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C-type lectins (CTLs) play important roles in innate immune system of crustaceans as pattern recognition receptors (PRRs). In this study, a novel CTL gene was obtained from the red swamp crayfish, Procambarus clarkii, designated as PcLec. PcLec encodes a peptide with 175 amino acids, with a signal peptide and a single carbohydrate recognition domain (CRD). The PcLec transcripts were specifically expressed in crayfish stomach and were induced by bacterial challenge. In vitro assays with recombinant PcLec protein revealed that it had bacterial binding activity, polysaccharide binding activity, bacterial agglutination activity, and antimicrobial activity. Most importantly, PcLec knockdown significantly impaired the survivability of crayfish upon oral infection with its pathogen A. hydrophila. According to these results, we infer that the PcLec plays a crucial role in antibacterial defense of crayfish.

Background Laurel wilt caused by Raffaelea lauricola is a lethal vascular disease of North American members of the Lauraceae plant family. This fungus and its primary ambrosia beetle vector Xyleborus glabratus originated from Asia; however, there is no report of laurel wilt causing widespread mortality on native Lauraceae trees in Asia. To gain insight into why R. lauricola is a tree-killing plant pathogen in North America, we generated and compared high quality draft genome assemblies of R. lauricola and its closely related non-pathogenic species R. aguacate. Results Relative to R. aguacate, the R. lauricola genome uniquely encodes several small-secreted proteins that are associated with virulence in other pathogens and is enriched in secondary metabolite biosynthetic clusters, particularly polyketide synthase (PKS), non-ribosomal peptide synthetase (NRPS) and PKS-NRPS anchored gene clusters. The two species also exhibit significant differences in secreted proteins including CAZymes that are associated with polysaccharide binding including the chitin binding CBM50 (LysM) domain. Transcriptomic comparisons of inoculated redbay trees and in vitro-grown fungal cultures further revealed a number of secreted protein genes, secondary metabolite clusters and alternative sulfur uptake and assimilation pathways that are coordinately up-regulated during infection. Conclusions Through these comparative analyses we have identified potential adaptations of R. lauricola that may enable it to colonize and cause disease on susceptible hosts. How these adaptations have interacted with co-evolved hosts in Asia, where little to no disease occurs, and non-co-evolved hosts in North America, where lethal wilt occurs, requires additional functional analysis of genes and pathways.

Genome data mining of the Antarctic yeast, Glaciozyma antarctica PI12 revealed an expansin-like protein encoding sequence (GaEXLX1). The GaEXLX1 protein is 24.8 kDa with a high alkaline pI of 9.81. Homology modeling of GaEXLX1 showed complete D1 and D2 domains of a conventional expansin. The protein exhibited 36% sequence similarity to Clavibacter michiganensis EXLX1 (PDB: 4JCW). Subsequently, a recombinant GaEXLX1 protein was produced using Escherichia coli expression system. Incubation with Avicel, filter paper and cotton fiber showed that the protein can disrupt the surface of crystalline and pure cellulose, suggesting a cell wall modification activity usually exhibited by expansin-like proteins. Binding assays displayed that GaEXLX1 can bind to polymeric substrates, including those postulated to be present in the sea ice ecosystem such as crab chitin and moss lichenan. GaEXLX1 may assist in the recognition and loosening of these substrates in the sea ice prior to hydrolysis by other extracellular enzymes. Similar loosening mechanism to classical expansin-like protein has been postulated for this psychrophilic protein based on several conserved residues of GaEXLX1 involved in binding interaction identified by docking analyses.

To date, COVID-19 is still a severe threat to public health, hence specific effective therapeutic drugs development against SARS-CoV-2 is urgent needed. 3CLpro and PLpro and RdRp are the enzymes required for the SARS-CoV-2 RNA synthesis. Therefore, binding to the enzyme may interfere the enzyme function. Before, we found that sulfated polysaccharide binding to 3CLpro might block the virus replication. Hence, we hypothesize that negative charged pectin glycan may also impede the virus replication. Here we show that 922 crude polysaccharide from Syzygium aromaticum may near completely block SARS-CoV-2 replication. The inhibition rate was 99.9% (EC50 : 0.90 μM). Interestingly, 922 can associates with 3CLpro, PLpro and RdRp. We further show that the homogeneous glycan 922211 from 922 may specifically attenuate 3CL protease activity. The IC50s of 922 and 922211 against 3CLpro are 4.73 ± 1.05 µM and 0.18 ± 0.01 µM, respectively. Monosaccharide composition analysis reveals that 922211 with molecular weight of 78.7 kDa is composed of rhamnose, galacturonic acid, galactose and arabinose in the molar ratio of 8.21 : 37.81 : 3.58 : 4.49. The structure characterization demonstrated that 922211 is a homogalacturonan linked to RG-I pectin polysaccharide. The linear homogalacturonan part in the backbone may be partly methyl esterified while RG-I type part bearing 1, 4-linked α-GalpA, 1, 4-linked α-GalpAOMe and 1, 2, 4-linked α-Rhap. There are four branches attached to C-1 or C4 position of Rhamnose glycosyl residues on the backbone. The branches are composed of 1, 3-linked β-Galp, terminal (T)-linked β-Galp, 1, 5-linked α-Araf, T-linked α-Araf, 4-linked α-GalpA and/or 4-linked β-GalpA. The above results suggest that 922 and 922211 might be a potential novel leading compound for anti-SARS-CoV-2 new drug development.

Significance Pseudomonas aeruginosa bacteria form antibiotic-tolerant biofilms that pose significant challenges in clinical settings. Overcoming these challenges requires fundamental insights into how biofilms are formed, combined with innovative strategies to disrupt biofilms. Electron cryotomography in situ data presented here reveal the arrangement of the key P. aeruginosa adhesin CdrA at biofilm cell–cell junctions. Guided by our imaging data, we raised and characterized a CdrA-specific nanobody binder capable of disrupting these cell–cell junctions, thereby increasing the efficacy of antibiotic-mediated bacterial killing in biofilms. Together these data provide a pathway for developing effective alternative bacterial infection treatment strategies., Many bacteria, including the major human pathogen Pseudomonas aeruginosa, are naturally found in multicellular, antibiotic-tolerant biofilm communities, in which cells are embedded in an extracellular matrix of polymeric molecules. Cell–cell interactions within P. aeruginosa biofilms are mediated by CdrA, a large, membrane-associated adhesin present in the extracellular matrix of biofilms, regulated by the cytoplasmic concentration of cyclic diguanylate. Here, using electron cryotomography of focused ion beam–milled specimens, we report the architecture of CdrA molecules in the extracellular matrix of P. aeruginosa biofilms at intact cell–cell junctions. Combining our in situ observations at cell–cell junctions with biochemistry, native mass spectrometry, and cellular imaging, we demonstrate that CdrA forms an extended structure that projects from the outer membrane to tether cells together via polysaccharide binding partners. We go on to show the functional importance of CdrA using custom single-domain antibody (nanobody) binders. Nanobodies targeting the tip of functional cell-surface CdrA molecules could be used to inhibit bacterial biofilm formation or disrupt preexisting biofilms in conjunction with bactericidal antibiotics. These results reveal a functional mechanism for cell–cell interactions within bacterial biofilms and highlight the promise of using inhibitors targeting biofilm cell–cell junctions to prevent or treat problematic, chronic bacterial infections.

