Search NASA⌕ Search

Engineering topics

Heiss, Christian

Publications and source records attributed to Heiss, Christian.

At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Characterization of the Neurospora crassa Galactosaminogalactan Biosynthetic Pathway

The Neurospora crassa genome has a gene cluster for the synthesis of galactosaminogalactan (GAG). The gene cluster includes the following: (1) UDP-glucose-4-epimerase to convert UDP-glucose and UDP-N-acetylglucosamine to UDP-galactose and UDP-N-acetylgalactosamine (NCU05133), (2) GAG synthase for the synthesis of an acetylated GAG (NCU05132), (3) GAG deacetylase (/NCW-1/NCU05137), (4) GH135-1, a GAG hydrolase with specificity for N-acetylgalactosamine-containing GAG (NCU05135), and (5) GH114-1, a galactosaminidase with specificity for galactosamine-containing GAG (NCU05136). The deacetylase was previously shown to be a major cell wall glycoprotein and given the name of NCW-1 (non-GPI anchored cell wall protein-1). Characterization of the polysaccharides found in the growth medium from the wild type and the GAG synthase mutant demonstrates that there is a major reduction in the levels of polysaccharides containing galactosamine and N-acetylgalactosamine in the mutant growth medium, providing evidence that the synthase is responsible for the production of a GAG. The analysis also indicates that there are other galactose-containing polysaccharides produced by the fungus. Phenotypic characterization of wild-type and mutant isolates showed that deacetylated GAG from the wild type can function as an adhesin to a glass surface and provides the fungal mat with tensile strength, demonstrating that the deacetylated GAG functions as an intercellular adhesive. The acetylated GAG produced by the deacetylase mutant was found to function as an adhesive for chitin, alumina, celite (diatomaceous earth), activated charcoal, and wheat leaf particulates.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

An exopolysaccharide pathway from a freshwater Sphingomonas isolate

Bacteria embellish their cell envelopes with a variety of specialized polysaccharides. Biosynthesis pathways for these glycans are complex, and final products vary greatly in their chemical structures, physical properties, and biological activities. This tremendous diversity comes from the ability to arrange complex pools of monosaccharide building blocks into polymers with many possible linkage configurations. Due to the complex chemistry of bacterial glycans, very few biosynthetic pathways have been defined in detail. As part of an initiative to characterize novel polysaccharide biosynthesis enzymes, we isolated a bacterium from Lake Michigan called Sphingomonas sp. LM7 that is proficient in exopolysaccharide (EPS) production. We identified genes that contribute to EPS biosynthesis in LM7 by screening a transposon mutant library for colonies displaying altered colony morphology. A gene cluster was identified that appears to encode a complete wzy/wzx-dependent polysaccharide assembly pathway. Deleting individual genes in this cluster caused a non-mucoid phenotype and a corresponding loss of EPS secretion, confirming the role of this gene cluster in polysaccharide production. We extracted EPS from LM7 cultures and determined that it contains a linear chain of 3- and 4-linked glucose, galactose, and glucuronic acid residues. Finally, we show that the EPS pathway in Sphingomonas sp. LM7 diverges from that of sphingan-family EPSs and adhesive polysaccharides such as the holdfast that are present in other Alphaproteobacteria. Our approach of characterizing complete biosynthetic pathways holds promise for engineering polysaccharides with valuable properties.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Pectic-AGP is a major form of Arabidopsis AGPs

As a key component in cell walls of numerous organisms ranging from green algae to higher plants, AGPs play principal roles in many biological processes such as cell-cell adhesion and regulating Ca 2+ signaling pathway as a Ca 2+ -capacitor. Consistently, AGP structures vary from species to species and from tissue to tissue. To understand the functions of AGPs, it is vital to know their structural differences relative to their location in the plant. Thus, AGPs were purified from different Arabidopsis tissues. Analyses of these AGPs demonstrated that the AGPs comprised covalently linked pectin and AGP, referred to as pectic-AGPs. Importantly, these pectic-AGPs were glycosylated with a remarkable variety of polysaccharides including homogalacturonan, rhamnogalacturonan-I, and type II arabinogalactan at different ratios and lengths. Furthermore, this result not only suggests that pectic-AGP is a major form of Arabidopsis AGPs, but also supports AGPs serve as crosslinkers covalently connecting pectins with structures tailored for tissue-specific functions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Permethylation as a strategy for high–molecular–weight polysaccharide structure analysis by nuclear magnetic resonance—Case study of Xylella fastidiosa extracellular polysaccharide

