Glycoproteomic characterization of carriers of the CD15/Lewisx epitope on Hodgkin's Reed-Sternberg cells

Abstract

Background: The Lewisx trisaccharide, also referred to as the CD15 antigen, is a diagnostic marker used to distinguish Hodgkin's lymphoma from other lymphocytic cancers. However, the role of such fucosylated structures remains poorly understood, in part because carriers of Lewisx structures on Hodgkin's Reed-Sternberg cells have not been identified.

Methods: GalMBP, an engineered carbohydrate-recognition protein that binds selectively to oligosaccharides with paired terminal galactose and fucose residues, has been used in conjunction with proteomic and glycomic analysis to identify glycoprotein carriers of Lewisx and related glycan structures in multiple Hodgkin's Reed-Sternberg cell lines.

Results: Multiple glycoproteins that bind to GalMBP and carry CD15/Lewisx have been identified in a panel of six Reed-Sternberg cell lines. The most commonly identified Lewisx-bearing glycoproteins are CD98hc, which was found in all six cell lines tested, and intercellular adhesion molecule-1 and DEC-205, which were detected in five and four of the lines, respectively. Thus, several of the most prominent cell adhesion molecules on 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 on Reed-Sternberg cells. Binding of DC-SIGN and other glycan-specific receptors to the Lewisx epitopes on CD98 and intercellular adhesion molecule-1 may facilitate interaction of the lymphoma cells with lymphocytes and myeloid cells in lymph nodes.

Figure 1

Analysis of GalMBP ligands from L-428 cells with anti-Lewis^x antibodies. Elution fractions from solubilized L-428 cells purified over a column of immobilized GalMBP were analyzed on 15% SDS-polyacrylamide gels followed by Coomassie blue staining or electroblotting onto nitrocellulose and probing with anti-Lewis^x antibodies and radiolabelled GalMBP. The different relative intensities of some of the bands suggest that GalMBP and the antibodies bind differentially to glycans.

Figure 2

Mass spectrometric analysis of N-linked glycans from GalMBP ligands on L-428 cells. Purified GalMBP ligands were digested with trypsin and N-linked glycans were released by peptide:N-glycosidase F. N-linked glycans were permethylated and subjected to Sep-Pak purification. A. The MALDI MS spectrum of one of the elution fractions from the Sep-Pak is shown. All labeled ions were subjected to MS/MS analysis. The schemes for each ion represent the most likely structure based on the fit between the calculated composition and the m/z (z = 1) ratio of the ions detected, taking into account the biosynthetic pathways of N-glycosylation in addition to the MS and MS/MS data collected. Structures in red boxes contain terminal fucose residues. B. An example of the MALDI MS/MS spectra, showing fragmentation of the 2591 molecular ion. The presence of major fragment ions at 1954 and 660 confirms the presence of terminal Lewis^x or Lewis^a structures. Specific evidence for the presence of Lewis^x structures is provided by elimination of fucose from the 3 position of GlcNAc to form the 2385 fragment ion. The presence of unfucosylated glycans that are suboptimal ligands for GalMBP, as well as high-mannose oligosaccharides that do not bind GalMBP at all, is presumed to reflect the presence of heavily glycosylated glycoproteins that also contain complex-type glycans bound by GalMBP as previously observed for breast cancer cells [6].

Figure 3

GalMBP ligands from L-428 cells. Cell extracts were fractionated on columns of immobilized GalMBP. Proteins present in elution fractions from the GalMBP column were resolved on 15% SDS-polyacrylamide gels, excised, and subjected to proteomic analysis by in-gel trypsin digestion. 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.

Figure 4

Glycoproteins found in multiple HRS cell lines. Proteins present in at least two cell lines in Additional file 2 are indicated by dots. Colored boxes highlight subgroups within the panel of cell lines.