Many bacteria, including the major human pathogen Pseudomonas aeruginosa, are naturally found in multicellular, antibiotic-tolerant biofilm communities, where cells are embedded in an extracellular matrix of polymeric molecules. Cell-cell interactions within P. aeruginosa biofilms are mediated by CdrA, a large, membrane-associated adhesin present in the extracellular matrix of biofilms, regulated by the cytoplasmic concentration of cyclic diguanylate. Here, using electron cryotomography of focused-ion beam milled specimens, we report the architecture of CdrA molecules in the extracellular matrix of P. aeruginosa biofilms at intact cell-cell junctions. Combining our in situ observations at cell-cell junctions with biochemistry, native mass spectrometry and cellular imaging, we demonstrate that CdrA forms an extended structure that projects from the outer membrane to tether cells together via polysaccharide binding partners. We go on to show the functional importance of CdrA using custom single-domain antibody (nanobody) binders. Nanobodies targeting the tip of functional cell-surface CdrA molecules could be used to inhibit bacterial biofilm formation or disrupt pre-existing biofilms in conjunction with bactericidal antibiotics. These results reveal a functional mechanism for cell-cell interactions within bacterial biofilms and highlight the promise of using inhibitors targeting biofilm cell-cell junctions to prevent or treat problematic, chronic bacterial infections.

Besides their catalysis, specific interactions between starch/glycogen processing enzymes and their substrates have been reported. Multiple branching enzyme (BE) isoforms, BE1, BE2, and BE3, have been found in a limited number of cyanobacterial species that are characterized by amylopectin accumulation. Seven surface binding sites (SBSs) located away from the active site have been identified in crystal structures of cyanobacterial BE1 from Crocosphaera subtropica (Cyanothece sp.) ATCC 51142 (51142BE1). In the present study, binding affinity toward amylopectin, amylose, and glycogen was investigated for wild-type 51142BE1 and its mutants (residues at SBSs important for sugar-binding were replaced by alanine). These enzymes showed retarded mobility during electrophoresis in non-denaturing polyacrylamide gels in the presence of polysaccharides. This was caused by interactions between the enzymes and the polysaccharides, enabling calculation of the dissociation constants (Kd values) of the enzymes toward the polysaccharides. Mutational analysis indicated that particular domains of the protein (domains A and C) were involved in the polysaccharide binding. Kd values toward the polysaccharides were also measured for 10 BE isoforms (five BE1, three BE2, and two BE3) from 5 cyanobacterial strains. All BEs displayed much lower Kd values (higher affinity) toward amylopectin and amylose than toward glycogen, as described for plant BEs. In addition, one BE2 displayed exceptionally high Kd values (low affinity), while two BE3 exhibited multiple Kd values to all polysaccharides. These results could be ascribed to sequence variations in the SBSs, irrespective of the catalytic specificity.

The complement C1r/C1s, Uegf, and Bmp1 (CUB) domains, which are most exclusively found in extracellular and plasma membrane-related proteins, are involved in various biological processes. In this study, a CUB domain-containing protein (designed as HcCDCP) was cloned and characterized from freshwater pearl mussel (Hyriopsis cumingii). The 2280 bp complete cDNA of the HcCDCP contained a 1002 bp open reading frame, which encoded a protein with 333 amino acids. The predicted HcCDCP protein contained a typical CUB domain and a transmembrane region. The tissue distribution analysis indicated that the HcCDCP was detected in all tissues, and the highest expression was found in hepatopancreas followed by gills. After infection with bacteria (i.e., Staphylococcus aureus and Vibrio parahaemolyticus), virus (white spot syndrome virus) and virus analogs (poly[I:C]), the mRNA level of the HcCDCP was significantly upregulated, suggesting that the HcCDCP might be involved in host immune defense response. The RNA interference revealed that the silencing of the HcCDCP could evidently inhibit the expression levels of lysozyme and tumor necrosis factor. Moreover, the recombinant protein of the CUB domain (rCUB) possessed binding capacity to eight different kinds of bacteria. The polysaccharide binding assay showed that the rCUB specifically bound to lipopolysaccharide, peptidoglycan, and D-mannose. This study provided valuable information for exploring the biological roles of CDCPs in the host defense system of mollusks.

Being biocompatible and biodegradable polymers, polysaccharides present a perspective material for drug delivery systems. This study aimed at unraveling the molecular details of interactions between rhamnogalacturonan I, brunched with galactan side chains, and RNase from Bacillus Intermedius, binase. FTIR- and NMR-spectroscopic analyses showed that binase interacts with side chains of the polysaccharide. In complexes with polysaccharide, the protein retains its native structure. The 2D-NMR techniques revealed eight protein residues responsive to polysaccharide binding. Further, computer simulations were carried out to provide the atomistic details of binase-polysaccharide complexes. Both blind and knowledge-based docking procedures elucidate the existence of epitopes on the binase surface with the preferential binding of galactan fragments. The refinement of these complexes by molecular dynamics simulations confirmed stable protein-polysaccharide interactions. The results of this study strengthen the knowledge on non-specific protein-carbohydrate interactions and outline the rhamnogalacturonan I as a possible matrix material for protein delivery systems.

Black poplar (Populus deltoides, P. nigra, and their hybrids) is the main poplar cultivars in China. It offers interesting options of large-scale biomass production for bioenergy due to its rapid growth and high yield. Poplar wood properties were associated with chemical components and physical structures during wood formation. In this study, five poplar cultivars, P. euramericana 'Zhonglin46' (Pe1), P. euramericana 'Guariento' (Pe2), P. nigra 'N179' (Pn1), P. deltoides 'Danhong' (Pd1), and P. deltoides 'Nanyang' (Pd2), were used to explore the molecular mechanism of xylem development. We analyzed the structural differences of developing xylem in the five cultivars and profiled the transcriptome-wide gene expression patterns through RNA sequencing. The cross sections of the developing xylem showed that the cell wall thickness of developed fiber in Pd1 was thickest and the number of xylem vessels of Pn1 was the least. A total of 10,331 differentially expressed genes were identified among 10 pairwise comparisons of the five cultivars, most of them were related to programmed cell death and secondary cell wall thickening. K-means cluster analysis and Gene Ontology enrichment analysis showed that the genes highly expressed in Pd1 were related to nucleotide decomposition, metabolic process, transferase, and microtubule cytoskeleton; whereas the genes highly expressed in Pn1 were involved in cell wall macromolecule decomposition and polysaccharide binding processes. Based on a weighted gene co-expression network analysis, a large number of candidate regulators for xylem development were identified. And their potential regulatory roles to cell wall biosynthesis genes were validated by a transient overexpression system. This study provides a set of promising candidate regulators for genetic engineering to improve feedstock and enhance biofuel conversion in the bioenergy crop Populus.

