In situ trans ligands of CD22 identified by glycan-protein photocross-linking-enabled proteomics.
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.
Ramya
T N C
TN
Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California 92037, USA.
Weerapana
Eranthie
E
Liao
Lujian
L
Zeng
Ying
Y
Tateno
Hiroaki
H
Liao
Liang
L
Yates
John R
JR
Cravatt
Benjamin F
BF
Paulson
James C
JC
D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects; D016466:CHO Cells; D002450:Cell Communication / Q000187:drug effects / Q000528:radiation effects; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects; D006224:Cricetinae; D003412:Cricetulus; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D007075:Immunoglobulin M / Q000276:immunology; D007150:Immunohistochemistry; D007553:Isotope Labeling; D008024:Ligands; D013058:Mass Spectrometry; D051379:Mice; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D051918:Sialic Acid Binding Ig-like Lectin 2 / Q000378:metabolism*; D012794:Sialic Acids / Q000378:metabolism; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D006023:Glycoproteins; D007075:Immunoglobulin M; D008024:Ligands; D051918:Sialic Acid Binding Ig-like Lectin 2; D012794:Sialic Acids