Small Oligosaccharides Research Articles

Modulating Heparanase Activity: Tuning Sulfation Pattern and Glycosidic Linkage of Oligosaccharides.

Heparanase cleaves polymeric heparan sulfate (HS) molecules into smaller oligosaccharides, allowing for release of angiogenic growth factors promoting tumor development and autoreactive immune cells to reach the insulin-producing β cells. Interaction of heparanase with HS chains is regulated by specific substrate sulfation sequences. We have synthesized 11 trisaccharides that are highly tunable in structure and sulfation pattern, allowing us to determine how heparanase recognizes HS substrate and selects a favorable cleavage site. Our study shows that (1) N-SO3- at +1 subsite and 6-O-SO3- at -2 subsite of trisaccharides are critical for heparanase recognition, (2) addition of 2-O-SO3- at the -1 subsite and of 3-O-SO3- to GlcN unit is not advantageous, and (3) the anomeric configuration (α or β) at the reducing end is crucial in controlling heparanase activity. Our study also illustrates that the α-trisaccharide having N- and 6-O-SO3- at -2 and +1 subsites inhibited heparanase and was resistant toward hydrolysis.

Effects of human milk oligosaccharides (HMOs) on gastrointestinal health.

Gastrointestinal (GI) health of newborns is maintained by the gastrointestinal microbiome, and the PNEI (psychoneuroendocrinoimmunology) system and can be altered by the delivery mode of the milk to the newborn such as breast versus bottle feeding, exposure to antibiotics in the milk, or exposure to environmental pollutants. The health of GI tract is particularly is maintained by breast feeding since the maternal milk provides micro- and macronutrients essential to growth, as well as multifunctional small oligosaccharides, particularly, 2'-fucosyllactose which maintain the GI health.

PSVII-34 Digestibility and growth performance of growing pigs, fed corn or wheat diets supplemented with xylanase

Abstract Corn and wheat contribute variable amounts of arabinoxylans (AX) to swine rations. Xylanase hydrolyzes AX into smaller oligosaccharides, potentially improving nutrient digestibility or fermentation. This experiment was designed to determine the response of growing pigs to exogenous xylanase supplementation in either a corn or wheat based diet. Two hundred pigs (7.99±2.8kg), 5 pigs/pen, 40 pens in 2 blocks (n = 10) were assigned to one of four treatments for 140±7d. Treatments, arranged in a 2x2 factorial, included grain (corn/wheat based) and xylanase (±0.01%,16 000 BXU/kg) as main effects. Pigs were on a five-phase feeding program; diets were isonitrogenous and isoenergetic within each phase. Growth and feed intake were determined weekly. Fecal samples were collected in phases two, four, and five. Growth, feed intake and feed efficiency were unaffected by xylanase (P > 0.10). Except for phase one, pigs receiving corn had improved (P < 0.05) ADG and ADFI relative to wheat; while G:F was improved only in the second phase (P < 0.05). Gross energy digestibility was higher in corn (88.2%) than wheat (87.2%) diets in the nursery phase (P < 0.05). In the finisher phase, xylanase improved energy digestibility of wheat diets (grain*xylanase, P < 0.05). During the nursery phase, percent fecal acetic acid was decreased with xylanase in corn; but increased in wheat diets (grain*xylanase, P < 0.05). Conversely, fecal isobutyric, butyric and isovaleric acid were decreased with xylanase in wheat diets only (grain*xylanase, P < 0.05). In the grower phase, fecal acetic acid was higher (P < 0.05) in wheat than corn diets, while isovaleric, valeric and caproic acid were reduced (P < 0.05). In the finisher phase, fecal propionic acid was lower in wheat diets (P < 0.05). During the finisher phase of growth, xylanase improved energy digestibility of pigs receiving wheat based diets, however this effect was not observed when pigs received corn based diets.

Structural insights into polysaccharide recognition by Flavobacteriumjohnsoniae dextranase, a member of glycoside hydrolase family 31.

