Semaphorin 3A binding to chondroitin sulfate E enhances the biological activity of the protein, and cross-links and rigidifies glycosaminoglycan matrices

Abstract

Semaphorin 3A (Sema3A) is a secreted protein that signals to cells through binding to neuropilin and plexin receptors and provides neurons with guidance cues key for axon pathfinding, and also controls cell migration in several other biological systems. Sema3A interacts with glycosaminoglycans (GAGs), an interaction that could localize the protein within tissues and involves the C-terminal domain of the protein. This domain comprises several furin cleavage sites that are processed during secretion and in previous works have hampered recombinant production of full-length wild type Sema3A, and the biochemical analysis of Sema3A interaction with GAGs. In this work, we have developed a strategy to purify the full-length protein in high yield and identified two sequences in the C-terminal domain, KRDRKQRRQR and KKGRNRR, which confer to the protein sub nM affinity for chondroitin sulfate and heparan sulfate polysaccharides. Using chemically defined oligosaccharides and solid phase binding assays, we report that Sema3A recognizes a (GlcA-GalNAc4S6S)2 motif but not a (GlcA2S-GalNAc6S)2 motif and is thus highly specific for type E chondroitin sulfate. Functionally, we found that Sema3A rigidified CS-E films that mimic the GAG presentation within extracellular matrices (ECMs), suggesting that Sema3A may have a previously unidentified function to cross-link and thus stabilize GAG-rich ECMs. Finally, we demonstrated that the full-length Sema3A is more potent at inhibiting neurite outgrowth than the truncated or mutant forms that were previously purified and that the GAG binding sites are required to achieve full activity. The results suggest that Sema3A can rigidify and cross-link GAG matrices, implicating Sema3A could function as an extracellular matrix organizer in addition to binding to and signaling through its cognate cell surface receptors.

An Extracellular Perspective on CNS Maturation: Perineuronal Nets and the Control of Plasticity

“…During adulthood, Sema3A in PNNs could stabilize existing synapses on the postsynaptic neuron. Finally, because it has been shown that Sema3A is able to rigidify CS-E-based matrices, it may cross-link PNN-GAGs, stabilizing the PNN structure [ 129 ].…”

Semaphorins in Adult Nervous System Plasticity and Disease

“…Scale bar: 2 mm (A), 5 µm (B). peptidic sequences located in the Sema3A C-terminal domain (Djerbal et al, 2019). Interestingly, one of these sequences is very similar to a sequence found in orthodenticle homeobox 2 (Otx2), which is involved in PNN formation and modulation of plasticity (Bernard and Prochiantz, 2016).…”

“…The most common CS-GAG isoform in the adult brain are: CS-A (C-4 sulfation on GalNAc), CS-C (C-6 sulfation on GalNAc), CS-D (C-2 sulfation on GlcA and a C-6 sulfation on GalNAc), and CS–E (C-4 and C-6 sulfation on GalNAc; Sugahara and Kitagawa, 2000 ). The interaction between Sema3A and PNNs is sulfation-dependent, with Sema3A interacting preferentially to CS-E extracted from adult brain PNNs ( Dick et al, 2013 ) via two specific peptidic sequences located in the Sema3A C-terminal domain ( Djerbal et al, 2019 ). Interestingly, one of these sequences is very similar to a sequence found in orthodenticle homeobox 2 (Otx2), which is involved in PNN formation and modulation of plasticity ( Bernard and Prochiantz, 2016 ).…”

“…Finally, Sema3A has been found to rigidify CS-E based matrices. Therefore, it may cross-link PNN-GAGs and thus contribute to PNN stability ( Djerbal et al, 2019 ).…”

Modelling and Refining Neuronal Circuits with Guidance Cues: Involvement of Semaphorins

“…It is possibly because of their firing properties that PV+ interneurons, concomitantly to their maturation, are enwrapped by a protective sheet of extracellular matrix, the perineuronal net (PNN) [ 14 , 15 , 16 ], which supposedly creates a protection from oxidative stress [ 17 ]. Accumulating evidence shows that SEMA3A, already expressed by these cells as they establish in the neocortex [ 18 ], binds to chondroitin sulfate proteoglycans, one of the main components of PNNs, to shape and probably stabilize the ensheathing matrix [ 19 , 20 , 21 , 22 ]. The enrichment of SEMA3A in this extracellular matrix may restrict neurite growth onto cell bodies of PV+ interneurons, which could explain why emergence of PNNs determines the closure of plasticity windows [ 23 ].…”