Broad and unspecific use of antibiotics accelerates spread of resistances. Sensitive and robust pathogen detection is thus important for a more targeted application. Bacteriophages contain a large repertoire of pathogen‐binding proteins. These tailspike proteins (TSP) often bind surface glycans and represent a promising design platform for specific pathogen sensors. We analysed bacteriophage Sf6 TSP that recognizes the O‐polysaccharide of dysentery‐causing Shigella flexneri to develop variants with increased sensitivity for sensor applications. Ligand polyrhamnose backbone conformations were obtained from 2D 1H,1H‐trNOESY NMR utilizing methine–methine and methine–methyl correlations. They agreed well with conformations obtained from molecular dynamics (MD), validating the method for further predictions. In a set of mutants, MD predicted ligand flexibilities that were in good correlation with binding strength as confirmed on immobilized S. flexneri O‐polysaccharide (PS) with surface plasmon resonance. In silico approaches combined with rapid screening on PS surfaces hence provide valuable strategies for TSP‐based pathogen sensor design., The way to go: Antibiotic resistance is increasing worldwide. By understanding and fine‐tuning protein–carbohydrate interaction, improved bacterial sensors for faster diagnostics can be developed for example based on bacteriophage tailspike proteins. We used molecular dynamics simulations, NMR spectroscopy and surface plasmon resonance experiments for the design of affinity improved Shigella flexneri O‐antigen binding tailspike proteins.

Intelectin is a recently characterized soluble galactofuranose-binding lectin that exists in species ranging from amphioxus to human. Interestingly, intelectin does not contain a canonical carbohydrate-recognition domain (CRD). Therefore, we designed serial deletions of intelectin in the Chinese amphioxus (Branchiostoma belcheri tsingtauense, AmphiITLN71469) in order to identify functional regions required for carbohydrate binding. Our results revealed that Domain 5 (aa 203–302) was able to bind lipopolysaccarides (LPS) or peptidoglycan (PGN) and agglutinate bacteria as efficiently as the full-length protein. Three dimensional (3D) atomic models of Domain 5 were generated by ab initio based program QUARK and by Iterative Threading Assembly Refinement (I-TASSER) programs, in which four amino acids mediating calcium-binding (G54-G55-G56-E91) were identified by hemagglutination assay. Furthermore, a striking functional conservation of Domain 5 was detected in zebrafish intelectin 1. Taken together, our findings identified for the first time a new CRD domain in intelectin, thereby providing new knowledge leading to a better understanding of pathogen-host interactions.

Lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP) existed in diversity of invertebrates including shrimp plays a crucial role in an innate immunity via mediating the recognition of invading pathogens. In this study, LGBP was cloned and characterized from the hepatopancreas of Litopenaeus vannamei , named as LvLGBP. Its full-length cDNA of 1282 bp contained an open reading frame (1101 bp) encoding a peptide of 367 amino acids. The LGBP primary structure contained a glycosyl hydrolase domain, two integrin binding motifs, two kinase C phosphorylation sites, and two polysaccharide recognition motifs which were identified as a polysaccharide binding motif and a β-1,3-glucan recognition motif. The LvLGBP transcripts were expressed mainly in the hepatopancreas. Upon challenge with Vibrio parahaemolyticus or white spot syndrome virus (WSSV), the LvLGBP mRNA expression was significantly up-regulated to reach a maximum at 48 h post injection. Its expression was also induced by lipopolysaccharide (LPS) or β-1,3-glucan stimulation. RNAi-based silencing resulted in the critical suppression of LvLGBP expression. Knockdown of LvLGBP gene with co-inoculation by V. parahaemolyticus or WSSV led to increase in the cumulative mortality and reduce in the median lethal time. Native LGBP was detected only in the hepatopancreas as verified by Western blotting. Purified LGBP from the hepatopancreas exhibited the agglutinating and binding activity towards Gram-negative bacterium V. parahaemolyticus with calcium-dependence. Its agglutinating activity was dominantly inhibited by LPS with higher potential than β-1,3-glucan. Purified LvLGBP could significantly activate the hemocyte phenoloxidase activity in the presence of LPS (12.9 folds), while slight activation was detected with β-1,3-glucan (2.0 folds). It could enhance the encapsulation by hemocytes but did not have antibacterial activity. These results provided evidence that LvLGBP might act as a pathogenic recognition protein to activate shrimp immune defense against invading pathogens via the agglutination, binding and enhancing encapsulation and phenoloxidase activity of the hemocytes.

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.

The differences in molecular mechanisms between osteochondroma and bizarre parosteal osteochondromatous proliferation (BPOP) remain to be fully elucidated. In the present study, the differentially expressed genes between BPOP and osteochondroma were obtained from the Gene Expression Omnibus online database, and the associations among these genes were analyzed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) online bioinformatics software. The results revealed several differentially expressed genes between human BPOP and osteochondroma. These differentially expressed genes were also enriched in different subgroups based on the analysis using DAVID online software, including ‘transforming growth factor β receptor signaling pathway’, ‘BMP signaling pathway’, ‘Wnt receptor signaling pathway’, ‘response to chemical stimulus’, ‘regulation of inflammatory response’, ‘response to stress’, ‘glycosaminoglycan binding’, ‘polysaccharide binding’, ‘extracellular matrix structural constituent’ and ‘growth factors binding’. Taken together, these findings led to the conclusion that different gene regulatory mechanisms exist between BPOP and osteochondroma. Environmental stimulation and inflammation may contribute to BPOP or osteochondroma, and differences in extracellular matrix may contribute to differences in biological characteristics between BPOP and osteochondroma.

Ovarian cancer is a major cause of mortality in women. However, the molecular events underlying the pathogenesis of the disease are yet to be fully elucidated. In the present study, an integrated bioinformatics analysis was performed to identify core genes involved in serous epithelial ovarian cancer. A total of three expression datasets were downloaded from the Gene Expression Omnibus database, and included 46 serous epithelial ovarian cancer and 30 ovarian surface epithelium samples. The three datasets were merged, and batch normalization was performed. The normalized merged data were subsequently analyzed for differentially expressed genes (DEGs). In total, 2,212 DEGs were identified, including 1,300 upregulated and 912 downregulated genes. Gene Ontology analysis revealed that these DEGs were primarily involved in 'regulation of cell cycle', 'mitosis', 'DNA packaging' and 'nucleosome assembly'. The main cellular components included 'extracellular region part', 'chromosome', 'extracellular matrix' and 'condensed chromosome kinetochore', whereas the molecular functions included 'Calcium ion binding', 'polysaccharide binding', 'enzyme inhibitor activity', 'growth factor activity', 'cyclin-dependent protein kinase regulator activity', 'microtubule motor activity' and 'Wnt receptor activity'. Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that these DEGs were predominantly involved in 'Wnt signaling pathway', 'pathways in cancer', 'PI3K-Akt signaling pathway', 'cell cycle', 'ECM-receptor interaction', 'p53 signaling pathway' and 'focal adhesion'. The 20 most significant DEGs were identified from the protein-protein interaction network, and Oncomine analysis of these core genes revealed that 13 were upregulated and two were downregulated in serous epithelial ovarian cancer. Survival analysis revealed that cyclin B1, polo like kinase 1, G protein subunit γ transducin 1 and G protein subunit γ 12 are key molecules that may be involved in the prognosis of serous epithelial ovarian cancer. These core genes may provide novel treatment targets, although their roles in the carcinogenesis and prognosis of serous epithelial ovarian cancer require further study.

