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Identification of Avian Wax Synthases
Background:
Bird species show a high degree of variation in the composition of their preen gland waxes. For instance, galliform birds like chicken contain fatty acid esters of 2,3-alkanediols, while Anseriformes like goose or Strigiformes like barn owl contain wax monoesters in their preen gland secretions. The final biosynthetic step is catalyzed by wax synthases (WS) which have been identified in pro- and eukaryotic organisms.
Results:
Sequence similarities enabled us to identify six cDNAs encoding putative wax synthesizing proteins in chicken and two from barn owl and goose. Expression studies in yeast under in vivo and in vitro conditions showed that three proteins from chicken performed WS activity while a sequence from chicken, goose and barn owl encoded a bifunctional enzyme catalyzing both wax ester and triacylglycerol synthesis. Mono- and bifunctional WS were found to differ in their substrate specificities especially with regard to branched-chain alcohols and acyl-CoA thioesters. According to the expression patterns of their transcripts and the properties of the enzymes, avian WS proteins might not be confined to preen glands.
Conclusions:
We provide direct evidence that avian preen glands possess both monofunctional and bifunctional WS proteins which have different expression patterns and WS activities with different substrate specificities.
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Enzymatic activity and immunoreactivity of Aca s 4, an alpha-amylase allergen from the storage mite Acarus siro
Background:
Enzymatic allergens of storage mites that contaminate stored food products are poorly characterized. We describe biochemical and immunological properties of the native alpha-amylase allergen Aca s 4 from Acarus siro, a medically important storage mite.
Results:
A. siro produced a high level of alpha-amylase activity attributed to Aca s 4. This enzyme was purified and identified by protein sequencing and LC-MS/MS analysis. Aca s 4 showed a distinct inhibition pattern and an unusual alpha-amylolytic activity with low sensitivity to activation by chloride ions. Homology modeling of Aca s 4 revealed a structural change in the chloride-binding site that may account for this activation pattern. Aca s 4 was recognized by IgE from house dust mite-sensitive patients, and potential epitopes for cross-reactivity with house dust mite group 4 allergens were found.
Conclusions:
We present the first protein-level characterization of a group 4 allergen from storage mites. Due to its high production and IgE reactivity, Aca s 4 is potentially relevant to allergic hypersensitivity.
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Hyaluronan synthase mediates dye translocation across liposomal membranes
Background:
Hyaluronan (HA) is made at the plasma membrane and secreted into the extracellular medium or matrix by phospolipid-dependent hyaluronan synthase (HAS), which is active as a monomer. Since the mechanism by which HA is translocated across membranes is still unresolved, we assessed the presence of an intraprotein pore within HAS by adding purified Streptococcus equisimilis HAS (SeHAS) to liposomes preloaded with the fluorophore Cascade Blue (CB).
Results:
CB translocation (efflux) was not observed with mock-purified material from empty vector control E. coli membranes, but was induced by SeHAS, purified from membranes, in a time- and dose-dependent manner. CB efflux was eliminated or greatly reduced when purified SeHAS was first treated under conditions that inhibit enzyme activity: heating, oxidization or cysteine modification with N-ethylmaleimide. Reduced CB efflux also occurred with SeHAS K48E or K48F mutants, in which alteration of K48 within membrane domain 2 causes decreased activity and HA product size. The above results used liposomes containing bovine cardiolipin (BCL). An earlier study testing many synthetic lipids found that the best activating lipid for SeHAS is tetraoleoyl cardiolipin (TO-CL) and that, in contrast, tetramyristoyl cardiolipin (TM-CL) is an inactivating lipid (Weigel et al, J. Biol. Chem. 281, 36542, 2006). Consistent with the effects of these CL species on SeHAS activity, CB efflux was more than 2-fold greater in liposomes made with TO-CL compared to TM-CL.
Conclusions:
The results indicate the presence of an intraprotein pore in HAS and support a model in which HA is translocated to the exterior by HAS itself.