Current practices for structural analysis of extremely large-molecular-weight polysaccharides via solution-state nuclear magnetic resonance (NMR) spectroscopy incorporate partial depolymerization protocols that enable polysaccharide solubilization in suitable solvents. Non-specific depolymerization techniques utilized for glycosidic bond cleavage, such as chemical degradation or ultrasonication, potentially generate structural fragments that can complicate complete and accurate characterization of polysaccharide structures. Utilization of appropriate enzymes for polysaccharide degradation, on the other hand, requires prior structural knowledge and optimal enzyme activity conditions that are not available to an analyst working with novel or unknown compounds. Herein, we describe an application of a permethylation strategy that allows the complete dissolution of intact polysaccharides for NMR structural characterization. This approach is utilized for NMR analysis of Xylella fastidiosa extracellular polysaccharide (EPS), which is essential for the virulence of the plant pathogen that affects multiple commercial crops and is responsible for multibillion dollar losses each year.

13C↗
DOE OSTI2023

Xylella fastidiosa modulates exopolysaccharide polymer length and the dynamics of biofilm development with a β-1,4-endoglucanase

Xylella fastidiosa is a Gram-negative bacterium that causes disease in many economically important crops. It colonizes the plant host xylem and the mouthparts of its insect vectors where it produces exopolysaccharide (EPS) and forms robust biofilms. Typically, the ability to form a biofilm enhances virulence, but X. fastidiosa does not fit neatly into that paradigm. Instead, X. fastidiosa enters into biofilms to attenuate its movement in the xylem, which, in turn, slows disease progression. In most of its over 600 known plant hosts, X. fastidiosa behaves as a benign commensal, but in some hosts like Vitis vinifera grapevines, it acts as a pathogen. Its ability to attenuate its own virulence in susceptible hosts may be a remnant of its commensal lifestyle in other hosts. Here, we demonstrate that X. fastidiosa utilizes a β-1,4 endoglucanase to cleave its self-produced β-1,4-glucan exopolysaccharide polymer to process it from a higher molecular weight to a lower molecular weight polymer. This processing mediates surface adherence of the cells and ultimately governs overall biofilm architecture, indicating enzymatic pruning of the EPS plays a key role in biofilm-mediated attenuation of X. fastidiosa in planta and, thus, is a key vestige that links its commensal behaviors to its parasitic behaviors in specific hosts.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Xylan-cellulose core structure of oat water-extractable β-glucan macromolecule: Insight into interactions and organization of the cell wall complex

Water-extractable β-glucan with high molar mass (HM) determines health benefits of oat food. Oat β-glucan was extracted by a standardized in vitro digestion method and co-existing water-extractable polysaccharide (WEP) fraction and its HM-arabinoxylan (HM-AX) subfraction were isolated to identify their highly acid-resistant subunit and investigate molecular interactions between constituent polymers. The WEP and HM-AX samples consisted of arabinoxylans (AXs) (74 and 76 %, respectively), however, cellulose constituted the secondary component (6.6 and 12.8 %, respectively). Multi-detection HPSEC along with specific enzymatic hydrolysis of AXs revealed the presence of the HM-xylan domain (16 and 34 %, respectively) built of numerous single- and multi-component populations with random coil and rod-like conformations, which were embedded in a xylan matrix with spherical conformation and controlled the macromolecular shape. Unlike single-component populations, the multi-component ones were resistant to hydrolytic action of AX-hydrolyzing enzymes and represented the subunits that anchor matrix polysaccharides onto cellulose surface. These results indicate that water-extractable β-glucan macromolecule comprises as integral element a cellulose core with two linking populations, HM-xylan and low molar mass glucomannan, which are surrounded by a feruloylated AX-arabinan-arabinogalactan composite and next laminated by β-glucan matrix. Here, the stiff cellulose-xylan backbone is the basis of HM β-glucan organization, controlled by its cellulose-like segments.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Structure, Function, and Application of Self–Healing Adhesives from Mistletoe Viscin