Glycoside hydrolase family (GH) 31 contains a large variety of enzymes, but the major members are enzymes that act on relatively small oligosaccharides such as α-glucosidase. Here, we determined the crystal structure of Flavobacteriumjohnsoniae dextranase (FjDex31A), an enzyme from F.johnsoniae that hydrolyzes a polysaccharide, dextran. FjDex31A is composed of four domains: an N-terminal domain, a catalytic domain, a proximal C-terminal domain, and a distal C-terminal domain, as observed in typical GH31 enzymes. However, the architecture of active site residues in FjDex31A, other than subsite -1, is markedly different from that of other GH31 enzymes. The FjDex31A structure in complex with isomaltotriose shows that Gly273 and Tyr524, both of which interact with an α-glucose residue at subsite -2, as well as Trp376 and Leu308-cisGln309, are especially unique to FjDex31A. Site-directed mutagenesis of Gly273 and Tyr524 resulted in a decrease in the hydrolysis of polysaccharides dextran and pullulan, as well as that of the disaccharide isomaltose. These results suggest that, regardless of the length of sugar chains of the substrates, binding of FjDex31A to the substrates at subsite -2 is likely to be important for its activity. DATABASE: Structural data are available in the Protein Data Bank under the accession numbers 6JR6, 6JR7, and 6JR8.

PSVI-25 Feed NSPase enzyme, Natugrain® TS, improves performance and carcass characteristics of grow-finishing pigs

Abstract In a separate data set reported on the same trial (Coelho, 2018) reported that Natugrain TS changed the microbiome by breaking down arabinoxylans into smaller oligosaccharides, decreasing intestinal E. coli and intestinal lesion score. This study data set evaluated the effect of a NSPase enzyme, Natugrain TS, on pig performance and carcass characteristics. A total of 3,750 weaned pigs (28 kg), (PIC 337 x Camborough), were used in a randomized complete block design with pen as the experimental unit, treatment as the fixed effect, and block as the random effect (25 pigs/pen x 5 treatments x 30 replications). Pigs were blocked by sex and weight and fed mash corn/soy/DDGs diets formulated to meet or exceed current NRC (2012) recommendations. Treatments were T1= 0 g/MT Natugrain TS, T2= 100 g/MT Natugrain TS, T3= 150 g/MT Natugrain TS, T4= 200 g/MT Natugrain TS and T5= 250 g/MT Natugrain TS. At the finishing phase, Natugrain TS significantly increased ADG up to 200 g/ton (841, 842, 847, 854 and 852 g, P < 0.05, respectively), decreased FCR up to a dose level of 200 g/ton (2.77, 2.76, 2.74, 2.72 and 2.73, P < 0.05, respectively), increased carcass yield up to a dose level of 200 g/ton (70.2, 70.6, 71.2, 71.4 and 71.2, P < 0.05, respectively), increased percent lean up to a dose level of 200 g/ton (50.1, 50.7, 51.2, 52.4 and 52.1%, P < 0.05, respectively) and decreased mortality up to a dose level of 200 g/ton (4.8, 3.6, 2.7, 1.9 and 2.1%, P < 0.05, respectively). In conclusion, Natugrain TS at 150 to 200 g/ton increased pig performance, carcass charateristics and decreased mortality.

Separation of Isomeric O-Glycans by Ion Mobility and Liquid Chromatography-Mass Spectrometry.