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Lipid-bound apolipoproteins in tyrosyl radical-oxidized HDL stabilize ABCA1 like lipid-free apolipoprotein A-I
Background:
ATP-binding cassette transporter A1 (ABCA1) mediates the lipidation of exchangeable apolipoproteins, the rate-limiting step in the formation of high density lipoproteins (HDL). We previously demonstrated that HDL oxidized ex vivo by peroxidase-generated tyrosyl radical (tyrosylated HDL, tyrHDL) increases the availability of cellular cholesterol for efflux and reduces the development of atherosclerosis when administered to apolipoprotein E-deficient mice as compared to treatment with control HDL.
Results:
In the current study we determined that tyrHDL requires functional ABCA1 for this enhanced activity. Like lipid-free apolipoprotein A-I (apoA-I), tyrHDL increases total and cell surface ABCA1, inhibits calpain-dependent and -independent proteolysis of ABCA1, and can be bound by cell surface ABCA1 in human skin fibroblasts. Additionally, tyrHDL apoproteins are susceptible to digestion by enteropeptidase like lipid-free apoA-I, but unlike lipid-bound apoA-I on HDL, which is resistant to proteolysis.
Conclusions:
These results provide the first evidence that lipid-bound apolipoproteins on the surface of spherical HDL particles can behave like lipid-free apoA-I to increase ABCA1 protein levels and activity.
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Topological characterisation and identification of critical domains within glucosyltransferase IV (GtrIV) of Shigella flexneri
Background:
The three bacteriophage genes gtrA, gtrB and gtr(type) are responsible for O-antigen glucosylation in Shigella flexneri. Both gtrA and gtrB have been demonstrated to be highly conserved and interchangeable among serotypes while gtr(type) was found to be specific to each serotype, leading to the hypothesis that the Gtr(type) proteins are responsible for attaching glucosyl groups to the O-antigen in a site- and serotype- specific manner. Based on the confirmed topologies of GtrI, GtrII and GtrV, such interaction and attachment of the glucosyl groups to the O-antigen has been postulated to occur in the periplasm.
Results:
In this study, the topology of GtrIV was experimentally determined by creating different fusions between GtrIV and a dual-reporter protein, PhoA/LacZ. This study shows that GtrIV consists of 8 transmembrane helices, 2 large periplasmic loops, 2 small cytoplasmic N- and C- terminal ends and a re-entrant loop that occurs between transmembrane helices III and IV. Though this topology differs from that of GtrI, GtrII, GtrV and GtrX, it is very similar to that of GtrIc. Furthermore, both the N-terminal periplasmic and the C-terminal periplasmic loops are important for GtrIV function as shown via a series of loop deletion experiments and the creation of chimeric proteins between GtrIV and its closest structural homologue, GtrIc.
Conclusion:
The current study provides the basis for elucidating the structure and mechanism of action of this important O-antigen modifying glucosyltransferase.
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Low affinity PEGylated hemoglobin from Trematomus bernacchii, a model for hemoglobin-based blood substitutes
Background:
Conjugation of human and animal hemoglobins with polyethylene glycol has been widely explored as a means to develop blood substitutes, a novel pharmaceutical class to be used in surgery or emergency medicine. However, PEGylation of human hemoglobin led to products with significantly different oxygen binding properties with respect to the unmodified tetramer and high NO dioxygenase reactivity, known causes of toxicity. These recent findings call for the biotechnological development of stable, low-affinity PEGylated hemoglobins with low NO dioxygenase reactivity.
Results:
To investigate the effects of PEGylation on protein structure and function, we compared the PEGylation products of human hemoglobin and Trematomus bernacchii hemoglobin, a natural variant endowed with a remarkably low oxygen affinity and high tetramer stability. We show that extension arm facilitated PEGylation chemistry based on the reaction of T. bernacchii hemoglobin with 2-iminothiolane and maleimido-functionalyzed polyethylene glycol (MW 5000 Da) leads to a tetraPEGylated product, more homogeneous than the corresponding derivative of human hemoglobin. PEGylated T. bernacchii hemoglobin largely retains the low affinity of the unmodified tetramer, with a p50 50 times higher than PEGylated human hemoglobin. Moreover, it is still sensitive to protons and the allosteric effector ATP, indicating the retention of allosteric regulation. It is also 10-fold less reactive towards nitrogen monoxide than PEGylated human hemoglobin.