Berries from the European Mistletoe (Viscum album) possess a sticky tissue called viscin that facilitates adhesion and germination onto host trees. Recent studies of viscin have demonstrated its adhesive capacity on a range of natural and synthetic surfaces including wood, skin, metals, and plastic. Yet, the underlying mechanisms remain poorly understood. Here, an investigation of the adhesive performance of mistletoe viscin is performed, demonstrating its hygroscopic nature and ability to self-heal following adhesive failure. It is identified that adhesion originates from a water-soluble adhesive component that can be extracted, isolated, and characterized independently. Lap shear mechanical testing indicates that the mistletoe adhesive extract (MAE) outperforms native viscin tissue, as well as gum arabic and arabinogalactan—common plant-based adhesives. Furthermore, humidity uptake experiments reveal that MAE can reversibly absorb nearly 100% of its mass in water from the atmosphere. In-depth spectroscopic and mass spectrometry investigations reveal a composition consisting primarily of an atypical arabinogalactan, with additional sugar alcohols. Finally, several proof-of-concept applications are demonstrated using MAE for hygro-responsive reversible adhesion between various surfaces including skin, plastic, PDMS, and paper, revealing that MAE holds potential as a biorenewable and reusable adhesive for applications in cosmetics, packaging, and potentially, tissue engineering.

36 MATERIALS SCIENCE↗
DOE OSTI2023

Genetic Characterization of the Acidic and Neutral Glycosphingolipid Biosynthetic Pathways in Neurospora crassa

Fungal glycosphingolipids (GSLs) are important membrane components which play a key role in vesicle trafficking. To assess the importance of GSLs in the fungal life cycle, we performed a mutant phenotypic study of the acidic and neutral GSL biosynthetic pathways in Neurospora crassa. GSL biosynthesis begins with two reactions leading up to the formation of dihydrosphingosine. The first of these reactions is catalyzed by serine palmitoyltransferase and generates 3-keto dihydrosphinganine. In N. crassa, this reaction is catalyzed by GSL-1 and GSL-2 and is required for viability. The second reaction is carried out by GSL-3, a 3-keto dihydrosphinoganine reductase to generate dihydrosphingosine, which is used for the synthesis of neutral and acidic GSLs. We found that deletion mutations in the acidic GSL pathway leading up to the formation of mannosylinositol-phosphoceramide are lethal, indicating that acidic GSLs are essential for viability in N. crassa. Once mannosylinositol-phosphoceramide is made, it is further modified by GSL-5, an inositol-phosphoceramide-B C26 hydroxylase, which adds a hydroxyl group to the amide-linked fatty acid. GSL-5 is not required for viability but gives a clear mutant phenotype affecting all stages of the life cycle. Our results show that the synthesis of mannosylinositol-phosphoceramide is required for viability and that the modification of the amide-linked fatty acid is important for acidic GSL functionality. We also examined the neutral GSL biosynthetic pathway and identified the presence of glucosylceramide. The deletion of neutral GSL biosynthetic genes affected hyphal morphology, vegetative growth rate, conidiation, and female development. Our results indicate that the synthesis of neutral GSLs is essential for normal growth and development of N. crassa.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

An Immunomodulatory Polysaccharide–Protein Complex Isolated from the Polypore Fungus Royoporus badius

Many wild edible polypore mushrooms have medicinal value. In this study, we investigate the potential medicinal properties of the wild polypore mushroom Royoporus badius collected from north-central British Columbia, Canada. Water extract from R. badius was found to exhibit potent immunomodulatory activity. The extract was purified using DEAE-Sephadex anion-exchange chromatography as well as Sephacryl S-500 and HPLC BioSEC5 size-exclusion chromatography, to yield a novel polysaccharide-protein complex (IMPP-Rb).IMPP-Rb has a peak maxima molecular weight (M p ) of 950 kDa. GC-MS analyses showed that IMPP-Rb is composed predominantly of glucose (49.2%), galactose (11.3%), mannose (10.8%), rhamnose (9.6%), and galacturonic acid (8.2%), with smaller amounts of xylose (5.2%), fucose (2.8%), N-acetyl glucosamine (1.8%), and arabinose (1.2%). IMPP-Rb has multiple linkages, with 4-Glcp, 4-Manp, 6-Manp, 3,4-Manp, 4-Xylp, and 2-Rhap being the most prominent. IMPP-Rb is capable of inducing many cytokines in vitro and the protein component is indispensable for its immunomodulatory activity. IMPP-Rb has potential application as an immuno-stimulatory agent with pharmaceutical value.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Structural characterization and biological activity of an α-glucan from the mollusk Marcia hiantina (Lamarck, 1818)

