Oxidation of the FAD cofactor to the 8-formyl-derivative in human electron-transferring flavoprotein.

The heterodimeric human (h) electron-transferring flavoprotein (ETF) transfers electrons from at least 13 different flavin dehydrogenases to the mitochondrial respiratory chain through a non-covalently bound FAD cofactor. Here, we describe the discovery of an irreversible and pH-dependent oxidation of the 8α-methyl group to 8-formyl-FAD (8f-FAD), which represents a unique chemical modification of a flavin cofactor in the human flavoproteome. Furthermore, a set of hETF variants revealed that several conserved amino acid residues in the FAD-binding pocket of electron-transferring flavoproteins are required for the conversion to the formyl group. Two of the variants generated in our study, namely αR249C and αT266M, cause glutaric aciduria type II, a severe inherited disease. Both of the variants showed impaired formation of 8f-FAD shedding new light on the potential molecular cause of disease development. Interestingly, the conversion of FAD to 8f-FAD yields a very stable flavin semiquinone that exhibited slightly lower rates of electron transfer in an artificial assay system than hETF containing FAD. In contrast, the formation of 8f-FAD enhanced the affinity to human dimethylglycine dehydrogenase 5-fold, indicating that formation of 8f-FAD modulates the interaction of hETF with client enzymes in the mitochondrial matrix. Thus, we hypothesize that the FAD cofactor bound to hETF is subject to oxidation in the alkaline (pH 8) environment of the mitochondrial matrix, which may modulate electron transport between client dehydrogenases and the respiratory chain. This discovery challenges the current concepts of electron transfer processes in mitochondria.

Structure and biochemical properties of recombinant human dimethylglycine dehydrogenase and comparison to the disease-related H109R variant.

The human dimethylglycine dehydrogenase (hDMGDH) is a flavin adenine dinucleotide (FAD)- and tetrahydrofolate (THF)-dependent, mitochondrial matrix enzyme taking part in choline degradation, one-carbon metabolism and electron transfer to the respiratory chain. The rare natural variant H109R causes dimethylglycine dehydrogenase deficiency leading to increased blood and urinary dimethylglycine concentrations. A detailed biochemical and structural characterization of hDMGDH was thus far hampered by insufficient heterologous expression of the protein. In the present study, we report the development of an intracellular, heterologous expression system in Komagataella phaffii (formerly known as Pichia pastoris) providing the opportunity to determine kinetic parameters, spectroscopic properties, thermostability, and the redox potential of hDMGDH. Moreover, we have successfully crystallized the wild-type enzyme and determined the structure to 3.1-Å resolution. The structure-based analysis of our biochemical data provided new insights into the kinetic properties of the enzyme in particular with respect to oxygen reactivity. A comparative study with the H109R variant demonstrated that the variant suffers from decreased protein stability, cofactor saturation, and substrate affinity. DATABASE: Structural data are available in the PDB database under the accession number 5L46.

High-quality production of human α-2,6-sialyltransferase in Pichia pastoris requires control over N-terminal truncations by host-inherent protease activities.

