Bioinformatics-based identification of GH12 endoxyloglucanases in citrus-pathogenic Penicillium spp.

Millions of tons of citrus peel waste are produced every year as a byproduct of the juice industry. Citrus peel is rich in pectin and xyloglucan, but while the pectin is extracted for use in the food industry, the xyloglucan is currently not valorized. To target hydrolytic degradation of citrus peel xyloglucan into oligosaccharides, we have used bioinformatics to identify three glycoside hydrolase 12 (GH12) endoxyloglucanases (EC 3.2.1.151) from the citrus fruit pathogens Penicillium italicum GL-Gan1 and Penicillium digitatum Pd1 and characterized them on xyloglucan obtained by alkaline extraction from citrus peel. The enzymes displayed pH-temperature optima of pH 4.6-5.3 and 35-37°C. PdGH12 from P. digitatum and PiGH12A from P. italicum share 84% sequence identity and displayed similar kinetics, although kcat was highest for PdGH12. In contrast, PiGH12B from P. italicum, which has the otherwise conserved Trp in subsite -4 replaced with a Tyr, displayed a 3 times higher KM and a 4 times lower kcat/KM than PiGH12A, but was the most thermostable enzyme of the three Penicillium-derived endoxyloglucanases. The benchmark enzyme AnGH12 from Aspergillus nidulans was more thermally stable and had a higher pH-temperature optimum than the enzymes from Penicillum spp. The difference in structure of the xyloglucan oligosaccharides extracted from citrus peel xyloglucan and tamarind xyloglucan by the new endoxyloglucanases was determined by LC-MS. The inclusion of citrus peel xyloglucan demonstrated that the endoxyloglucanases liberated fucosylated xyloglucan oligomers, implying that these enzymes have the potential to upgrade citrus peel residues to produce oligomers useful as intermediates or bioactive compounds.

Bridging DNA contacts allow Dps from E. coli to condense DNA.

The DNA-binding protein from starved cells (Dps) plays a crucial role in maintaining bacterial cell viability during periods of stress. Dps is a nucleoid-associated protein that interacts with DNA to create biomolecular condensates in live bacteria. Purified Dps protein can also rapidly form large complexes when combined with DNA in vitro. However, the mechanism that allows these complexes to nucleate on DNA remains unclear. Here, we examine how DNA topology influences the formation of Dps-DNA complexes. We find that DNA supercoils offer the most preferred template for the nucleation of condensed Dps structures. More generally, bridging contacts between different regions of DNA can facilitate the nucleation of condensed Dps structures. In contrast, Dps shows little affinity for stretched linear DNA before it is relaxed. Once DNA is condensed, Dps forms a stable complex that can form inter-strand contacts with nearby DNA, even without free Dps present in solution. Taken together, our results establish the important role played by bridging contacts between DNA strands in nucleating and stabilizing Dps complexes.

Bridging DNA contacts allow Dps from E. coli to condense DNA.

The DNA-binding protein from starved cells (Dps) plays a crucial role in maintaining bacterial cell viability during periods of stress. Dps is a nucleoid-associated protein that interacts with DNA to create biomolecular condensates in live bacteria. Purified Dps protein can also rapidly form large complexes when combined with DNA in vitro. However, the mechanism that allows these complexes to nucleate on DNA remains unclear. Here, we examine how DNA topology influences the formation of Dps-DNA complexes. We find that DNA supercoils offer the most preferred template for the nucleation of condensed Dps structures. More generally, bridging contacts between different regions of DNA can facilitate the nucleation of condensed Dps structures. In contrast, Dps shows little affinity for stretched linear DNA before it is relaxed. Once DNA is condensed, Dps forms a stable complex that can form inter-strand contacts with nearby DNA, even without free Dps present in solution. Taken together, our results establish the important role played by bridging contacts between DNA strands in nucleating and stabilizing Dps complexes.

Relationships of crystallinity and reaction rates for enzymatic degradation of poly (ethylene terephthalate), PET.