Starch is the major energy storage carbohydrate in photosynthetic eukaryotes. Several enzymes are involved in building highly organized semi-crystalline starch granules, including starch-synthase III (SSIII), which is widely conserved in photosynthetic organisms. This enzyme catalyzes the extension of the α-1,4 glucan chain and plays a regulatory role in the synthesis of starch. Interestingly, unlike most plants, the unicellular green alga Ostreococcus tauri has three SSIII isoforms. In the present study, we describe the structure and function of OsttaSSIII-B, which has a similar modular organization to SSIII in higher plants, comprising three putative starch-binding domains (SBDs) at the N-terminal region and a C-terminal catalytic domain (CD). Purified recombinant OsttaSSIII-B displayed a high affinity toward branched polysaccharides such as glycogen and amylopectin, and to ADP-glucose. Lower catalytic activity was detected for the CD lacking the associated SBDs, suggesting that they are necessary for enzyme function. Moreover, analysis of enzyme kinetic and polysaccharide-binding parameters of site-directed mutants with modified conserved aromatic amino acid residues W122, Y124, F138, Y147, W279, and W304, belonging to the SBDs, revealed their importance for polysaccharide binding and SS activity. Our results suggest that OT_ostta13g01200 encodes a functional SSIII comprising three SBD domains that are critical for enzyme function. Fil: Barchiesi, Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina Fil: Velazquez, Maria Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro de Estudios Fotosintéticos y Bioquímicos. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Centro de Estudios Fotosintéticos y Bioquímicos; Argentina Fil: Palopoli, Nicolás. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina Fil: Iglesias, Alberto Alvaro. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina Fil: Gomez-Casati, Diego F.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro de Estudios Fotosintéticos y Bioquímicos. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Centro de Estudios Fotosintéticos y Bioquímicos; Argentina Fil: Ballicora, Miguel Angel. Loyola University Chicago; Estados Unidos Fil: Busi, María Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina

Goats are an important source of fibers. In the present study microarray technology was used to investigate the potential genes primarily involved in hair and cashmere growth in the Laiwu black goat. A total of 655 genes differentially expressed in body (hair‑growing) and groin (hairless) skin were identified, and their potential association with hair and cashmere growth was analyzed. The majority of genes associated with hair growth regulation could be assigned to intracellular, intracellular organelle, membrane‑bound vesicle, cytoplasmic vesicle, pattern binding, heparin binding, polysaccharide binding, glycosaminoglycan binding and cytoplasmic membrane‑bound vesicle categories. Numerous genes upregulated in body compared with groin skin contained common motifs for nuclear factor 1A, Yi, E2 factor (E2F) and cyclic adenosine monophosphate response element binding (CREB)/CREBβ binding sites in their promoter region. The promoter region of certain genes downregulated in body compared with groin skin contained three common regions with LF‑A1, Yi, E2F, Collier/Olfactory‑1/early B‑cell factor 1, peroxisome proliferator‑activated receptor α or U sites. Thus, the present study identified molecules in the cashmere‑bearing skin area of the Laiwu black goat, which may contribute to hair and cashmere traits.

Sialic acid - binding immunoglobulin - like lectins (Siglecs) are members of the largest superfamily of immune receptors; they recognize sialic acid and are mainly expressed in immune cells. Studies on mammals indicate that Streptococcus agalactiae (GBS) evade immune reactions by interacting with the host immune cells via the sialic acid of sialylated capsular polysaccharides. However, it is currently unknown if fish-derived GBS can interact with Siglecs to evade host immunity. In this study, we examined the binding of FITC-GBS with neutrophils to determine the presence of receptors that binds with GBS. Furthermore, 3 Siglec-like genes, (OnSiglec-1-like/-4b-like/-14-like) from the neutrophils cDNA were screened by PCR. All the genes had specific domains (immunostimulation and immunosuppression domains), conserved amino acid residues, and sialic acid polysaccharide binding sites that are found in mammalian Siglecs. Flow cytometry of Siglecs-like/COS-7 cells and ELISA of Siglecs/Ex-Fc fusion proteins confirmed that 3 Siglecs-like have high binding activity with GBS. Erythrocytes adhesion assays and sialylated glycans binding assay confirmed that 3 Siglecs-like bind to sialic acid polysaccharides. Siglecs-like had high expression levels in the spleen, gill, and kidney in Oreochromis niloticus by qPCR. After experimental infection, Siglec-1-like/-14-like showed a significant upregulated initially and later downregulated in liver, spleen, kidney, and gill. However, Siglec-4b-like was downregulated in most tissues, except that in liver. The results indicate that 3 OnSiglecs-like may recognize GBS sialylated capsular polysaccharides. GBS infections led to significant changes in Siglecs-like expression in immune-related tissues. However, immunostimulation or immunosuppression via the recognition of GBS by different Siglecs-like molecules requires additional studies.

To retain its biological activity and conformation as well as to preserve the multiple protein components, a xylanolytic multi-enzyme complex (XMEC) was isolated from a growth culture of Tepidimicrobium xylanilyticum BT14 to homogeneity by a mild isolation method using ultrafiltration, polysaccharide binding affinity, and gel filtration chromatographic techniques. The isolated XMEC was active at neutral and alkaline pH, showing 100% of the maximal xylanase activity at pH 6.5 and more than 50% at pH 9.0. It exhibited a temperature optimum at 60 °C (pH 6.5) and maintained its full activity at 60 °C for 180 min; its half-life at 70 °C was up to 90 min. The XMEC was highly resistant to various metal ions, including Ca 2+ , Mn 2+ , K + , Ni 2+ , Zn 2+ , Mg 2+ and Fe 2+ , but not to Cu 2+ and Hg 2+ . It hydrolysed xylans including birchwood xylan, oat spelt xylan, and highly substituted rye arabinoxylan to principally yield xylobiose and xylotriose. The presence of sugars including xylose, glucose and cellobiose, as well as the presence of ethanol stimulated the xylanase activity. These results indicate that the XMEC has potential in such xylan-related bioconversion industries as bioethanol production.

