Browsing by Author "Li, Lei"

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    Characterization of a fatty acid elongase condensing enzyme by site-directed mutagenesis and biochemical analysis
    (2014) Hernandez-Buquer, Selene; Long, Eric C. (Eric Charles); Blacklock, Brenda J.; Li, Lei
    Fatty acid elongation is the extension of de novo synthesized fatty acids through a series of four reactions analogous to those of fatty acid synthase. ELOs catalyze the first reaction in the elongation pathway through the condensation of an acyl group with a two carbon unit derived from malonyl-CoA. This study uses the condensing enzyme, EloA, from the cellular slime mold, Dictyostelium discoideum as a model for the family of ELOs. EloA has substrate specificity for monounsaturated and saturated C16 fatty acids and catalyzes the elongation of 16:1Δ9 to 18:1Δ11. Site-directed mutagenesis was used to change residues highly conserved among the ELO family to examine their potential role in the condensation reaction. Mutant EloAs were expressed in yeast and fatty acid methyl esters prepared from total cellular lipids were analyzed by gas chromatography/mass spectrometry. Sixteen out of twenty mutants had a decrease in 18:1Δ11 production when compared to the wild-type EloA with little to no activity observed in ten mutants, four mutants had within 20% of wild-type activity, and six mutants had 10-60% of wild-type activity. Immunoblot studies using anti-EloA serum were used to determine if the differences in elongation activity were related to changes in protein expression for each mutant. Analysis of immunoblots indicated that those mutants with little to no activity, with the exception of T130A and Q203A, had x comparable protein expression to the wild-type. Further research included the solubilization of the His6-ELoA fusion protein and preliminary work toward the isolation of the tagged protein and the use of a radiolabeled condensation assay to determine the activity of the eluted protein. Preliminary results indicated that the protein was solubilized but the eluted protein showed no activity when examined by a condensation assay. The work presented here contributes to a better understanding of the role of certain amino acid residues in the activity of EloA and serves as a stepping-stone for future EloA isolation work.
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    Chemical Modifications of the Capsid for Redirecting and Improving the Efficacy of Adeno-Associated Virus Vectors
    (Mary Ann Liebert, 2021-12) Lam, Anh Kim; Frabutt, Dylan A.; Li, Lei; Xiao, Weidong; Pediatrics, School of Medicine
    Adeno-associated virus (AAV) vector-directed gene therapy is one of the most exciting modalities of biotechnology as more applications enter clinical stage. Although AAV vectors generally feature low toxicity, high stability, and long-lasting transgene expression, potential challenging issues of AAV include high vector dose, limited tissue tropism, and the host immune response and inflammation, which are all related to the capsid protein. To overcome these challenges, various strategies have been developed to engineer AAV capsids. Apart from widely employed genetic engineering of capsid protein, powerful and versatile chemical modification strategies are underexploited. This minireview summarizes recent advances and our perspectives for future direction in AAV capsid chemical modification to enhance its therapeutic use for gene therapy.
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    Convergent chemoenzymatic synthesis of O-GalNAc rare cores 5, 7, 8 and their sialylated forms
    (Royal Society of Chemistry, 2023-01-18) Gadi, Madhusudhan Reddy; Chen, Congcong; Bao, Shumin; Wang, Shuaishuai; Guo, Yuxi; Han, Jinghua; Xiao, Weidong; Li, Lei; Pediatrics, School of Medicine
    All O-GalNAc glycans are derived from 8 cores with 2 or 3 monosaccharides linked via α- or β-glycosidic bonds. While chemical and chemoenzymatic syntheses of β-linked cores 1-4 and 6 and derived glycans have been well developed, the preparation of α-linked rare cores 5, 7, and 8 is challenging due to the presence of this 1,2-cis linkage. Meanwhile, the biosynthesis and functional roles of these structures are poorly understood. Herein, we synthesize 3 α-linked rare cores with exclusive α-configuration from a versatile precursor through multifaceted chemical modulations. Efficient regioselective α2-6sialylion of the rare cores was then achieved by Photobacterium damselae α2-6sialyltransferase-catalyzed reactions. These structures, together with β-linked cores 1-4 and 6, and their sialylated forms, were fabricated into a comprehensive O-GalNAc core microarray to profile the binding of clinically important GalNAc-specific lectins. It is found that only Tn, (sialyl-)core 5, and core 7 are the binders of WFL, VVL, and SBA, while DBA only recognized (sialyl-)core 5, and Jacalin is the only lectin that binds core 8. In addition, activity assays of human α-N-acetylgalactosaminide α2-6sialyltransferases (ST6GalNAcTs) towards the cores suggested that ST6GalNAc1 may be involved in the biosynthesis of previously identified sialyl-core 5 and sialyl-core 8 glycans. In conclusion, we provide efficient routes to access α-linked O-GalNAc rare cores and derived structures, which are valuable tools for functional glycomics studies of mucin O-glycans.
