Isolation and Characterization of a Novel Lysine Racemase from a Soil Metagenomic Library

A lysine racemase (lyr) gene was isolated from a soil metagenome by functional complementation for the first time by using Escherichia coli BCRC 51734 cells as the host and d-lysine as the selection agent. The lyr gene consisted of a 1,182-bp nucleotide sequence encoding a protein of 393 amino acids with a molecular mass of about 42.7 kDa. The enzyme exhibited higher specific activity toward lysine in the l-lysine-to-d-lysine direction than in the reverse reaction.Amino acids are the building blocks of proteins and play an important role in the regulation of the metabolism of living organisms. Among two enantiomers of naturally occurring amino acids, l-amino acids are predominant in living organisms, while d-amino acids are found in both free and bound states in various organisms like bacteria (36), yeasts (35), plants (47), insects (11), mammals (17), bivalves (39), and fish (28). The d-amino acids are mostly endogenous and produced by racemization from their counterparts by the action of a racemase. Thus, the amino acid racemases are involved in d-amino acid metabolism (29, 46). Since the discovery of alanine racemase in 1951 (42), several racemases toward amino acids, such as those for glutamate, threonine, serine, aspartate, methionine, proline, arginine, and phenylalanine, have been reported in bacteria, archaea, and eukaryotes, including mammals (1, 2, 15, 30, 31, 44). They are classified into two groups: pyridoxal 5′-phosphate (PLP)-dependent and PLP-independent enzymes (9, 36).Lysine racemase (Lyr, EC 5.1.1.5) was first reported in Proteus vulgaris ATCC 4669 (19) and proposed to be involved in the lysine degradation of bacterial cells (5, 19). Catabolism of lysine occurs via two parallel pathways. In one of the pathways, δ-aminovalerate is the key metabolite, whereas in the other l-lysine is racemized to d-lysine, and l-pipecolate and α-aminoadipate (AMA) are the key metabolites (5). d-Lysine catabolism proceeds through a series of cyclized intermediates which are necessary to regenerate an α-amino acid and comprise the following metabolites (AMA pathway): d-lysine→α-keto-ɛ-amino caproate→Δ¹-piperideine-2-carboxylate→pipecolate→Δ¹-piperideine-6-carboxylate→α-amino-δ-formylcaproate→α-AMA→α-ketoadipate (6, 7, 12, 27). The final product is converted to α-ketoglutarate via a series of coenzyme A derivatives and subsequently participates as an intermediate in the Krebs cycle. This pathway suggests that the biological function of d-lysine in the bacteria is that of a carbon or nitrogen source. Racemization of added l-lysine to d-lysine by whole cells of Proteus spp. and Escherichia spp. (19) and by the cell extract of Pseudomonas putida ATCC 15070 (5, 20) has been found. However, the enzyme has not been purified to homogeneity, and thus, its molecular and catalytic characteristics, including its gene structure, have not been elucidated. In this study, we explored a metagenomic library constructed from a garden soil to isolate a novel Lyr enzyme. After expression in Escherichia coli, the purified enzyme was characterized in terms of optimal pH and temperature, thermal stability, and racemization activity.     