This work is part of a 10-year project to examine thawing permafrost peatlands and is the first virome-particle-based approach to characterize viruses in these systems. This method yielded >2-fold-more viral populations (vOTUs) per gigabase of metagenome than vOTUs derived from bulk-soil metagenomes from the same site (J. B. Emerson, S. Roux, J. R. Brum, B. Bolduc, et al., Nat Microbiol 3:870–880, 2018, https://doi.org/10.1038/s41564-018-0190-y). We compared the ecology of the recovered vOTUs along a permafrost thaw gradient and found (i) habitat specificity, (ii) a shift in viral community identity from soil-like to aquatic-like viruses, (iii) infection of dominant microbial hosts, and (iv) carriage of host metabolic genes. These vOTUs can impact ecosystem carbon processing via top-down (inferred from lysing dominant microbial hosts) and bottom-up (inferred from carriage of auxiliary metabolic genes) controls. This work serves as a foundation which future studies can build upon to increase our understanding of the soil virosphere and how viruses affect soil ecosystem services., Rapidly thawing permafrost harbors ∼30 to 50% of global soil carbon, and the fate of this carbon remains unknown. Microorganisms will play a central role in its fate, and their viruses could modulate that impact via induced mortality and metabolic controls. Because of the challenges of recovering viruses from soils, little is known about soil viruses or their role(s) in microbial biogeochemical cycling. Here, we describe 53 viral populations (viral operational taxonomic units [vOTUs]) recovered from seven quantitatively derived (i.e., not multiple-displacement-amplified) viral-particle metagenomes (viromes) along a permafrost thaw gradient at the Stordalen Mire field site in northern Sweden. Only 15% of these vOTUs had genetic similarity to publicly available viruses in the RefSeq database, and ∼30% of the genes could be annotated, supporting the concept of soils as reservoirs of substantial undescribed viral genetic diversity. The vOTUs exhibited distinct ecology, with different distributions along the thaw gradient habitats, and a shift from soil-virus-like assemblages in the dry palsas to aquatic-virus-like assemblages in the inundated fen. Seventeen vOTUs were linked to microbial hosts (in silico), implicating viruses in infecting abundant microbial lineages from Acidobacteria, Verrucomicrobia, and Deltaproteobacteria, including those encoding key biogeochemical functions such as organic matter degradation. Thirty auxiliary metabolic genes (AMGs) were identified and suggested virus-mediated modulation of central carbon metabolism, soil organic matter degradation, polysaccharide binding, and regulation of sporulation. Together, these findings suggest that these soil viruses have distinct ecology, impact host-mediated biogeochemistry, and likely impact ecosystem function in the rapidly changing Arctic. IMPORTANCE This work is part of a 10-year project to examine thawing permafrost peatlands and is the first virome-particle-based approach to characterize viruses in these systems. This method yielded >2-fold-more viral populations (vOTUs) per gigabase of metagenome than vOTUs derived from bulk-soil metagenomes from the same site (J. B. Emerson, S. Roux, J. R. Brum, B. Bolduc, et al., Nat Microbiol 3:870–880, 2018, https://doi.org/10.1038/s41564-018-0190-y). We compared the ecology of the recovered vOTUs along a permafrost thaw gradient and found (i) habitat specificity, (ii) a shift in viral community identity from soil-like to aquatic-like viruses, (iii) infection of dominant microbial hosts, and (iv) carriage of host metabolic genes. These vOTUs can impact ecosystem carbon processing via top-down (inferred from lysing dominant microbial hosts) and bottom-up (inferred from carriage of auxiliary metabolic genes) controls. This work serves as a foundation which future studies can build upon to increase our understanding of the soil virosphere and how viruses affect soil ecosystem services.

Breast carcinoma is one of the most common types of malignant neoplasms, and is associated with high rates of morbidity and mortality. Altered gene expression is critical in the development of breast cancer. To identify the important differentially expressed genes and microRNAs in breast carcinoma, mRNA (GSE26910, GSE42568, and GSE89116) and microRNA (GSE35412) microarray datasets were downloaded from the Gene Expression Omnibus database. The differentially expressed microRNA expression data were extracted with GEO2R online software. The DAVID online database was used to perform a function and pathway enrichment analysis of the key identified differentially expressed genes. A protein-protein interaction (PPI) network was constructed using the STRING online database, and visualized in Cytoscape software. The effect of the expression level of the key identified genes on overall survival (OS) time was analyzed by using the Kaplan-Meier Plotter online database. Furthermore, the online miRNA databases TargetScan, microT-CDS, and TarBase were used to identify the target genes of the differentially expressed miRNAs. A total of 254 differentially expressed genes were identified, which were enriched in cell adhesion, polysaccharide binding, extracellular region part and ECM-receptor interactions. The PPI network contained 250 nodes and 375 edges. Five differentially expressed genes were found to be significantly negatively correlated with the differentially expressed miRNAs, which were potentially also target genes for miRNAs. Four of the five genes, including AKAP12, SOPB, TCF7L2, COL12A1 and TXNIP were downregulated, and were associated with the OS of patients with breast carcinoma. In addition, a total of 130 differentially expressed miRNAs were identified. In conclusion, these results constitute a novel model for miRNA-mRNA differential expression patterns, and further studies may provide potential targets for diagnosing and understanding the mechanisms of breast carcinoma.

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, a significant stress response is induced during gastrointestinal transit and upon bile exposure, a normal component of human digestion. To overcome the bactericidal effects of bile, many enteric pathogens form a biofilm hypothesized to permit survival when transiting through the small intestine. Here we present methodologies to define biofilm formation through solid-phase adherence assays as well as extracellular polymeric substance (EPS) matrix detection and visualization. Furthermore, biofilm dispersion assessment is presented to mimic the analysis of events triggering release of bacteria during the infection process. Crystal violet staining is used to detect adherent bacteria in a high-throughput 96-well plate adherence assay. EPS production assessment is determined by two assays, namely microscopy staining of the EPS matrix and semi-quantitative analysis with a fluorescently-conjugated polysaccharide binding lectin. Finally, biofilm dispersion is measured through colony counts and plating. Positive data from multiple assays support the characterization of biofilms and can be utilized to identify bile salt-induced biofilm formation in other bacterial strains.

Expansins are a family of proteins with plant cell wall remodeling-activity, which bind cell wall components through hydrophobic and electrostatic interactions. A shallow area on the surface of the protein serves as the polysaccharide binding site (PBS) and it is composed of conserved residues. However, electric charge differences on the opposite face of the PBS produce basic, neutral, or acidic proteins. An analysis of forty-four bacterial expansins, homologues of BsEXLX1, revealed two main groups defined by: (a) the presence or absence of disulfide bonds; and (b) by the proteins isoelectric point (pI). We determined the location of the residues responsible for the pI on the structure of representative expansins. Our results suggest that the electric charge at the opposite site of the PBS may help in substrate differentiation among expansins from different species; in addition, electrostatic polarization between the front and the back of the molecule could affect expansin activity on cellulose.

Affinity electrophoresis has long been used to study the interaction between proteins and large soluble ligands. The technique has been found to have great utility for the examination of polysaccharide binding by proteins, particularly carbohydrate binding modules (CBMs). In recent years, carbohydrate surface binding sites of proteins mostly enzymes have also been investigated by this method. Here, we describe a protocol for identifying binding interactions between enzyme catalytic modules and a variety of carbohydrate ligands.

Pattern recognition molecules play an important role in innate immunity by recognizing conserved molecular patterns that are present on the surface of invading microorganisms. In this study, a lipopolysaccharide and β-1,3-glucan binding protein (LGBP) gene was cloned from the hard clam Meretrix meretrix (designated as Mm-LGBP) by the expressed sequence tags and rapid amplification of cDNA ends method. The cDNA was 1827 bp in length, consisting of a 71-bp 5'-terminal untranslated region, a 62-bp 3'UTR, and a 1734-bp open reading frame encoding a 577-amino acid polypeptide with an estimated molecular mass of 60.7 kDa and a theoretical isoelectric point of 5.56. Characteristic potential polysaccharide binding, cell adhesion, and glucanase motifs were identified in the Mm-LGBP, indicating that Mm-LGBP should be a new member of the LGBP family. Quantitative real-time polymerase chain reaction was developed to detect the mRNA expression level of Mm-LGBP in 6 different tissues. Higher-level mRNA expression of Mm-LGBP was detected in the gill and digestive gland tissues. The upregulation of Mm-LGBP mRNA after Vibrio anguillarum challenge showed that Mm-LGBP play a pivotal role in antibacterial immunity.

