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Homomultimeric complexes of CD22 in B cells revealed by protein-glycan cross-linking

Nature Chemical Biology volume 1, pages 93–97 (2005)Cite this article

Abstract

CD22 is a negative regulator of B-cell receptor signaling, an activity mediated by recruitment of SH2 domain–containing phosphatase 1 through a phosphorylated immunoreceptor tyrosine inhibitory motif in its cytoplasmic domain1. As in other members of the sialic acid–binding immunoglobulin-like lectin, or siglec, family, the extracellular N-terminal immunoglobulin domain of CD22 binds to glycan ligands containing sialic acid, which are highly expressed on B-cell glycoproteins2. B-cell glycoproteins bind to CD22 in cis and 'mask' the ligand-binding domain3, modulating its activity as a regulator of B-cell signaling4,5,6. To assess cell-surface cis ligand interactions, we developed a new method for in situ photoaffinity cross-linking of glycan ligands to CD22. Notably, CD45, surfaceIgM (sIgM) and other glycoproteins that bind to CD22 in vitro7,8 do not appear to be important cis ligands of CD22 in situ. Instead, CD22 seems to recognize glycans of neighboring CD22 molecules as cis ligands, forming homomultimeric complexes.

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Figure 1: Introduction of a carbohydrate photoaffinity cross-linker into cell-surface glycoprotein ligands in situ.
Figure 2: Cross-linking of 9-AAz-NeuAc–containing glycans to CD22-Fc chimera in vitro, or endogenous B-cell membrane–bound CD22 in situ.
Figure 3: Differential recognition of B-cell glycoproteins by CD22 in vitro and in situ.
Figure 4: CD22 forms homomultimeric complexes with glycans on neighboring CD22 molecules.

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Acknowledgements

We wish to thank M. Pawlita for the K20 cell line, A. Varki for the plasmid encoding for CD22-Fc plasmid, O. Blixt for the Galβ1-4GlcNAcβ-Lc-Lc-biotin, H. Li and M. Iufer for expert technical assistance, L.K. Allin for preparation of enzymes used in synthesis, and A. Tran-Crie for assistance in manuscript preparation. This work was funded by the US National Institutes of Health grants GM60938 and AI050143 and a Wenner-Gren Foundation fellowship to P.B.

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  1. Shoufa Han and Brian E Collins: Both authors contributed equally to this work.

Authors and Affiliations

  1. Departments of Molecular Biology and Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, MEM-L71, La Jolla, 92037, California, USA

    Shoufa Han, Brian E Collins, Per Bengtson & James C Paulson

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Correspondence to James C Paulson.

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Supplementary information

Supplementary Fig. 1

Requirement of UV light for crosslinking of CD22 in situ. (PDF 592 kb)

Supplementary Fig. 2

Demonstration of equivalent biotinylation of cell preparations used for SNA and CD22-Fc immunoprecipitations in. (PDF 434 kb)

Supplementary Fig. 3

CD45, CD19, and PMCA can be immunoprecipitated by CD22-Fc in a sialic acid dependent manner. (PDF 561 kb)

Supplementary Fig. 4

Absence of CD22 crosslinking to CD19 or CD45. (PDF 419 kb)

Supplementary Fig. 5

Comparison of CD22 crosslinking in resting and activated K20 cells. (PDF 699 kb)

Supplementary Fig. 6

CD22 interacts in cis with glycans on neighboring CD22 molecules to form homo-multimeric complexes. (PDF 488 kb)

Supplementary Methods (PDF 219 kb)

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Han, S., Collins, B., Bengtson, P. et al. Homomultimeric complexes of CD22 in B cells revealed by protein-glycan cross-linking. Nat Chem Biol 1, 93–97 (2005). https://doi.org/10.1038/nchembio713

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