Figure 1. 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).
Figure 2. 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).
Figure 3. 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 NFLAG-
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 N-FLAGmalectin
after deletion of the predicted N-terminal signal peptide.
Note that N-FLAG-malectin also diffuses to the nucleus (asterisks)
and can be found in protein aggregates (arrow heads). Bar, 10 m.
Malectin Binds Glc2-N-glycan
Figure 4. 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 -helices (1–3) and -strands (1–12), 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 (-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.
Figure 5. 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. and 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.
Figure S1: Sequence alignment of animal malectin proteins vs. Arabidopsis thaliana and Oryza
sativa RLK proteins. (A) Alignment of representative arabidopsis and rice RLK-malectin-like
domains with the malectin core domain of X. laevis, humans, rat and mouse. Sequences are labelled
with UniProtKB/TrEMBL accession numbers. (XENLA = Xenopus laevis, DROME = Drosophila
melanogaster, SCHJA = Schistosoma japonicum, CAEEL = Caenorhabditis elegans, CRYPV =
Cryptospridium parvum, ARATH = Arabidopsis thaliana, ORYSA = Oryza sativa). The aromatic
residues and the aspartate that in X. laevis mediate interactions with the glucose residues (Fig.4D)
are marked by red crosses, and are not conserved in plants. (B) Domain topologies of plant and
animal proteins that contain the malectin core domain. The two topologies among plant RLKs are
shown. Labels: SP - signal peptide; TM- transmembrane helix; LRR - leucine rich repeat: STKinase
– serine/threonine receptor-like kinase.
Figure S2: Malectin binding to glucose disaccharides studied by isothermal titration
calorimetry. Kojibiose (A), nigerose (B) and maltose (C). The raw data are shown in the upper
panel, and the integrated heat data, corrected for dilution, are shown in the lower panel.
ITC measurements were carried out using a VP-ITC Mircocal clorimeter (Mircocal, Northhampton,
MA, USA) in 20mM phosphate buffer (pH 6.8), 150mM KCl and 1mM TCEP. A typical titration
consisted of injecting 10μl of the sugar into the malectin sample, at time intervals of 5min, to ensure
that the titration peak returned to the baseline.
Figure S3: NOEs between malectin and nigerose. (A) Part of a 13C-edited half-filtered-NOESY
experiment (mixing time 150 ms) showing intermolecular NOEs between malectin and nigerose.
(B) Structure of α-nigerose.
Figure S4: Microarray analyses of the interactions of malectin with Glc1-, Glc2- and Glc3-high
mannose N-glycans and gluco-oligosaccharide probes. The oligosaccharide probes were printed
as duplicate spots and binding was assayed with malectin at 20, 5, 1 and 0.5 μg/ml (panels A to D,
respectively). Numerical scores are shown for the binding signals [means of duplicate values at 2
and 7 fmol/spot, (blue and red bars, respectively) with error bars]. At a malectin concentration of 20
μg/ml, the binding signals for the Glc2-high mannose N-glycan probe, both at 2 and 7 fmol, were
too high to be accurately quantified (asterisk in A) and were annotated as >> 50000 in Table 1.
Other oligosaccharide probes tested included the glucose disaccharides kojibiose (Glcα1-2Glc)
nigerose (Glcα1-3Glc), maltodextrins (Glcα1-4Glc, dp 2-7); and oligosaccharides from dextran
(isomalto) (Glcα1-6Glc, dp 2-7); laminarin (Glcβ1-3Glc, dp 2-7); cellulose (Glcβ1-4Glc, dp 2-6);
and pustulan (Glcβ1-6Glc, dp 2-7). Abbreviations G3N, G2N and G1N designate
Glc3Man7(D1)GlcNAc, Glc2Man7(D1)GlcNAc and Glc1Man9GlcNAc2 N-glycan probes,
respectively; dp, degree of polymerization of the gluco-oligomers.
mlec (malectin) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 20, dorsal view, anterior left, dorsal up.
mlec (malectin) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior left, dorsal up.
mlec (malectin) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 41, lateral view, anterior left, dorsal up.