Antimicrobial peptides are a pivotal component of the invertebrate innate immune system. In this study, we identified a lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP) gene from the pacific abalone Haliotis discus hannai (HDH), which is involved in the pattern recognition mechanism and plays avital role in the defense mechanism of invertebrates immune system. The HDH-LGBP cDNA consisted of a 1263-bp open reading frame (ORF) encoding a polypeptide of 420 amino acids, with a 20-amino-acid signal sequence. The molecular mass of the protein portion was 45.5 kDa, and the predicted isoelectric point of the mature protein was 4.93. Characteristic potential polysaccharide binding motif, glucanase motif, and β-glucan recognition motif were identified in the LGBP of HDH. We used its polysaccharide-binding motif sequence to design two novel antimicrobial peptide analogs (HDH-LGBP-A1 and HDH-LGBP-A2). By substituting a positively charged amino acid and amidation at the C-terminus, the pI and net charge of the HDH-LGBP increased, and the proteins formed an α-helical structure. The HDH-LGBP analogs exhibited antibacterial and antifungal activity, with minimal effective concentrations ranging from 0.008 to 2.2 μg/mL. Additionally, both were toxic against human cervix (HeLa), lung (A549), and colon (HCT 116) carcinoma cell lines but not much on human umbilical vein cell (HUVEC). Fluorescence-activated cell sorter (FACS) analysis showed that HDH-LGBP analogs disturb the cancer cell membrane and cause apoptotic cell death. These results suggest the use of HDH-LGBP analogs as multifunctional drugs.

Glucoamylases are responsible for hydrolysis of starch and polysaccharides to yield β-d-glucose. Rhizopus oryzae glucoamylase (RoGA) is composed of an N-terminal starch binding domain (SBD) and a C-terminal catalytic domain connected by an O-glycosylated linker. Two carbohydrate binding sites in RoSBD have been identified, site I is created by three highly conserved aromatic residues, Trp47, Tyr83, and Tyr94, and site II is built up by Tyr32 and Phe58. Here, the two crystal structures of RoSBD in complex with only α-(1,6)-linked isomaltotriose (RoSBD-isoG3) and isomaltotetraose (RoSBD-isoG4) have been determined at 1.2 and 1.3 A, respectively. Interestingly, site II binding is observed in both complexes, while site I binding is only found in the RoSBD-isoG4 complex. Hence, site II acts as the recognition binding site for carbohydrate and site I accommodates site II to bind isoG4. Site I participates in sugar binding only when the number of glucosyl units of oligosaccharides is more than three. Taken together, two carbohydrate binding sites in RoSBD cooperate to reinforce binding mode of glucoamylase with polysaccharides as well as the starch. Proteins 2014; 82:1079–1085. © 2013 Wiley Periodicals, Inc.

Cellular behaviors and a xylanolytic-cellulolytic enzyme system of Tepidimicrobium xylanilyticum BT14 towards xylan-rich plant biomass degradation were characterized. During the exponential growth phase, the bacterial cells were bound tightly to the growth substrate where the degradation zones appeared mostly around the cells, indicating that the xylanolytic-cellulolytic enzyme system was linked to the cell surfaces. Interestingly, several cells appeared to secrete extracellular matrix to connect to their neighbors, and the matrix disappeared when cells passed to the stationary growth phase. Cationized-ferritin staining resulted in a dense assembly of bulbs, protuberance-like structures on the growing bacterial cell surfaces. The cell-associated proteins derived by sonication contained predominated xylanase with relatively low carboxymethyl-cellulase (CMCase) activities, suggesting that the xylanolytic-cellulolytic enzyme system occurred as a cell-associated enzyme. By means of gel-filtration chromatography, a high molecular mass protein with the estimated size of 2000 kDa was retrieved from the cell-associated enzymes, and it appeared as a single protein band on non-denaturing gel. However, more bands were obtained after the protein was boiled with sodium dodecyl sulfate and β-mercaptoethanol – which contained 4 xylanases and one CMCase – suggesting that these proteins were organized as a multienzyme complex (MEC) in natural form. Additionally, the predominated xylanolytic MEC preferred binding to cellulose rather than xylan.

The liver is the most frequent site of metastasis with a 5-year survival rate of only 20-40%. In this work, hyaluronate (HA)-death receptor 5 antibody (DR5 Ab) conjugate was synthesized as a dual targeting therapeutic agent to treat liver metastasis. Dual targeting was achieved by DR5 Ab, a humanized agonistic monoclonal antibody binding to DR5 frequently overexpressed in many kinds of cancer cells, and by HA, a natural polysaccharide binding to HA receptors highly expressed in both the liver and cancer cells. Thiol end-modified HA was site-specifically conjugated to N-glycan on Fc region of oxidized DR5 Ab using a heterobifunctional linker of 3-(2-pyridyldithio)propionyl hydrazide (PDPH). The successful synthesis of HA-DR5 Ab conjugate was confirmed by (1)H NMR, purpald assay, dynamic light scattering (DLS), and high-performance liquid chromatography (HPLC). In vitro analysis of HA-DR5 Ab conjugate revealed that the conjugation of HA to DR5 Ab did not affect the binding affinity and anticancer efficacy of DR5 Ab. Remarkably, according to in vivo bioimaging study, HA-DR5 Ab conjugate appeared to be highly accumulated in the liver and dramatically effective in inhibiting the tumor growth in liver metastasis model mice.

Study of recalcitrant polysaccharide degradation by bacterial systems is critical for understanding biological processes such as global carbon cycling, nutritional contributions of the human gut microbiome, and the production of renewable fuels and chemicals. One bacterium that has a robust ability to degrade polysaccharides is the Gram-negative saprophyte Cellvibrio japonicus. A bacterium with a circuitous history, C. japonicus underwent several taxonomy changes from an initially described Pseudomonas sp. Most of the enzymes described in the pre-genomics era have also been renamed. This review aims to consolidate the biochemical, structural, and genetic data published on C. japonicus and its remarkable ability to degrade cellulose, xylan, and pectin substrates. Initially, C. japonicus carbohydrate-active enzymes were studied biochemically and structurally for their novel polysaccharide binding and degradation characteristics, while more recent systems biology approaches have begun to unravel the complex regulation required for lignocellulose degradation in an environmental context. Also included is a discussion for the future of C. japonicus as a model system, with emphasis on current areas unexplored in terms of polysaccharide degradation and emerging directions for C. japonicus in both environmental and biotechnological applications.

Transposon insertions in Geobacter sulfurreducens GSU1501, part of an ATP-dependent exporter within an operon of polysaccharide biosynthesis genes, were previously shown to eliminate insoluble Fe(III) reduction and use of an electrode as an electron acceptor. Replacement of GSU1501 with a kanamycin resistance cassette produced a similarly defective mutant, which could be partially complemented by expression of GSU1500 to GSU1505 in trans . The Δ1501 mutant demonstrated limited cell-cell agglutination, enhanced attachment to negatively charged surfaces, and poor attachment to positively charged poly- d -lysine- or Fe(III)-coated surfaces. Wild-type and mutant cells attached to graphite electrodes, but when electrodes were poised at an oxidizing potential inducing a positive surface charge (+0.24 V versus the standard hydrogen electrode [SHE]), Δ1501 mutant cells detached. Scanning electron microscopy revealed fibrils surrounding wild-type G. sulfurreducens which were absent from the Δ1501 mutant. Similar amounts of type IV pili and pilus-associated cytochromes were detected on both cell types, but shearing released a stable matrix of c -type cytochromes and other proteins bound to polysaccharides. The matrix from the mutant contained 60% less sugar and was nearly devoid of c -type cytochromes such as OmcZ. The addition of wild-type extracellular matrix to Δ1501 cultures restored agglutination and Fe(III) reduction. The polysaccharide binding dye Congo red preferentially bound wild-type cells and extracellular matrix material over mutant cells, and Congo red inhibited agglutination and Fe(III) reduction by wild-type cells. These results demonstrate a crucial role for the xap (extracellular anchoring polysaccharide) locus in metal oxide attachment, cell-cell agglutination, and localization of essential cytochromes beyond the Geobacter outer membrane.

