinA but not the dystonia-associated torsinA ΔE mutant. Our data suggest that printor may serve as a cofactor of torsinA and provide a new molecular target for understanding and treating dystonia.