4E) by PNGase F treatment and analyzed by graphitized carbon nanoLC-ion trap-MS/MS (Figure 5); glycan compositions and structural features were confirmed by tandem mass spectrometry (not shown). Rabbit polyclonal to ANXA8L2 are shown for those carrying the six major N-glycans (schematics above Kira8 Hydrochloride with symbols as in Fig. 5). For site 1 (N59) they were detected as double peaks due to partial oxidization (ox) of methionine giving a 16 Da mass shift. B. Glycosylation analysis of site 2 (N59) and 3 (N116) of galectin-1 C3S bound and unbound haptoglobin. Tryptic glycopeptides containing the two sites were identified by nano-LC-ESI-ion trap-MS, and MS peaks are shown for those carrying Kira8 Hydrochloride the major detected combinations of N-glycans (schematics above mass spectra).(PDF) pone.0026560.s003.pdf (1004K) GUID:?5430681C-F16F-4ED1-865C-E7634E4BCFC7 Figure S4: Tandem mass spectrometry of glycopeptide M54-K77 carrying a biantennary, non-sialylated N-glycan. The quadruple protonated glycopeptide was subjected to ion trap-MS/MS analysis.(TIF) pone.0026560.s004.tif (460K) GUID:?5A71E43D-7A06-42B2-9680-3C4B33349742 Figure S5: Affinity chromatography of serum and haptoglobin on galectin-1 C3S mutants. (A) Serum from a healthy individual was analyzed using galectin-1 C3S and three further mutants in site B. Only the lactose eluted fractions are shown. Conditions are identical as in Fig. 2. (B). SDS-PAGE of peak bound (BF) and unbound (N-BF) fractions from the experiment of (A). The unfractionated serum (St.fr.) and size markers are shown to the left. All bound fractions showed the same pattern of proteins with -2-macroglobulin, IgM and haptoglobin as major species, except that mutant N34D bound less of all, most clearly visible for haptoglobin. (C) Affinity chromatography on galectin-1 C3S and C3S N34D of serum treated or not treated with neuraminidase. N34D binds much less untreated serum glycoproteins, but neuraminidase restores binding to equal levels as for galectin-1 C3S. (D) Affinity chromatography of haptoglobin (from pooled human plasma (Sigma-Aldrich)) on galectin-1 C3S and C3S N34D.(TIF) pone.0026560.s005.tif (1.0M) GUID:?FA9D5DEC-C43E-4CC1-8204-9B33A1E84620 Figure S6: Human transferrin analyzed by affinity chromatography on galectin-1 C3S or galectin-3. 2 mg of human transferrin were analyzed Kira8 Hydrochloride using galectin-1 and galectin-3 coupled 1 ml affinity columns. As predicted galectin-1 does not bind human transferrin, while approximately 6% bind galectin-3 (top panel). The galectin-3 non-binding transferrin was treated or not treated with neuraminidase (NA) (0.1 mol 1 h at 37C), and again analyzed on galectin-3 or galectin-1 coupled affinity columns. Removal of sialylations generated an additional 4% of galectin-3 binding transferrin (middle panel), but only traces (about 1.5%) of galectin-1 binding transferrin (bottom panel).(TIF) pone.0026560.s006.tif (564K) GUID:?420ECD8D-29BE-48DB-B514-D5CA3E3C307C Figure S7: Analysis of haptoglobin-haemoglobin complex in macrophages. A) Native gel electrophoresis of haptoglobin-haemoglobin complex. Haptoglobin (left) moved into the gel, while haemoglobin (middle) did not. For the haptoglobin-haemoglobin complex (right) the free haptoglobin band had disappeared from the gel, indicating that the complex had formed. B) Light microscope images (40) of THP-1 cells, untreated, induced to differentiate with PMA (5 days), or in addition IL-4 (2 days) for alternatively activation. Untreated cells were transferred to a polylysine glass slide and allowed to sediment for 15 min at room temperature before microscopy, while treated cells were grown directly on coverslips. Scale bar represents 50 m. C) Uptake of Hp-Hb complex in differentiated and activated THP-1 cells. THP-1 grown in the presence of PMA (5 days) or PMA (5 days) +IL-4 (2 days) were incubated with 0.2 m NHS-sulphorhodamine conjugated galectin-1 non-binding haptoglobin in complex with haemoglobin for 30 minutes. Cells were set in formaldehyde and examined by fluorescence microscopy (40). Nuclei had been stained blue with Hoechst. Range bar symbolizes 50 m. All microscopy pictures had been taken using a Nikon Eclipse TE2000-E fluorescence microscope.(TIF) pone.0026560.s007.tif (1.4M) GUID:?78D289E5-6E9B-48A4-816D-0D79675C759D Amount S8: Receiver operating feature (ROC) curve analysis for different measured parameters to tell apart sera from breasts cancer individuals from controls. A) Focus of galectin-1 destined haptoglobin, B) percentage of galectin-1 destined IgM, and C) proportion from the percentages of galectin-1 destined haptoglobin and IgM. The region beneath the curve (AUC) signifies the discriminatory power from the assessed parameter. A worth 0.90 is known as excellent.(TIF) pone.0026560.s008.tif (174K) GUID:?B5949CBB-1B96-4B67-8C1B-9F81D09D9BEB Desk S1: Topics and respective quantity of galectin-1 ligands in sera. (TIF) pone.0026560.s009.tif (1.5M) GUID:?81F03656-77F0-4451-AADF-E1EB6DEBCEF3 Desk S2: Comparative affinity of galectin-1 proteins for little saccharides and haptoglobin. (DOCX) pone.0026560.s010.docx (16K) GUID:?3247EC49-C789-450C-95D2-EB8B8C80100C Abstract Sera from 25 metastatic breast cancer individuals and 25 healthful controls were put through affinity chromatography using immobilized galectin-1. Serum in the healthy subjects included typically 1.2 mg per ml (range 0.7C2.2) galectin-1 binding glycoproteins, whereas serum in the breast cancer sufferers contained typically 2.2 mg/ml.