Identification and characterization of a mouse dipeptidase that hydrolyzes L-carnosine

L-Carnosine is a bioactive dipeptide present in mammalian tissues including the central nervous system. We have recently shown that L-carnosine is involved in the regulation of energy homeostasis through the autonomic nervous system, but the mechanisms for its biosynthesis and degradation have not yet been fully elucidated. Here we report the biochemical and immunohistochemical characterization of a mammalian protein that has a 17% overall amino acid sequence homology with a Lactobacilus carnosinase, PepV. A recombinant protein expressed in E. coli has the enzymatic ability to digest L-carnosine and various other dipeptides, and this activity is inhibited by bestatin. It requires Mn2+ for enzymatic activity and its effect is reversible. Immunohistochemical analysis showed that a few neuronal populations express this protein at very high levels. It is highly expressed in the parafascicular nucleus of the thalamus, tuberomammillary nucleus of the hypothalamus and the mitral cell layer of the olfactory bulb. In addition, neuronal processes, but not cell bodies, are stained in the striatum. In all these areas, the protein did not colocalize with the glial fibrilary acidic protein. These results suggest that a peptidase that digests L-carnosine is enriched in several specific neuronal populations in the central nervous system.     

Cloning and characterization of a novel human alkaline ceramidase. A mammalian enzyme that hydrolyzes phytoceramide

Ceramidases are enzymes involved in regulating cellular levels of ceramides, sphingoid bases, and their phosphates. Based on sequence homology to the yeast alkaline ceramidases YPC1p (Mao, C., Xu, R., Bielawska, A., and Obeid, L. M. (2000) J. Biol. Chem. 275, 6876--6884) and YDC1p (Mao, C., Xu, R., Bielawska, A., Szulc, Z. M., and Obeid, L. M. (2000) J. Biol Chem. 275, 31369--31378), we report the identification and cloning of a cDNA encoding for a novel human alkaline ceramidase (aPHC) that hydrolyzes phytoceramide selectively. Northern blot analysis showed that aPHC was ubiquitously expressed, with the highest expression in placenta. Green fluorescent protein tagging showed that it was localized in both the Golgi apparatus and endoplasmic reticulum. Overexpression of aPHC in mammalian cells elevated in vitro ceramidase activity toward N-4-nitrobenz-2-oxa-1,3-diazole-C(12)-phytoceramide. Its expression in a yeast mutant strain devoid of any ceramidase activity restored the ceramidase activity and caused an increase in the hydrolysis of phytoceramide in yeast cells, thus leading to the decreased biosynthesis of sphingolipids. These data collectively suggest that, similar to the yeast phytoceramidase YPC1p, aPHC has phytoceramidase activity both in vitro and in cells; hence, it is a functional homolog of the yeast phytoceramidase YPC1p. However, in contrast to YPC1p, aPHC exhibited no reverse activity of ceramidase either in vitro or in cells. Biochemical characterization showed that aPHC had a pH optimum of 9.5, was activated by Ca(2+), but was inhibited by Zn(2+) and sphingosine. Substrate specificity showed that aPHC hydrolyzed phytoceramide preferentially. Together, these data demonstrate that aPHC is a novel human alkaline phytoceramidase, the first mammalian alkaline ceramidase to be identified as being specific for the hydrolysis of phytoceramide.     

Identification of a Saccharomyces cerevisiae glucosidase that hydrolyzes flavonoid glucosides

Baker's yeast (Saccharomyces cerevisiae) whole-cell bioconversions of naringenin 7-O-β-glucoside revealed considerable β-glucosidase activity, which impairs any strategy to generate or modify flavonoid glucosides in yeast transformants. Up to 10 putative glycoside hydrolases annotated in the S. cerevisiae genome database were overexpressed with His tags in yeast cells. Examination of these recombinant, partially purified polypeptides for hydrolytic activity with synthetic chromogenic α- or β-glucosides identified three efficient β-glucosidases (EXG1, SPR1, and YIR007W), which were further assayed with natural flavonoid β-glucoside substrates and product verification by thin-layer chromatography (TLC) or high-performance liquid chromatography (HPLC). Preferential hydrolysis of 7- or 4'-O-glucosides of isoflavones, flavonols, flavones, and flavanones was observed in vitro with all three glucosidases, while anthocyanins were also accepted as substrates. The glucosidase activities of EXG1 and SPR1 were completely abolished by Val168Tyr mutation, which confirmed the relevance of this residue, as reported for other glucosidases. Most importantly, biotransformation experiments with knockout yeast strains revealed that only EXG1 knockout strains lost the capability to hydrolyze flavonoid glucosides.     

