Characterization and Structural Analysis of a Novel Carbohydrate-Binding Module from Family 96 with Chondroitin Sulfate-Specific Binding Capacity.

Chondroitin sulfate (CS) is the predominant glycosaminoglycan within the human body and is widely applied in various industries. Carbohydrate-binding modules (CBMs) possessing the capacity for carbohydrate recognition are verified to be important tools for polysaccharide investigation. Only one CS-specific CBM, PhCBM100, has hitherto been characterized. In the present study, two CBM96 domains present in the same putative PL8_3 chondroitin AC lyase were discovered and recombinantly expressed. The results of microtiter plate assays and affinity gel electrophoresis assays showed that the two corresponding proteins, DmCBM96-1 and DmCBM96-2, bind specifically to CSs. The crystal structure of DmCBM96-1 was determined at a 2.20 Å resolution. It adopts a ß-sandwich fold comprising two antiparallel ß-sheets, showing structural similarities to TM6-N4, which is the founding member of the CBM96 family. Site mutagenesis analysis revealed that the residues of Arg27, Lys45, Tyr51, Arg53, and Arg157 are critical for CS binding. The characterization of the two CBM96 proteins demonstrates the diverse ligand specificity of the CBM96 family and provides promising tools for CS investigation.

Structural investigation of Fun168A unraveling the recognition mechanism of endo-1,3-fucanase towards sulfated fucan.

BACKGROUND: Sulfated fucan has gained interest due to its various physiological activities. Endo-1,3-fucanases are valuable tools for investigating the structure and establishing structure-activity relationships of sulfated fucan. However, the substrate recognition mechanism of endo-1,3-fucanases towards sulfated fucan remains unclear, limiting the application of endo-1,3-fucanases in sulfated fucan research. SCOPE AND APPROACH: This study presented the first crystal structure of endo-1,3-fucanase (Fun168A) and its complex with the tetrasaccharide product, utilizing X-ray diffraction techniques. The novel subsite specificity of Fun168A was identified through glycomics and nuclear magnetic resonance (NMR). KEY FINDINGS AND CONCLUSIONS: The structure of Fun168A was determined at 1.92 Å. Residues D206 and E264 acted as the nucleophile and general acid/base, respectively. Notably, Fun168A strategically positioned a series of polar residues at the subsites ranging from -2 to +3, enabling interactions with the sulfate groups of sulfated fucan through salt bridges or hydrogen bonds. Based on the structure of Fun168A and its substrate recognition mechanisms, the novel subsite specificities at the -2 and +2 subsites of Fun168A were identified. Overall, this study provided insight into the structure and substrate recognition mechanism of endo-1,3-fucanase for the first time and offered a valuable tool for further research and development of sulfated fucan.

The α-linkage in funoran and agarose could be hydrolyzed by a GH96 family enzyme: Discovery of the α-funoranase.

Agarans represent a group of galactans extracted from red algae. Funoran and agarose are the two major types and commercially applied polysaccharides of agaran. Although the glycoside hydrolases targeting ß-glycosidic bonds of agaran have been widely investigated, those capable of degrading α-glycosidic bonds of agarose were limited, and the enzyme degrading α-linkages of funoran has not been reported till now. In this study, a GH96 family enzyme BiAF96A_Aq from a marine bacterium Aquimarina sp. AD1 was heterologously expressed in Escherichia coli. BiAF96A_Aq exhibited dual activities towards the characteristic structure of funoran and agarose, underscoring the multifunctionality of GH96 family members. Glycomics and NMR analysis revealed that BiAF96A_Aq hydrolyzed the α-1,3 glycosidic bonds between 3,6-anhydro-α-l-galactopyranose (LA) and ß-d-galactopyranose-6-sulfate (G6S) of funoran, as well as LA and ß-d-galactopyranose (G) of agarose, through an endo-acting manner. The end products of BiAF96A_Aq were majorly composed of disaccharides and tetrasaccharides. The identification of the activity of BiAF96A_Aq on funoran indicated the first discovery of the funoran hydrolase for α-1,3 linkage. Considering the novel catalytic reaction, we proposed to name this activity as "α-funoranase" and recommended the assignment of a dedicated EC number for its classification.