Delivery of Human Immunodeficiency Virus Vaccine Vectors to the Intestine Induces Enhanced Mucosal Cellular Immunity

Effective vaccines for human immunodeficiency virus type 1 (HIV-1) will likely need to stimulate protective immunity in the intestinal mucosa, where HIV-1 infection causes severe CD4⁺ T-cell depletion. While replication-competent recombinant adenovirus (rAd) vectors can stimulate adenovirus-specific mucosal immunity after replication, oral delivery of replication-defective rAd vectors encoding specific immunogens has proven challenging. In this study, we have systematically identified barriers to effective gut delivery of rAd vectors and identified sites and strategies to induce potent cellular and humoral immunity. Vector-mediated gene transfer by rAd5 was susceptible to low-pH buffer, gastric and pancreatic proteases, and extracellular mucins. Using ex vivo organ explants, we found that transduction with rAd5 was highest in the ileum and colon among all intestinal segments. Transgene expression was 100-fold higher after direct surgical introduction into the ileum than after oral gavage, with rAd5 showing greater potency than the rAd35 or the rAd41 vector. A single immunization of rAd5 encoding HIV-1 gp140B to the ileum stimulated potent CD8⁺ T-cell responses in the intestinal and systemic compartments, and these responses were further enhanced by intramuscular rAd5 boosting. These studies suggest that induction of primary immune responses by rAd5 gut immunization and subsequent systemic boosting elicits potent antigen-specific gut mucosal responses.Human immunodeficiency virus type 1 (HIV-1) infection is characterized by uncontrolled virus replication and cytopathicity in the intestinal mucosa, the site of major T-cell depletion after primary infection. The gastrointestinal (GI) tract is the predominant site of a pronounced CD4⁺ T-cell loss in the early stages of HIV infection and simian immunodeficiency virus (SIV) infection in the nonhuman primate model (3, 23, 26, 43). It has been suggested that a mucosal vaccine which generates HIV-specific CD8⁺ T cells in the gut could prevent the loss of CD4⁺ cells in gut-associated lymphoid tissue, establishment of infection, or spread of virus (13, 34). Therefore, targeted delivery of vaccines to the GI tract to stimulate mucosal responses has the potential to improve the efficacy of immune protection against HIV-1; however, the site of gene-based transduction and the barriers to vaccine delivery have not been well defined.Adenoviruses (Ads) have been used extensively as vectors for both gene transfer and vaccine development. They offer several advantages as tools for vaccine delivery, such as the ability to transduce both dividing and nondividing cells, relative safety and stability in vivo, ease of production in high titers, and lack of integration (2, 35). These vectors are promising because parenteral administration in both animals and humans has been shown to generate strong and long-lasting humoral and cellular immune responses. The immune responses surpass those achieved with other types of gene vectors and genetic vaccines (5, 38, 46). As a result, recombinant Ad (rAd) vectors have been developed and tested as vaccine vehicles to immunize against a number of pathogens (4, 10, 15, 18, 41).Orally (p.o.) delivered vaccines are attractive in theory because of their ease of administration and potential to deliver antigen to gut-associated lymphoid tissue, permitting induction of immune responses in both mucosal and systemic compartments. At the same time, p.o. delivery of replication-defective rAd vectors has posed a challenge and has met with variable levels of success. Immunization with rAd5 encoding rabies virus antigens, influenza virus antigens, or other antigens has generated some protection against infection in animal models (9, 27, 31, 39, 41), but p.o. immunization has elicited much lower CD8⁺ T-cell responses than systemic delivery (33), and a much higher dose is required to induce immune responses (37). We have recently shown in an HIV vaccine model that rAd41, a human enteric Ad-based vector, induced potent CD8⁺ T-cell responses in both systemic and mucosal compartments when primed p.o. or in the ileum (17). The previous study showed that rAd41 vectors delivered through direct ileal injection elicited mucosal cell immunity, but whether other rAd vectors could stimulate these responses and which factors affected delivery and immunogenicity were unknown. In this report, we have investigated the mechanisms associated with the low immunogenicity of rAd5 dosed through the p.o. route in mice. The purpose was to identify barriers to effective delivery of rAd vectors to gut tissues and to ascertain sites and strategies for induction of potent cellular and humoral immunity. To investigate the mechanism of the low immunogenicity of rAd vectors through the p.o. route and develop effective delivery of rAd5 and rare serotype rAd35 vectors as gut mucosal HIV vaccines, we have analyzed the obstacles to p.o. immunization, characterized vector transgene expression, and systematically compared immune responses induced by p.o. and local immunization strategies. These studies demonstrated that the higher immune responses were strongly associated with higher gene expression in the intestine and support further study of gut mucosal immunization in SIV challenge models as a potential HIV vaccine strategy.     