Glycosylation is one of the most important post-translational modifications essential for modulating biological functions on cellular surfaces and within cells. Glycan structures are not predictable from the genome since their biosynthesis is nontemplate driven and subject to multiple sequential and competitive glycosyltransferases/glycosidases. From a structural viewpoint, their analysis presents a particular challenge in terms of sensitivity and structural characterization. Porous graphitized carbon liquid chromatography coupled mass spectrometry (PGCLC-MS) is arguably the gold-standard for the structural characterization of glycoconjugates, especially complex mixtures typical in biological samples. This high performance is due in large part to chromatographic separation of isomers and the information delivered by collision induced fragmentation of each glycan in the mass spectrometer. More recently, ion mobility mass spectrometry (IM-MS) has emerged as an effective tool for gas-phase separation of isomeric oligosaccharides that has been demonstrated with small oligosaccharides and N-glycans. Here, we present a direct comparison of the IM- and LC-separation of O-glycans from porcine gastric and human salivary mucins. Our results identify structures, which are resolved by PGCLC and/or IM, validating the combination of the two methods. Taken together, the incorporation of both techniques into a single platform would be powerful and undoubtedly valuable for determining the full glycome of unknown samples.

Isolation and Identification of Cellulose Degrading Bacteria from Banana Peel Compost

Cellulolytic bacteria are bacteria that have the ability to hydrolyze cellulose complexes into smaller oligosaccharides and eventually become glucose. Glucose is used as a carbon and energy source for bacterial growth. This study was conducted to isolate the cellulose degrading bacteria from banana peel compost that produce cellulose enzymes based on the clear zone that visible around the colony. The cellulolytic activity was determined by the ability of bacteria to hydrolyze the Carboxymethyl Cellulose (CMC) substrate. Determination of cellulolytic activity is known based on cellulolytic index calculation, the diameter total minus the diameter of the colony and divided by the diameter of the colony. The result of five bacterial isolates was found but only one bacterium had the potential to be a cellulose degradation. Based on the Microbact Gram-Negative Identification System, the bacterium is Burkholderia cepacia. These bacteria have an important role in nature as decomposers of various complex compounds, such as cellulose, hemicellulose, lignin, and pectin.

Tuning the Product Spectrum of a Glycoside Hydrolase Enzyme by a Combination of Site-Directed Mutagenesis and Tyrosine-Specific Chemical Modification.

Selective chemical modification of proteins plays a pivotal role for the rational design of enzymes with novel and specific functionalities. In this study, a strategic combination of genetic and chemical engineering paves the way for systematic construction of biocatalysts by tuning the product spectrum of a levansucrase from Bacillus megaterium (Bm-LS), which typically produces small levan-like oligosaccharides. The implementation of site-directed mutagenesis followed by a tyrosine-specific modification enabled control of the product synthesis: depending on the position, the modification provoked either enrichment of short oligosaccharides (up to 800 % in some cases) or triggered the formation of high molecular weight polymer. The chemical modification can recover polymerization ability in variants with defective oligosaccharide binding motifs. Molecular dynamic (MD) simulations provided insights into the effect of modifying non-native tyrosine residues on product specificity.

Hierarchical and programmable one-pot synthesis of oligosaccharides

The programmable one-pot oligosaccharide synthesis method was designed to enable the rapid synthesis of a large number of oligosaccharides, using the software Optimer to search Building BLocks (BBLs) with defined relative reactivity values (RRVs) to be used sequentially in the one-pot reaction. However, there were only about 50 BBLs with measured RRVs in the original library and the method could only synthesize small oligosaccharides due to the RRV ordering requirement. Here, we increase the library to include 154 validated BBLs and more than 50,000 virtual BBLs with predicted RRVs by machine learning. We also develop the software Auto-CHO to accommodate more data handling and support hierarchical one-pot synthesis using fragments as BBLs generated by the one-pot synthesis. This advanced programmable one-pot method provides potential synthetic solutions for complex glycans with four successful examples demonstrated in this work.