Identification and characterization of a novel protein that regulates RNA-protein interaction

In a previous study [Nachaliel et al., 1993], we identified an RNA-binding protein (RBP) in FTO-2B rat hepatoma cells whose activity was stimulated upon the dissociation of a protein factor. We report in this article that the RBP is a complex protein of about 400 kDa, composed of RNA-binding subunit(s) (RBS), and regulatory subunit(s) (RS). We purified the RS to near-homogeneity (Mr approximately 25,000) and determined the amino acid sequence of a peptide derived from RS. On the basis of this sequence information, the cDNA for RS was obtained. Recombinant RS protein expressed in Escherichia coli had the capacity to bind RBS and inhibit its RNA-binding activity. The cDNA contains the complete coding sequence because the recombinant protein has the same electrophoretic mobility as that of the native RS in SDS-polyacrylamide gels. Sequence comparison showed that RS is almost identical to DJ-1, a recently discovered protein with an oncogenic potential, and CAP1, a rat sperm protein. However, the protein does not contain any known motifs that can provide a clue as to its exact function. Indirect immunofluorescence analyses showed that in addition to the cytoplasm, where RS is associated with microtubular filaments, the polypeptide is localized to the cell nucleus. The possible role of RS is discussed.     

Identification and characterization of a novel (S)-ketoprofen-specific esterase

A new (S)-ketoprofen specific esterase (EST-Y29) was identified from a metagenome library from environmental samples, which showed homologies with class C-beta lactamase, penicillin binding protein, and other lipases/esterases. In order to investigate the biochemical and biophysical properties, the recombinant protein was overexpressed, purified to homogeneity, and characterized. This EST-Y29 has high catalytic activity against p-nitrophenyl esters of short fatty acids (C(2) and C(4)) and alpha-naphthyl acetate with activation energy of 30.4 kJ/mol. We have further characterized EST-Y29 using high performance liquid chromatography (HPLC), circular dichroism (CD), dynamic light scattering (DLS) and size exclusion chromatography (SEC).     

Identification and characterization of a novel Nogo-interacting mitochondrial protein (NIMP)

Nogo is a potent inhibitor of regeneration following spinal cord injury. To develop a better understanding of the mechanisms responsible for regenerative failure we used a yeast two-hybrid approach to try and identify proteins that interact with Nogo. We identified a novel mitochondrial protein designated Nogo-interacting mitochondrial protein (NIMP) in a screen of an adult human brain cDNA library. This interaction was confirmed by co-immunoprecipitation in both brain tissue (endogenous) and transfected HEK293T cells (overexpressed). In support of these studies we demonstrate that Nogo interacts with the UQCRC1 and UQCRC2 components of complex III, within the mitochondrial respiratory chain. The mitochondrial localization of NIMP was evidenced by confocal image analysis and western blot analysis of isolated mitochondria. NIMP is highly conserved and ubiquitously expressed in mitochondria-enriched tissues. Within the CNS, NIMP-like immunoreactivity is present in neurons and astrocytes. These data suggest that NIMP is a novel mitochondrial protein that interacts with Nogo. The interaction of Nogo with mitochondrial proteins may provide insight into the mechanisms for Nogo-induced inhibition of neurite growth.     