Lipid Phosphate Phosphatase 3 Stabilization of β-Catenin Induces Endothelial Cell Migration and Formation of Branching Point Structures

Endothelial cell (EC) migration, cell-cell adhesion, and the formation of branching point structures are considered hallmarks of angiogenesis; however, the underlying mechanisms of these processes are not well understood. Lipid phosphate phosphatase 3 (LPP3) is a recently described p120-catenin-associated integrin ligand localized in adherens junctions (AJs) of ECs. Here, we tested the hypothesis that LPP3 stimulates β-catenin/lymphoid enhancer binding factor 1 (β-catenin/LEF-1) to induce EC migration and formation of branching point structures. In subconfluent ECs, LPP3 induced expression of fibronectin via β-catenin/LEF-1 signaling in a phosphatase and tensin homologue (PTEN)-dependent manner. In confluent ECs, depletion of p120-catenin restored LPP3-mediated β-catenin/LEF-1 signaling. Depletion of LPP3 resulted in destabilization of β-catenin, which in turn reduced fibronectin synthesis and deposition, which resulted in inhibition of EC migration. Accordingly, reexpression of β-catenin but not p120-catenin in LPP3-depleted ECs restored de novo synthesis of fibronectin, which mediated EC migration and formation of branching point structures. In confluent ECs, however, a fraction of p120-catenin associated and colocalized with LPP3 at the plasma membrane, via the C-terminal cytoplasmic domain, thereby limiting the ability of LPP3 to stimulate β-catenin/LEF-1 signaling. Thus, our study identified a key role for LPP3 in orchestrating PTEN-mediated β-catenin/LEF-1 signaling in EC migration, cell-cell adhesion, and formation of branching point structures.Angiogenesis, the formation of new blood vessels, involves several well-coordinated cellular processes, including endothelial cell (EC) migration, synthesis and deposition of extracellular matrix proteins, such as fibronectin, cell-cell adhesion, and formation of branching point structures (1-3, 19, 33); however, less is known about the underlying mechanisms of these processes (6, 8, 12, 14, 16, 17). For example, adherens junctions (AJs), which mediate cell-cell adhesion between ECs, may be involved in limiting the extent of cell migration (2, 14, 38, 40). VE-cadherin, a protein found in AJs, is a single-pass transmembrane polypeptide responsible for calcium-dependent homophilic interactions through its extracellular domains (2, 38, 40). The VE-cadherin cytoplasmic domain interacts with the Armadillo domain-containing proteins, β-catenin, γ-catenin (plakoglobin), and p120-catenin (p120ctn) (2, 15, 38, 40, 43). Genetic and biochemical evidence documents a crucial role of β-catenin in regulating cell adhesion as well as proliferation secondary to the central position of β-catenin in the Wnt signaling pathway (13, 16, 25, 31, 44). In addition, the juxtamembrane protein p120ctn regulates AJ stability via binding to VE-cadherin (2, 7, 9, 15, 21, 28, 32, 43). The absence of regulation or inappropriate regulation of β-catenin and VE-cadherin functions is linked to cardiovascular disease and tumor progression (2, 6).We previously identified lipid phosphate phosphatase 3 (LPP3), also known as phosphatidic acid phosphatase 2b (PAP2b), in a functional assay of angiogenesis (18, 19, 41, 42). LPP3 not only exhibits lipid phosphatase activity but also functions as a cell-associated integrin ligand (18, 19, 35, 41, 42). The known LPPs (LPP1, LPP2, and LPP3) (20-23) are six transmembrane domain-containing plasma membrane-bound enzymes that dephosphorylate sphingosine-1-phosphate (S1P) and its structural homologues, and thus, these phosphatases generate lipid mediators (4, 5, 23, 35, 39). All LPPs, which contain a single N-glycosylation site and a putative lipid phosphatase motif, are situated such that their N and C termini are within the cell (4, 5, 22, 23, 35, 39). Only the LPP3 isoform contains an Arg-Gly-Asp (RGD) sequence in the second extracellular loop, and this RGD sequence enables LPP3 to bind integrins (18, 19, 22). Transfection experiments with green fluorescent protein (GFP)-tagged LPP1 and LPP3 showed that LPP1 is apically sorted, whereas LPP3 colocalized with E-cadherin at cell-cell contact sites with other Madin-Darby canine kidney (MDCK) cells (22). Mutagenesis and domain swapping experiments established that LPP1 contains an apical targeting signal sequence (FDKTRL) in its N-terminal segment. In contrast, LPP3 contains a dityrosine (109Y/110Y) basolateral sorting motif (22). Interestingly, conventional deletion of Lpp3 is embryonic lethal, since the Lpp3 gene plays a critical role in extraembryonic vasculogenesis independent of its lipid phosphatase activity (11). In addition, an LPP3-neutralizing antibody was shown to prevent cell-cell interactions (19, 42) and angiogenesis (42). Here, we addressed the hypothesis that LPP3 plays a key role in EC migration, cell-cell adhesion, and formation of branching point structures by stimulating β-catenin/lymphoid enhancer binding factor 1 (β-catenin/LEF-1) signaling.     