Conclusions:
These results indicate that PEGylated hemoglobins, provided that a suitable starting hemoglobin variant is chosen, can cover a wide range of oxygen-binding properties, potentially meeting the functional requirements of blood substitutes in terms of oxygen affinity, tetramer stability and NO dioxygenase reactivity.
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Computational Identification and Experimental Characterization of Substrate Binding Determinants of Nucleotide Pyrophosphatase/Phosphodiesterase 7
Background:
Nucleotide pyrophosphatase/phosphodiesterase 7 (NPP7) is the only member of the mammalian NPP enzyme family that has been confirmed to act as a sphingomyelinase, hydrolyzing sphingomyelin (SM) to form phosphocholine and ceramide. NPP7 additionally hydrolyzes lysophosphatidylcholine (LPC), a substrate preference shared with the NPP2/autotaxin(ATX) and NPP6 mammalian family members. This study utilizes a synergistic combination of molecular modeling validated by experimental site-directed mutagenesis to explore the molecular basis for the unique ability of NPP7 to hydrolyze SM.
Results:
The catalytic function of NPP7 against SM, LPC, platelet activating factor (PAF) and para-nitrophenylphosphorylcholine (pNPPC) is impaired in the F275A mutant relative to wild type NPP7, but different impacts are noted for mutations at other sites. These results are consistent with a previously described role of F275 to interact with the choline headgroup, where all substrates share a common functionality. The L107F mutation showed enhanced hydrolysis of LPC, PAF and pNPPC but reduced hydrolysis of SM. Modeling suggests this difference can be explained by the gain of cation-pi interactions with the choline headgroups of all four substrates, opposed by increased steric crowding against the sphingoid tail of SM. Modeling also revealed that the long and flexible hydrophobic tails of substrates exhibit considerable dynamic flexibility in the binding pocket, reducing the entropic penalty that might otherwise be incurred upon substrate binding.
Conclusions:
Substrate recognition by NPP7 includes several important contributions, ranging from cation-pi interactions between F275 and the choline headgroup of all substrates, to tail-group binding pockets that accommodate the inherent flexibility of the lipid hydrophobic tails. Two contributions to the unique ability of NPP7 to hydrolyze SM were identified. First, the second hydrophobic tail of SM occupies a second hydrophobic binding pocket. Second, the leucine residue present at position 107 contrasts with a conserved phenylalanine in NPP enzymes that do not utilize SM as a substrate, consistent with the observed reduction in SM hydrolysis by the NPP7-L107F mutant.
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Fatty Acyl-CoA Reductases of Birds
Background:
Birds clean and lubricate their feathers with waxes that are produced in the uropygial gland, a holocrine gland located on their back above the tail. The type and the composition of the secreted wax esters are dependent on the bird species, for instance the wax ester secretion of goose contains branched-chain fatty acids and unbranched fatty alcohols, whereas that of barn owl contains fatty acids and alcohols both of which are branched. Alcohol-forming fatty acyl-CoA reductases (FAR) catalyze the reduction of activated acyl groups to fatty alcohols that can be esterified with acyl-CoA thioesters forming wax esters.
Results:
cDNA sequences encoding fatty acyl-CoA reductases were cloned from the uropygial glands of barn owl (Tyto alba), domestic chicken (Gallus gallus domesticus) and domestic goose (Anser anser domesticus). Heterologous expression in Saccharomyces cerevisiae showed that they encode membrane associated enzymes which catalyze a NADPH dependent reduction of acyl-CoA thioesters to fatty alcohols. By feeding studies of transgenic yeast cultures and in vitro enzyme assays with membrane fractions of transgenic yeast cells two groups of isozymes with different properties were identified, termed FAR1 and FAR2. The FAR1 group mainly synthesized 1-hexadecanol and accepted substrates in the range between 14 and 18 carbon atoms, whereas the FAR2 group preferred stearoyl-CoA and accepted substrates between 16 and 20 carbon atoms. Expression studies with tissues of domestic chicken indicated that FAR transcripts were not restricted to the uropygial gland.
