Identifying Function Determining Residues in Neuroimmune Semaphorin 4A

Abstract

Semaphorin 4A (Sema4A) exerts a stabilizing effect on human Treg cells in PBMC and CD4+ T cell cultures by engaging Plexin B1. Sema4A deficient mice display enhanced allergic airway inflammation accompanied by fewer Treg cells, while Sema4D deficient mice displayed reduced inflammation and increased Treg cell numbers even though both Sema4 subfamily members engage Plexin B1. The main objectives of this study were: 1. To compare the in vitro effects of Sema4A and Sema4D proteins on human Treg cells; and 2. To identify function-determining residues in Sema4A critical for binding to Plexin B1 based on Sema4D homology modeling. We report here that Sema4A and Sema4D display opposite effects on human Treg cells in in vitro PBMC cultures; Sema4D inhibited the CD4+CD25+Foxp3+ cell numbers and CD25/Foxp3 expression. Sema4A and Sema4D competitively bind to Plexin B1 in vitro and hence may be doing so in vivo as well. Bayesian Partitioning with Pattern Selection (BPPS) partitioned 4505 Sema domains from diverse organisms into subgroups based on distinguishing sequence patterns that are likely responsible for functional differences. BPPS groups Sema3 and Sema4 into one family and further separates Sema4A and Sema4D into distinct subfamilies. Residues distinctive of the Sema3,4 family and of Sema4A (and by homology of Sema4D) tend to cluster around the Plexin B1 binding site. This suggests that the residues both common to and distinctive of Sema4A and Sema4D may mediate binding to Plexin B1, with subfamily residues mediating functional specificity. We mutated the Sema4A-specific residues M198 and F223 to alanine; notably, F223 in Sema4A corresponds to alanine in Sema4D. Mutant proteins were assayed for Plexin B1-binding and Treg stimulation activities. The F223A mutant was unable to stimulate Treg stability in in vitro PBMC cultures despite binding Plexin B1 with an affinity similar to the WT protein. This research is a first step in generating potent mutant Sema4A molecules with stimulatory function for Treg cells with a view to designing immunotherapeutics for asthma.

Plexin B1 controls Treg numbers, limits allergic airway inflammation, and regulates mucins

“…We reported previously that Plexin B1 plays a critical role in human Treg cell stability and function in vitro ( 7 , 8 ). To investigate the role of Plexin B1 signaling in Treg cell stability and function in vivo , Plexin B1 KO mice were generated at Cyagen ( Supplementary Figure S1A ).…”

“…While Sema4A deficient mice displayed enhanced allergic airway inflammation accompanied by fewer Treg cells, Sema4D deficient mice displayed reduced inflammation and increased Treg cell numbers even though both Sema4 subfamily members can engage Plexin B1 ( 10 , 23 , 24 ). Moreover, Sema4A and Sema4D display opposite effects on human Treg cells in in vitro cultures of peripheral blood mononuclear cells (PBMC); Sema4D inhibited CD4+CD25+Foxp3+ cell numbers and CD25/Foxp3 expression ( 7 ).…”

“…Therefore, the in vivo stimulation of Treg cells can provide a beneficial immunosuppressive effect in overexaggerated asthmatic lung response (3)(4)(5)(6). Our current study focuses on the stimulatory effect of neuroimmune semaphorin 4A (Sema4A) on Treg cells by utilizing its receptor Plexin B1 (7,8).…”

An antagonistic monoclonal anti–Plexin-B1 antibody exerts therapeutic effects in mouse models of postmenopausal osteoporosis and multiple sclerosis

“…Conversely, Plexin-B1 has been demonstrated to bind to other semaphorins in addition to Sema4D, that is, to Sema3C and Sema4A ( 48 , 49 ), which is of particular relevance in cancer cells: the Sema3C-induced activation of Plexin-B1 on prostate cancer cells promotes cancer growth through transactivation of receptor tyrosine kinases, such as the EGF receptor, ErbB-2 (HER2), and the hepatocyte growth factor receptor (c-Met) ( 48 ); the binding of Plexin-B1 to transmembrane Sema4A on cancer and on dendritic cells triggers a reverse signaling pathway, which controls cell migration via the scaffold protein Scrib ( 50 ). Of note, Sema4D and Sema4A have been shown to compete for binding to Plexin-B1 ( 51 ), suggesting that they share similar binding interfaces on Plexin-B1; this implicates that RbPLX7 might block binding of Plexin-B1 to both Sema4D as well as to Sema4A. In summary, it seems more than likely that the biological effects of anti-Sema4D and anti–Plexin-B1 antibodies are overlapping but are not identical.…”