Andes Virus Recognition of Human and Syrian Hamster β3 Integrins Is Determined by an L33P Substitution in the PSI Domain

Andes virus (ANDV) causes a fatal hantavirus pulmonary syndrome (HPS) in humans and Syrian hamsters. Human α_vβ₃ integrins are receptors for several pathogenic hantaviruses, and the function of α_vβ₃ integrins on endothelial cells suggests a role for α_vβ₃ in hantavirus directed vascular permeability. We determined here that ANDV infection of human endothelial cells or Syrian hamster-derived BHK-21 cells was selectively inhibited by the high-affinity α_vβ₃ integrin ligand vitronectin and by antibodies to α_vβ₃ integrins. Further, antibodies to the β₃ integrin PSI domain, as well as PSI domain polypeptides derived from human and Syrian hamster β₃ subunits, but not murine or bovine β₃, inhibited ANDV infection of both BHK-21 and human endothelial cells. These findings suggest that ANDV interacts with β₃ subunits through PSI domain residues conserved in both Syrian hamster and human β₃ integrins. Sequencing the Syrian hamster β₃ integrin PSI domain revealed eight differences between Syrian hamster and human β₃ integrins. Analysis of residues within the PSI domains of human, Syrian hamster, murine, and bovine β₃ integrins identified unique proline substitutions at residues 32 and 33 of murine and bovine PSI domains that could determine ANDV recognition. Mutagenizing the human β₃ PSI domain to contain the L33P substitution present in bovine β₃ integrin abolished the ability of the PSI domain to inhibit ANDV infectivity. Conversely, mutagenizing either the bovine PSI domain, P33L, or the murine PSI domain, S32P, to the residue present human β₃ permitted PSI mutants to inhibit ANDV infection. Similarly, CHO cells transfected with the full-length bovine β₃ integrin containing the P33L mutation permitted infection by ANDV. These findings indicate that human and Syrian hamster α_vβ₃ integrins are key receptors for ANDV and that specific residues within the β₃ integrin PSI domain are required for ANDV infection. Since L33P is a naturally occurring human β₃ polymorphism, these findings further suggest the importance of specific β₃ integrin residues in hantavirus infection. These findings rationalize determining the role of β₃ integrins in hantavirus pathogenesis in the Syrian hamster model.Hantaviruses persistently infect specific small mammal hosts and are spread to humans by the inhalation of aerosolized excreted virus (41, 42). Hantaviruses predominantly infect endothelial cells and cause one of two vascular leak-based diseases: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) (41). Hantavirus diseases are characterized by increased vascular permeability and acute thrombocytopenia in the absence of endothelial cell lysis (36, 41, 42, 54). In general, hantaviruses are not spread from person to person; however, the Andes hantavirus (ANDV) is an exception, since there are several reports of person-to-person transmission of ANDV infection (11, 37, 47, 52). ANDV is also unique in its ability to cause an HPS-like disease in Syrian hamsters and serves as the best-characterized hantavirus disease model with a long onset, symptoms, and pathogenesis nearly identical to that of HPS patients (20, 21, 50).Hantavirus infection of the endothelium alters endothelial cell barrier functions through direct and immunological responses (8, 14). Although the means by which hantaviruses cause pulmonary edema or hemorrhagic disease has been widely conjectured, the mechanisms by which hantaviruses elicit pathogenic human responses have yet to be defined. Hantaviruses coat the surface of infected VeroE6 cells days after infection (17), and this further suggests that dynamic hantavirus interactions with immune and endothelial cells are likely to contribute to viral pathogenesis. Hantavirus pathogenesis has been suggested to involve CD8⁺ T cells, tumor necrosis factor alpha or other cytokines, viremia, and the dysregulation of β₃ integrins (7, 8, 13-16, 25-28, 32, 34, 38, 44-46). However, these responses have not been demonstrated to contribute