The therapeutic effect of aucubin-supplemented hyaluronic acid on interleukin-1beta-stimulated human articular chondrocytes

BackgroundInjection of exogenous hyaluronic acid (HA) into the joint capsule improves symptoms of early stage osteoarthritis (OA). However, reactive oxygen species degrade HA into small oligosaccharides that can elicit pro-inflammatory responses. Likewise, disturbance of the antioxidant enzyme system and generation of oxidative stress by pro-inflammatory cytokines worsen knee OA. Accordingly, we proposed the use of aucubin, an antioxidant and anti-inflammatory compound, as a versatile adjuvant to HA for treating OA. MethodsPrimary human chondrocytes were cultured in media supplemented with aucubin in a series of concentrations (0, 0.01, 0.1, 1, and 10 μg/ml) to study dose-dependent toxicity. We then evaluated the therapeutic effects of HA (100 μg/ml) supplemented with aucubin (10 μg/ml) on interleukin-1 beta (IL-1β, 10 ng/ml)-stimulated chondrocytes. ResultsThe use of aucubin did not change cell viability or alter lactate dehydrogenase release to normal chondrocytes. Although the proliferation and sulfated glycosaminoglycan production were not affected, aucubin partially restored the hypertrophic transformation of chondrocytes. Relative to treatment with HA or aucubin alone, real-time PCR revealed that aucubin-supplemented HA down-regulated the mRNA levels of tumor necrosis factor-alpha (TNF-α), corrected collagen type 1 and aggrecan, and up-regulated tissue inhibitor of metalloproteinase 1. Moreover, ELISA testing also showed a reduced TNF-α production. Although superoxide dismutases activity was still distributed, aucubin restored total antioxidant capacity of IL-1β-stimulated chondrocytes. Western blotting further showed that aucubin inhibited cyclooxygenase-2 and regulated the nuclear factor (erythroid-derived 2)-like 2 pathway. ConclusionAucubin can enhance the anti-catabolic and anti-inflammatory effects of HA on OA chondrocytes.

An F-type lectin domain directs the activity of Streptosporangium roseum alpha-l-fucosidase

F-type lectins are phylogenetically widespread but selectively distributed fucose-binding lectins with L-fucose- and calcium-binding sequence motifs and an F-type lectin fold. Bacterial F-type lectin domains frequently occur in tandem with various protein domains in diverse architectures, indicating a possible role in directing enzyme activities or other biological functions to distinct fucosylated niches. Here, we report the biochemical characterization of a Streptosporangium roseum protein containing an F-type lectin domain in tandem with an NPCBM-associated domain and a family GH 29A alpha-l-fucosidase domain. We show that the F-type lectin domain of this protein recognizes fucosylated glycans in both α and β linkages but has high affinity for a Fuc-α-1,2-Gal motif and that the alpha-l-fucosidase domain displays hydrolytic activity on glycan substrates with α1-2 and α1-4 linked fucose. We also show that the F-type lectin domain does not have any effect on the activity of the cis-positioned alpha-l-fucosidase domain with the synthetic substrate, 4-Methylumbelliferyl-alpha-l-fucopyranoside or on inhibition of this activity by l-fucose or deoxyfuconojirimycin hydrochloride. However, the F-type lectin domain together with the NPCBM-associated domain enhances the activity of the cis-positioned alpha-l-fucosidase domain for soluble fucosylated oligosaccharide substrates. While there are many reports of glycoside hydrolase activity towards insoluble and soluble polysaccharides being enhanced by cis-positioned carbohydrate binding modules on the polypeptide, this is the first report, to our knowledge, of enhancement of activity towards aqueous, freely diffusible, small oligosaccharides. We propose a model involving structural stabilization and a bind-and-jump action mediated by the F-type lectin domain to rationalize our findings.

Efficient conversion of chitosan into chitooligosaccharides by a chitosanolytic activity from Bacillus thuringiensis

Starting from a commercial preparation of Bacillus thuringiensis var. aizawai, widely employed as biopesticide in agriculture, we obtained a chitosanolytic activity by washing the cells with concentrated sodium acetate. The optimum pH and temperature values for this enzyme were 6.0 and 60 °C, respectively. The enzymatic extract efficiently hydrolyzed various chitosans with different deacetylation degrees (DD) into small oligosaccharides. Combining mass spectrometry (ESI-Q-TOF) and high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), we observed the formation of fully deacetylated chitooligosaccharides [fdCOS, (GlcN)2-5] and, to a lesser extent, partially acetylated chitooligosaccharides [paCOS, GlcNAc-(GlcN)1-3]. A 10 g/L solution of chitosan with 600–800 kDa and DD ≥ 90% was fully converted into oligosaccharides in 55 h. Most of the products were fdCOS: 1.6 g/L of chitobiose, 1.7 g/L of chitotriose, 5 g/L of chitotetraose and 1.4 g/L of chitopentaose.

