Human Follicular Lymphoma Cells Contain Oligomannose Glycans in the Antigen-binding Site of the B-cell Receptor

Yusuke Mimura,Freda K. Stevenson,David J. Harvey,Natalia Zabalegui,Catherine M. Radcliffe,Yoshinobu Kimura,Pauline M. Rudd,Mercedes Rodriguez-Calvillo,James N. Arnold,Susana Inogès,Mark R. Wormald,David M. Suter,Ascensión López-Díaz de Cerio,Louise Royle,Maurizio Bendandi,C. Ian Mockridge,Raymond A. Dwek,Robert B. Sim,Kathleen N. Potter

doi:10.1074/jbc.m602690200

Abstract

Expression of surface immunoglobulin appears critical for the growth and survival of B-cell lymphomas. In follicular lymphoma, we found previously that the Ig variable (V) regions in the B-cell receptor express a strikingly high incidence of N-glycosylation sequons, NX(S/T). These potential glycosylation sites are introduced by somatic mutation and are lymphoma-specific, pointing to their involvement in tumor pathogenesis. Analysis of the V region sugars from lymphoma-derived IgG/IgM reveals that they are mostly oligomannose and, remarkably, are located in the antigen-binding site, possibly precluding conventional antigen binding. The Fc region contains complex glycans, confirming that the normal glycan processing pathway is intact. Binding studies indicate that the oligomannose glycans occupying the V regions are accessible to mannose-binding lectin. These findings suggest a potential contribution to lymphoma pathogenesis involving antigen-independent interaction of surface immunoglobulin of the B-cell receptor with mannose-binding molecules of innate immunity in the germinal center.

Highlights

Expression of sIg8 is critical for the survival of normal B-cells in the periphery, even in the absence of antigen [1], indicating
Binding studies indicate that the oligomannose glycans occupying the V regions are accessible to mannose-binding lectin. These findings suggest a potential contribution to lymphoma pathogenesis involving antigen-independent interaction of surface immunoglobulin of the B-cell receptor with mannose-binding molecules of innate immunity in the germinal center
Sites generated by somatic mutation are frequent and possibly mandatory in FL, but they exist in other germinal center (GC)-associated lymphomas [11]

Summary

EXPERIMENTAL PROCEDURES

Idiotypic Ig Production and Identification of Tumor-derived Gene Sequences—Six patients with Stage IVA FL had surgical biopsy at first relapse following chemotherapy; five expressed sIgG, and one expressed sIgM. N-linked Glycan Analysis—15 ␮g of five FLIgG samples (FL2, -4, -11, -31, and -32), one FLIgM (FL3), CLL1, normal human serum NIgG, and NIgM were run on 10% SDS-polyacrylamide gels [20, 21]. Papain Digestion of IgG and Fab/Fc Separation—Digestion of IgG samples FL2, FL4, FL31, and NIgG (100 ␮g) was performed with 1 ␮g of papain (Sigma) in 250 ␮l of 0.1 M phosphate buffer containing 2 mM EDTA, 12 mM cysteine (pH 7) for 16 h. To test the completion of the digestion, aliquots (4 ␮l) were treated with 50 mM iodoacetamide at 4 °C for 30 min to inactivate papain, added to nonreducing SDS-sample buffer preheated to 100 °C, and heated at 100 °C for 3 min. MBL Purification and Binding Assay—Rabbit anti-MBL polyclonal antiserum was depleted of any anti-mannan antibodies on a mannan-agarose resin (catalog number M9917; Sigma) and pre-equilibrated in phosphate-buffered saline, JOURNAL OF BIOLOGICAL CHEMISTRY

INGSN SNTSS RNCSS RNSSS SNCSR KNETW GNISS YNSSN LNCTV GNISS TNLTV

RESULTS

Peak number

Peak assignments and percentage abundance of glycans of NIgM and FLIgM