Proteins present in bands 1-16, identified by mass spectrometry following trypsin digestion, are listed in the right hand column and in Additional file 1. In an alternative approach to the proteomic analysis, glycoproteins purified on GalMBP were identified directly by trypsin digestion of the pooled elution fractions. in conjunction with proteomic and glycomic analysis to identify glycoprotein service providers of Lewisx and related glycan constructions in multiple Hodgkin’s Reed-Sternberg cell lines. Results Multiple glycoproteins ZM39923 that bind to GalMBP and carry CD15/Lewisx have been recognized in a panel of six Reed-Sternberg cell lines. The most commonly recognized Lewisx-bearing glycoproteins are CD98hc, which was found in all six cell lines tested, and intercellular adhesion molecule-1 and DEC-205, which were recognized in five and four of the lines, respectively. Thus, several of the most prominent cell adhesion molecules within the lymphomas carry this characteristic glycan epitope. In addition, the Hodgkin’s Reed-Sternberg cell lines can be grouped into subsets based on the presence or absence of less common Lewisx-bearing glycoproteins. Conclusions CD98 and intercellular adhesion molecule-1 are major carriers of CD15/Lewisx ZM39923 on Reed-Sternberg cells. Binding of DC-SIGN and additional glycan-specific receptors to the Lewisx epitopes on CD98 and intercellular adhesion molecule-1 may facilitate connection of the lymphoma cells with lymphocytes and myeloid cells in lymph nodes. Background The Lewisx blood group epitope, also referred to as the CD15 antigen, has been reported on many different cancers and malignancy cell lines including Hodgkins lymphomas, a common form of lymphocytic malignancy. The presence of Lewisx has been used like a marker for the neoplastic tumour cells of Hodgkins lymphoma, referred to as Hodgkins Reed-Sternberg (HRS) cells. HRS cells form a relatively small ZM39923 populace of the tumour mass, with the remaining cells consisting of non-neoplastic reactive cells including T lymphocytes, granulocytes, macrophages and plasma cells [1,2]. Crosslinking of HRS cell-surface molecules comprising Lewisx, using anti-Lewisx antibodies, stimulates cellular signaling through the tyrosine phosphorylation of proteins including c-Cbl [3], suggesting that recognition of protein service providers of Lewisx on HRS cells may provide insight into how cellular activation is accomplished. The C-type (Ca2+-dependent) carbohydrate-recognition website of serum mannose-binding protein, which normally binds to mannose-containing oligosaccharides characteristic of pathogens, can be re-engineered to bind galactose-containing glycans [4,5]. ZM39923 Glycan array analysis reveals the modified protein, referred to as galactose-specific ZM39923 mannose-binding protien (GalMBP), binds preferentially to oligosaccharides in which terminal galactose residues are adjacent to terminal fucose residues, as with the Lewisx blood group epitope [6]. The specificity of GalMBP indicated that it would be a useful tool for probing the way that Lewisx is definitely presented on the surface of Reed-Sternberg cells. By combining affinity purification on immobilized GalMBP with glycomics and proteomics, several cell surface molecules on HRS cells have STMY been found to carry the Lewisx epitope, with the weighty chain of CD98 being a common carrier on multiple HRS cell lines. Methods Cell tradition HRS cell lines L-428, KMH-2, L-1236, L-540, HDLM-2 and U-HO1 were purchased from your DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH), Braunschweig, Germany, which offered characterization using antibody reactivity of cell surface markers, PCR of minisatellite markers, isoelectric focusing of malate dehydrogenase and aspartate aminotransferase, and cytogenetics. Cell lines L-428, KM-H2 and L-1236 were cultivated in RPMI-1640 medium supplemented with 10% fetal calf serum, 2 mM glutamine, 100 U/ml penicillin, and 100 g/ml streptomycin. Cell lines L-540 and HDLM-2 were cultivated in the same medium but with 20% fetal calf serum. Cell collection U-HO1 was produced in 1:4 Iscove’s altered Dulbecco’s medium:RPMI-1640 medium supplemented with 20% fetal calf serum, 2 mM glutamine, 100 U/ml penicillin, and 100 g/ml streptomycin. Purification of membrane glycoproteins on immobilized GalMBP Cells produced to 0.5 – 1 106 cells/ml in 100 ml of medium were harvested by centrifugation at.
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