Biocatalytic degradation of plastic waste is anticipated to play an important role in future recycling systems. However, enzymatic degradation of crystalline poly (ethylene terephthalate) (PET) remains consistently poor. Herein, we employed functional assays to elucidate the molecular underpinnings of this limitation. This included utilizing complementary activity assays to monitor the degradation of PET disks with varying crystallinity (XC ), as well as determining enzymatic kinetic parameters for soluble PET fragments. The results indicate that an efficient PET-hydrolase, LCCICCG , operates through an endolytic mode of action, and that its activity is limited by conformational constraints in the PET polymer. Such constraints become more pronounced at high XC values, and this limits the density of productive sites on the PET surface. Endolytic chain-scissions are the dominant reaction type in the initial stage, and this means that little or no soluble organic product are released. However, endolytic cuts gradually and locally promote chain mobility and hence the density of attack sites on the surface. This leads to an upward concave progress curve; a behavior sometimes termed lag-phase kinetics.

Three dimensional printed biofilms: Fabrication, design and future biomedical and environmental applications.

Three dimensional printing has emerged as a widely acceptable strategy for the fabrication of mammalian cell laden constructs with complex microenvironments for tissue engineering and regenerative medicine. More recently 3D printed living materials containing microorganisms have been developed and matured into living biofilms. The potential for engineered 3D biofilms as in vitro models for biomedical applications, such as antimicrobial susceptibility testing, and environmental applications, such as bioleaching, bioremediation, and wastewater purification, is extensive but the need for an in-depth understanding of the structure-function relationship between the complex construct and the microorganism response still exists. This review discusses 3D printing fabrication methods for engineered biofilms with specific structural features. Next, it highlights the importance of bioink compositions and 3D bioarchitecture design. Finally, a brief overview of current and potential applications of 3D printed biofilms in environmental and biomedical fields is discussed.

Enzymatic degradation of poly(ethylene terephthalate) (PET): Identifying the cause of the hypersensitive enzyme kinetic response to increased PET crystallinity.

Plastic pollution poses a significant environmental challenge, with poly(ethylene terephthalate) (PET) being a major contributor due to its extensive use in single use applications such as plastic bottles and other packaging material. Enzymatic degradation of PET offers a promising solution for PET recycling, but the enzyme kinetics in relation to the degree of crystallinity (XC) of the PET substrate are poorly understood. In this study, we investigated the hypersensitive enzyme kinetic response on PET at XC from ∼8.5-12% at 50 °C using the benchmark PET hydrolysing enzyme LCCICCG. We observed a substantial reduction in the maximal enzymatic reaction rate (invVmax) with increasing XC, corresponding to a 3-fold reduction in invVmax when the XC of PET increased from 8.6% to 12.2%. The kinetic analysis revealed that the level of the Mobile Amorphous Fraction (XMAF) was a better descriptor for the enzymatic degradation rate response than XC (or (100%-XC)). By continuous monitoring of the enzymatic reaction progress, we quantified the lag phase prolongation in addition to the steady-state kinetic rates (vss) of the reactions and found that the duration of the lag phase of a reaction could be predicted from the vss and XC by multiple linear regression modeling. The linear correlation between the duration of the lag phase and the vss of the enzymatic PET degradation affirmed that the LCCICCG worked via a random/endo-type enzymatic attack pattern. The longer lag phase at increased XC of PET is proposed to be due to increased substrate entanglement density as well as unproductive enzyme binding to the crystalline regions of PET. The findings enhance our understanding of PET enzymatic degradation kinetics and its dependence on substrate composition, i.e., XMAF and XC.

Glucuronan lyases from family PL7 use a Tyr/Tyr syn ß-elimination catalytic mechanism for glucuronan breakdown.

TpPL7A and TpPL7B, members of CAZy family PL7, act as ß-glucuronan lyases. TpPL7A diverges by lacking the catalytic histidine, identified as the Brønsted base in PL7 alginate lyases. Our research, including TpPL7A's crystal structure, and mutagenesis studies, reveals a shared syn-ß-elimination mechanism with a single tyrosine serving as both base and acid catalyst. This mechanism may extend to subfamily PL7_4 glucuronan lyases.

Bioinformatics and functional selection of GH77 4-α-glucanotransferases for potato starch modification.