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    Cryptic resolution sites in the vector plasmid lead to the heterogeneities in the rAAV vectors
    (Wiley, 2023) Zhang, Junping; Chrzanowski, Matthew; Frabutt, Dylan A.; Lam, Anh K.; Mulcrone, Patrick L.; Li, Lei; Konkle, Barbara A.; Miao, Carol H.; Xiao, Weidong; Pediatrics, School of Medicine
    Recombinant adeno-associated virus (rAAV) vectors carry a cassette of interest retaining only the inverted terminal repeats (ITRs) from the wild-type virus. Conventional rAAV production primarily uses a vector plasmid as well as helper genes essential for AAV replication and packaging. Nevertheless, plasmid backbone related contaminants have been a major source of vector heterogeneity. The mechanism driving the contamination phenomenon has yet to be elucidated. Here we identified cryptic resolution sites in the plasmid backbone as a key source for producing snapback genomes, which leads to the increase of vector genome heterogeneity in encapsidated virions. By using a single ITR plasmid as a model molecule and mapping subgenomic particles, we found that there exist a few typical DNA break hotspots in the vector DNA plasmid backbone, for example, on the ampicillin DNA element, called aberrant rescue sites. DNA around these specific breakage sites may assume some typical secondary structures. Similar to normal AAV vectors, plasmid DNA with a single ITR was able to rescue and replicate efficiently. These subgenomic DNA species significantly compete for trans factors required for rAAV rescue, replication, and packaging. The replication of single ITR contaminants during AAV production is independent of size. Packaging of these species is greatly affected by its size. A single ITR and a cryptic resolution site in the plasmid work synergistically, likely causing a source of plasmid backbone contamination.
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    Discrimination of color copier/laser printer toners by Raman spectroscopy and subsequent chemometric analysis
    (2013-11-20) Feldmann, Jeanna Marie; Goodpaster, John V. (John Vincent); Siegel, Jay A.; Li, Lei; Picard, Christine; Muhoberac, Barry B.
    Toner analysis has become an area of increased interest due to the wide availability of laser printers and photocopiers. Toner is most often encountered on paper in questioned document analysis. Because of this, it is important to develop methods that limit the interference of paper without damaging or destroying the document. Previous research using Fourier transform infrared spectroscopy (FTIR) has differentiated toners based on their polymer resin components. However, Raman spectroscopy and chemometric analysis are not typically used for the examination of this material. Raman spectroscopy is a popular tool for the chemical analysis of pigmented samples and was used to characterize cyan, yellow, and magenta toners. Analyses were performed using a dispersive micro-Raman spectrometer equipped with a 785nm diode laser, a CCD detector, and an objective at 20X magnification. One hundred samples of each color toner were collected. Three different and separate methods were developed for cyan, yellow, and magenta toners on paper to optimize results. Further analysis of the magenta toners was excluded due to a weak signal and significant paper interference. The data collected from the analyses of the blue and yellow toners was then processed using a combination of statistical procedures, including principal component analysis (PCA), agglomerative hierarchal clustering (AHC), and discriminative analysis (DA). Ninety-six blue toners were analyzed by PCA and three classes of spectra were suggested. Discriminant analysis showed that the three classes were well-differentiated with a cross-validation accuracy of 100% for the training set and 100% cross-validation accuracy for the external validation set. Eighty-eight yellow toners were analyzed by AHC and four classes of spectra were suggested. Discriminant analysis showed good differentiation between the classes with a cross-validation accuracy of 95.45% for the training set, but showed poor differentiation for the external validation set with a cross-validation accuracy of 72%. While these toners were able to be discriminated, no correlation could be made between the manufacturer, printer make and model, and the toner sample.
