A cationic motif upstream Engrailed2 homeodomain controls cell internalization through selective interaction with heparan sulfates

Abstract

Engrailed2 (En2) is a transcription factor that transfers from cell to cell through unconventional pathways. The poorly understood internalization mechanism of this cationic protein is proposed to require an initial interaction with cell-surface glycosaminoglycans (GAGs). To decipher the role of GAGs in En2 internalization, we have quantified the entry of its homeodomain region in model cells that differ in their content in cell-surface GAGs. The binding specificity to GAGs and the influence of this interaction on the structure and dynamics of En2 was also investigated at the amino acid level. Our results show that a high-affinity GAG-binding sequence (RKPKKKNPNKEDKRPR), upstream of the homeodomain, controls En2 internalization through selective interactions with highly-sulfated heparan sulfate GAGs. Our data underline the functional importance of the intrinsically disordered basic region upstream of En2 internalization domain, and demonstrate the critical role of GAGs as an entry gate, finely tuning homeoprotein capacity to internalize into cells.

ENGRAILED‐1 transcription factor has a paracrine neurotrophic activity on adult spinal α‐motoneurons

“…This RK‐OTX2 domain peptide (RKQRRERTTFTRAQL) competes for OTX2 binding to PV cells and antagonizes its specific internalization upon infusion or injection into the cerebral cortex (Beurdeley et al , 2012 ). A similar GAG‐binding domain was recently described in EN2 (Cardon et al , 2023 ) and sequence comparisons between OTX2, EN2, and several other HPs identified putative GAG‐binding sites in many of them (examples in Fig 7B , left panel). In the case of EN1, this domain (RKLKKKKNEKEDKRPRTAF), thereafter RK‐EN1, is present between residues 292 and 310 of hEN1.…”

“…Although we have not directly demonstrated that EN1 binds GAGs, the conservation of the GAG-binding domain and the analogy with OTX2 (Beurdeley et al, 2012) and EN2 (Cardon et al, 2023) support a specific association of EN1 with MN-expressed GAGs. If this is confirmed by future studies, it will be important to identify the molecular nature of this GAG as done for OTX2, EN2, and also VAX1 (Kim et al, 2014).…”

“…The role of this domain in the specific targeting of EN1 and the presence of EN1 binding sites at the MN surface are both illustrated by the ability of the peptide to abolish EN1‐specific uptake by ventral spinal cord MNs. Although we have not directly demonstrated that EN1 binds GAGs, the conservation of the GAG‐binding domain and the analogy with OTX2 (Beurdeley et al , 2012 ) and EN2 (Cardon et al , 2023 ) support a specific association of EN1 with MN‐expressed GAGs. If this is confirmed by future studies, it will be important to identify the molecular nature of this GAG as done for OTX2, EN2, and also VAX1 (Kim et al , 2014 ).…”

“…homeodomain (Beurdeley et al, 2012;Miyata et al, 2012). Interestingly, similar putative GAG-binding domains are present upstream of the homeodomain in many HPs (examples in Fig 7B ), including EN2 (Cardon et al, 2023) and EN1 for which this putative GAGbinding sequence is RKLKKKKNEKEDKRP. The role of this domain in the specific targeting of EN1 and the presence of EN1 binding sites at the MN surface are both illustrated by the ability of the peptide to abolish EN1-specific uptake by ventral spinal cord MNs.…”

“…Data information: The internalization experiments were performed once with each antibody. Left panel gives examples of putative glycosaminoglycan (GAG)‐binding domain in 11 homeoprotein transcription factors. Based on the alignment and on published work on OTX2 (Beurdeley et al , 2012) and EN2 (preprint: Cardon et al , 2023), a putative EN1 GAG‐binding domain (RK‐EN1) was designed. Middle panel quantifies the inhibitory effect of RK‐EN1 on hEN1 capture (86/8 antibody) by ChAT+ cells demonstrating that RK‐EN1 in a 1 to 20 ratio reduces the % of EN1‐positive MNs (ChAT+) from 50 to less than 10%.…”