Biochemical characterization and cleavage specificities analyses of three endo-1,3-fucanases within glycoside hydrolase family 174.

Sulfated fucans have garnered extensive research interest in recent decades due to their varied bioactivity. Fucanases are important tools for investigating sulfated fucans. This study reported the bioinformatic analysis and biochemical properties of three GH174 family endo-1,3-fucanases. Wherein, Fun174Rm and Fun174Sb showed the highest optimal reaction temperature among the reported fucanases, and Fun174Sb possessed favorable thermostability and catalysis efficiency. Fun174Rm displayed a random endo-acting manner, while Fun174Ri and Fun174Sb hydrolyzed sulfated fucan in processive manners. UPLC-MS and NMR analyses confirmed that the three enzymes catalyze cleavage of the α(1 â†’ 3)-bonds between Fucp2S and Fucp2S in the sulfated fucan from Isostichopus badionotus. These enzymes demonstrated novel cleavage specificities, which could accept α-Fucp2S residues at subsites -1 and + 1. The acquiring of these biotechnological tools would be beneficial to the in-depth research of sulfated fucans.

Analysis of unsaturated alginate oligosaccharides using high-performance anion exchange chromatography coupled with mass spectrometry.

Alginate is a commercially important polysaccharide composed of mannuronic acid and its C5 differential isomer guluronic acid. Comprehensive research on alginate and alginate lyases requires efficient and precise analytical methods for alginate oligosaccharides. In this research, high-performance anion exchange chromatography (HPAEC) in parallel with pulsed amperometric detection (PAD) and mass spectrometry (MS) was applied to the analysis of oligosaccharides obtained by alginate lyase. By optimizing the chromatographic conditions including mobile phase concentration, flow rate, and elution gradient, the analysis of a single sample could be completed in 30 min. Seven unsaturated alginate oligosaccharides were separated and identified through their analysis time observed with PAD, including all structurally different unsaturated disaccharides and trisaccharides. The quantitative analysis of seven oligosaccharides was performed based on the quantitative capability of PAD. The method exhibited adequate linearity and precision parameters. All the calibration curves showed good linearity at least in the concentration range of 0.002 to 0.1 mg/mL. The HPAEC-PAD/MS method provides a general and efficient online method to analyze alginate oligosaccharides.

Characterization and structural identification of a family 16 carbohydrate-binding module (CBM): First structural insights into porphyran-binding CBM.

Porphyran is a favorable functional polysaccharide widely distributed in Porphyra. It displays a linear structure majorly constituted by alternating 1,4-linked α-l-galactopyranose-6-sulfate (L6S) and 1,3-linked ß-d-galactopyranose (G) units. Carbohydrate-binding modules (CBMs) are desired tools for the investigation and application of polysaccharides, including in situ visualization, on site and specific assay, and functionalization of biomaterials. However, only one porphyran-binding CBM has been hitherto reported, and its structural knowledge is lacking. Herein, a novel CBM16 family domain from a marine bacterium Aquimarina sp. BL5 was discovered and expressed. The recombinant protein AmCBM16 exhibited the desired specificity for porphyran. Bio-layer interferometry assay revealed that the protein binds to porphyran tetrasaccharide (L6S-G)2 with an association constant of 1.3 × 103 M-1. The structure of AmCBM16 was resolved by the X-ray crystallography, which displays a ß-sandwich fold with two antiparallel ß-sheets constituted by 10 ß-strands. Site-directed mutagenesis analysis demonstrated that the residues Gly-30, Trp-31, Lys-88, Lys-123, Phe-125, and Phe-127 play dominant roles in AmCBM16 binding. This study provides the first structural insights into porphyran-binding CBM.

