N-Linked Glycosylation Regulates CD22 Organization and Function

Abstract

The organization and clustering of cell surface proteins plays a critical role in controlling receptor signaling; however, the biophysical mechanisms regulating these parameters are not well understood. Elucidating these mechanisms is highly significant to our understanding of immune function in health and disease, given the importance of B cell receptor (BCR) signaling in directing B cells to produce antibodies for the clearance of pathogens, and the potential deleterious effects of dysregulated BCR signaling, such as in B cell malignancies or autoimmune disease. One of main inhibitory co-receptors on B cells is CD22, a sialic-acid binding protein, which interacts homotypically with other sialylated CD22 molecules, as well as heterotypically with IgM and CD45. Although the importance of CD22 in attenuating BCR signaling is well established, we still do not fully understand what mediates CD22 organization and association to BCRs. CD22 is highly glycosylated, containing 12 N-linked glycosylation sites on its extracellular domain, the function of which remain to be resolved. We were interested in how these glycosylation sites mediate homotypic vs. heterotypic interactions. To this end, we mutated five out of the six N-linked glycosylation residues on CD22 localized closest to the sialic acid binding site. Glycan site N101 was not mutated as this resulted in lack of CD22 expression. We used dual-color super-resolution imaging to investigate the impact of altered glycosylation of CD22 on the nanoscale organization of CD22 and its association with BCR. We show that mutation of these five glycosylation sites increased the clustering tendency of CD22 and resulted in higher density CD22 nanoclusters. Consistent with these findings of altered CD22 organization, we found that mutation of N-glycan sites attenuated CD22 phosphorylation upon BCR stimulation, and consequently, increased BCR signaling. Importantly, we identified that these sites may be ligands for the soluble secreted lectin, galectin-9, and are necessary for galectin-9 mediated inhibition of BCR signaling. Taken together, these findings implicate N-linked glycosylation in the organization and function of CD22, likely through regulating heterotypic interactions between CD22 and its binding partners.

Characterization of Sialic Acid-Binding Immunoglobulin-Type Lectins in Fish Reveals Teleost-Specific Structures and Expression Patterns

“…Regarding the sequence comparison of the V-set Ig-like domain, which is responsible for sialic acid-binding ( Figure 4A), N 101 seems to be conserved from mammals to lower vertebrates (N 105 in fishes), while N 67 and N 135 seem to be absent. Recently, Wasim et al determined that mutations of N 67 , N 112 , N 135 , N 164 and N 231 resulted in a higher density of CD22 nanoclusters, along with a decreased CD22-phosphorylation rate and an increased B-cell signaling, culminating in a reduced functionality of CD22 [52]. Therefore, we took a closer look at the N-glycosylation sites in the CD22 orthologs from maraena whitefish and rainbow trout.…”

Application of electrochemical biosensors in tumor cell detection

“…Therefore, biosensors constructed on the basis of specific reactions between antigens and antibodies, specific recognition function of adapters and the cell impedance principle have successfully achieved high‐sensitivity linear detection of various cancer cells, including human liver cancer cell line HepG2, human breast cancer cell line MCF‐7, small cell lung cancer, lung adenocarcinoma, squamous cell cancer, skin cancer, prostate cancer and breast cancer . In addition, glycosyls on the cell surface play an important role in cell recognition, adhesion, immune response, and the occurrence and migration of cancer cells . Therefore, the cell electrochemical sensor based on surface‐specific glycosyls of cancer cells has also successfully achieved the specific recognition and linear detection of HeLa cells, polysaccharide‐rich K562 leukemia cells, and MDA‐MB‐231 cells …”

“…[39][40][41] In addition, glycosyls on the cell surface play an important role in cell recognition, adhesion, immune response, and the occurrence and migration of cancer cells. [42][43][44] Therefore, the cell electrochemical sensor based on surface-specific glycosyls of cancer cells has also successfully achieved the specific recognition and linear detection of HeLa cells, polysaccharide-rich K562 leukemia cells, and MDA-MB-231 cells. 14,[45][46][47][48] Folic acid (FA) receptor is a cell surface receptor that is excessively expressed on most human tumor cells and rarely expressed or not at all in normal organs.…”

Biological and Technical Challenges in Unraveling the Role of N-Glycans in Immune Receptor Regulation

“…T cells from mice deficient in the glycosyltransferase GnT-V show a lack of complex branching N-glycans, which reduces Galectin-3 binding and enhances TCR clustering, resulting in autoimmunity (Demetriou et al, 2001;Chen et al, 2007). On B cells, BCR signaling is influenced by Galectin-9 dependent N-glycan mediated lateral interactions between the BCR and CD22, an ITIM bearing member of the sialic acid-binding immunoglobulin-like lectin (Siglec) family (Wasim et al, 2019). CD22 is heavily glycosylated and forms homo-oligomers by binding glycans of neighboring CD22 molecules in cis on the B cell membrane (Han et al, 2005).…”

“…CD22 is heavily glycosylated and forms homo-oligomers by binding glycans of neighboring CD22 molecules in cis on the B cell membrane (Han et al, 2005). CD22 function as inhibitor of BCR signaling is hampered by removing five of its 12 glycosylation sites (Wasim et al, 2019). These glycans on CD22 may be bound by Galectin-9 and crosslinked with the BCR, thus explaining the inhibitory function of CD22 and Galectin-9 on BCR signaling (Cao et al, 2018;Wasim et al, 2019).…”