Broad expression of malectin in embryonic and adult X. laevis. (A) Analysis of embryos by whole mount in situ hybridization showing malectin expression in the anterior neuroectoderm (ne) and neural crest (nc) at stages 18 and 20 (A1, A2), and at later stages, e.g., 32, in the hatching gland (hg), retina (re), otic vesicle (ot), epibranchial placodes (eb), pronephros (pn), and the tail tip (tp); anterior (a); posterior (p). At stage 41 (A4), transcripts are detected in the liver (li), dorsal and ventral pancreas (dp and vp), branchial arches (ba), and the proctodeum (pd). (B) Analysis of embryonic expression of malectin by RT-PCR showing expression in the oocyte (stage VI) and continued expression throughout development, and the contrasting expression of protein disulfide isomerase, xPDIp, only from late tadpole stage 39 onward. Abbreviations: stage VI oocyte (VI); unfertilized egg (0) and fertilized egg (1). (C) The expression analysis in adult tissue by semiquantitative RT-PCR showing a broad distribution of malectin in comparison with xPDIp, which is detected in pancreas and stomach only. Additional abbreviations not defined in A: gall bladder (gb); heart (he); intestine (in); kidney (ki); lung (lu); muscle (mu); pancreas (pa); stomach (st).

Sequence alignment of malectin proteins in animals. Malectin proteins are composed of an N-terminal signal peptide (SP, AA 1-26), a C-terminal transmembrane helix (TM; AA 255-274) and a highly conserved central part of ∼190 residues followed by an acidic, glutamate-rich region. The secondary structure elements derived from the experimental structure (see Figure 4A) are shown on top of the amino acid sequence; and the four aromatic residues (Y67, Y89, Y116, and F117) and D186 mediating the carbohydrate interaction are marked by red crosses (Xen, Xenopus laevis [100/100]; Hum, Homo sapiens [89/95]; Mou, Mus musculus [86/94]; Hen, Gallus gallus [84/96], Fly, Drosophila melanogaster [41/58]; Aed, Aedes aegyptii [44/62]; Cae, Caenorhabditis elegans [36/58]; Sch, Schistosoma japonicum [42/59]; Nem, Nematostella vectensis [51/69]). The bracketed numbers represent the percentage amino acid conservation in comparison with the X. laevis malectin protein (identities/similarities).

ER localization of malectin. (A) Schematic drawing of the FLAG-tagged malectin constructs used for transient transfection experiments. N-FLAG-malectin represents the full-length protein including a FLAG-tag (F) between the predicted N-terminal signal peptide (red, SP; AA 1-26) and the conserved lectin-like domain (blue, LLD; AA 27-213) that is followed by a hydrophobic C-terminal domain (yellow, HD; AA 255-274). The deletion construct ΔN-FLAG-malectin lacks the N-terminal SP. (B) Immunofluorescence analysis of U-2 OS cells for FLAG-tagged malectin (green) and the ER marker calnexin (red) showing that malectin and calnexin colocalize in ER structures (arrows indicate presence of both markers in the nuclear envelope). (C) Cytoplasmic localization of ΔN-FLAG-malectin after deletion of the predicted N-terminal signal peptide. Note that ΔN-FLAG-malectin also diffuses to the nucleus (asterisks) and can be found in protein aggregates (arrow heads). Bar, 10 μm.

Structure of the main domain of malectin and of the malectin–nigerose complex. (A) Ensemble of the 10 lowest energy structures (out of 100 calculated) after water refinement. The four loops (L1–L4), which could only be assigned in the presence of a carbohydrate ligand, are highlighted in green: L1, G62-G68; L2, T86-N90; L3, E114-A118; and L4, Y185-N187. (B) Ribbon representation of malectin. Secondary structure elements are colored in red and blue for α-helices (α1–α3) and β-strands (β1–β12), respectively. (C) Ensemble of the 10 lowest energy structures of the malectin–nigerose complex. Only the ligand-binding pocket is shown. The four aromatic residues and D186 mediating the interaction are relatively well defined. Yellow, Y89; cyan, Y67; magenta, Y116; blue, F117; and brown, D186. Nigerose is presented in green (D). Detailed view of the malectin–nigerose interaction. Nigerose is sandwiched by Y67, Y89, Y116, and F117. The nonreducing and reducing residues of nigerose are labeled Glc-A and Glc-B, respectively. Oxygen atoms of the carbohydrate are highlighted as red spheres. The orange arrow points to the oxygen atom of the C-2 hydroxyl group of Glc-A, where the outermost glucose residue of Glc₃-N-glycan would be attached. The magenta arrow highlights the oxygen atom of the C-1 hydroxyl group of Glc-B (α-form), where the polymannose part of the Glc₂-N-glycan would be continued. The oxygen atom of the equatorial C-2 hydroxyl group of Glc-B is marked by the yellow arrow. If at the Glc-B position there was a mannose residue, as in Glc₁-N-glycan, the stacking interaction would be hindered as there would be an axial hydroxyl group pointing toward Y116 and F117. (E) Schematic drawing of the Glc₃-Man₉-N-glycan. Glucose is depicted as green circles, mannose as yellow squares, and GlcNAc as blue circles.

One dimensional STD spectra for glucose and glucose disaccharides: glucose (A), cellobiose (B), maltose (C) nigerose (D), kojibiose (E), and isomaltose (F). Structures of the tested carbohydrates are depicted on the left of the corresponding spectra. α and β denote the stereoisomers of the anomeric center of the reducing-end glucose ring. The ring protons of the nonreducing residues are labeled 1–6, and those of the reducing residues 1′–6′. Assignment tables of the carbohydrate ligands are in Supplemental Table S4.

High selectivity of malectin binding to the Glc₂-N-glycan revealed by carbohydrate microarray analyses. In total, 335 oligosaccharide probes (Supplemental Table S6) were printed as duplicate spots. Malectin binding was assayed at 5 μg/ml. Numerical scores are shown of the binding signals (means of duplicate values at 7 fmol/spot, with error bars; the error bars represent half of the difference between the two values). The glucosylated N-glycan sequences are annotated. Inset, expanded region of the microarray highlighting the selective binding of malectin to the Glc₂-N-glycan probe in contrast with the broad binding profile of ConA. Abbreviations for the probes: G₃N, Glc₃Man₇(D1)GlcNAc; G₂N, Glc₂Man₇(D1)GlcNAc; G₁N, Glc₁Man₉GlcNAc_2; M₉N, Man₉GlcNAc₂; and M₇N, Man₇(D1)GlcNAc₂. These five probes were arrayed at positions 162–165 and at position 168, respectively.