Identification of a novel intracellular cholesteryl ester hydrolase (carboxylesterase 3) in human macrophages: compensatory increase in its expression after carboxylesterase 1 silencing

Cholesteryl ester (CE) hydrolysis is the rate-limiting step in the removal of free cholesterol (FC) from macrophage foam cells, and several enzymes have been identified as intracellular CE hydrolases in human macrophages. We have previously reported the antiatherogenic role of a carboxylesterase [carboxylesterase 1 (CES1)], and the objective of the present study was to determine the contribution of CES1 to total CE hydrolytic activity in human macrophages. Two approaches, namely, immune depletion and short hairpin (sh)RNA-mediated knockdown, were used. Immuneprecipitation by a CES1-specific antibody resulted in a 70-80% decrease in enzyme activity, indicating that CES1 is responsible for >70% of the total CE hydrolytic activity. THP1-shRNA cells were generated by stably transfecting human THP1 cells with four different CES1-specific shRNA vectors. Despite a significant (>90%) reduction in CES1 expression both at the mRNA and protein levels, CES1 knockdown neither decreased intracellular CE hydrolysis nor decreased FC efflux. Examination of the underlying mechanisms for the observed lack of effects of CES1 knockdown revealed a compensatory increase in the expression of a novel CES, CES3, which is only expressed at <30% of the level of CES1 in human macrophages. Transient overexpression of CES3 led to an increase in CE hydrolytic activity, mobilization of intracellular lipid droplets, and a reduction in cellular CE content, establishing CES3 as a bona fide CE hydrolase. This study provides the first evidence of functional compensation whereby increased expression of CES3 restores intracellular CE hydrolytic activity and FC efflux in CES1-deficient cells. Furthermore, these data support the concept that intracellular CE hydrolysis is a multienzyme process.     

Identification and characterization of novel immunomodulatory bursal-derived pentapeptide-II (BPP-II)

The bursa of Fabricius, the acknowledged central humoral immune organ, plays a vital role in B lymphocyte differentiation. However, there are few reports of the molecular basis of the mechanism on immune induction and potential antitumor activity of bursal-derived peptides. In this paper, a novel bursal-derived pentapeptide-II (BPP-II, MTLTG) was isolated and exerted immunomodulatory functions on antibody responses in vitro. Gene microarray analyses demonstrated that BPP-II regulated expression of 2478 genes in a mouse-derived hybridoma cell line. Immune-related gene ontology functional procedures were employed for further functional analysis. Furthermore, the majority of BPP-II-regulated pathways were associated with immune responses and tumor processes. Moreover, BPP-II exhibited immunomodulatory effects on antigen-specific immune responses in vivo, including enhancement of avian influenza virus (H9N2 subtype)-specific antibody and cytokine production and modification of T cell immunophenotypes and lymphocyte proliferation. Finally, BPP-II triggered p53 expression and stabilization and selectively inhibited tumor cell proliferation. These data identified the multifunctional factor, BPP-II, as a novel biomaterial representing an important linking between the humoral central immune system and immune induction, including antitumor. Information generated in this study elucidates further the mechanisms involved in humoral immune system and represents the potential basis of effective immunotherapeutic strategies for treating human tumors and immune improvement.     

Identification of an enzymatic activity that hydrolyzes protein-bound ADP-ribose in skeletal muscle

An enzymatic activity present in high-speed supernatant fluids of rat skeletal muscle was found that catalyzes the release of ADP-ribose from ADP-ribosylated-modified lysozyme. The nature of the product was proved by chromatographic studies and proton nuclear magnetic resonance spectroscopy. The enzyme activity is stimulated by Mg2+, dithioerythritol, and flouride. These results and those published earlier (Soman, G., Mickelson, J.R., Louis, C.F., and Graves, D.J. (1984) Biochem. Biophys. Res. Commun. 120, 973-980) show that ADP-ribosylation is a reversible process in skeletal muscle.     

Identification and characterization of a novel outer membrane protein (OMP J) of Moraxella catarrhalis that exists in two major forms

