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Open Access Publications from the University of California

Scholarly Works (5 results)

  • Thesis
  • Peer Reviewed
UC Riverside Electronic Theses and Dissertations (2015)

Heparin is unbranched anionic polysaccharide that is part of the glycosaminoglycan family. Although its structural microheterogeneity allows heparin to mediate a variety of biological processes by binding to over 400 different proteins, it also makes structural characterization challenging. This dissertation focuses on the separation and isolation of unique heparin oligosaccharides and their structure elucidation using nuclear magnetic resonance (NMR) and mass spectrometry. The impact of sequence on secondary structure is evaluated through detection of specific intramolecular hydrogen bonds.

Separation of the low-molecular-weight heparin enoxaparin was accomplished using size-exclusion chromatography (SEC) followed by strong anion-exchange high-performance liquid chromatography (SAX-HPLC). Typically, size-similar SEC fractions are combined prior to SAX-HPLC separation. However, screening individual SEC fractions using their sulfamate 1H and 15N chemical shifts allowed for SAX-HPLC resolution of the SEC fractions containing 3-O-sulfated oligosaccharides and led to the isolation and characterization of a unique 3-O-sulfated heparin tetrasaccharide.

The effect of structure, particularly glucosamine 3-O-sulfation, on hydroxyl proton hydrogen bonding was explored. Experimental conditions were optimized for the NMR detection and identification of heparin hydroxyl protons. Solvent exchange rates were determined using exchange spectroscopy (EXSY). Structurally specific differences in the hydroxyl proton exchange rates of the hydroxyl protons were explored for Arixtra, a synthetic pentasaccharide that mimics the Antithrombin-III binding sequence, and for isolated oligosaccharides that do not contain a 3-O-sulfated glucosamine. Transverse relaxation rates (R2) measured for the Arixtra hydroxyl protons using the Carr-Purcell-Meiboom-Gill experiment are compared with EXSY solvent exchange rates. Because R2 values are sensitive to factors other than exchange, good correlations with the EXSY exchange rates were not uniformly obtained.

Experimental conditions are reported allowing the 1H NMR observation of the amine protons of glucosamine. Exchange properties of amine protons in monosaccharides differing only by 3-O-sulfation are compared. Amine group acidity (pKa) was measured to probe participation of the amine group in a salt bridge. Although no evidence of a salt bridge between the amine and sulfate groups was found in the 3-O-sulfated glucosamine (GlcN3S) monosaccharide, these experiments demonstrate the feasibility of probing for structurally important salt bridges in longer oligosaccharides.