Investigation of the Presence of Fibrillin in Sea Cucumber (Apostichopus japonicus) Body Wall by Utilizing Targeted Proteomics and Visualization Strategies.

Fibrillin is an important structural protein in connective tissues. The presence of fibrillin in sea cucumber Apostichopus japonicus is still poorly understood, which limits our understanding of the role of fibrillin in the A. japonicus microstructure. The aim of this study was to clarify the presence of fibrillin in the sea cucumber A. japonicus body wall. Herein, the presence of fibrillin in sea cucumber A. japonicus was investigated by utilizing targeted proteomics and visualization strategies. The contents of three different isoforms of fibrillin with high abundance in A. japonicus were determined to be 0.96, 2.54, and 0.15 µg/g (wet base), respectively. The amino acid sequence of fibrillin (GeneBank number: PIK56741.1) that started at position 631 and ended at position 921 was selected for cloning and expressing antigen. An anti-A. japonicus fibrillin antibody with a titer greater than 1:64 000 was successfully obtained. It was observed that the distribution of fibrillin in the A. japonicus body wall was scattered and dispersed in the form of fibril bundles at the microscale. It further observed that fibrillin was present near collagen fibrils and some entangled outside the collagen fibrils at the nanoscale. Moreover, the stoichiometry of the most dominant collagen and fibrillin molecules in A. japonicus was determined to be approximately 250:1. These results contribute to an understanding of the role of fibrillin in the sea cucumber microstructure.

Identification and structural characterization of a novel chondroitin sulfate-specific carbohydrate-binding module: The first member of a new family, CBM100.

Chondroitin sulfate is a biologically and commercially important polysaccharide with a variety of applications. Carbohydrate-binding module (CBM) is an important class of carbohydrate-binding protein, which could be utilized as a promising tool for the applications of polysaccharides. In the present study, an unknown function domain was explored from a putative chondroitin sulfate lyase in PL29 family. Recombinant PhCBM100 demonstrated binding capacity to chondroitin sulfates with Ka values of 2.1 ± 0.2 × 106 M-1 and 6.0 ± 0.1 × 106 M-1 to chondroitin sulfate A and chondroitin sulfate C, respectively. The 1.55 Å resolution X-ray crystal structure of PhCBM100 exhibited a ß-sandwich fold formed by two antiparallel ß-sheets. A binding groove in PhCBM100 interacting with chondroitin sulfate was subsequently identified, and the potential of PhCBM100 for visualization of chondroitin sulfate was evaluated. PhCBM100 is the first characterized chondroitin sulfate-specific CBM. The novelty of PhCBM100 proposed a new CBM family of CBM100.

Characterization of a novel endo-1,3-fucanase from marine bacterium Wenyingzhuangia fucanilytica reveals the presence of diversity within glycoside hydrolase family 168.

Sulfated fucans attract increasing research interests in recent decades for their various physiological activities. Fucanases are indispensable tools for the investigation of sulfated fucans. Herein, a novel GH168 family endo-1,3-fucanase was cloned from the genome of marine bacterium Wenyingzhuangia fucanilytica. The expressed protein Fun168D was a processive endo-acting enzyme. Ultra performance liquid chromatography-high resolution mass spectrum and NMR analyses revealed that the enzyme cleaved the α-1 â†’ 3 bonds between α-l-Fucp(2OSO3-) and α-l-Fucp(2OSO3-) in sulfated fucan from Isostichopus badionotus, and α-1 â†’ 3 bonds between α-l-Fucp(2OSO3-) and α-l-Fucp(2,4OSO3-) in sulfated fucan from Holothuria tubulosa. Fun168D would prefer to accept α-l-Fucp(2,4OSO3-) than α-l-Fucp(2OSO3-) at subsite +1, and could tolerate the absence of fucose residue at subsite +2. The novel cleavage specificity and hydrolysis pattern revealed the presence of diversity within the GH168 family, which would facilitate the development of diverse biotechnological tools for the molecule tailoring of sulfated fucan.

