Directed Evolution of Lectins with Sugar-binding Specificity for 6-Sulfo-galactose

Abstract

Background: 6-sulfo-galactose is a potential marker of various diseases, but no useful probes to detect this glycoepitope have been available. Results: Mutants with novel affinity for 6-sulfo-galactose were engineered from an R-type galactose-binding lectin. Conclusion:We succeeded in providing a method to create probes for 6-sulfo-galactose by a molecular evolutionary strategy. Significance: The tools developed in our study will be especially useful in the context of sulfoglycomics.

Tailoring GalNAcα1-3Galβ-specific lectins from a multi-specific fungal galectin: dramatic change of carbohydrate specificity by a single amino-acid substitution

“…The sugar-binding activity of ACG mutants was analysed by glycoconjugate microarray on the basis of methods described previously [16]. Briefly, ACG mutants with an N-terminal FLAG tag in pET27b prepared as described above were expressed under the induction of 1 mM IPTG in BL21-CondonPlus (DE3)-RIL for 24 h at 20 • C. Subsequently, cells were harvested and washed with PBS( − ) three times before lysis by suspension in a 1:5 culture volume of BugBuster ® HT Protein Extraction Reagent (Novagen).…”

“…Recombinant ACG is stable and can be easily produced in Escherichia coli, allowing it to be used for protein engineering. In the present study, we first targeted all of the β-galactose-binding residues of ACG to assess their importance by replacing each of them with alanine (alanine mutagenesis) and analysed their binding activity by a high-throughput screening method using an evanescent-type glycoconjugate microarray [16]. To reduce the background affinity to LacNAc, they focused on Glu 86 , a residue involved in both terminal galactose and penultimate GlcNAc binding in the context of LacNAc.…”

Recognition of the Thomsen-Friedenreich Pancarcinoma Carbohydrate Antigen by a Lamprey Variable Lymphocyte Receptor

“…Most often, monoclonal antibodies are isolated from mice immunized with the glycan of interest, but this process is laborious, and most human glycans are not particularly immunogenic in mice, probably due to their conserved structures in these species, which gives rise to self-tolerance. Other strategies include directed evolution of antibodies (7) or lectins (3,8), carbohydrate binding peptides (9), and glycan-specific aptamers (10), but none has proven generally useful.…”

The Lectin Frontier Database (LfDB), and Data Generation Based on Frontal Affinity Chromatography

“…Xenopus galectin-VIIa [81] Human, chicken, nematode, sponge, fungal galectrins [36] Human galectin-9 [82] Nematode galectin LEC-1 and N-and C-CRDs [83] C-type PF00059 C-type α/β-fold Atlantic salmon serum [84] calcium-dependent Sea cucumber lectin CEL-IV [85] DC-SIGN, DC-SIGNR, and LSECtin [86] Langerin to sulfated and mannsylated glycans [87] MGL1, MGL2, and their mutants [88] Atlantic salmon C-type lectin receptor C (SCLRC) [89] Acorn barnacle Megabalanus rosa, Balanus rostatus [90] R-type PF00652 β-trefoil Mutants of earthworm lectin EW29Ch [91] ricin B chain-related RCA120/RCA-I [58] Phytopathogenic Sclerotinia sclerotiorum [92] Jacalin-related PF01419 β-prism I Human ZG16 p [93] Mannose-binding type Jacalin-related lectins [37] Jacalin [94] Castanea crenata agglutinin (CCA) [95] Cycas revoluta leaf lectin (CRLL) [95] GNA-related monocot-type PF04152 β-prism II Two-domain GNA-related lectins [96] Sea urchin egg PF02140 α/β-fold with two long Shishamo smelt Osmerus (Spirinchus) lanceolatus [97] Fungal fucose-specific PF07938 6-bladed β-propeller Mushroom Pholiota squarrosa [104] Aspergillus oryzae [105] AAL-like Fungal fruit body lectin PF07367 α/β-sandwich Agaricus bisporus agglutinin (ABA) [106] Others Mussel, Mytilus galloprovincialis [107] orphan or family Mushroom Hygrophorus russula [108] unidentified Eggs of Japanese sea hare Aplysia kurodai [109] Feather star Oxycomanthus japonicas [110] Coronate moon turban Turbo (Lunella)…”