Family 42 carbohydrate-binding modules display multiple arabinoxylan-binding interfaces presenting different ligand affinities

Materials with novel and enhanced functionalities can be obtained by modifying cellulose with a range of biomolecules. This functionalization can deliver tailored cellulose-based materials with enhanced physical and chemical properties and control of biological interactions that match specific applications. One of the foundations for the success of such biomaterials is to efficiently control the capacity to combine relevant biomolecules into cellulose materials in such a way that the desired functionality is attained. In this context, our main goal was to develop bi-functional biomolecular constructs for the precise modification of cellulose hydrogels with bioactive molecules of interest. The main idea was to use biomolecular engineering techniques to generate and purify different recombinant fusions of carbohydrate binding modules (CBMs) with significant biological entities. Specifically, CBM-based fusions were designed to enable the bridging of proteins or oligonucleotides with cellulose hydrogels. The work focused on constructs that combine a family 3 CBM derived from the cellulosomal-scaffolding protein A from Clostridium thermocellum (CBM3) with the following: (i) an N-terminal green fluorescent protein (GFP) domain (GFP-CBM3); (ii) a double Z domain that recognizes IgG antibodies; and (iii) a C-terminal cysteine (CBM3C). The ability of the CBM fusions to bind and/or anchor their counterparts onto the surface of cellulose hydrogels was evaluated with pull-down assays. Capture of GFP-CBM3 by cellulose was first demonstrated qualitatively by fluorescence microscopy. The binding of the fusion proteins, the capture of antibodies (by ZZ-CBM3), and the grafting of an oligonucleotide (to CBM3C) were successfully demonstrated. The bioactive cellulose platform described here enables the precise anchoring of different biomolecules onto cellulose hydrogels and could contribute significatively to the development of advanced medical diagnostic sensors or specialized biomaterials, among others.

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Integrated Counts of Carbohydrate-Active Protein Domains as Metabolic Readouts to Distinguish Probiotic Biology and Human Fecal Metagenomes

Scientific Reports ◽

10.1038/s41598-019-53173-7 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Hong-Hsing Liu ◽

Yu-Chen Lin ◽

Chen-Shuan Chung ◽

Kevin Liu ◽

Ya-Hui Chang ◽

...

Keyword(s):

Markov Models ◽

Protein Domains ◽

Degradation Pathway ◽

The Other ◽

Carbohydrate Binding ◽

Gram Stain ◽

Active Protein ◽

Metabolic Potential ◽

Independent Validation ◽

Carbohydrate Binding Modules

AbstractBowel microbiota is a “metaorgan” of metabolisms on which quantitative readouts must be performed before interventions can be introduced and evaluated. The study of the effects of probiotic Clostridium butyricum MIYAIRI 588 (CBM588) on intestine transplantees indicated an increased percentage of the “other glycan degradation” pathway in 16S-rRNA-inferred metagenomes. To verify the prediction, a scoring system of carbohydrate metabolisms derived from shotgun metagenomes was developed using hidden Markov models. A significant correlation (R = 0.9, p < 0.015) between both modalities was demonstrated. An independent validation revealed a strong complementarity (R = −0.97, p < 0.002) between the scores and the abundance of “glycogen degradation” in bacteria communities. On applying the system to bacteria genomes, CBM588 had only 1 match and ranked higher than the other 8 bacteria evaluated. The gram-stain properties were significantly correlated to the scores (p < 5 × 10−4). The distributions of the scored protein domains indicated that CBM588 had a considerably higher (p < 10−5) proportion of carbohydrate-binding modules than other bacteria, which suggested the superior ability of CBM588 to access carbohydrates as a metabolic driver to the bowel microbiome. These results demonstrated the use of integrated counts of protein domains as a feasible readout for metabolic potential within bacteria genomes and human metagenomes.

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Rotavirus VP8*: Phylogeny, Host Range, and Interaction with Histo-Blood Group Antigens

Journal of Virology ◽

10.1128/jvi.00979-12 ◽

2012 ◽

Vol 86 (18) ◽

pp. 9899-9910 ◽

Cited By ~ 107

Author(s):

Yang Liu ◽

Pengwei Huang ◽

Ming Tan ◽

Yiliu Liu ◽

Jacek Biesiada ◽

...