Marcia hiantina (Mollusca, Bivalvia) (Lamarck, 1818), is an edible clam mainly distributed along the tropical coastal regions. Recent researches have demonstrated that clams can possess compounds, including polysaccharides, with a wide range of biological actions including antioxidant, immunomodulatory and antitumor activities. Here an α-glucan was isolated from M. hiantina by hot water, purified by anion exchange chromatography, and its structure was characterized by a combination of multiple nuclear magnetic resonance (NMR) methods (1D 1 H, 1 H- 1 H COSY, 1 H- 1 H TOCSY, 1 H- 1 H NOESY, 1 H- 13 C HSQC and 1 H- 13 C HSQC-NOESY spectra), gas chromatography-mass spectrometry, and high performance size exclusion chromatography (HPSEC). The analysis from NMR, monosaccharide composition, methylation analyses and HPSEC combined with multi angle light scattering (MALS) of M. hiantina-derived α-glycan confirmed a branched polysaccharide exclusively composed of glucose (Glc), mostly 4-linked in its backbone, branched occasionally at 6-positions, and having a molecular weight of ~ 570 kDa. The mollusk α-glucan was subjected to four cell-based assays: (i) viability of three cell lines (RAW264.7, HaCaT, and HT-29), (ii) activity on lipopolysaccharide (LPS)-induced prostaglandin production in RAW264.7 cells, (iii) inhibitory activities of in H 2 O 2 - and LPS-induced reactive oxygen species (ROS) production in HMC3 cells, and (iv) HaCaT cell proliferation. Furthermore, results have indicated no cytotoxicity, potent inhibition of both H 2 O 2 - and LPS-induced ROS, and potent cell proliferative activity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Most of the rhamnogalacturonan-I from cultured Arabidopsis cell walls is covalently linked to arabinogalactan-protein

To characterize a purified rhamnogalacturonan-I (RG-I) containing both RG-I and arabinogalactan-protein (AGP) types of glycosyl residues, an AGP-specific β-1,3-galactanase that can cleave the AG backbone and release the AG sidechain was applied to this material. Carbohydrate analysis and NMR spectroscopy verified that the galactanase-released carbohydrate consists of RG-I covalently attached to the AG sidechain, proving a covalent linkage between RG-I and AGP. Size exclusion chromatography-multiangle light scattering-refractive index detection revealed that the galactanase-released RG-I has an average molecular weight of 41.6 kDa, which, together with the percentage of pectic sugars suggests an RG-I-AGP comprising one AGP covalently linked to two RG-I glycans. Carbohydrate analysis and NMR results of the RG-I-AGP, the galactanase-released glycans, and the RG lyase-released glycans demonstrated that the attached RG-I glycans are decorated with α-1,5-arabinan, β-1,4-galactan, xylose, and 4-O-Me-xylose sidechains. Furthermore, our measurement suggests that the covalently linked RG-I-AGP is the major component of the traditionally prepared RG-I.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Isolation and characterization of an anti-proliferative polysaccharide from the North American fungus Echinodontium tinctorium

Abstract A novel polysaccharide EtGIPL1a was purified from fruiting bodies of Echinodontium tinctorium , a fungus unique to western North America. EtGIPL1a has an estimated weight average molecular weight of 275 kDa and is composed of glucose (54.3%), galactose (19.6%), mannose (11.1%), fucose (10.3%), glucuronic acid (4%), and rhamnose (0.6%). It has multiple glycosidic linkages, with 3-Glc p (28.9%), 6-Glc p (18.3%), 3,6-Glc p (13%), 4-Glc p A (9.2%), 6-Gal p (3.9%), 2,6-Gal p (2.6%), 3-Fuc p (2.5%), 6-Man p (2.4%) being the most prominent, and unsubstituted glucose (15.3%), mannose (1.3%) and fucose (0.9%) as major terminal sugars. EtGIPL1a has a backbone containing mostly 3-substituted β -glucopyranose with 4-substituted glucopyranosyluronic acid. EtGIPL1a showed anti-proliferative activity against multiple cancer cell lines, with IC 50 ranging from 50.6 to 1446 nM. Flow cytometry analyses confirmed that apoptosis induction is one mechanism for its anti-proliferative activity. EtGIPL1a should be further investigated for its potential anti-cancer activity in animal models, and for its possible utility in differentiation cancer therapy.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Structure of Lipopolysaccharide from Liberibacter crescens Is Low Molecular Weight and Offers Insight into Candidatus Liberibacter Biology