BACKGROUND: α-2,6-sialyltransferase catalyzes the terminal step of complex N-glycan biosynthesis on human glycoproteins, attaching sialic acid to outermost galactosyl residues on otherwise fully assembled branched glycans. This "capping" of N-glycans is critical for therapeutic efficacy of pharmaceutical glycoproteins, making the degree of sialylation an important parameter of glycoprotein quality control. Expression of recombinant glycoproteins in mammalian cells usually delivers heterogeneous N-glycans, with a minor degree of sialylation. In-vitro chemo-enzymatic glycoengineering of the N-glycans provides an elegant solution to increase the degree of sialylation for analytical purposes but also possibly for modification of therapeutic proteins. RESULTS: Human α-2,6-sialyltransferase (ST6Gal-I) was secretory expressed in P.pastoris KM71H. ST6Gal-I featuring complete deletion of both the N-terminal cytoplasmic tail and the transmembrane domain, and also partial truncation of the stem region up to residue 108 were expressed N-terminally fused to a His or FLAG-Tag. FLAG-tagged proteins proved much more resistant to proteolysis during production than the corresponding His-tagged proteins. Because volumetric transferase activity measured on small-molecule and native glycoprotein acceptor substrates did not correlate to ST6Gal-I in the supernatant, enzymes were purified and characterized in their action on non-sialylated protein-linked and released N-glycans, and the respective N-terminal sequences were determined by automated Edman degradation. Irrespective of deletion construct used (Δ27, Δ48, Δ62, Δ89), isolated proteins showed N-terminal processing to a highly similar degree, with prominent truncations at residue 108 - 114, whereby only Δ108ST6Gal-I retained activity. FLAG-tagged Δ108ST6Gal-I was therefore produced and obtained with a yield of 4.5 mg protein/L medium. The protein was isolated and shown by MS to be intact. Purified enzyme exhibited useful activity (0.18 U/mg) for sialylation of different substrates. CONCLUSIONS: Functional expression of human ST6Gal-I as secretory protein in P.pastoris necessitates that N-terminal truncations promoted by host-inherent proteases be tightly controlled. N-terminal FLAG-Tag contributes extra stability to the N-terminal region as compared to N-terminal His-Tag. Proteolytic degradation proceeds up to residues 108 - 114 and of the resulting short-form variants, only Δ108ST6Gal-I seems to be active. FLAG-Δ108ST6Gal-I transfers sialic acids to monoclonal antibody substrate with sufficient yields, and because it is stably produced in P.pastoris, it is identified here as an interesting glycoengineering catalyst.

Production of the sesquiterpenoid (+)-nootkatone by metabolic engineering of Pichia pastoris.

The sesquiterpenoid (+)-nootkatone is a highly demanded and highly valued aroma compound naturally found in grapefruit, pummelo or Nootka cypress tree. Extraction of (+)-nootkatone from plant material or its production by chemical synthesis suffers from low yields and the use of environmentally harmful methods, respectively. Lately, major attention has been paid to biotechnological approaches, using cell extracts or whole-cell systems for the production of (+)-nootkatone. In our study, the yeast Pichia pastoris initially was applied as whole-cell biocatalyst for the production of (+)-nootkatone from (+)-valencene, the abundant aroma compound of oranges. Therefore, we generated a strain co-expressing the premnaspirodiene oxygenase of Hyoscyamus muticus (HPO) and the Arabidopsis thaliana cytochrome P450 reductase (CPR) that hydroxylated extracellularly added (+)-valencene. Intracellular production of (+)-valencene by co-expression of valencene synthase from Callitropsis nootkatensis resolved the phase-transfer issues of (+)-valencene. Bi-phasic cultivations of P. pastoris resulted in the production of trans-nootkatol, which was oxidized to (+)-nootkatone by an intrinsic P. pastoris activity. Additional overexpression of a P. pastoris alcohol dehydrogenase and truncated hydroxy-methylglutaryl-CoA reductase (tHmg1p) significantly enhanced the (+)-nootkatone yield to 208mg L(-1) cell culture in bioreactor cultivations. Thus, metabolically engineered yeast P. pastoris represents a valuable, whole-cell system for high-level production of (+)-nootkatone from simple carbon sources.

Association of Toll-like receptor 9 haplotypes with chronic periodontitis in Czech population.