Quantitative Top-Down Mass Spectrometry Identifies Proteoforms Differentially Released during Mechanical Stimulation of Mouse Skin.

Mechanotransduction refers to the processes whereby mechanical stimuli are converted into electrochemical signals that allow for the sensation of our surrounding environment through touch. Despite its fundamental role in our daily lives, the molecular and cellular mechanisms of mechanotransduction are not yet well-defined. Previous data suggest that keratinocytes may release factors that activate or modulate cutaneous sensory neuron terminals, including small molecules, lipids, peptides, proteins, and oligosaccharides. This study presents a first step toward identifying soluble mediators of keratinocyte-sensory neuron communication by evaluating the potential for top-down mass spectrometry to identify proteoforms released during 1 min of mechanical stimulation of mouse skin from naı̈ve animals. Overall, this study identified 47 proteoforms in the secretome of mouse hind paw skin, of which 14 were differentially released during mechanical stimulation, and includes proteins with known and previously unknown relevance to mechanotransduction. Finally, this study outlines a bioinformatic workflow that merges output from two complementary analysis platforms for top-down data and demonstrates the utility of this workflow for integrating quantitative and qualitative data.

Breaking the limits in analyzing carbohydrate recognition by NMR: Resolving Branch‐ Selective Interaction of a Tetraantennary N‐Glycan with lectins

N‐glycans are ubiquitous in nature and functionalize glycoproteins. Protein glycosylation is required for proper biological and biophysical function and often, alterations in glycosylation are related to diseases.[1]Complex glycosylation patterns containing multiantennary N‐glycans are typically found in mature glycoproteins. However, the structural characterization of these glycans is rather challenging. Usually, NMR and X‐ray diffraction techniques fail to provide specific answers on the structure and molecular recognition features due to the intrinsic attributes of the glycan. The properties of the glycosidic bond and especially the presence of 1–6 linkages endow a large flexibility to the molecule. This feature often precludes the crystallization of complex carbohydrates and/or hampers the detection of enough electron density for most of the glycan part in the X‐ray diffraction analysis of glycoproteins. Moreover, the standard use of the corresponding fitting programs to deduce the three‐dimensional structures frequently give rise to incorrect structures of the corresponding saccharide moieties.[2] Thus, any advance in this area is of fundamental value.As a promising approach, carbohydrates conjugated to lanthanide‐binding tags have recently revealed high potential toward this aim. [3,4] In its vicinity, a complexed paramagnetic ion induces significant chemical shift changes of the NMR signals of the glycan due to dipolar interactions involving the unpaired electron of the metal. These pseudocontact shifts (PCS) depend on the distance between each proton and the metal (PCS are proportional to 1/r3). [5] This methodology has first been applied to the study of small oligosaccharides (di‐, tri‐and tetrasaccharides) [6–10], then to N‐glycans. The conformational properties of complex‐type biantennary and high‐mannose‐type N‐glycans were elucidated and could be resolved in each case down to the level of individual branches.[3,4] Proceeding from this experimental basis, we herein extend this concept to the level of high‐degree branching, and show that it is possible to experimentally characterize the conformational behavior and recognition properties of a galactosylated complex‐type tetraantennary N‐glycan, which is not possible by standard NMR methodologies due to chemical shift degeneracy of the glycan. We have then tested the hypothesis of branch selectivity for two N‐acetyllactosamine‐binding lectins, Datura stramonium seed lectin (DSL) and Ricinus Communis agglutinin (RCA 120). [11]Support or Funding InformationWe thank Agencia Estatal de Investigacion and MINECO of Spain (Grants CTQ2016‐76263‐P, CTQ2015‐64597‐C2‐1P, CTQ2015‐64597‐C2‐2P, CTQ2015‐64624‐R and the SEV‐2016‐0644 Severo Ochoa Excellence Acreditation) and the Deutsche Forschungsgemeinschaft for financial support.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Novel Bifunctional Endolytic Alginate Lyase with Variable Alginate-Degrading Modes and Versatile Monosaccharide-Producing Properties.