The characteristic structure of funoran could be hydrolyzed by a GH86 family enzyme (Aga86A_Wa): Discovery of the funoran hydrolase.

Funoran, agarose and porphyran all belong to agaran, and share the similar skeleton. Although the glycoside hydrolase for agarose and porphyran, i.e. agarase and porphyranase, have been extensively studied, the enzyme hydrolyzing funoran has not been reported hitherto. The crystal structure of a previously characterized GH86 ß-agarase Aga86A_Wa showed a large cavity at subsite -1, which implied its ability to accommodate sulfate ester group. By using glycomics and NMR analysis, the activity of Aga86A_Wa on the characteristic structure of funoran was validated, which signified the first discovery of funoran hydrolase, i.e. funoranase. Aga86A_Wa hydrolyzed the ß-1,4 glycosidic bond between ß-d-galactopyranose-6-sulfate (G6S) and 3,6-anhydro-α-l-galactopyranose (LA) unit of funoran, and released disaccharide LA-G6S as the predominant end product. Considering the hydrolysis pattern, we proposed to name the activity represented by Aga86A_Wa on funoran as "ß-funoranase" and suggested to assign it an EC number.

Sulfated fucan could serve as a species marker of sea cucumber with endo-1,3-fucanase as the essential tool.

In the past few decades, sulfated fucan from sea cucumber had attracted considerable interest owing to its abundant physiological activities. Nevertheless, its potential for species discrimination had not been investigated. Herein, particular attention was given to sea cucumber Apostichopus japonicus, Acaudina molpadioides, Holothuria hilla, Holothuria tubulosa, Isostichopus badionotus and Thelenota ananas to examine the feasibility of sulfated fucan as a species marker of sea cucumber. The enzymatic fingerprint suggested that sulfated fucan exhibited significant interspecific discrepancy and intraspecific stability, which revealed that sulfated fucan could serve as the species marker of sea cucumber, by utilizing the overexpressed endo-1,3-fucanase Fun168A and the ultra-performance liquid chromatography-high resolution mass spectrum. Moreover, oligosaccharide profile of sulfated fucan was determined. The oligosaccharide profile combined with hierarchical clustering analysis and principal components analysis further confirmed that sulfated fucan could serve as a marker with a satisfying performance. Besides, load factor analysis showed that the minor structure of sulfated fucan also contributed to the sea cucumber discrimination, besides the major structure. The overexpressed fucanase played an indispensable role in the discrimination, due to its specificity and high activity. The study would lead to a new strategy for species discrimination of sea cucumber based on sulfated fucan.

Discovery of a sulfated fucan-specific carbohydrate-binding module: The first member of a new carbohydrate-binding module family.

Sulfated fucan is an important functional polysaccharide with various physiological activities. Carbohydrate-binding module (CBM) is a representative class of carbohydrate-binding protein, which could be employed as a favorable tool for the investigations and applications of polysaccharides. Nevertheless, only one confirmed sulfated fucan-binding CBM has been hitherto reported. In the present study, an unknown domain with a predicted ß-sandwich fold was discovered from a fucanase Fun174A, and further cloned and heterologously expressed in Escherichia coli. The expressed protein Fun174A-CBM displayed a specific binding capacity to sulfated fucan. The bio-layer interferometry assays showed that the protein could bind to the sulfated fucan tetrasaccharide with an affinity constant of 2.83 × 10-4 M. Fun174A-CBM shared no significant sequence similarity to any identified CBMs, indicating that it represents a new CBM family. The discovery of Fun174-CBM and the novel CBM family would be beneficial to the investigations of sulfated fucan-binding proteins.

Characterization of an endo-1,3-fucanase from marine bacterium Wenyingzhuangia aestuarii: The first member of a novel glycoside hydrolase family GH174.