Conclusion:
The data of our study suggest that the identified and characterized avian FAR isozymes, FAR1 and FAR2, can be involved in wax ester biosynthesis and in other pathways like ether lipid synthesis.
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Roles of quaternary structure and cysteine residues in the activity of human serine racemase
Background:
D-serine is an important coagonist at the NR1 subunit of the NMDA receptor class of glutamate receptors. It is chiefly synthesized in the CNS by serine racemase (SR). Regulation of SR activity is still poorly understood. As step toward developing reagents and methods for investigating SR in vitro, we analyzed structure-function relationships of a recombinant enzyme of human sequence.
Results:
Michaelis-Menten kinetic analysis indicated a KM value of 14 mM and Vmax value of 3.66 μmol·mg-1·hr-1 when L-serine was used as a substrate for purified SR. Gel-filtration chromatography and protein cross-linking experiments revealed that dimer is the major oligomeric form of recombinant SR in aqueous solution, though the proportions of monomer, tetramer, and larger aggregates differed somewhat with the specific buffer used. These buffers also altered activity in a manner correlating with the relative abundance of dimer. Activity assays showed that the dimeric gel-filtration fraction held the highest activity. Chemical reduction with DTT increased the activity of SR by elevating Vmax; cystamine, a reagent that blocks sulfhydryl groups, abolished SR activity. Gel-filtration chromatography and western blot analysis indicated that DTT enhanced the recovery of noncovalent SR dimer.
Conclusions:
These data suggest that SR is most active as a noncovalent dimer containing one or more free sulfhydryls in the enzyme's active center or a modulatory site. Buffer composition and reduction/oxidation status during preparation can dramatically impact interpretations of SR activity. These findings also highlight the possibility that SR is sensitive to oxidative stress in vivo.
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Bifunctional TaqII restriction endonuclease: redefining the prototype DNA recognition site and establishing the Fidelity Index for partial cleaving.
Background:
The TaqII enzyme is a member of the Thermus sp. enzyme family that we propounded previously within Type IIS restriction endonucleases, containing related thermophilic bifunctional endonucleases-methyltransferases from various Thermus sp.: TaqII, Tth111II, TthHB27I, TspGWI, TspDTI and TsoI. These enzymes show significant nucleotide and amino acid sequence similarities, a rare phenomenon among restriction endonucleases, along with similarities in biochemical properties, molecular size, DNA recognition sequences and cleavage sites. They also feature some characteristics of Types I and III.
Results:
Barker et al. reported the Type IIS/IIC restriction endonuclease TaqII as recognizing two distinct cognate site variants (5' -GACCGA-3' and 5'-CACCCA-3') while cleaving 11/9 nucleotides downstream. We used four independent methods, namely, shotgun cloning and sequencing, restriction pattern analysis, digestion of particular custom substrates and GeneScan analysis, to demonstrate that the recombinant enzyme recognizes only 5' -GACCGA-3' sites and cleaves 11/9 nucleotides downstream. We did not observe any 5'-CACCCA-3' cleavage under a variety of conditions and site arrangements tested. We also characterized the enzyme biochemically and established new digestion conditions optimal for practical enzyme applications. Finally, we developed and propose a new version of the Fidelity Index - the Fidelity Index for Partial Cleavage (FI-PC).
Conclusions:
The DNA recognition sequence of the bifunctional prototype TaqII endonuclease-methyltransferase from Thermus aquaticus has been redefined as recognizing only 5' -GACCGA-3' cognate sites. The reaction conditions (pH and salt concentrations) were designed either to minimize (pH = 8.0 and 10 mM ammonium sulphate) or to enhance star activity (pH = 6.0 and no salt). Redefinition of the recognition site and reaction conditions makes this prototype endonuclease a useful tool for DNA manipulation; as yet, this enzyme has no practical applications. The extension of the Fidelity Index will be helpful for DNA manipulation with enzymes only partially cleaving DNA.
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