Additional File 1:
Creators: name: Pang, Yue
name: Gou, Meng
name: Yang, Kai
name: Lu, Jiali
name: Han, Yinglun
name: Teng, Hongming
name: Li, Changzhi
name: Wang, Haina
name: Liu, Caigang
name: Zhang, Kejia
name: Yang, Yongliang
name: Li, Qingwei
Publisher: name: figshare
Year: 2019
Subjects: subject: Biophysics
subject: Biochemistry
subject: Microbiology
subject: FOS: Biological sciences
subject: Cell Biology
subject: Genetics
subject: Molecular Biology
subject: Biotechnology
subject: Chemical Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Cancer
subject: Infectious Diseases
subject: FOS: Health sciences
subject: Plant Biology

description: 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)