4-α-glucanotransferases (4αGTs, EC 2.4.1.25) from glycoside hydrolase family 77 (GH77) catalyze chain elongation of starch amylopectin chains and can be utilized to structurally modify starch to tailor its gelation properties. The potential relationship between the structural design of 4αGTs and functional starch modification is unknown. Here, family GH77 was mined in silico for enzyme candidates based on sub-grouping guided by Conserved Unique Peptide Patterns (CUPP) bioinformatics categorization. From + 12,000 protein sequences a representative set of 27 4αGTs, representing four different domain architectures, different bacterial origins and diverse CUPP groups, was selected for heterologous expression and further study. Most of the enzymes catalyzed starch modification, but their efficacies varied substantially. Five of the 4αGTs were characterized in detail, and their action was compared to that of the industrial benchmark enzyme, Tt4αGT (CUPP 77_1.2), from Thermus thermophilus. Reaction optima of the five 4αGTs ranged from ∼40-60 °C and pH 7.3-9.0. Several were stable for a minimum 4 h at 70 °C. Domain architecture type A proteins, consisting only of a catalytic domain, had high thermal stability and high starch modification ability. All five novel 4αGTs (and Tt4αGT) induced enhanced gelling of potato starch. One, At4αGT from Azospirillum thermophilum (CUPP 77_2.4), displayed distinct starch modifying abilities, whereas T24αGT from Thermus sp. 2.9 (CUPP 77_1.2) modified the starch similarly to Tt4αGT, but slightly more effectively. T24αGT and At4αGT are thus interesting candidates for industrial starch modification. A model is proposed to explain the link between the 4αGT induced molecular modifications and macroscopic starch gelation.

Solid-State Fermented Plant Foods as New Protein Sources.

The current animal-based production of protein-rich foods is unsustainable, especially in light of continued population growth. New alternative proteinaceous foods are therefore required. Solid-state fermented plant foods from Africa and Asia include several mold- and Bacillus-fermented foods such as tempeh, sufu, and natto. These fermentations improve the protein digestibility of the plant food materials while also creating unique textures, flavors, and taste sensations. Understanding the nature of these transformations is of crucial interest to inspire the development of new plant-protein foods. In this review, we describe the conversions taking place in the plant food matrix as a result of these solid-state fermentations. We also summarize how these (nonlactic) plant food fermentations can lead to desirable flavor properties, such as kokumi and umami sensations, and improve the protein quality by removing antinutritional factors and producing additional essential amino acids in these foods. Expected final online publication date for the Annual Review of Food Science and Technology, Volume 15 is April 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Significance of poly(ethylene terephthalate) (PET) substrate crystallinity on enzymatic degradation.

Poly(ethylene terephthalate) (PET) is a semi-crystalline plastic polyester material with a global production volume of 83 Mt/year. PET is mainly used in textiles, but also widely used for packaging materials, notably plastic bottles, and is a major contributor to environmental plastic waste accumulation. Now that enzymes have been demonstrated to catalyze PET degradation, new options for sustainable bio-recycling of PET materials via enzymatic catalysis have emerged. The enzymatic degradation rate is strongly influenced by the properties of PET, notably the degree of crystallinity, XC. The higher the XC of the PET material, the slower the enzymatic rate. Crystallization of PET, resulting in increased XC, is induced thermally (via heating) and/or mechanically (via stretching), and the XC of most PET plastic bottles and microplastics exceeds what currently known enzymes can readily degrade. The enzymatic action occurs at the surface of the insoluble PET material and improves when the polyester chain mobility increases. The chain mobility increases drastically when the temperature exceeds the glass transition temperature, Tg, which is ∼40 °C at the surface layer of PET. Since PET crystallization starts at 70 °C, the ideal temperature for enzymatic degradation is just below 70 °C to balance high chain mobility and enzymatic reaction activation without inducing crystal formation. This paper reviews the current understanding on the properties of PET as an enzyme substrate and summarizes the most recent knowledge of how the crystalline and amorphous regions of PET form, and how the XC and the Tg impact the efficiency of enzymatic PET degradation.