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    The Enzyme-Mediated Direct Reversal of a Dithymine Photoproduct in Germinating Endospores
    (MDPI, 2013-06-25) Yang, Linlin; Li, Lei; Chemistry and Chemical Biology, School of Science
    Spore photoproduct lyase (SPL) repairs a special thymine dimer, 5-thyminyl-5,6-dihydrothymine, which is commonly called spore photoproduct, or SP, in germinating endospores. SP is the exclusive DNA photo-damaging product found in endospores; its generation and swift repair by SPL are responsible for the spores’ extremely high UV resistance. Early in vivo studies suggested that SPL utilizes a direct reversal strategy to repair SP in the absence of light. Recently, it has been established that SPL belongs to the radical S-adenosylmethionine (SAM) superfamily. The enzymes in this superfamily utilize a tri-cysteine CXXXCXXC motif to bind a [4Fe-4S] cluster. The cluster provides an electron to the S-adenosylmethionine (SAM) to reductively cleave its C5′-S bond, generating a reactive 5′-deoxyadenosyl (5′-dA) radical. This 5′-dA radical abstracts the proR hydrogen atom from the C6 carbon of SP to initiate the repair process; the resulting SP radical subsequently fragments to generate a putative thymine methyl radical, which accepts a back-donated H atom to yield the repaired TpT. The H atom donor is suggested to be a conserved cysteine141 in B. subtilis SPL; the resulting thiyl radical likely interacts with a neighboring tyrosine99 before oxidizing the 5′-dA to 5′-dA radical and, subsequently, regenerating SAM. These findings suggest SPL to be the first enzyme in the large radical SAM superfamily (>44,000 members) to utilize a radical transfer pathway for catalysis; its study should shed light on the mechanistic understanding of the SAM regeneration process in other members of the superfamily.
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    EPR Study of UV-irradiated Thymidine Microcrystals Supports Radical Intermediates in Spore Photoproduct Formation
    (ACS, 2016-09) Hayes, Ellen C.; Jian, Yajun; Stoll, Stefan; Li, Lei; Department of Physics, School of Science
    Spore photoproduct is a thymidine dimer formed when bacterial endospore DNA is exposed to ultraviolet (UV) radiation. The mechanism of formation of this thymidine dimer has been proposed to proceed through a radical-pair intermediate. The intermediate forms when a methyl-group hydrogen atom of one thymidine nucleobase is transferred to the C6 position of an adjacent thymidine nucleobase, forming two species, the TCH2 and TH radicals, respectively. Using a series of thymidine isotopologues and electron paramagnetic resonance (EPR) spectroscopy, we show that microcrystals of thymidine exposed to UV radiation produce these two radical species. We observe three sources that donate the additional hydrogen at the C6 position of the TH radical. One of the three sources is the methyl group of another thymidine molecule in a significant fraction of the TH species. This lends support to the radical-pair intermediate proposed in the formation of spore photoproduct.
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    Examining the base stacking interaction in a dinucleotide context via reversible cyclobutane dimer analogue formation under UV irradiation
    (Royal Society of Chemistry, 2013-11-14) Liu, Degang; Li, Lei; Chemistry & Chemical Biology, School of Science
    Substituted tolyl groups are considered as close isosteres of the thymine (T) residue. They can be recognized by DNA polymerases as if they were thymine. Although these toluene derivatives are relatively inert toward radical additions, our recent finding suggests that the dinucleotide analogue TpTo (To = 2'-deoxy-1-(3-tolyl)-β-D-ribofuranose) supports an ortho photocycloaddition reaction upon UV irradiation, producing two cyclobutane pyrimidine dimer (CPD) analogues 2 and 3. Our report here further shows that formation of these CPD species is reversible under UVC irradiation, resembling the photochemical property of the CPD species formed between two Ts. Analyzing the stability of these CPD analogues suggests that one (2) is more stable than the other (3). The TpTo conformer responsible for 2 formation is also more stable than that responsible for 3 formation, as indicated by the Gibbs free energy change calculated from the constructed Bordwell thermodynamic cycle. These different stabilities are not due to the varying photochemical properties, as proved by quantum yields determined from the corresponding photoreactions. Instead, they are ascribed to the different stacking interaction between the T and the To rings both in the TpTo dinucleotide as well as in the formed CPD analogues. Factors contributing to the ring stacking interactions are also discussed. Our proof-of-concept approach suggests that a carefully designed Bordwell cycle coupled with reversible CPD formations under UV irradiation can be very useful in studying DNA base interactions.