Endo-type alginate lyases usually degrade alginate completely into various size-defined unsaturated oligosaccharide products (≥disaccharides), while exoenzymes primarily produce monosaccharide products including saturated mannuronate (M) and guluronate (G) units and particularly unsaturated Δ units. Recently, two bifunctional alginate lyases have been identified as endolytic but M- and G-producing with variable action modes. However, endolytic Δ-producing alginate lyases remain undiscovered. Herein, a new Flammeovirga protein, Aly2, was classified into the polysaccharide lyase 7 superfamily. The recombinant enzyme and its truncated protein showed similar stable biochemical characteristics. Using different sugar chains as testing substrates, we demonstrated that the two enzymes are bifunctional while G-preferring, endolytic whereas monosaccharide-producing. Furthermore, the catalytic module of Aly2 can vary the action modes depending on the terminus type, molecular size, and M/G content of the substrate, thereby yielding different levels of M, G, and Δ units. Notably, the enzymes preferentially produce Δ units when digesting small size-defined oligosaccharide substrates, particularly the smallest substrate (unsaturated tetrasaccharide fractions). Deletion of the non-catalytic region of Aly2 caused weak changes in the action modes and biochemical characteristics. This study provided extended insights into alginate lyase groups with variable action modes for accurate enzyme use.

TGFβ counteracts LYVE-1-mediated induction of lymphangiogenesis by small hyaluronan oligosaccharides.

During tissue injury, inflammation, and tumor growth, enhanced production and degradation of the extracellular matrix glycosaminoglycan hyaluronan (HA) can lead to the accumulation of small HA (sHA) oligosaccharides. We have previously reported that accumulation of sHA in colorectal tumors correlates with lymphatic invasion and lymph node metastasis, and therefore, investigated here are the effects of sHA on the lymphatic endothelium. Using cultured primary lymphatic endothelial cells (LECs) and ex vivo and in vivo lymphangiogenesis assays, we found that in contrast to high-molecular-weight HA (HMW-HA), sHA of 4-25 disaccharides in length can promote the proliferation of LECs and lymphangiogenesis in a manner that is dependent on their size and concentration. At pathophysiologically relevant concentrations found in tumor interstitial fluid, sHA is pro-proliferative, acts synergistically with VEGF-C and FGF-2, and stimulates the outgrowth of lymphatic capillaries in ex vivo lymphangiogenesis assays. In vivo, intradermally injected sHA acts together with VEGF-C to increase lymphatic vessel density. Higher concentrations of sHA were found to induce expression of the anti-lymphangiogenic cytokine TGFβ in LECs, which serves to counter-regulate sHA-induced LEC proliferation and lymphangiogenesis. Using appropriate knockout mice and blocking antibodies, we found that the effects of sHA are mediated by the sialylated form of the lymphatic HA receptor LYVE-1, but not by CD44 or TLR-4. These data are consistent with the notion that accumulation of sHA in tumors may contribute to tumor-induced lymphangiogenesis, leading to increased dissemination to regional lymph nodes. KEY MESSAGES : sHA promotes lymphangiogenesis primarily through increased LEC proliferation sHA induces proliferation in a narrow concentration window due to upregulated TGFβ Smaller HA oligosaccharides more potently induce proliferation than larger ones VEGF-C and FGF-2-induced LEC proliferation and lymphangiogenesis is augmented by sHA Sialylated LYVE-1, but not CD44 or TLR-4, mediate the effects of sHA on LEC.

