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Additional file 1: Table S1. LC–MS analysis of tryptic-digested peptides of LSSL. Table S2. The full names and abbreviation of species used in the analysis.

Figure S1. Localization of LIP in the lipid raft microdomains of cancer cell membranes. Cell lines and primary leukocyte cells isolated from particular individuals (normal and diseased, isolated from peripheral blood) using Ficoll lymphocyte separation medium at a density of 1.077 g/mL. The cells were incubated with Alexa488-tagged LIP (1 μg/mL) at 37 °C for 30 min and subjected to flow cytometric analysis. Histogram showing statistics of the above results. Means ± SDs are shown (n = 3 per group). Figure S2. Comparative analysis of mannose-specific jacalin-related lectin (mJRL) family members. (A) Sequence alignment of mJRL family members. The residues in the primary carbohydrate binding site are shaded yellow. The residues in a second potential carbohydrate binding site are shaded cyan. Dln1 is from zebrafish (PDB code 4ZNO) [13]; Heltuba is from Helianthus tuberosus (PDB code 1C3K) [51]; GRFT (antiviral lectin griffithsin) is from the red alga Griffithsia sp. (PDB code 3LL0) [52]; Banlec is from banana lectin (PDB code 3MIT) [31]; and ZG16p is from human pancreatic lectin (PDB code 3VY7) [53]. The sequences were aligned with Clustal W [54]. (B) Top view of the superimposed lectin module of several mJRL proteins. The 12-stranded β-sheets are labeled. L1 is the GG loop. L6 is the ligand-binding loop. L4 is the ligand recognition loop. L2 and L3 are the ligand-binding loop and GG loop of the putative second binding site. L5 was also found to be involved in ligand binding in Banlec. LIP is shown in sandy brown. Dln1 is shown in blue (sucrose) and magenta (mannose). GRFT is shown in green. Banlec is shown in salmon. Heltuba is shown in purple. ZG16p is shown in gray. Figure S3. Sialylated antennary N-glycan specificity of LIP. (A) 100 N-glycan identification list. The numbers of complex and hybrid NgGlycans, high-mannose N-glycans and Neu5Gc N-glycans are 81, 10 and 9, respectively. (B) Typical binding of LIP assay result from the 100 N-Glycan Array. 103: Biotinylated mannose (0.01 mg/mL), 104: Human IgG (0.01 mg/mL), 105: Mouse IgG (0.1 mg/mL) as a positive control. (C) The N-glycosidase F-treated aqueous fraction from MCF-7, K562 cells and human leukocytes after Triton X-114 phase separation was digested with trypsin and analyzed by MS/MS. The general scheme and chemical treatments used in this study (left pane). (D) Distribution profile of lipid raft components in DRMs by sucrose density gradient centrifugation. Cells were lysed in MBS buffer containing 1% Triton X-100 at 4 °C. Lysates were fractionated by sucrose gradient centrifugation, and 7 fractions were collected from the top of the centrifuge tube. A sample from each fraction was subjected to dot immunoblotting analysis using antibodies to flotillin-I to confirm the fraction of lipid raft components. (E) MCF-7 and K562 cells and human leukocytes were examined with anti-Neu5Gc antibodies in culture containing either fetal bovine serum or human serum. Values are the means of three independent experiments. Means ± SDs are shown. Figure S4. The effect of PI-PLC on GPI-APs and SM. (A) MALDI-TOF MS spectra of SM standard treated with PI-PLC and sphingomyelinase (SMase). After treatment with PI-PLC and SMase, the content of the SM standard (m/z = 703.56) decreased. (B) The general scheme of GPI-APs and SM structure in mammalian cells. Figure S5. Relative expression of the mRNA of SM-related synthetases in different tumor cells measured by real-time PCR. Figure S6. Protein expression and purification. The LIP mutants were constructed using a Site-directed Mutagenesis Kit (Thermo Scientific) and then were expressed, refolded and purified following the same procedures as for wild-type LIP protein. (PDF 1456 kb)

Table S1. Cytocidal activities of LIP against various tumor cells. Table S2. The effect of LIP on normal and primary cells. Table S3. Data collection and refinement statistics for LIP. One crystal was used for each structure. Values in parentheses are for the highest resolution shell. Rmerge=ΣhΣi|Ih,i-Ih|/ΣhΣiIh,i, where Ih is the mean intensity of the i observations of symmetry related reflections of h. R=Σ|Fobs-Fcalc|/ΣFobs, where Fcalc is the calculated protein structure factor from the atomic model (Rfree was calculated with 5% of the reflections selected randomly). Table S4. Data of Z scores and RMSDs for each PDB comparison. Table S5. Binding free energy calculation by MM/PBSA method after molecular dynamics (MD) simulation by Gromacs. ∆Evdw: van der Waal energy; ∆Eele: electrostatic energy; ∆GPB: polar salvation energy; ∆GSA: non-polar salvation energy;∆Gbinding: binding energy. (DOCX 28 kb)

Figure S1. The distribution of mass errors is near zero and most of them are less than 20Â ppm, which means the mass accuracy of the MS data fits the requirement and has a good QC validation of MS data. Figure S2. The protein composition of buccal gland secretion from lampreys fed for 10Â min and 60Â min was detected by 2D-PAGE. Table S1. The distribution of identified proteins on biological process (A), molecular function (B) and cellular component (C) during fasting and feeding stages. The top ten terms on biological process, molecular function, and cellular component were listed. Excel The identified protein species in the buccal gland secretion of lampreys during fasting and feeding stages in Ensembl lamprey and NCBI databases. (PDF 927 kb)