Sulfated fucans are important marine polysaccharides with various biological and biomedical activities. Fucanases are favorable tools to establish the structure-activity relationships of sulfated fucans. Herein, gene fun174A was discovered from the genome of marine bacterium Wenyingzhuangia aestuarii OF219, and none of the pre-defined glycosidic hydrolase domains were predicted in the protein sequence of Fun174A. Recombinant Fun174A demonstrated a low optimal reaction pH at 5.5. It might degrade sulfated fucans in an endo-processive manner. Glycomics and NMR analyses proved that it specifically hydrolyzed α-1,3-l-fucoside bonds between 2-O-sulfated and non-sulfated fucose residues in the sulfated fucan from sea cucumber Isostichopus badionotus. D119, E120 and E218 were critical for the activity of Fun174A, as identified by site-directed mutagenesis. Three homologs of Fun174A were confirmed to exhibit endo-1,3-fucanase activities. The novelty on sequences of Fun174A and its homologs reveals a new glycoside hydrolase family, GH174.

Structural characterization on a ß-agarase Aga86A_Wa from Wenyingzhuangia aestuarii reveals the prevalent methyl-galactose accommodation capacity of GH86 enzymes at subsite -1.

Agarans are sulfated galactans extracted from red algae with high structural complexity, of which natural methylation often occurs on the O-6 position of its ß-d-galactopyranose units. Although many agaran degrading enzymes, including agarases and porphyranases, have been characterized, little attention has been paid to the tolerance of methyl groups at cleavage subsites. In this study, the structure of GH86 ß-agarase Aga86A_Wa from Wenyingzhuangia aestuarii was determined by X-ray crystallography and investigated from a structural biology perspective. The structure indicated that an accommodation pocket formed by F367, Y280, and Q326 at subsite -1 contributes to the methyl-galactose tolerance of Aga86A_Wa. Furthermore, we found that similar accommodation pockets were present in the structures of two other GH86 enzymes BuGH86 from Bacteroides uniformis and BpGH86A from Phocaeicola plebeius, and their previously undisclosed methyl-galactose tolerance was verified, validating the function of the pockets. Phylogenetic analysis, structural modeling, and hydrolysis product characterization suggested that the methyl-galactose accommodation capacity at subsite -1 was prevalent in GH86 members. These findings achieve a better understanding of the function and mechanism of GH86 agaran degrading enzymes, and will facilitate the precise preparation of agaran oligosaccharides by employing defined tools.

Characterization of a sulfated fucan-specific carbohydrate-binding module: A promising tool for investigating sulfated fucans.

Sulfated fucans are important polysaccharides with diverse biological and biomedical activities. Carbohydrate-binding modules (CBMs) could serve as beneficial tools for the investigation of polysaccharides. Nevertheless, no sulfated fucan-binding CBM has been hitherto discovered. In the present study, a novel CBM47 domain was cloned from the marine bacterium Wenyingzhuangia fucanilytica, and heterologously expressed in Escherichia coli. The expressed protein WfCBM47 exhibited a specific binding capacity to sulfated fucans with the backbone composed of 1,3-α-l-fucopyranose residues. Furthermore, a fluorescent probe was successfully constructed by fusing WfCBM47 with a green fluorescent protein, based on which the in situ visualization of sulfated fucan in the sea cucumber (Apostichopus japonicus) body wall was implemented for the first time. The discovery of WfCBM47 provided a promising tool for future investigations on sulfated fucans.

Overexpression of long non-coding RNA LINC00158 inhibits neuronal apoptosis by promoting autophagy in spinal cord injury.