Biochemical Characteristics and Variable Alginate-Degrading Modes of a Novel Bifunctional Endolytic Alginate Lyase.

ABSTRACTBifunctional alginate lyases can efficiently degrade alginate comprised of mannuronate (M) and guluronate (G), but their substrate-degrading modes have not been thoroughly elucidated to date. In this study, we present Aly1 as a novel bifunctional endolytic alginate lyase of the genus Flammeovirga. The recombinant enzyme showed optimal activity at 50°C and pH 6.0. The enzyme produced unsaturated disaccharide (UDP2) and trisaccharide fractions as the final main alginate digests. Primary substrate preference tests and further structure identification of various size-defined final oligosaccharide products demonstrated that Aly1 is a bifunctional alginate lyase and prefers G to M. Tetrasaccharide-size fractions are the smallest substrates, and M, G, and UDP2 fractions are the minimal product types. Remarkably, Aly1 can vary its substrate-degrading modes in accordance with the terminus types, molecular sizes, and M/G contents of alginate substrates, producing a series of small size-defined saturated oligosaccharide products from the nonreducing ends of single or different saturated sugar chains and yielding unsaturated products in distinct but restricted patterns. The action mode changes can be partially inhibited by fluorescent labeling at the reducing ends of oligosaccharide substrates. Deletion of the noncatalytic region (NCR) of Aly1 caused weak changes of biochemical characteristics but increased the degradation proportions of small size-defined saturated M-enriched oligosaccharide substrates and unsaturated tetrasaccharide fractions without any size changes of degradable oligosaccharides, thereby enhancing the M preference and enzyme activity. Therefore, our results provided insight into the variable action mode of a novel bifunctional endolytic alginate lyase to inform accurate enzyme use.IMPORTANCE The elucidated endolytic alginate lyases usually degrade substrates into various size-defined unsaturated oligosaccharide products (≥UDP2), and exolytic enzymes yield primarily unsaturated monosaccharide products. However, it is poorly understood whether endolytic enzymes can produce monosaccharide product types when degrading alginate. In this study, we demonstrated that Aly1, a bifunctional alginate lyase of Flammeovirga sp. strain MY04, is endolytic and monosaccharide producing. Using various sugar chains as testing substrates, we also proved that key factors causing Aly1's action mode changes are the terminus types, molecular sizes, and M/G contents of substrates. Furthermore, the NCR fragment's effects on Aly1's biochemical characteristics and alginate-degrading modes and corresponding mechanisms were discovered by gene truncation and enzyme comparison. In summary, this study provides a novel bifunctional endolytic tool and a variable action mode for accurate use in alginate degradation.

Novel polysaccharide binding to the N-terminal tail of galectin-3 is likely modulated by proline isomerization.

Interactions between galectins and polysaccharides are crucial to many biological processes, and yet these are some of the least understood, usually being limited to studies with small saccharides and short oligosaccharides. The present study is focused on human galectin-3 (Gal-3) interactions with a 60 kDa rhamnogalacturonan RG-I-4 that we use as a model to garner information as to how galectins interact with large polysaccharides, as well as to develop this agent as a therapeutic against human disease. Gal-3 is unique among galectins, because as the only chimera-type, it has a long N-terminal tail (NT) that has long puzzled investigators due to its dynamic, disordered nature and presence of numerous prolines. Here, we use 15N-1H heteronuclear single quantum coherence NMR spectroscopy to demonstrate that multiple sites on RG-I-4 provide epitopes for binding to three sites on 15N-labeled Gal-3, two within its carbohydrate recognition domain (CRD) and one at a novel site within the NT encompassing the first 40 residues that are highly conserved among all species of Gal-3. Moreover, strong binding of RG-I-4 to the Gal-3 NT occurs on a very slow time scale, suggesting that it may be mediated by cis-trans proline isomerization, a well-recognized modulator of many biological activities. The NT binding epitope within RG-I-4 appears to reside primarily in the side chains of the polysaccharide, some of which are galactans. Our results provide new insight into the role of the NT in Gal-3 function.