AIM: Toll-like receptors (TLRs) belong to the pattern recognition receptors family of signal molecules that recognize conserved microbial structures. The aim of this study was to analyse polymorphisms in the TLR genes and their association with chronic periodontitis (CP). MATERIAL AND METHODS: Two polymorphisms (2408G/A, i.e. Arg753Gln and -16934A/T) in TLR-2 and three variants (-1486C/T, -1237C/T and+2848A/G) in the TLR-9 genes were studied in 222 patients with CP and 259 unrelated controls. All polymorphisms were detected using the polymerase chain reaction-restriction fragment length polymorphism methods. Subgingival bacterial colonization was investigated by the VariOr Dento test. RESULTS: No significant differences were found in allele and genotype frequencies of all polymorphisms between patients and controls. Nevertheless, complex analysis revealed differences in TLR9 haplotype frequencies between both groups (p=0.001). Specifically, the haplotype T(-1486)/T(-1237)/A(2848) was significantly more frequent (9.6%versus 2.8%, p<0.000001) and the haplotype T(-1486)/T(-1237)/G (2848) of the TLR9 gene was less frequent (35.9%versus 43.3%, p=0.01) in patients than in controls. However, no significant relationships between periodontal pathogens, TLR polymorphisms and CP were found. CONCLUSIONS: In conclusion, although no significant role of the TLR2 gene in periodontitis was found, our results indicate that TLR9 haplotypes may be associated with susceptibility to CP.

The association of interleukin-4 haplotypes with chronic periodontitis in a Czech population.

BACKGROUND: Cytokine gene polymorphisms are known to influence the susceptibility and disease course of many chronic disorders. Recently, interleukin (IL)-4 gene polymorphisms were associated with aggressive periodontitis. The aim of this study was to test differences in the distribution of the IL-4 alleles, genotypes, and haplotypes between patients with chronic periodontitis (CP) and healthy controls in a Czech population. METHODS: The association study was conducted using an age- and smoking status-matched case-control design in patients with CP (n = 194) and healthy controls (n = 158) using the polymerase chain reaction-restriction fragment length polymorphism methods for the -590C/T, -33C/T, and intron 3 variable number tandem repeat (VNTR) variants of the IL-4 gene. RESULTS: No significant differences between patients and controls were found in allele and genotype frequencies of all three polymorphisms. Nevertheless, complex analysis revealed significant differences in haplotype frequencies between the groups (P = 0.005). The haplotype T(-590)/T(-33)/allele 2 VNTR (70 base pairs)(2) of the IL-4 gene was significantly more frequent in patients with CP than in controls (17.0% versus 11.0%; odds ratio = 1.85; 95% confidence interval: 1.19 to 2.87). CONCLUSION: The three polymorphisms in the IL-4 gene act in a cooperative fashion and suggest that the high-production IL-4 haplotype was associated with an increased risk for CP in the Czech population.

Effects of activated and nonactivated platelet-rich plasma on proliferation of human osteoblasts in vitro.

PURPOSE: The purpose of this study was to evaluate the effect of progressively increasing concentrations of activated and nonactivated platelet-rich plasma (PRP) on proliferation of human osteoblasts in vitro. MATERIALS AND METHODS: Human osteoblasts (hFOB 1.19) obtained from the American Type Culture Collection (ATCC, Manassas, VA) were used in the experiment. PRP was obtained from a 28-year-old healthy male volunteer by means of a Haemonetics gradient density cell separator (Haemonetics, Munich, Germany). Human thrombin was used to activate PRP. Three independent experiments were conducted. Samples containing 10% (0.38x increase in platelet count), 25% (0.95x increase in platelet count), 50% (1.95x increase in platelet count), and 75% (2.86x increase in platelet count) of activated PRP and nonactivated PRP were prepared including controls. After culture periods of 24, 48, and 72 hours osteoblast proliferation was evaluated by counting the number of cells using a Multisizer 3 Coulter Counter (Beckman Coulter, Inc, Fullerton, CA). RESULTS: After 24, 48, and 72 hours of incubation, the number of cells in the control group (without PRP) was higher than that of cells in samples containing activated or nonactivated PRP. Osteoblasts with 10% activated PRP (0.38x increase in platelet count) had the highest viability of all samples containing PRP. CONCLUSIONS: Activated PRP resulted in higher proliferation of osteoblasts compared with nonactivated PRP at concentrations of 10% (0.38x increase in platelet count) and 25% (0.95x increase in platelet count) in culture. This study failed to show significant increases in proliferation of human osteoblasts treated with activated or nonactivated PRP compared with controls in vitro.