Spinal cord injury (SCI) is a common central nervous system disease. It is reported that long non-coding RNA LINC00158 is involved in the process of SCI. The purpose of this study was to explore the biological role of LINC00158 in the SCI. First, we established a rat SCI model by surgical method and evaluated the motor function of rats by the Basso-Beattie-Bresnahan locomotor rating scale. The results showed that the expression of LINC00158 decreased and apoptotic cells increased in the SCI model rats. Meanwhile, we found the upregulated LC3-II/LC3-I, Beclin-1, and p62 in the SCI rats. Then, primary rat spinal cord neurons were exposed to oxygen/glucose deprivation (OGD) as an in vitro cell model of SCI. After OGD treatment, the expression of LINC00158 decreased significantly and the apoptosis of spinal cord neurons increased. OGD treatment resulted in upregulation of LC3-II/LC3-I and Beclin-1 and downregulation of p62 in primary spinal cord neurons, which could be eliminated by overexpression of LINC00158. 3-Methyladenine and chloroquine (autophagy inhibitor) reversed the inhibitory effect of LINC00158 overexpression on apoptosis of primary spinal cord neurons. In conclusion, this study demonstrated that LINC00158 overexpression repressed neuronal apoptosis by promoting autophagy, suggesting that LINC00158 may be a potential therapeutic target in the SCI.

Utilizing heterologously overexpressed endo-1,3-fucanase to investigate the structure of sulfated fucan from sea cucumber (Holothuria hilla).

Sea cucumber sulfated fucan (SC-FUC) attracted increasing interests in the recent decades. Endo-1,3-fucanase has been employed in the structural clarification and structure-function relationship investigations of SC-FUC. Nevertheless, the preparation of wild-type endo-1,3-fucanase is costly and time-consuming, which hinders its further utilization. In this study, a heterologously overexpressed endo-1,3-fucanase (FunA) was introduced into structural identification of SC-FUC. FunA was efficiently prepared within one day and utilized in the investigation of sulfated fucan from sea cucumber Holothuria hilla (Hh-FUC). By using enzymatic degradation, glycomics and NMR analysis, the major structure of Hh-FUC was identified to be composed of a tetrasaccharide repeating unit →3-α-l-Fucp-1 â†’ 3-α-l-Fucp2,4(OSO3-)-1 â†’ 3-α-l-Fucp2(OSO3-)-1 â†’ 3-α-l-Fucp2(OSO3-)-1→. Due to the efficient acquisition of enzyme and the superior oligosaccharide recovery, 0.6 mL of E. coli broth and 10 mg of Hh-FUC were sufficient for the structural identification. The results demonstrated the superiority of heterologously overexpressed fucanase over its wild-type enzyme in structural investigation of sulfated fucan.

Cloning, Heterologous Expression, and Characterization of a ßκ-Carrageenase From Marine Bacterium Wenyingzhuangia funcanilytica: A Specific Enzyme for the Hybrid Carrageenan-Furcellaran.

Carrageenan is a group of important food polysaccharides with high structural heterogeneity. Furcellaran is a typical hybrid carrageenan, which contains the structure consisted of alternative ß-carrageenan and κ-carrageenan motifs. Although several furcellaran-hydrolyzing enzymes have been characterized, their specificity for the glycosidic linkage was still unclear. In this study, we cloned, expressed, and characterized a novel GH16_13 furcellaran-hydrolyzing enzyme Cgbk16A_Wf from the marine bacterium Wenyingzhuangia fucanilytica CZ1127. Cgbk16A_Wf exhibited its maximum activity at 50°C and pH 6.0 and showed high thermal stability. The oligosaccharides in enzymatic products were identified by liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) and nuclear magnetic resonance (NMR) spectroscopy. It was confirmed that Cgbk16A_Wf specifically cleaves the ß-1,4 linkages between ß-carrageenan and κ-carrageenan motifs from non-reducing end to reducing end. Considering the structural heterogeneity of carrageenan and for the unambiguous indication of the specificity, we recommended to name the furcellaran-hydrolyzing activity represented by Cgbk16A as "ßκ-carrageenase" instead of "furcellaranase".