Divergent Modes of Glycan Recognition by a New Family of Carbohydrate-binding Modules

Abstract

The genomes of myonecrotic Clostridium perfringens isolates contain genes encoding a large and fascinating array of highly modular glycoside hydrolase enzymes. Although the catalytic activities of many of these enzymes are somewhat predictable based on their amino acid sequences, the functions of their abundant ancillary modules are not and remain poorly studied. Here, we present the structural and functional analysis of a new family of ancillary carbohydrate-binding modules (CBMs), CBM51, which was previously annotated in data bases as the novel putative CBM domain. The high resolution crystal structures of two CBM51 members, GH95CBM51 and GH98CBM51, from a putative family 95 ␣-fucosidase and from a family 98 blood group A/B antigen-specific endo-␤-galactosidase, respectively, showed them to have highly similar ␤-sandwich folds. However, GH95CBM51 was shown by glycan microarray screening, isothermal titration calorimetry, and x-ray crystallography to bind galactose residues, whereas the same analyses of GH98CBM51 revealed specificity for the blood group A/B antigens through non-conserved interactions. Overall, this work identifies a new family of CBMs with many members having apparent specificity for eukaryotic glycans, in keeping with the glycan-rich environment C. perfringens would experience in its host. However, a wider bioinformatic analysis of this CBM family also indicated a large number of members in non-pathogenic environmental bacteria, suggesting a role in the recognition of environmental glycans.Carbohydrates have critical functions in numerous biological events, including, for example, the movement and interactions of cells and proteins in animals, the recycling of plant cell wall carbohydrates, and the interactions between hosts and disease-causing organisms. Central to the role of carbohydrates in biological processes are protein-carbohydrate interactions. Non-catalytic carbohydrate-binding proteins (e.g. lectins, antibodies, and transport proteins) and catalytic carbohydrate-active enzymes are finely tuned to recognize particular carbohydrate structural motifs. The information content of glycans is realized through the specificity of non-catalytic carbohydratebinding proteins (like lectins and antibodies), whereas carbohydrate-active enzymes change the information content and often unlock the energy contained within these molecules.Carbohydrate-binding modules (CBMs) 3 are a comparatively new class of non-catalytic carbohydrate-recognizing polypeptide that are generally defined by their presence as ancillary modules in larger, multimodular carbohydrate-active enzymes such as glycoside hydrolases, glycosyltransferases, and polysaccharide lyases (1). In the context of these enzymes, the role of CBMs is to specifically bind the carbohydrate substrate and hold the enzyme in proximity to the substrate, allowing catalysis to proceed more efficiently (2). The number of CBM families, which are defined on the basis of amino acid sequence similarity, has grown to the current number of 50 (www.cazy. org). ...

The Overall Architecture and Receptor Binding of Pneumococcal Carbohydrate-Antigen-Hydrolyzing Enzymes

“…15 Furthermore, Sp3GH98 is known to have catalytic specificity for the BGA and BGB antigens, indicating that the two CBMs from Sp3GH98 might be specific for the BGA and/ or BGB antigens. To test this hypothesis, we cloned the gene fragments encoding separate CBM51-1 and CBM51-2 polypeptides and produced and purified the polypeptides.…”

“…Yet, another option is that the inactivated CBM is vestigial, resulting from mutations that did not significantly impact the activity of the enzyme, and it serves no significant purpose in a protein that might be considered an evolutionary precursor to a version of the enzyme that contains a single CBM. Indeed, the homolog of Sp3GH98 from C. perfringens, which has the same specificity as Sp3GH98, possesses only single N-terminal CBM51 that binds blood group antigens, 15 supporting the idea that only a single functional CBM is necessary. Nevertheless, the overall implication of CBM51-2 lacking carbohydrate binding activity is that this tandem is very unlikely to have an increased affinity for clustered glycans through an avidity effect.…”

Differential Recognition and Hydrolysis of Host Carbohydrate Antigens by Streptococcus pneumoniae Family 98 Glycoside Hydrolases

“…pneumoniae GH98 Enzymes-The GH98 enzymes from S. pneumoniae TIGR4 (Sp4GH98) and SP3-BS71 (Sp3GH98) are 1038-and 1005-amino acid, respectively, multimodular proteins with predicted classical N-terminal Gram-positive secretion signal sequences. Sp4GH98 possesses three C-terminal family 47 carbohydrate-binding modules (33), whereas Sp3GH98 has two N-terminal modules that have sequence identity with family 51 carbohydrate-binding modules (34). The N terminus of Sp4GH98 and the C terminus of Sp3GH98 contain a domain of ϳ650 amino acids that is predicted on the basis of sequence alignments with a known family 98 glycoside hydrolase, the GH98 endogalactosidase E-ABase from Clostridium perfringens (35), to house the catalytic activity.…”

Structural basis of Clostridium perfringens toxin complex formation

“…Recent structural and functional characterization of CBMs associated with the NanJ sialidase (9) and CpGH95 (20) indicates that these modules target their respective catalytic domains to their eukaryotic carbohydrate targets, further supporting a role of these carbohydrate-active enzymes in the host-pathogen relationship. It therefore appears that this complex arsenal of C. perfringens glycoside hydrolases contributes to the bacterium's virulence through tissue/glycan destruction to promote spread, potentiating the activity of the major toxins and providing a source of nutrition for the bacterium.…”