The anticoagulant behavior of sulfated polysaccharides from seaweeds is reviewed based on their chemical structures. Analysis of the literature suggested that the driving force for the formation of the sulfated polysaccharide/ protein complex is the non-specific polar interaction between the negatively and positively charged groups in the polysaccharide and protein, respectively and that the complex is further stabilized by short-range interactions. The polysaccharide binding site should be able to go through the following conformational steps in the formation of the complex: random coil→ordered conformation→low distortion of this conformation to form a complementary fitting structure with the protein backbone. The sulfated monosaccharide units with the highest potential for anticoagulant activity should have two sulfate groups and a glycosidic linkage on the pyranose ring with C-2, C-3 and C-4 in 2S, 3R, 4R or 2R, 3S, 4S configurations for galactose, fucose and arabinose and 2S, 3S, 4R, for rhamnose. Three distributions of these substituents appear: 3-linked 2,4-disulfated units, 4-linked 2,3-disulfated units and 2-linked 3,4-disulfated residues. These types of units have the possibility, through the equilibrium of the chair conformations, to place their sulfate groups in adequate spacial positions to interact with basic groups of the protein. The anticoagulant activity is mainly attributed to thrombin inhibition mediated by antithrombin and/or heparin cofactor II, with different effectivenesses depending of the compound. Other mechanisms are also proposed and these differences could be attributed to the diversity of structures of the polysaccharides evaluated and to the fact that one compound may have more than one target protease. Fil: Ciancia, Marina. Universidad de Buenos Aires. Facultad de Agronomía. Departamento de Biología Aplicada y Alimentos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; Argentina Fil: Quintana, Irene Luisa. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Cerezo, Alberto. Universidad de Buenos Aires. Facultad de Agronomía. Departamento de Biología Aplicada y Alimentos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; Argentina

Killer lectin-like receptors NKG2D and CD94 on natural killer cells trigger cytotoxicity through binding of glycans on target cells including sialyl Lewis X antigen. We previously reported that NKG2D and CD94 recognize alpha2,3-linked NeuAc on multi-antennary N-glycans. Here we further investigated polysaccharide binding by these receptors, using glutathione-S-transferase-fused extracellular domains of NKG2D AA 73-216 (rNKG2Dlec) and CD94 AA 68-179 (rCD94lec). We found that rNKG2Dlec and rCD94lec bind in a dose-dependent manner to plates coated with heparin-conjugated bovine serum albumin (heparin-BSA). Binding to heparin-BSA was suppressed by soluble sulfate-containing polysaccharides, but minimally impacted by 2-O-, 6-O-, and 2-N-desulfated heparin. Mutagenesis revealed that (152)Y and (199)Y of NKG2D and (144)F, (160)N, and (166)C of CD94 were critical for binding to heparin-BSA. The present manuscript provides the first evidence that NKG2D and CD94 bind to heparin and sulfate-containing polysaccharides.

Starch is degraded by amylases that frequently have a modular structure composed of a catalytic domain and at least one non-catalytic domain that is involved in polysaccharide binding. The C-terminal domain from the Lactobacillus amylovorus alpha-amylase has an unusual architecture composed of five tandem starch-binding domains (SBDs). These domains belong to family 26 in the carbohydrate-binding modules (CBM) classification. It has been reported that members of this family have only one site for starch binding, where aromatic amino acids perform the binding function. In SBDs, fold similarities are better conserved than sequences; nevertheless, it is possible to identify in CBM26 members at least two aromatic residues highly conserved. We attempt to explain polysaccharide recognition for the L. amylovorus alpha-amylase SBD through site-directed mutagenesis of aromatic amino acids. Three amino acids were identified as essential for binding, two tyrosines and one tryptophan. Y18L and Y20L mutations were found to decrease the SBD binding capacity, but unexpectedly, the mutation at W32L led to a total loss of affinity, either with linear or ramified substrates. The critical role of Trp 32 in substrate binding confirms the presence of just one binding site in each alpha-amylase SBD.

Pattern recognition molecules play an important role in innate immunity by recognizing common epitopes on the surface of invading microorganism. A pattern recognition protein (PRP) was isolated from a disk abalone, Haliotis discus discus, normalized cDNA library. It encodes 420 amino acids (aa) including a 20 aa signal peptide sequence. The mature protein has an estimated molecular mass of 45kDa and predicted pI of 5.0. The deduced aa sequence showed greatest homology (50%) with a beta-glucan recognition protein (BGRP) of the freshwater snail Biomphalaria glabrata. Characteristic potential polysaccharide binding, cell adhesion, and glucanase motifs, similar to invertebrate PRP motifs were also found in HdPRP. Reverse transcription-polymerase chain reaction (RT-PCR) results showed that the HdPRP was constitutively expressed in the gill, mantle, digestive tract, hepatopancreas and hemocytes, suggesting an innate immune role in these tissues. Animals injected with Vibrio alginolyticus bacteria showed that the mRNA expression was increased at 12h post-injection in the gill and continued until 48h. Abalone CuZn superoxide dismutase (HdCuZnSOD) gene expression was analyzed to detect any oxidative stress after exposure to different pathogen-associated molecular patterns (PAMP). This analysis showed that mRNA expression was significantly increased in Vibrio, lipopolysaccharides (LPS) and beta-1,3-glucan-injected animals compared to controls. Abalone PRP can recognize different PAMP and may activate different genes involved in the defence against these pathogens. It acts as an acute inducible protein that could play an important role in the abalone immune defence mechanism.

GA (glucoamylase) hydrolyses starch and polysaccharides to β-D-glucose. RoGA (Rhizopus oryzae GA) consists of two functional domains, an N-terminal SBD (starch-binding domain) and a C-terminal catalytic domain, which are connected by an O-glycosylated linker. In the present study, the crystal structures of the SBD from RoGA (RoGACBM21) and the complexes with β-cyclodextrin (SBD–βCD) and maltoheptaose (SBD–G7) were determined. Two carbohydrate binding sites, I (Trp47) and II (Tyr32), were resolved and their binding was co-operative. Besides the hydrophobic interaction, two unique polyN loops comprising consecutive asparagine residues also participate in the sugar binding. A conformational change in Tyr32 was observed between unliganded and liganded SBDs. To elucidate the mechanism of polysaccharide binding, a number of mutants were constructed and characterized by a quantitative binding isotherm and Scatchard analysis. A possible binding path for long-chain polysaccharides in RoGACBM21 was proposed.

Novel compounds presenting anticoagulant activity, such as sulfated polysaccharides, open new perspectives in medicine. Elucidation of the molecular mechanism behind this activity is desirable by itself, as well as because it allows for the design of novel compounds. In the present study, we investigated the action of an algal sulfated galactan, which potentiates alpha-thrombin inactivation by antithrombin. Our results indicate the following: 1) both the sulfated galactan and heparin potentiate protease inactivation by antithrombin at similar molar concentrations, however they differ markedly in the molecular size required for their activities; 2) this galactan interacts predominantly with exosite II on alpha-thrombin and, similar to heparin, catalyzes the formation of a covalent complex between antithrombin and the protease; 3) the sulfated galactan has a higher affinity for alpha-thrombin than for antithrombin. We propose that the preferred pathway of sulfated galactan-induced inactivation of alpha-thrombin by antithrombin starts with the polysaccharide binding to the protease through a high-affinity interaction. Antithrombin is then added to the complex and the protease is inactivated by covalent interactions. Finally, the antithrombin-alpha-thrombin covalent complex dissociates from the polysaccharide chain. This mechanism resembles the action of heparin with low affinity for antithrombin, as opposed to heparin with high affinity for serpin.