Structural Characterization and Cytotoxic Activity Evaluation of Ulvan Polysaccharides Extracted from the Green Algae Ulva papenfussii.

Ulvan, a sulfated heteropolysaccharide with structural and functional properties of interest for various uses, was extracted from the green seaweed Ulva papenfussii. U. papenfussii is an unexplored Ulva species found in the South China Sea along the central coast of Vietnam. Based on dry weight, the ulvan yield was ~15% (w/w) and the ulvan had a sulfate content of 13.4 wt%. The compositional constitution encompassed L-Rhamnose (Rhap), D-Xylose (Xylp), D-Glucuronic acid (GlcAp), L-Iduronic acid (IdoAp), D-Galactose (Galp), and D-Glucose (Glcp) with a molar ratio of 1:0.19:0.35:0.52:0.05:0.11, respectively. The structure of ulvan was determined using High-Performance Liquid Chromatography (HPLC), Fourier Transform Infrared Spectroscopy (FT-IR), and Nuclear Magnetic Resonance spectroscopy (NMR) methods. The results showed that the extracted ulvan comprised a mixture of two different structural forms, namely ("A3s") with the repeating disaccharide [→4)-ß-D-GlcAp-(1→4)-α-L-Rhap 3S-(1→]n, and ("B3s") with the repeating disaccharide [→4)-α-L-IdoAp-(1→4)-α-L-Rhap 3S(1→]n. The relative abundance of A3s, and B3s was 1:1.5, respectively. The potential anticarcinogenic attributes of ulvan were evaluated against a trilogy of human cancer cell lineages. Concomitantly, Quantitative Structure-Activity Relationship (QSAR) modeling was also conducted to predict potential adverse reactions stemming from pharmacological interactions. The ulvan showed significant antitumor growth activity against hepatocellular carcinoma (IC50 ≈ 90 µg/mL), human breast cancer cells (IC50 ≈ 85 µg/mL), and cervical cancer cells (IC50 ≈ 67 µg/mL). The QSAR models demonstrated acceptable predictive power, and seven toxicity indications confirmed the safety of ulvan, warranting its candidacy for further in vivo testing and applications as a biologically active pharmaceutical source for human disease treatment.

Structural and functional characterization of the novel endo-α(1,4)-fucoidanase Mef1 from the marine bacterium Muricauda eckloniae.

Fucoidanases (EC 3.2.1.-) catalyze the hydrolysis of glycosidic bonds between fucose residues in fucoidans. Fucoidans are a compositionally and structurally diverse class of fucose-containing sulfated polysaccharides that are primarily found in brown seaweeds. Here, the structural characterization of a novel endo-α(1,4)-fucoidanase, Mef1, from the marine bacterium Muricauda eckloniae is presented, showing sequence similarity to members of glycoside hydrolase family 107. Using carbohydrate polyacrylamide gel electrophoresis and nuclear magnetic resonance analyses, it is shown that the fucoidanase Mef1 catalyzes the cleavage of α(1,4)-linkages between fucose residues sulfated on C2 in the structure [-3)-α-L-Fucp2S-(1,4)-α-L-Fucp2S-(1-]n in fucoidan from Fucus evanescens. Kinetic analysis of Mef1 activity by Fourier transform infrared spectroscopy revealed that the specific Mef1 fucoidanase activity (Uf) on F. evanescens fucoidan was 0.1 × 10-3 Uf µM-1. By crystal structure determination of Mef1 at 1.8 Šresolution, a single-domain organization comprising a (ß/α)8-barrel domain was determined. The active site was in an extended, positively charged groove that is likely to be designed to accommodate the binding of the negatively charged, sulfated fucoidan substrate. The active site of Mef1 comprises the amino acids His270 and Asp187, providing acid/base and nucleophile groups, respectively, for the hydrolysis of glycosidic bonds in the fucoidan backbone. Electron densities were identified for two possible Ca2+ ions in the enzyme, one of which is partially exposed to the active-site groove, while the other is very tightly coordinated. A water wire was discovered leading from the exterior of the Mef1 enzyme into the active site, passing the tightly coordinated Ca2+ site.