Characterization of the Paenibacillus beijingensis DSM 24997 GtfD and its glucan polymer products representing a new glycoside hydrolase 70 subfamily of 4,6-α-glucanotransferase enzymes.

Previously we have reported that the Gram-negative bacterium Azotobacter chroococcum NCIMB 8003 uses the 4,6-α-glucanotransferase GtfD to convert maltodextrins and starch into a reuteran-like polymer consisting of (α1→4) glucan chains connected by alternating (α1→4)/(α1→6) linkages and (α1→4,6) branching points. This enzyme constituted the single evidence for this reaction and product specificity in the GH70 family, mostly containing glucansucrases encoded by lactic acid bacteria (http://www.CAZy.org). In this work, 4 additional GtfD-like proteins were identified in taxonomically diverse plant-associated bacteria forming a new GH70 subfamily with intermediate characteristics between the evolutionary related GH13 and GH70 families. The GtfD enzyme encoded by Paenibacillus beijingensis DSM 24997 was characterized providing the first example of a reuteran-like polymer synthesizing 4,6-α-glucanotransferase in a Gram-positive bacterium. Whereas the A. chroococcum GtfD activity on amylose resulted in the synthesis of a high molecular polymer, in addition to maltose and other small oligosaccharides, two reuteran-like polymer distributions are produced by P. beijingensis GtfD: a high-molecular mass polymer and a low-molecular mass polymer with an average Mw of 27 MDa and 19 kDa, respectively. Compared to the A. chroooccum GtfD product, both P. beijingensis GtfD polymers contain longer linear (α1→4) sequences in their structure reflecting a preference for transfer of even longer glucan chains by this enzyme. Overall, this study provides new insights into the evolutionary history of GH70 enzymes, and enlarges the diversity of natural enzymes that can be applied for modification of the starch present in food into less and/or more slowly digestible carbohydrate structures.

6-Mer Hyaluronan Oligosaccharides Modulate Neuroinflammation and α-Synuclein Expression in Neuron-Like SH-SY5Y Cells.

Several studies have shown the degradation of the extracellular matrix at the site of neuroinflammation and increased release of degradation products of glycosaminoglycans. Among these, low molecular weight fragments of hyaluronan (HA) may play a key role in the events leading to neuroinflammation and/or neuronal degeneration. Small HA fragments are able to induce inflammation by stimulating both TLR-2 and TLR-4 as well as CD44 receptors. This stimulation culminates in the nuclear translocation of NF-kB that in turn induces the production of pro-inflammatory intermediates such as TNF-α and IL-1β. The potential of HA fragments, as mediators of inflammation, it has been poorly investigated in neuron-like SH-SY5Y cells so the aim of this study was to investigate the neuroinflammatory effects of very small HA oligosaccharides, the involvement of TLR-2, TLR-4, and CD44 and the production of α-synuclein in such cells. The addition of HA fragments to cell cultures up-regulated TLR-2, TLR-4, and CD44 levels, induced NF-kB activity and increased both TNF-α and IL-β as well as α-synuclein production. On blocking the activity of TLR-2, TLR-4, and CD44 the levels of inflammatory parameters and of α-synuclein were significantly reduced. Since several data have shown as α-synuclein, produced from neurons, is able to initiate ex novo or to maintain an existing neuroinflammatory response, which has been suggested as one of the principal components involved in neurodegenerative pathologies, as PD, we suggest that HA pathways should be given careful consideration when devising future anti-neuroinflammatory strategies to defend against the onset of neurodegenerative disorders. J. Cell. Biochem. 117: 2835-2843, 2016. © 2016 Wiley Periodicals, Inc.