Keyword(s):

Sialic Acid ◽

Blood Group ◽

Binding Assay ◽

Distal Portion ◽

Carbohydrate Binding ◽

Blood Group Antigens ◽

Independent Manner ◽

Viral Attachment ◽

Mutagenesis Study ◽

Binding Interfaces

The distal portion of rotavirus (RV) VP4 spike protein (VP8*) is implicated in binding to cellular receptors, thereby facilitating viral attachment and entry. While VP8* of some animal RVs engage sialic acid, human RVs often attach to and enter cells in a sialic acid-independent manner. A recent study demonstrated that the major human RVs (P[4], P[6], and P[8]) recognize human histo-blood group antigens (HBGAs). In this study, we performed a phylogenetic analysis of RVs and showed further variations of RV interaction with HBGAs. On the basis of the VP8* sequences, RVs are grouped into five P genogroups (P[I] to P[V]), of which P[I], P[IV], and P[V] mainly infect animals, P[II] infects humans, and P[III] infects both animals and humans. The sialic acid-dependent RVs (P[1], P[2], P[3], and P[7]) form a subcluster within P[I], while all three major P genotypes of human RVs (P[4], P[6], and P[8]) are clustered in P[II]. We then characterized three human RVs (P[9], P[14], and P[25]) in P[III] and observed a new pattern of binding to the type A antigen which is distinct from that of the P[II] RVs. The binding was demonstrated by hemagglutination and saliva binding assay using recombinant VP8* and native RVs. Homology modeling and mutagenesis study showed that the locations of the carbohydrate binding interfaces are shared with the sialic acid-dependent RVs, although different amino acids are involved. The P[III] VP8* proteins also bind the A antigens of the porcine and bovine mucins, suggesting the A antigen as a possible factor for cross-species transmission of RVs. Our study suggests that HBGAs play an important role in RV infection and evolution.

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Versatile derivatives of carbohydrate-binding modules for imaging of complex carbohydrates approaching the molecular level of resolution

BioTechniques ◽

10.2144/000112244 ◽

2006 ◽

Vol 41 (4) ◽

pp. 435-443 ◽

Cited By ~ 61

Author(s):

Shi-You Ding ◽

Qi Xu ◽

Mursheda K. Ali ◽

John O. Baker ◽

Edward A. Bayer ◽

...

Keyword(s):

Molecular Level ◽

Carbohydrate Binding ◽

Carbohydrate Binding Modules ◽

Complex Carbohydrates ◽

Derivatives Of

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Functional Residue Prediction by Multiple Sequence Alignment for Carbohydrate Binding Modules

Proceedings of the 11th Joint Conference on Information Sciences (JCIS) ◽

10.2991/jcis.2008.93 ◽

2008 ◽

Author(s):

WI Chou ◽

WY Chou ◽

SC Lin ◽

TY Jiang ◽

CY Tang ◽

...

Keyword(s):

Sequence Alignment ◽

Multiple Sequence Alignment ◽

Carbohydrate Binding ◽

Multiple Sequence ◽

Functional Residue ◽

Carbohydrate Binding Modules ◽

Functional Residue Prediction

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Novel clostridial cell-surface hemicellulose-binding CBM3 proteins

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x21002764 ◽

2021 ◽

Vol 77 (4) ◽

Author(s):

Almog Hershko Rimon ◽

Oded Livnah ◽

Inna Rozman Grinberg ◽

Lizett Ortiz de Ora ◽

Oren Yaniv ◽

...

Keyword(s):