Polyphenol Oxidase Products Are Priming Agents for LPMO Peroxygenase Activity.

Polyphenol oxidases catalyze the hydroxylation of monophenols to diphenols, which are reducing agents for lytic polysaccharide monooxygenases (LPMOs) in their degradation of cellulose. In particular, the polyphenol oxidase MtPPO7 from Myceliophthora thermophila converts lignocellulose-derived monophenols, and under the new perspective of the peroxygenase reaction catalyzed by LPMOs, we aim to differentiate the role of the catalytic products of MtPPO7 in priming and fueling of LPMO activity. Exemplified by the activity of MtPPO7 towards guaiacol and by using the benchmark LPMO NcAA9C from Neurospora crassa we show that MtPPO7 catalytic products provide the initial electron for the reduction of Cu(II) to Cu(I) but cannot provide the required reducing power for continuous fueling of the LPMO. The priming reaction is shown to occur with catalytic amounts of MtPPO7 products and those compounds do not generate substantial amounts of H2 O2 in situ to fuel the LPMO peroxygenase activity. Reducing agents with a low propensity to generate H2 O2 can provide the means for controlling the LPMO catalysis through exogenous H2 O2 and thereby minimize any enzyme inactivation.

New Insight into the Substrate Selectivity of Bovine Milk γ-glutamyl Transferase via Structural and Molecular Dynamics Predictions.

Bovine milk γ-glutamyltransferase (BoGGT) can produce γ-glutamyl peptides using L-glutamine as a donor substrate, and the transpeptidase activity is highly dependent on both γ-glutamyl donors and acceptors. To explore the molecular mechanism behind the donor and acceptor substrate preferences for BoGGT, molecular docking and molecular dynamic simulations were performed with L-glutamine and L-γ-glutamyl-p-nitroanilide (γ-GpNA) as donors. Ser450 is a crucial residue for the interactions between BoGGT and donors. BoGGT forms more hydrogen bonds with L-glutamine than γ-GpNA, promoting the binding affinity between BoGGT and L-glutamine. Gly379, Ile399, and Asn400 are crucial residues for the interactions between the BoGGT intermediate and acceptors. The BoGGT intermediate forms more hydrogen bonds with Val-Gly than L-methionine and L-leucine, which can promote the transfer of the γ-glutamyl group from the intermediate to Val-Gly. This study reveals the critical residues responsible for the interactions of donors and acceptors with the BoGGT and provides a new understanding of the substrate selectivity and catalytic mechanism of GGT.

Impact of Enzymatically Extracted High Molecular Weight Fucoidan on Lipopolysaccharide-Induced Endothelial Activation and Leukocyte Adhesion.

The endothelial cell lining creates an interface between circulating blood and adjoining tissue and forms one of the most critical barriers and targets for therapeutical intervention. Recent studies suggest that fucoidans, sulfated and fucose-rich polysaccharides from brown seaweed, show multiple promising biological effects, including anti-inflammatory properties. However, their biological activity is determined by chemical characteristics such as molecular weight, sulfation degree, and molecular structure, which vary depending on the source, species, and harvesting and isolation method. In this study, we investigated the impact of high molecular weight (HMW) fucoidan extract on endothelial cell activation and interaction with primary monocytes (MNCs) in lipopolysaccharide (LPS)-induced inflammation. Gentle enzyme-assisted extraction combined with fractionation by ion exchange chromatography resulted in well-defined and pure fucoidan fractions. FE_F3, with a molecular weight ranging from 110 to 800 kDa and a sulfate content of 39%, was chosen for further investigation of its anti-inflammatory potential. We observed that along with higher purity of fucoidan fractions, the inflammatory response in endothelial mono- and co-cultures with MNCs was reduced in a dose-dependent manner when testing two different concentrations. This was demonstrated by a decrease in IL-6 and ICAM-1 on gene and protein levels and a reduced gene expression of TLR-4, GSK3ß and NF-kB. Expression of selectins and, consequently, the adhesion of monocytes to the endothelial monolayer was reduced after fucoidan treatment. These data indicate that the anti-inflammatory effect of fucoidans increases with their purity and suggest that fucoidans might be useful in limiting the inflammatory response of endothelial cells in cases of LPS-induced bacterial infection.

