In situ trans ligands of CD22 identified by glycan-protein photocross-linking-enabled proteomics

Mol Cell Proteomics. 2010 Jun;9(6):1339-51. doi: 10.1074/mcp.M900461-MCP200. Epub 2010 Feb 19.

Abstract

CD22, a regulator of B-cell signaling, is a siglec that recognizes the sequence NeuAcalpha2-6Gal on glycoprotein glycans as ligands. CD22 interactions with glycoproteins on the same cell (in cis) and apposing cells (in trans) modulate its activity in B-cell receptor signaling. Although CD22 predominantly recognizes neighboring CD22 molecules as cis ligands on B-cells, little is known about the trans ligands on apposing cells. We conducted a proteomics scale study to identify candidate trans ligands of CD22 on B-cells by UV photocross-linking CD22-Fc chimera bound to B-cell glycoproteins engineered to carry sialic acids with a 9-aryl azide moiety. Using mass spectrometry-based quantitative proteomics to analyze the cross-linked products, 27 glycoproteins were identified as candidate trans ligands. Next, CD22 expressed on the surface of one cell was photocross-linked to glycoproteins on apposing B-cells followed by immunochemical analysis of the products with antibodies to the candidate ligands. Of the many candidate ligands, only the B-cell receptor IgM was found to be a major in situ trans ligand of CD22 that is selectively redistributed to the site of cell contact upon interaction with CD22 on the apposing cell.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • B-Lymphocytes / cytology
  • B-Lymphocytes / drug effects
  • B-Lymphocytes / metabolism
  • B-Lymphocytes / radiation effects
  • CHO Cells
  • Cell Communication / drug effects
  • Cell Communication / radiation effects
  • Cell Membrane / drug effects
  • Cell Membrane / metabolism
  • Cell Membrane / radiation effects
  • Cricetinae
  • Cricetulus
  • Cross-Linking Reagents / pharmacology*
  • Glycoproteins / metabolism*
  • Humans
  • Immunoglobulin M / immunology
  • Immunohistochemistry
  • Isotope Labeling
  • Ligands
  • Mass Spectrometry
  • Mice
  • Proteomics / methods*
  • Reproducibility of Results
  • Sialic Acid Binding Ig-like Lectin 2 / metabolism*
  • Sialic Acids / metabolism
  • Ultraviolet Rays*

Substances

  • Cross-Linking Reagents
  • Glycoproteins
  • Immunoglobulin M
  • Ligands
  • Sialic Acid Binding Ig-like Lectin 2
  • Sialic Acids