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    Exploring the mechanism of action of spore photoproduct lyase
    (2014-08-27) Nelson, Renae; Li, Lei; Long, Eric C. (Eric Charles); McLeish, Michael J.
    Spore photoproduct lyase (SPL) is a radical SAM (S-adenosylmethionine) enzyme that is responsible for the repair of the DNA UV damage product 5-thyminyl-5,6-dihydrothymine (also called spore photoproduct, SP) in the early germination phase of bacterial endospores. SPL initiates the SP repair process using 5'-dA• (5'-deoxyadenosyl radical) generated by SAM cleavage to abstract the H6proR atom which results in a thymine allylic radical. These studies provide strong evidence that the TpT radical likely receives an H atom from an intrinsic H atom donor, C141 in B. subtilis SPL. I have shown that C141 can be alkylated in native SPL by iodoacetamide treatment indicating that it is accessible to the TpT radical. Activity studies demonstrate a 3-fold slower repair rate of SP by C141A which produces TpTSO2 - and TpT simultaneously with no lag phase observed for TpTSO2- formation. Additionally, formation of both products shows a Dvmax kinetic isotope effect (KIE) of 1.7 ± 0.2 which is smaller than the DVmax KIE of 2.8 ± 0.3 for the WT SPL reaction. Removal of the intrinsic H atom donor by this single mutation disrupts the rate-limiting process in the enzyme catalysis. Moreover, C141A exhibits ~0.4 turnover compared to the > 5 turnovers in the WT SPL reaction. In Y97 and Y99 studies, structural and biochemical data suggest that these two tyrosine residues are also crucial in enzyme catalysis. It is suggested that Y99 in B. subtilis SPL uses a novel hydrogen atom transfer pathway utilizing a pair of cysteinetyrosine residues to regenerate SAM. The second tyrosine, Y97, structurally assists in SAM binding and may also contribute to SAM regeneration by interacting with radical intermediates to lower the energy barrier for the second H-abstraction step.
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    Fast and high-throughput LC-MS characterization, and peptide mapping of engineered AAV capsids using LC-MS/MS
    (Elsevier, 2022-09-24) Lam, Anh K.; Zhang, Junping; Frabutt, Dylan; Mulcrone, Patrick L.; Li, Lei; Zeng, Lifan; Herzog, Roland W.; Xiao, Weidong; Pediatrics, School of Medicine
    Adeno-associated virus (AAV) has emerged as a leading platform for gene therapy. With the skyrocketing rate of AAV research and the prevalence of many new engineered capsids being investigated in preclinical and clinical trials, capsid characterization plays a vital role in serotype confirmation and quality control. Further, peptide mapping the capsid proteins might inevitably be a future requirement by regulatory agencies since it is a critical step in good manufacturing practice (GMP) for biotherapeutic characterization. To overcome many challenges that traditional methods like SDS-PAGE and western blots carry, liquid chromatography and mass spectrometry (LC-MS) allows high resolution and sensitivity with great accuracy in characterizing the AAV capsid proteins. Our optimized LC-MS method provides quick sample preparation, a fast and high-throughput 4-min run, and high sensitivity, which allows for very efficient characterization of wild-type and engineered capsids. This study also reports the usage of LC-MS/MS peptide mapping of AAV capsid proteins to determine the most accessible lysine residues targeted by chemical modifications. Our detailed protocols are anticipated to promote the development and discovery of AAV variants with high accuracy and efficiency.