Human Fc Receptor–Like 5 Binds Intact IgG via Mechanisms Distinct from Those of Fc Receptors

Abstract

Fc receptor-like 5 (FCRL5) regulates BCR signaling and has been reported to bind aggregated IgG. Using surface plasmon resonance, we analyzed the interaction of native IgG samples with FCRL5, revealing a complex binding mechanism, where isotype is just one factor. FCRL5 bound IgG1 and IgG4 with approximately 1 μM KD, while the interaction with IgG3 was a magnitude weaker. However, IgG2 samples displayed a wide range of affinities, indicating that additional factors affect binding. We used a panel of 19 anti-FCRL5 mAbs with defined reactivity to identify domains involved in ligand binding. Six mAbs blocked IgG binding, indicating critical roles of FCRL5 domains 1 and 3, as well as epitopes at the domain 1/2 and domain 2/3 boundaries. We found that only glycosylated IgG containing both Fab arms and the Fc region bound with high affinity. Furthermore, the presence of sialic acid in the IgG carbohydrate altered FCRL5 binding. The interaction of IgG and FCRL5 consisted of two kinetic components, suggesting a complex binding mechanism. We established that the IgG-Fc and IgG-F(ab’)2 fragments bind FCRL5 independently but with low affinity, revealing the mechanism behind the two-step binding of whole IgG. This complex binding mechanism is distinct from that of Fc-receptors, which bind through the Fc. We propose that FCRL5 is a new type of receptor that recognizes intact IgG, possibly enabling B cells to sense immunoglobulin quality. Recognition of undamaged IgG molecules by FCRL5 could allow B cells to engage recently produced antibodies.

Developing the IVIG biomimetic, Hexa-Fc, for drug and vaccine applications

“…Human B cells are known to express two FcγRs for IgG, FcγRIIb and FcRL55622. To determine if these receptors could contribute to the interaction of hexa-Fc with B cells, and to overcome issues of background binding observed with isolated B cells, we evaluated the extent of binding of hexa-Fc to 293 cells transiently expressing these proteins or control CD200R and FcRL4 receptors by FACS6.…”

“…Since the interaction of IVIG with FcRL522, FcγRIIb28 and DC-SIGN91129 has been attributed to direct and/or indirect effects of sialic acid on the Fc, we next investigated the nature of the N -glycans on hexa-Fc and compared them with two different IVIG preparations (GammaGard™ or Malawian IVIG) and the dimeric-Fc (Figure 4). MS analysis of hexa-Fc revealed a paucity of sialylated structures but enrichment for high mannose glycans (Man 5 GlcNAc 2 , Man 6 GlcNAc 2 ) (Figure 4D).…”

Highly parallel characterization of IgG Fc binding interactions

“…These results are consistent with recent publications. 45,46 Interestingly, oligomannose-sensitive binding of FcRL5 has not been previously observed.…”

Fc Receptor–like 5 Expression Distinguishes Two Distinct Subsets of Human Circulating Tissue–like Memory B Cells

“…Using a chimeric receptor containing the cytoplasmic tail of FCRL5, crosslinking FCRL5 and the BCR was shown to recruit SHP-1 to the two ITIM motifs of FCRL5, resulting in reduced BCR-induced calcium mobilization and protein tyrosine phos-phorylation (20). We and others reported that FCRL5 binds intact IgG via a complex mechanism (21,22), and therefore immune complexes may link FCRL5 to the BCR, potentially blocking B cell activation similarly to the inhibitory FcgRIIB. Potential activating functions were also proposed for FCRL5 (23), similarly to FCRL3 (24) and FCRL4 (25), suggesting dual regulatory capacities.…”