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 alpha-fucosidase and from a family 98 blood group A/B antigen-specific endo-beta-galactosidase, respectively, showed them to have highly similar beta-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.
Highlights
Wall carbohydrates, and the interactions between hosts and disease-causing organisms
The Carbohydrate-binding modules (CBMs) appear to have a role in recognizing the information content of cellular glycans, which allows the enzymatic activity of the virulence factor to be appropriately directed
Among its minor toxins are a battery of carbohydrate-active enzymes of the class glycoside hydrolase, which are thought to aid in major toxin delivery, tissue destruction, 3 The abbreviations used are: CBMs, carbohydrate-binding modules; NPCBM, novel putative CBM; galactose or D-N-acetylgalactosamine (GalNAc), D-N-acetylgalactosamine
Summary
Wall carbohydrates, and the interactions between hosts and disease-causing organisms. NPCBM domains show no amino acid sequence identity to known carbohydrate-binding proteins but were hypothesized by Rigden [12] to be CBMs based on their context (i.e. in carbohydrate-active enzymes) and their proposed -sandwich fold, which is common to CBMs. To test the hypothesized carbohydrate-binding function of these modules, we performed structural and functional studies on the NPCBMs from two different multimodular clostridial enzymes, one a hypothesized family 95 ␣-L-fucosidase (CPF_2129 from C. perfringens strain ATCC 13124) and the other a confirmed family 98 blood group A/B antigen-specific endo--D-galactosidase (EabC from C. perfringens strain ATCC 10543) (Fig. 1) [13]. GH98CBM51 was clearly the more specific module, showing binding only to glycans bearing the blood group A or B antigen trisaccharide determinants (Fig. 2B).
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