Summary The molecular and functional characteristics of natural antibody from the preimmune repertoire have not been explored in detail in man. We describe seven human IgM monoclonal antibodies selected on the basis of pneumococcal polysaccharide binding that share both molecular and functional characteristics with natural antibody, suggesting a common B cell lineage origin. Unlike class-switched antibodies, which are serotype-specific, the antibodies were polyreactive and bound all pneumococcal polysaccharide capsular serotypes tested. Some bound endogenous antigens, including blood group antigens and intermediate filament proteins. All the antibodies used unmutated heavy chain V (IGHV) that are expressed at an increased frequency in the elderly and in the preimmune repertoire. The CDR3 was characterized by long length (mean aa 18·4 (±4·2) and selective use of IGHD6 (P

The non-catalytic, family 11 carbohydrate binding module (CtCBM11) belonging to a bifunctional cellulosomal cellulase from Clostridium thermocellum was hyper-expressed in E. coli and functionally characterized. Affinity electrophoresis of CtCBM11 on nondenaturing PAGE containing cellulosic polysaccharides showed binding with β-glucan, lichenan, hydroxyethyl cellulose and carboxymethyl cellulose. In order to elucidate the involvement of conserved aromatic residues Tyr 22, Trp 65 and Tyr 129 in the polysaccharide binding, site-directed mutagenesis was carried out and the residues were changed to alanine. The results of affinity electrophoresis and binding adsorption isotherms showed that of the three mutants Y22A, W65A and Y129A of CtCBM11, two mutants Y22A and Y129A showed no or reduced binding affinity with polysaccharides. These results showed that tyrosine residue 22 and 129 are involved in the polysaccharide binding. These residues are present in the putative binding cleft and play a critical role in the recognition of all the ligands recognized by the protein.

Helicobacter pylori alpha1,3-fucosyltransferase (FucT) is involved in catalysis to produce the Lewis x trisaccharide, the major component of the bacteria's lipopolysaccharides, which has been suggested to mimic the surface sugars in gastric epithelium to escape host immune surveillance. We report here three x-ray crystal structures of FucT, including the FucT.GDP-fucose and FucT.GDP complexes. The protein structure is typical of the glycosyltransferase-B family despite little sequence homology. We identified a number of catalytically important residues, including Glu-95, which serves as the general base, and Glu-249, which stabilizes the developing oxonium ion during catalysis. The residues Arg-195, Tyr-246, Glu-249, and Lys-250 serve to interact with the donor substrate, GDP-fucose. Variations in the protein and ligand conformations, as well as a possible FucT dimer, were also observed. We propose a catalytic mechanism and a model of polysaccharide binding not only to explain the observed variations in H. pylori lipopolysaccharides, but also to facilitate the development of potent inhibitors.

A factorial experimental design (5×3×2) was used to investigate the effects of non-starch polysaccharide binding agents on physical properties of single-screw extrusion. Extrusion cooking trials were performed with an ingredient blend for yellow perch, fortified with five non-starch polysaccharide binding agents including three plant-origin gums (guar, wheat gluten, carboxymethyl cellulose (CMC)) and two microbial-origin exopolysaccharide gums (xanthan and pullulan), with three levels of gum inclusion (3, 6, and 10%), and two levels of screw speed (100 and 150 rpm). Effects of the independent variables on extrudate characteristics were extensively analyzed and included density, expansion ratio, water absorption and solubility indices, pellet durability, and color. Increasing gum level from 3 to 10% led to a considerable increase in unit density of extrudates for xanthan, guar, wheat gluten, CMC, and pullulan by 39.6%, 21%, 11.4%, 30%, and 19.7%, respectively. The minimum (357 kg m-3) and maximum (607 kg m-3) bulk densities were observed for the diets produced with 6% guar at 150 rpm and 10% xanthan at 100 rpm, respectively. The mean values of expansion ratio for the diets containing exopolysaccharide gums were slightly smaller than those of the other diets. Increasing gum inclusion level increased the expansion ratio of the extrudates using xanthan, wheat gluten and pullulan but reduced the expandability of diets used guar gum; increased levels did not change the expansion ratio of extrudates containing CMC. At the highest levels of gum inclusion and screw speed, both pullulan and wheat gluten gums provided better expandability of the feed extrudate. Exopolysaccharide gums resulted in extrudates with significantly higher pellet durability and water solubility indices. Overall, the addition of 6 to 10% non-starch exopolysaccharides could improve the pellet durability of aqua feed extrudates. A future study investigating the effects of feed composition and additional extrusion processing conditions on the physical parameters of these products in aquafeeds would be appropriate.

Previous results showed that the cell-surface esterase from Acinetobacter venetianus RAG-1 enhances the emulsification properties of the polymeric bioemulsifier emulsan and its deproteinated derivative apoemulsan (Bach H, Berdichevsky Y, Gutnick D (2003) An exocellular protein from the oil-degrading microbe Acinetobacter venetianus RAG-1 enhances the emulsifying activity of the polymeric bioemulsifier emulsan. Appl Environ Microbiol 69:2608–2615). Here we show that in the presence of the his-tagged recombinant esterase from RAG-1, 18 different polysaccharides from microbial, plant, insect and synthetic sources formed hexadecane-in-water emulsions. Emulsifying activities were distributed over a 13-fold range from over 4800 U/mg protein/mg polysaccharide in the case of apoemulsan to 370 U/mg protein/mg polysaccharide in the case of alginic acid. The stability of the emulsions ranged between 95 and 58%. Emulsions formed in the presence of seven of the polysaccharides exhibited stabilities of over 80%. The esterase from A. calcoaceticus BD4, which shows sequence homology to the RAG-1 esterase, was inactive in emulsification enhancement. The sequence of the RAG-1 esterase was shown to contain two conserved peptide sequences previously shown to be implicated in carbohydrate/polysaccharide binding. A hypothetical model illustrating a possible mode of interaction between the esterase, the apoemulsan and the oil droplet is presented. The complex is presumed to generate a series of “coated” oil droplets which are restricted in their ability to coalesce resulting in a relatively stable emulsion.

Glucosidic bonds from different non-soluble polysaccharides such as starch, cellulose and xylan are hydrolyzed by amylases, cellulases and xylanases, respectively. These enzymes are produced by microorganisms. They have a modular structure that is composed of a catalytic domain and at least one non-catalytic domain that is involved in polysaccharide binding. Starch-binding modules are present in microbial enzymes that are involved in starch metabolism; these are classified into several different families on the basis of their amino acid sequence similarities. Such binding domains promote attachment to the substrate and increase its concentration at the active site of the enzyme, which allows microorganisms to degrade non-soluble starch. Fold similarities are better conserved than sequences; nevertheless, it is possible to notice two evolutionary clusters of microbial starch-binding domains. These domains have enormous potential as tags for protein immobilization, as well as for the tailoring of enzymes that play a part in polysaccharide metabolism.