Moraxella catarrhalis is a common commensal of the human respiratory tract that has been associated with a number of disease states, including acute otitis media in children and exacerbations of chronic obstructive pulmonary disease in adults. During studies to investigate the outer membrane proteins of this bacterium, two novel major proteins, of approximately 19 kDa and 16 kDa (named OMP J1 and OMP J2, respectively), were identified. Further analysis indicated that these two proteins possessed almost identical gene sequences, apart from two insertion/deletion events in predicted external loops present within the putative barrel-like structure of the proteins. The development of a PCR screening strategy found a 100% (96/96) incidence for the genes encoding the OMP J1 and OMP J2 proteins within a set of geographically diverse M. catarrhalis isolates, as well as a significant association of OMP J1/OMP J2 with both the genetic lineage and the complement resistance phenotype (Fisher's exact test; P < 0.01). Experiments using two DeltaompJ2 mutants (one complement resistant and the other complement sensitive) indicated that both were less easily cleared from the lungs of mice than were their isogenic wild-type counterparts, with a significant difference in bacterial clearance being observed for the complement-resistant isolate but not for its isogenic DeltaompJ2 mutant (unpaired Student's t test; P < 0.001 and P = 0.32). In this publication, we characterize a novel outer membrane protein of Moraxella catarrhalis which exists in two variant forms associated with particular genetic lineages, and both forms are suggested to contribute to bacterial clearance from the lungs.     

Purification and characterization of a lactonase from Burkholderia sp. R-711, that hydrolyzes (R)-5-oxo-2-tetrahydrofurancarboxylic acid

A lactonase hydrolyzing (R)-5-oxo-2-tetrahydrofurancarboxylic acid to D-alpha-hydroxyglutaric acid was purified 170-fold with 2% recovery to near homogeneity from crude extracts of Burkholderia sp. R-711, which had been isolated as a bacterium able to assimilate (R)-5-oxo-2-tetrahydrofurancarboxylic acid. The molecular mass was estimated to be 33 kDa by gel filtration. The purified preparation migrated as a single band of molecular mass 38 kDa upon SDS-PAGE. The maximum activity was observed at pH 7.0-8.0 and 35-40 degrees C. The enzyme required no added cofactors or metal ions; the activity was inhibited to 60-100% by SH-blocking reagents, but was not affected by metal-chelating reagents. The enzyme showed lower activity and affinity toward (S)-5-oxo-2-tetrahydrofurancarboxylic acid, but did not act on other natural and synthetic lactones tested.     

Partial purification and characterization of a rat kidney neutral endopeptidase that hydrolyzes succinyl trialanine-4-nitroanilide

The activity for the hydrolysis of succinyl trialanine-4-nitroanilide was higher in kidney homogenates of female rats and mice than in those of male rats and mice. An enzyme hydrolyzing the above substrate was extracted from female rat kidney homogenate and partially purified by means of gel filtration on Sepharose 4B, anion-exchange chromatography on DEAE-Sepharose CL-6B and affinity chromatography on carbobenzoxy-L-Ala-L-Ala-D-Ala-polylysine-agarose. The purified enzyme cleaved the bond between succinyl dialanine and alanine-4-nitroanilide of the substrate and showed a Km value of 3.3 mM at the optimal pH of 7.5. The activity was increased by Ca2+ and Mg2+, but inhibited by EDTA. With oxidized insulin B chain as a substrate, the enzyme cleaved the carbonyl bonds of Ala-14, Tyr-16 and Gly-23 efficiently, and those of His-5 and His-10 less efficiently.     

A β-glucosidase in feline kidney that hydrolyzes amygdalin (Laetrile)

A β-glucosidase has been demonstrated in cat, rat, and rabbit kidney tissue that catalyzes the hydrolytic cleavage of the terminal glucose residue of amygdalin (mandelonitrile-β-gentiobioside). The enzyme was partially purified from feline kidney and its properties were determined. Although the natural substrates for the enzyme are unknown at this time, this β-glucosidase requires an aryl or unsaturated alkyl aglycone moiety for catalytic function. The unusual species and tissue distribution of the enzyme have considerable implications for cancer chemotherapeutic trials with amygdalin (Laetrile).     

