Abstract
The core α1–6 fucosylation-specific lectin from a mushroom Pholiota squarrosa (PhoSL) is a potential tool for precise diagnosis of cancers. This lectin consists of only 40 amino acids and can be chemically synthesized. We showed here that a synthesized PhoSL peptide formed a trimer by gel filtration and chemical cross-linking assays, and determined a structure of the PhoSL trimer by NMR. The structure possesses a β-prism motif with a three-fold rotational symmetry, where three antiparallel β-sheets are tightly connected by swapping of β-strands. A triad of Trp residues comprises the structural core, forming NH–π electrostatic interactions among the indole rings. NMR analysis with an excess amount of fucose revealed the structural basis for the molecular recognition. Namely, fucose deeply enters a pocket formed at a junction of β-sheet edges, with the methyl group placed at the bottom. It forms a number of hydrophobic and hydrogen-bonding interactions with PhoSL residues. In spite of partial similarities to the structures of other functionally related lectins, the arrangement of the antiparallel β-sheets in the PhoSL trimer is novel as a structural scaffold, and thus defines a novel type of lectin structure.
Highlights
Fucosylation is a common modification in glycoproteins, and is classified into several types with regard to the modes of linkage, i.e., α1–2, α1–3, α1–4, and α1–6
Researchers started to use PhoSL for detecting core fucosylation in diagnosis of cancers and related diseases, i.e., colorectal, prostate, pancreatic, and liver cancers as well as a chronic pancreatitis that is a background of pancreatic ductal adenocarcinoma[5,6,7,8,9]
Three bands were observed in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) (Fig. 1C)
Summary
Backbone Nuclear Overhauser effect (NOE) connections revealed that PhoSL possessed a secondary structure rich in β-strands (Fig. 2A,B). In the structure calculation, ambiguities with regard to this distinction were introduced (see Materials and Methods section; Table 1) This solution structure revealed a β-prism scaffold with a clear three-fold rotational symmetry (Fig. 3B) It consists of three β-sheets each containing essentially the four β-strands. Because of the reductive condition during the NMR measurements, we observed Hγ protons of these Cys residues and NOEs with other residues (Fig. S1B) Considering their relative positions, we can expect a disulfide bridge under an oxidative condition, which is likely to contribute to the structural stability.
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