Biotransformation of ginsenoside Rb1 and Rd to four rare ginsenosides and evaluation of their anti-melanogenic effects.

Improving physiological activity of primary ginsenosides through biotransformation is of great significance for food applications. In this study, gynostapenoside XVII, gynostapenoside LXXV, ginsenoside F2, and ginsenoside CK were obtained by enzymolysis of an accessible extract composed of ginsenoside Rb1 and Rd. Their effects on melanin content and tyrosinase activity were compared in vitro, and molecular docking simulation was employed to elucidate the interaction between tyrosinase and individual saponin. The results indicated that four rare ginsenosides decreased tyrosinase activity, melanin content and microphthalmia-associated transcription factor (MITF) expression level, more greatly than their primary ginsenosides, and they were more readily to bind with ASP10 and GLY68 at active site of tyrosinase to inhibit tyrosinase activity as well. These findings suggested that the rare ginsenosides obtained by enzymolysis had excellent anti-melanogenic effect, which could expand the application of ginsenosides in the field of functional foods and health supplements.

Conserved unique peptide patterns (CUPP) online platform 2.0: implementation of +1000 JGI fungal genomes.

Carbohydrate-processing enzymes, CAZymes, are classified into families based on sequence and three-dimensional fold. Because many CAZyme families contain members of diverse molecular function (different EC-numbers), sophisticated tools are required to further delineate these enzymes. Such delineation is provided by the peptide-based clustering method CUPP, Conserved Unique Peptide Patterns. CUPP operates synergistically with the CAZy family/subfamily categorizations to allow systematic exploration of CAZymes by defining small protein groups with shared sequence motifs. The updated CUPP library contains 21,930 of such motif groups including 3,842,628 proteins. The new implementation of the CUPP-webserver, https://cupp.info/, now includes all published fungal and algal genomes from the Joint Genome Institute (JGI), genome resources MycoCosm and PhycoCosm, dynamically subdivided into motif groups of CAZymes. This allows users to browse the JGI portals for specific predicted functions or specific protein families from genome sequences. Thus, a genome can be searched for proteins having specific characteristics. All JGI proteins have a hyperlink to a summary page which links to the predicted gene splicing including which regions have RNA support. The new CUPP implementation also includes an update of the annotation algorithm that uses only a fourth of the RAM while enabling multi-threading, providing an annotation speed below 1 ms/protein.

New insights to diversity and enzyme-substrate interactions of fungal glucuronoyl esterases.

Glucuronoyl esterases (GEs) (EC 3.1.1.117) catalyze the cleavage of ester-linked lignin-carbohydrate complexes that has high impact on the plant cell wall integrity. The GEs are among the very few known types of hydrolytic enzymes that act at the interface of lignin, or which may potentially interact with lignin itself. In this review, we provide the latest update of the current knowledge on GEs with a special focus on the fungal variants. In addition, we have established the phylogenetic relationship between all GEs and this reveals that the fungal enzymes largely fall into one major branch, together with only a minor subset of bacterial enzymes. About 22% of the fungal proteins carry an additional domain, which is almost exclusively a CBM1 binding domain. We address how GEs may interact with the lignin-side of their substrate by molecular docking experiments based on the known structure of the Cerrena unicolor GE (CuGE). The docking studies indicate that there are no direct interactions between the enzyme and the lignin polymer, that the lignin-moiety is facing away from the protein surface and that an elongated carbon-chain between the ester-linkage and the first phenyl of lignin is preferable. Much basic research on these enzymes has been done over the past 15 years, but the next big step forward for these enzymes is connected to application and how these enzymes can facilitate the use of lignocellulose as a renewable resource. KEY POINTS: Fungal GEs are closely related and are sometimes linked to a binding module Molecular docking suggests good accommodation of lignin-like substructures GEs could be among the first expressed enzymes during fungal growth on biomass.

Establishment of specific age-related macular degeneration relevant gene expression panels using porcine retinal pigment epithelium for assessing fucoidan bioactivity.