to hantavirus pathogenesis, and in some cases there are conflicting data on their involvement (18, 25-28, 34, 35, 44, 45, 48). Immune complex deposition clearly contributes to HFRS patient disease and renal sequelae (4, 7), but it is unclear what triggers vascular permeability in HPS and HFRS diseases or why hemorrhage occurs in HFRS patients but not in HPS patients (8, 36, 54). Acute thrombocytopenia is common to both diseases, and platelet dysfunction resulting from defective platelet aggregation is reported in HFRS patients (7, 8).Pathogenic hantaviruses have in common their ability to interact with α_IIbβ₃ and α_vβ₃ integrins present on platelets and endothelial cells (13, 16), and β₃ integrins have primary roles in regulating vascular integrity (1, 2, 6, 19, 22, 39, 40). Consistent with the presence of cell surface displayed virus (17), pathogenic hantaviruses uniquely block α_vβ₃ directed endothelial cell migration and enhance endothelial cell permeability for 3 to 5 days postinfection (14, 15). Pathogenic hantaviruses dysregulate β₃ integrin functions by binding domains present at the apex of inactive β₃ integrin conformers (38). α_vβ₃ forms a complex with vascular endothelial cell growth factor receptor 2 (VEGFR2) and normally regulates VEGF-directed endothelial cell permeability (2, 3, 10, 39, 40). However, both β₃ integrin knockouts and hantavirus-infected endothelial cells result in increased VEGF-induced permeability, presumably by disrupting VEGFR2-β₃ integrin complex formation (2, 14, 19, 39, 40). This suggests that at least one means for hantaviruses to increase vascular permeability occurs through interactions with β₃ integrins that are required for normal platelet and endothelial cell functions.α_vβ₃ and α_IIbβ₃ integrins exist in two conformations: an active extended conformation where the ligand binding head domain is present at the apex of the heterodimer and a basal, inactive bent conformation where the globular head of the integrin is folded toward the cell membrane (30, 53, 55). Pathogenic HTN and NY-1 hantaviruses bind to the N-terminal plexin-semaphorin-integrin (PSI) domain of β₃ integrin subunits and are selective for bent, inactive α_vβ₃ integrin conformers (38). Pathogenic hantavirus binding to inactive α_vβ₃ integrins is consistent with the selective inhibitory effect of hantaviruses on α_vβ₃ function and endothelial cell permeability (14, 15, 38). Although the mechanism of hantavirus induced vascular permeability has yet to be defined, there is a clear role for β₃ integrin dysfunction in vascular permeability deficits (5, 6, 22, 29, 39, 40, 51) which make an understanding of hantavirus interactions with β₃ subunits important for both entry and disease processes.The similarity between HPS disease in humans and Syrian hamsters (20, 21) suggests that pathogenic mechanisms of ANDV disease are likely to be coincident. Curiously, other hantaviruses (Sin Nombre virus [SNV] and Hantaan virus [HTNV]) are restricted in Syrian hamsters and fail to cause disease in this animal, even though they are prominent causes of human disease (50). Although the host range restriction for SNV and HTNV in Syrian hamsters has not been defined (33), the pathogenesis of ANDV in Syrian hamsters suggests that both human and Syrian hamster β₃ integrins may similarly be used by ANDV and contribute to pathogenesis.We demonstrate here that ANDV infection of the Syrian hamster BHK-21 cell line and human endothelial cells is dependent on α_vβ₃ and inhibited by α_vβ₃ specific ligands and antibodies. Further, polypeptides expressing the N-terminal 53 residues of human and Syrian hamster β₃ subunits block ANDV infection. This further indicates that ANDV interaction with the N-terminal 53 residues of both human and Syrian hamster β₃ integrins is required for viral entry. We also demonstrate that ANDV recognition of human and Syrian hamster β₃ integrins is determined by proline substitutions at residues 32/33 within the β₃ integrin PSI domain. These results define unique ANDV interactions with human and Syrian hamster β₃ integrins.     