Identification and characterization of novel lysine-independent apolipoprotein(a)-binding sites in fibrin(ogen) alphaC-domains

Accumulation of lipoprotein(a) (Lp(a)) in atherosclerotic plaques is mediated through interaction of fibrin-(ogen) deposits with the apolipoprotein(a) (apo(a)) moiety of Lp(a). It was suggested that because apo(a) competes with plasminogen for binding to fibrin, causing inhibition of fibrinolysis, it could also promote atherothrombosis. Because the fibrin(ogen) alphaC-domains bind plasminogen and tissue-type plasminogen activator with high affinity in a Lys-dependent manner, we hypothesized that they could also bind apo(a). To test this hypothesis, we studied the interaction between the recombinant apo(a) A10 isoform and the recombinant alphaC-fragment (Aalpha-(221-610)) corresponding to the alphaC-domain by enzyme-linked immunosorbent assay and surface plasmon resonance. Both methods revealed a high affinity interaction (Kd = 19-21 nm) between the immobilized alphaC-fragment and apo(a), indicating that the former contains an apo(a)-binding site. This affinity was comparable to that of apo(a) for fibrin. At the same time, no interaction was observed between soluble fibrinogen and immobilized apo(a), suggesting that, in the former, this and other apo(a)-binding sites are cryptic. Further experiments with truncated recombinant variants of the alphaC-fragment allowed localization of the apo(a)-binding site to the Aalpha-(392-610) region. The presence of epsilon-aminocaproic acid only slightly inhibited binding of apo(a) to the alphaC-fragment, indicating the Lys-independent nature of their interaction. In agreement, the influence of plasminogen or tissue-type plasminogen activator on binding of apo(a) to the alphaC-fragment was minimal. These results indicate that the alphaC-domains contain novel high affinity apo(a)-binding sites that may provide a Lys-independent mechanism for bringing Lp(a) to places of fibrin deposition such as injured vessels or atherosclerotic lesions.     

Identification and characterization of a novel human myeloid inhibitory C-type lectin-like receptor (MICL) that is predominantly expressed on granulocytes and monocytes

Inhibitory and activatory C-type lectin-like receptors play an important role in immunity through the regulation of leukocytes. Here, we report the identification and characterization of a novel myeloid inhibitory C-type lectin-like receptor (MICL) whose expression is primarily restricted to granulocytes and monocytes. This receptor, which contains a single C-type lectin-like domain and a cytoplasmic immunoreceptor tyrosine-based inhibitory motif, is related to LOX-1 (lectin-like receptor for oxidized low density lipoprotein-1) and the beta-glucan receptor (Dectin-1) and is variably spliced and highly N-glycosylated. We demonstrate that it preferentially associates with the signaling phosphatases SHP-1 and SHP-2, but not with SHIP. Novel chimeric analyses with a construct combining MICL and the beta-glucan receptor show that MICL can inhibit cellular activation through its cytoplasmic immunoreceptor tyrosine-based inhibitory motif. These data suggest that MICL is a negative regulator of granulocyte and monocyte function.     

Identification and characterization of a novel polysaccharide deacetylase C (PdaC) from Bacillus subtilis

Cell wall metabolism and cell wall modification are very important processes that bacteria use to adjust to various environmental conditions. One of the main modifications is deacetylation of peptidoglycan. The polysaccharide deacetylase homologue, Bacillus subtilis YjeA (renamed PdaC), was characterized and found to be a unique deacetylase. The pdaC deletion mutant was sensitive to lysozyme treatment, indicating that PdaC acts as a deacetylase. The purified recombinant and truncated PdaC from Escherichia coli deacetylated B. subtilis peptidoglycan and its polymer, (-GlcNAc-MurNAc[-L-Ala-D-Glu]-)(n). Surprisingly, RP-HPLC and ESI-MS/MS analyses showed that the enzyme deacetylates N-acetylmuramic acid (MurNAc) not GlcNAc from the polymer. Contrary to Streptococcus pneumoniae PgdA, which shows high amino acid sequence similarity with PdaC and is a zinc-dependent GlcNAc deacetylase toward peptidoglycan, there was less dependence on zinc ion for deacetylation of peptidoglycan by PdaC than other metal ions (Mn(2+), Mg(2+), Ca(2+)). The kinetic values of the activity toward B. subtilis peptidoglycan were K(m) = 4.8 mM and k(cat) = 0.32 s(-1). PdaC also deacetylated N-acetylglucosamine (GlcNAc) oligomers with a K(m) = 12.3 mM and k(cat) = 0.24 s(-1) toward GlcNAc(4). Therefore, PdaC has GlcNAc deacetylase activity toward GlcNAc oligomers and MurNAc deacetylase activity toward B. subtilis peptidoglycan.