Carbohydrate recognition by an architecturally complex α-N-acetylglucosaminidase from Clostridium perfringens.

Elizabeth Ficko-Blean,Robert J Woods,Matthew Tessier,Alisdair B Boraston,Michael D Suits,Christopher P Stuart,Melissa Cid,Maria Gasset

doi:10.1371/journal.pone.0033524

Abstract

CpGH89 is a large multimodular enzyme produced by the human and animal pathogen Clostridium perfringens. The catalytic activity of this exo-α-d-N-acetylglucosaminidase is directed towards a rare carbohydrate motif, N-acetyl-β-d-glucosamine-α-1,4-d-galactose, which is displayed on the class III mucins deep within the gastric mucosa. In addition to the family 89 glycoside hydrolase catalytic module this enzyme has six modules that share sequence similarity to the family 32 carbohydrate-binding modules (CBM32s), suggesting the enzyme has considerable capacity to adhere to carbohydrates. Here we suggest that two of the modules, CBM32-1 and CBM32-6, are not functional as carbohydrate-binding modules (CBMs) and demonstrate that three of the CBMs, CBM32-3, CBM32-4, and CBM32-5, are indeed capable of binding carbohydrates. CBM32-3 and CBM32-4 have a novel binding specificity for N-acetyl-β-d-glucosamine-α-1,4-d-galactose, which thus complements the specificity of the catalytic module. The X-ray crystal structure of CBM32-4 in complex with this disaccharide reveals a mode of recognition that is based primarily on accommodation of the unique bent shape of this sugar. In contrast, as revealed by a series of X-ray crystal structures and quantitative binding studies, CBM32-5 displays the structural and functional features of galactose binding that is commonly associated with CBM family 32. The functional CBM32s that CpGH89 contains suggest the possibility for multivalent binding events and the partitioning of this enzyme to highly specific regions within the gastrointestinal tract.

Highlights

Mucins are heavily O-glycosylated glycoproteins that act to protect the epithelia from harmful bacteria by forming a biophysical barrier to infection as well as supporting innate and adaptive immunity [1]
Among the enzymes that C. perfringens employs to cope with the mucosal surface are the glycoside hydrolases, which have varying catalytic specificities that reflect the diversity in host glycans; these include, but are not limited to, neuraminidases (GH33)[3,4], exo- and endo-b-N-acetylglucosaminidases (GH84 and GH85)[5,6,7], an endo-a-N-acetylgalactosaminidase (GH101)[8,9], as well as CpGH89, which is an exo-a-Nacetylglucosaminidase [10,11]
The strict conservation of residues involved in galactose recognition suggested that CBM32-5 belongs to the galactose binding group of family 32 carbohydrate-binding modules (CBMs) [7,27,28]

Summary

Introduction

Mucins are heavily O-glycosylated glycoproteins that act to protect the epithelia from harmful bacteria by forming a biophysical barrier to infection as well as supporting innate and adaptive immunity [1]. The addition of galactose or GalNAc to CBM32-5 perturbed the UV absorption of this protein in a manner consistent with the involvement of tyrosine residues in carbohydrate binding [29](Figure 2B).

Results

Conclusion