Potential Application of N-Carbamoyl-β-Alanine Amidohydrolase from Agrobacterium tumefaciens C58 for β-Amino Acid Production

An N-carbamoyl-β-alanine amidohydrolase of industrial interest from Agrobacterium tumefaciens C58 (βcar_At) has been characterized. βcar_At is most active at 30°C and pH 8.0 with N-carbamoyl-β-alanine as a substrate. The purified enzyme is completely inactivated by the metal-chelating agent 8-hydroxyquinoline-5-sulfonic acid (HQSA), and activity is restored by the addition of divalent metal ions, such as Mn²⁺, Ni²⁺, and Co²⁺. The native enzyme is a homodimer with a molecular mass of 90 kDa from pH 5.5 to 9.0. The enzyme has a broad substrate spectrum and hydrolyzes nonsubstituted N-carbamoyl-α-, -β-, -γ-, and -δ-amino acids, with the greatest catalytic efficiency for N-carbamoyl-β-alanine. βcar_At also recognizes substrate analogues substituted with sulfonic and phosphonic acid groups to produce the β-amino acids taurine and ciliatine, respectively. βcar_At is able to produce monosubstituted β²- and β³-amino acids, showing better catalytic efficiency (k_cat/K_m) for the production of the former. For both types of monosubstituted substrates, the enzyme hydrolyzes N-carbamoyl-β-amino acids with a short aliphatic side chain better than those with aromatic rings. These properties make βcar_At an outstanding candidate for application in the biotechnology industry.N-Carbamoyl-β-alanine amidohydrolase (NCβAA) (EC 3.5.1.6), also known as β-alanine synthase or β-ureidopropionase, catalyzes the third and final step of reductive pyrimidine degradation. In this reaction, N-carbamoyl-β-alanine or N-carbamoyl-β-aminoisobutyric acid is irreversibly hydrolyzed to CO₂, NH₃, and β-alanine or β-aminoisobutyric acid, respectively (43). Eukaryotic NCβAAs have been purified from several sources (10, 25, 33, 39, 42, 44). Nevertheless, only two prokaryotic NCβAAs, belonging to the Clostridium and Pseudomonas genera (4, 29), have been purified to date, although this activity has been inferred for several microorganisms due to the appearance of the reductive pathway of pyrimidine degradation (38, 45). Pseudomonas NCβAA is also able to hydrolyze l-N-carbamoyl-α-amino acids, and indeed, this activity is widespread in the bacterial kingdom (3, 23, 26, 46).β-Amino acids have unique pharmacological properties, and their utility as building blocks of β-peptides, pharmaceutical compounds, and natural products is of growing interest (14). β-Alanine, a natural β-amino acid, is a precursor of coenzyme A and pantothenic acid in bacteria and fungi (vitamin B₅) (7). β-Alanine is widely distributed in the central nervous systems of vertebrates and is a structural analogue of γ-amino-n-butyric acid and glycine, major inhibitory neurotransmitters, suggesting that it may be involved in synaptic transmissions (20). Another important natural β-amino acid is taurine (2-aminoethanesulfonic acid), which plays an important role in several essential processes, such as membrane stabilization, osmoregulation, glucose metabolism, antioxidation, and development of the central nervous system and the retina (9, 28, 33). 2-Aminoethylphosphonate, the most common naturally occurring phosphonate, also known as ciliatine, is an important precursor used in the biosynthesis of phosphonolipids, phosphonoproteins, and phosphonoglycans (5). β-Homoalanine (β-aminobutyric acid) has been used successfully for the design of nonnatural ligands for therapeutic application against autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, or autoimmune uveitis (30). Substituted β-amino acids can be denominated β², β³, and β^2,3, depending on the position of the side chain(s) (R) on the amino acid skeleton (18). β²-Amino acids are not yet as readily available as their β³-counterparts, as they must be prepared using multistep procedures (17).We decided to characterize NCβAA (β-carbamoylase) from Agrobacterium tumefaciens C58 (βcar_At) after showing that some dihydropyrimidinases belonging to the Arthrobacter and Sinorhizobium genera are able to hydrolyze different 5- or 6-substituted dihydrouracils to the corresponding N-carbamoyl-β-amino acids (18, 22). If βcar_At could decarbamoylate the reaction products of dihydrouracils, different β-amino acids would be obtained enzymatically in the same way that α-amino acids are produced via the hydantoinase process (6, 21). We therefore describe the physical, biochemical, kinetic, and substrate specificity properties of recombinant βcar_At.