PURPOSE: Age-related macular degeneration (AMD) is the leading cause of severe vision loss in industrialized nations. Important factors in pathogenesis are oxidative stress, inflammation, and, in the wet form of AMD, angiogenesis. Fucoidans, sulfated polysaccharides from brown algae, may have antioxidant, anti-inflammatory, and antiangiogenic effects. In this study, we established specific gene expression panels for inflammation, oxidative stress and angiogenesis in porcine retinal pigment epithelium (RPE), and investigated the effect of fucoidans on gene expression under different noxious agents. METHODS: Primary porcine RPE cells cultured for at least 14 days were used. Using viability assays with tetrazolium bromide and real-time polymerase chain reaction of marker genes, positive controls were established for appropriate concentrations and exposure times of selected noxious agents (lipopolysaccharide (LPS), H2O2, CoCl2). Three different AMD relevant gene panels specific for porcine RPE for inflammation, oxidative stress, and angiogenesis were established, and the influence of fucoidans (mainly Fucus vesiculosus; FV) on gene expression was investigated. RESULTS: The following was shown by gene expression analyses: (1) Inflammation panel: Expression of 18 genes was affected under LPS (three days). Among them, LPS increased genes for interleukin 1 receptor 2, interleukin 8, cyclooxygenase-2 and vascular cell adhesion protein 1 expression which were diminished when FV was present. (2) Oxidative stress panel: Under stimulation of H2O2 (one day) and LPS (one day), expression of a total of 15 genes was affected. LPS induced increase in genes for superoxide dismutase-1, C-X-C motif chemokine 10, and CC chemokine ligand-5 expression was not detected when FV was present. (3) Angiogenesis panel: Under stimulation with CoCl2 (three days) expression of six genes was affected, with the increase of genes for angiopoietin 2, vascular endothelial growth factor receptor-1, and follistatin being diminished when FV was present. CONCLUSION: Three specific gene expression panels for porcine RPE that map genes for three of the major pathological factors of AMD, inflammation, oxidative stress, and angiogenesis, were established. Further, we demonstrated that fucoidans can reduce stress related gene activation in all of these three major pathogenic pathways. This study is another indication that fucoidans can act on different pathomechanisms of AMD simultaneously, which provides further evidence for fucoidans as a possible drug for treatment and prevention of AMD.

Substrate specificity and transglycosylation capacity of α-L-fucosidases across GH29 assessed by bioinformatics-assisted selection of functional diversity.

Glycoside hydrolase family 29 (GH29) encompasses α-L-fucosidases, i.e. enzymes that catalyze the hydrolytic release of fucose from fucosylated glycans, including N- and O-linked glycans on proteins, and these α-L-fucosidases clearly play important roles in biology. GH29 enzymes work via a retaining exo-action mechanism, and some can catalyze transfucosylation. There is no formal subfamily division of GH29 α-L-fucosidases, but they are nonetheless divided into two subfamilies: GH29A having a range of substrate specificities and GH29B having narrower substrate specificity. However, the sequence traits that determine the substrate specificity and transglycosylation ability of GH29 enzymes are not well characterized. Here, we present a new functional map of family GH29 members based on peptide-motif clustering via CUPP (conserved unique peptide patterns) and compare the substrate specificity and transglycosylation activity of 21 representative α-L-fucosidases across the 53 CUPP groups identified. The 21 enzymes exhibited different enzymatic rates on 8 test substrates, CNP-Fuc, 2'FL, 3FL, Lewisa, Lewisx, Fuc-α1,6-GlcNAc, Fuc-α1,3-GlcNAc, and Fuc-α1,4-GlcNAc. Certain CUPP groups clearly harbored a particular type of enzymes, e.g. the majority of the enzymes having activity on Lewisa or Lewisx categorized in the same CUPP clusters. In general, CUPP was useful for resolving GH29 into functional diversity subgroups when considering hydrolytic activity. In contrast, the transglycosylation capacity of GH29 α-L-fucosidases was distributed across a range of CUPP groups. Transglycosylation thus appears to be a common trait among these enzymes and not readily predicted from sequence comparison.