    Cover page: Molecular Level Characterization of Heparin Structure
    • Article
    • Peer Reviewed
    UC Santa Cruz Previously Published Works (2022)
    Recent NMR studies of the exchangeable protons of GAGs in aqueous solution, including those of the amide, sulfamate, and hydroxyl moieties, have demonstrated potential for the detection of intramolecular hydrogen bonds providing insights into secondary structure preferences. GAG amide protons are observable by NMR over wide pH and temperature ranges; however, specific solution conditions are required to reduce the exchange rate of the sulfamate and hydroxyl protons and allow their detection by NMR. Building on the vast body of knowledge on detection of hydrogen bonds in peptides and proteins, a variety of methods can be used to identify hydrogen bonds in GAGs including temperature coefficient measurements, evaluation of chemical shift differences between oligo- and monosaccharides, and relative exchange rates measured through line shape analysis and EXSY spectra. Emerging strategies to allow direct detection of hydrogen bonds through heteronuclear couplings offer promise for the future. Molecular dynamic simulations are important in this effort both to predict and confirm hydrogen bond donors and acceptors.
      Cover page: Methods for Measuring Exchangeable Protons in Glycosaminoglycans
      • Article
      • Peer Reviewed
      UC Riverside Previously Published Works (2015)
      The filamentous fungus Neurospora crassa is a long-studied eukaryotic microbial system amenable to heterologous expression of native and foreign proteins. However, relatively few highly tunable promoters have been developed for this species. In this study, we compare the tcu-1 and nit-6 promoters for controlled expression of a GFP reporter gene in N. crassa. Although the copper-regulated tcu-1 has been previously characterized, this is the first investigation exploring nitrogen-controlled nit-6 for expression of heterologous genes in N. crassa. We determined that fragments corresponding to 1.5-kb fragments upstream of the tcu-1 and nit-6 open reading frames are needed for optimal repression and expression of GFP mRNA and protein. nit-6 was repressed using concentrations of glutamine from 2 to 20 mM and induced in medium containing 0.5-20 mM nitrate as the nitrogen source. Highest levels of expression were achieved within 3 hr of induction for each promoter and GFP mRNA could not be detected within 1 hr after transfer to repressing conditions using the nit-6 promoter. We also performed metabolic profiling experiments using proton NMR to identify changes in metabolite levels under inducing and repressing conditions for each promoter. The results demonstrate that conditions used to regulate tcu-1 do not significantly change the primary metabolome and that the differences between inducing and repressing conditions for nit-6 can be accounted for by growth under nitrate or glutamine as a nitrogen source. Our findings demonstrate that nit-6 is a tunable promoter that joins tcu-1 as a choice for regulation of gene expression in N. crassa.
        Cover page: Metabolic Impacts of Using Nitrogen and Copper-Regulated Promoters to Regulate Gene Expression in Neurospora crassa
        • Article
        • Peer Reviewed
        UC Riverside Previously Published Works (2015)
        The filamentous fungus Neurospora crassa is a long-studied eukaryotic microbial system amenable to heterologous expression of native and foreign proteins. However, relatively few highly tunable promoters have been developed for this species. In this study, we compare the tcu-1 and nit-6 promoters for controlled expression of a GFP reporter gene in N. crassa. Although the copper-regulated tcu-1 has been previously characterized, this is the first investigation exploring nitrogen-controlled nit-6 for expression of heterologous genes in N. crassa. We determined that fragments corresponding to 1.5-kb fragments upstream of the tcu-1 and nit-6 open reading frames are needed for optimal repression and expression of GFP mRNA and protein. nit-6 was repressed using concentrations of glutamine from 2 to 20 mM and induced in medium containing 0.5-20 mM nitrate as the nitrogen source. Highest levels of expression were achieved within 3 hr of induction for each promoter and GFP mRNA could not be detected within 1 hr after transfer to repressing conditions using the nit-6 promoter. We also performed metabolic profiling experiments using proton NMR to identify changes in metabolite levels under inducing and repressing conditions for each promoter. The results demonstrate that conditions used to regulate tcu-1 do not significantly change the primary metabolome and that the differences between inducing and repressing conditions for nit-6 can be accounted for by growth under nitrate or glutamine as a nitrogen source. Our findings demonstrate that nit-6 is a tunable promoter that joins tcu-1 as a choice for regulation of gene expression in N. crassa.
          Cover page: Metabolic Impacts of Using Nitrogen and Copper-Regulated Promoters to Regulate Gene Expression in Neurospora crassa.
          • Article
          • Peer Reviewed
          UC Riverside Previously Published Works (2015)
          Compstatin peptides are complement inhibitors that bind and inhibit cleavage of complement C3. Peptide binding is enhanced by hydrophobic interactions; however, poor solubility promotes aggregation in aqueous environments. We have designed new compstatin peptides derived from the W4A9 sequence (Ac-ICVWQDWGAHRCT-NH2, cyclized between C2 and C12), based on structural, computational, and experimental studies. Furthermore, we developed and utilized a computational framework for the design of peptides containing non-natural amino acids. These new compstatin peptides contain polar N-terminal extensions and non-natural amino acid substitutions at positions 4 and 9. Peptides with α-modified non-natural alanine analogs at position 9, as well as peptides containing only N-terminal polar extensions, exhibited similar activity compared to W4A9, as quantified via ELISA, hemolytic, and cell-based assays, and showed improved solubility, as measured by UV absorbance and reverse-phase HPLC experiments. Because of their potency and solubility, these peptides are promising candidates for therapeutic development in numerous complement-mediated diseases.
            Cover page: New Compstatin Peptides Containing N‑Terminal Extensions and Non-Natural Amino Acids Exhibit Potent Complement Inhibition and Improved Solubility Characteristics
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