Characterization of a high-affinity sialic acid-specific CBM40 from Clostridium perfringens and engineering of a divalent form

Abstract

CBMs (carbohydrate-binding modules) are a class of polypeptides usually associated with carbohydrate-active enzymatic sites. We have characterized a new member of the CBM40 family, coded from a section of the gene NanI from Clostridium perfringens Glycan arrays revealed its preference towards α(2,3)-linked sialosides, which was confirmed and quantified by calorimetric studies. The CBM40 binds to α(2,3)-sialyl-lactose with a Kd of ∼30 μM, the highest affinity value for this class of proteins. Inspired by lectins' structure and their arrangement as multimeric proteins, we have engineered a dimeric form of the CBM, and using SPR (surface plasmon resonance) we have observed 6-11-fold binding increases due to the avidity affect. The structures of the CBM, resolved by X-ray crystallography, in complex with α(2,3)- or α(2,6)-sialyl-lactose explain its binding specificity and unusually strong binding.

XBP1s activation can globally remodel N-glycan structure distribution patterns

“…The α2,6-sialic acid-specific lectins SNA-I, TJA-I, and PSL-I showed an average 29% decrease in binding relative to the vehicle-treated control. Our engineered recombinant lectin diCBM40, which binds both α2,3and α2,6-linked sialic acids with similar affinity (22), showed a 35% loss in binding, further supporting a decrease in α2,6-sialylation. XBP1s activation also decreased bisecting N-glycans, as indicated by decreasing PHA-E signal ( Fig.…”

Tailor-made Janus lectin with dual avidity assembles glycoconjugate multilayers and crosslinks protocells

“… 18 We previously demonstrated that RSL can be engineered, resulting in neoRSL with controlled number of binding sites. 18 , 19 As for sialic acid, a CBM from the NanI sialidase of Clostridium perfringens ATCC13124 (CBM40_NanI) was selected as the protein candidate to be fused to RSL due to the relatively strong affinity for sialylated oligosaccharides ( K D = 32 μM for 3′-siallyllactose), 20 the ease of expression, and the accessibility of both C-ter and N-ter extremities for engineering ( Fig. 1C ).…”

Structure and engineering of tandem repeat lectins

“…Creating such multivalent receptors through artificially constructed tandem repeats is a route of interest. It generally strongly enhances the receptor affinity for its ligand [54,55]. It has also been demonstrated that expression as tandem-repeat sequences improves the production of recombinant algal lectins in bacterial systems [56].…”