Calcium Binding ◽

Three Dimensional ◽

Dimensional Structure ◽

Carbohydrate Binding ◽

Unexpected Finding ◽

Calcium Binding Site ◽

Three Dimensional Structure ◽

Loop Region ◽

Carbohydrate Binding Modules ◽

Cellulose Binding

A novel member of the family 3 carbohydrate-binding modules (CBM3s) is encoded by a gene (Cthe_0271) in Clostridium thermocellum which is the most highly expressed gene in the bacterium during its growth on several types of biomass substrates. Surprisingly, CtCBM3-0271 binds to at least two different types of xylan, instead of the common binding of CBM3s to cellulosic substrates. CtCBM3-0271 was crystallized and its three-dimensional structure was solved and refined to a resolution of 1.8 Å. In order to learn more about the role of this type of CBM3, a comparative study with its orthologue from Clostridium clariflavum (encoded by the Clocl_1192 gene) was performed, and the three-dimensional structure of CcCBM3-1192 was determined to 1.6 Å resolution. Carbohydrate binding by CcCBM3-1192 was found to be similar to that by CtCBM3-0271; both exhibited binding to xylan rather than to cellulose. Comparative structural analysis of the two CBM3s provided a clear functional correlation of structure and binding, in which the two CBM3s lack the required number of binding residues in their cellulose-binding strips and thus lack cellulose-binding capabilities. This is an enigma, as CtCBM3-0271 was reported to be a highly expressed protein when the bacterium was grown on cellulose. An additional unexpected finding was that CcCBM3-1192 does not contain the calcium ion that was considered to play a structural stabilizing role in the CBM3 family. Despite the lack of calcium, the five residues that form the calcium-binding site are conserved. The absence of calcium results in conformational changes in two loops of the CcCBM3-1192 structure. In this context, superposition of the non-calcium-binding CcCBM3-1192 with CtCBM3-0271 and other calcium-binding CBM3s reveals a much broader two-loop region in the former compared with CtCBM3-0271.

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Carbohydrate-Binding Modules in Plant Cell Wall-Degrading Enzymes

Trends in Glycoscience and Glycotechnology ◽

10.4052/tigg.1403.1e ◽

2016 ◽

Vol 28 (161) ◽

pp. E49-E53

Author(s):

Shuichi Karita

Keyword(s):

Cell Wall ◽

Plant Cell ◽

Plant Cell Wall ◽

Carbohydrate Binding ◽

Cell Wall Degrading Enzymes ◽

Degrading Enzymes ◽

Carbohydrate Binding Modules

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A unique combination of glycoside hydrolases in Streptococcus suis specifically and sequentially acts on host-derived αGal-epitope glycans

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011977 ◽

2020 ◽

Vol 295 (31) ◽

pp. 10638-10652

Author(s):

Ping Chen ◽

Ran Liu ◽

Mengmeng Huang ◽

Jinlu Zhu ◽

Dong Wei ◽

...

Keyword(s):

Wide Spectrum ◽

Streptococcus Suis ◽

Glycoside Hydrolases ◽

Carbohydrate Binding ◽

Unique Combination ◽

Enzymatic Assays ◽

Tracheal Epithelial Cells ◽

Carbohydrate Binding Modules ◽

Host Signaling ◽

Important Host

Infections by many bacterial pathogens rely on their ability to degrade host glycans by producing glycoside hydrolases (GHs). Here, we discovered a conserved multifunctional GH, SsGalNagA, containing a unique combination of two family 32 carbohydrate-binding modules (CBM), a GH16 domain and a GH20 domain, in the zoonotic pathogen Streptococcus suis 05ZYH33. Enzymatic assays revealed that the SsCBM-GH16 domain displays endo-(β1,4)-galactosidase activity specifically toward the host-derived αGal epitope Gal(α1,3)Gal(β1,4)Glc(NAc)-R, whereas the SsGH20 domain has a wide spectrum of exo-β-N-acetylhexosaminidase activities, including exo-(β1,3)-N-acetylglucosaminidase activity, and employs this activity to act in tandem with SsCBM-GH16 on the αGal-epitope glycan. Further, we found that the CBM32 domain adjacent to the SsGH16 domain is indispensable for SsGH16 catalytic activity. Surface plasmon resonance experiments uncovered that both CBM32 domains specifically bind to αGal-epitope glycan, and together they had a KD of 3.5 mm toward a pentasaccharide αGal-epitope glycan. Cell-binding and αGal epitope removal assays revealed that SsGalNagA efficiently binds to both swine erythrocytes and tracheal epithelial cells and removes the αGal epitope from these cells, suggesting that SsGalNagA functions in nutrient acquisition or alters host signaling in S. suis. Both binding and removal activities were blocked by an αGal-epitope glycan. SsGalNagA is the first enzyme reported to sequentially act on a glycan containing the αGal epitope. These findings shed detailed light on the evolution of GHs and an important host-pathogen interaction.

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