Huanglongbing (HLB) disease, also known as citrus greening disease, was first reported in the US in 2005. Since then, the disease has decimated the citrus industry in Florida, resulting in billions of dollars in crop losses and the destruction of thousands of acres of citrus groves. The causative agent of citrus greening disease is the phloem limited pathogen Candidatus Liberibacter asiaticus. As it has not been cultured, very little is known about the structural biology of the organism. Liberibacter are part of the Rhizobiaceae family, which includes nitrogen-fixing symbionts of legumes as well as the Agrobacterium plant pathogens. To better understand the Liberibacter genus, a closely related culturable bacterium (Liberibacter crescens or Lcr) has attracted attention as a model organism for structural and functional genomics of Liberibacters. Given that the structure of lipopolysaccharides (LPS) from Gram-negative bacteria plays a crucial role in mediating host-pathogen interactions, we sought to characterize the LPS from Lcr. We found that the major lipid A component of the LPS consisted of a pentaacylated molecule with a β-6-GlcN disaccharide backbone lacking phosphate. The polysaccharide portion of the LPS was unusual compared to previously described members of the Rhizobiaceae family in that it contained ribofuranosyl residues. The LPS structure presented here allows us to extrapolate known LPS structure/function relationships to members of the Liberibacter genus which cannot yet be cultured. It also offers insights into the biology of the organism and how they manage to effectively attack citrus trees.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Glycosylation of SARS-CoV-2: structural and functional insights

The COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Similar to other coronaviruses, its particles are composed of four structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins. S, E, and M proteins are glycosylated, and the N protein is phosphorylated. The S protein is involved in the interaction with the host receptor human angiotensin-converting enzyme 2 (hACE2), which is also heavily glycosylated. Recent studies have revealed several other potential host receptors or factors that can increase or modulate the SARS-CoV-2 infection. Interestingly, most of these molecules bear carbohydrate residues. While glycans acquired by the viruses through the hijacking of the host machinery help the viruses in their infectivity, they also play roles in immune evasion or modulation. Here, glycans play complex roles in viral pathobiology, both on their own and in association with carrier biomolecules, such as proteins or glycosaminoglycans (GAGs). Understanding these roles in detail can help in developing suitable strategies for prevention and therapy of COVID-19. In this review, we sought to emphasize the interplay of SARS-CoV-2 glycosylated proteins and their host receptors in viral attachment, entry, replication, and infection. Moreover, the implications for future therapeutic interventions targeting these glycosylated biomolecules are also discussed in detail.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Granulibacter bethesdensis, a Pathogen from Patients with Chronic Granulomatous Disease, Produces a Penta-Acylated Hypostimulatory Glycero-D-talo-oct-2-ulosonic Acid–Lipid A Glycolipid (Ko-Lipid A)

Granulibacter bethesdensis can infect patients with chronic granulomatous disease, an immunodeficiency caused by reduced phagocyte NADPH oxidase function. Intact G. bethesdensis (Gb) is hypostimulatory compared to Escherichia coli, i.e., cytokine production in human blood requires 10–100 times more G. bethesdensis CFU/mL than E. coli. To better understand the pathogenicity of G. bethesdensis, we isolated its lipopolysaccharide (GbLPS) and characterized its lipid A. Unlike with typical Enterobacteriaceae, the release of presumptive Gb lipid A from its LPS required a strong acid. NMR and mass spectrometry demonstrated that the carbohydrate portion of the isolated glycolipid consists of α-Manp-(1→4)-β-GlcpN3N-(1→6)-α-GlcpN-(1⇿1)-α-GlcpA tetra-saccharide substituted with five acyl chains: the amide-linked N-3′ 14:0(3-OH), N-2′ 16:0(3-O16:0), and N-2 18:0(3-OH) and the ester-linked O-3 14:0(3-OH) and 16:0. The identification of glycero-d-talo-oct-2-ulosonic acid (Ko) as the first constituent of the core region of the LPS that is covalently attached to GlcpN3N of the lipid backbone may account for the acid resistance of GbLPS. In addition, the presence of Ko and only five acyl chains may explain the >10-fold lower proinflammatory potency of GbKo–lipidA compared to E. coli lipid A, as measured by cytokine induction in human blood. These unusual structural properties of the G.bethesdensis Ko–lipid A glycolipid likely contribute to immune evasion during pathogenesis and resistance to antimicrobial peptides.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