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.

Fig. 1. Structure and recognition sequences of En2 region 183-259.

a Solution structure of the homeodomain of chicken En2 (region 200-259, PDB code 3ZOB). b Sequence alignment of En2 region 183-259 (chicken gene numbering) with Otx2 region 19-97 (numbering is identical for chicken and mammals). Note that these regions are strongly conserved (99% sequence identity between chicken and human En2, and 100% sequence identity between chicken and human Otx2). Positively charged, negatively charged, polar, apolar, and aromatic amino acids are colored in blue, red, green, black, and orange, respectively. The putative nuclear localization sequence (NLS) of En2 and the RK doublet of Otx2 that was shown to be crucial for the binding to highly sulfated CS are highlighted by rectangles. c Topology of En2 proteins and peptide used in this study. d Structure of representative disaccharide units of heparan sulfates (HS), chondroitin sulfates (CS), and heparin (HI).

Fig. 2. Quantitation of ExtHD and HD internalization in cells and impact of GAGs content.

a Quantity (pmoles) of internalized ExtHD and HD incubated at 7 µM with 10⁶ CHO-K1 or GAG^deficient pgsA-745 cells for 1 h at 37 °C (n = 27 and 18 for CHO-K1 and pgsA-745 cells, respectively). CHO-K1 express similar levels of HS and mono-sulfated chondroitins; pgsA-745 express 10–30-fold lower GAGs levels than CHO-K1. b Quantity of internalized ExtHD incubated at 7 µM with cells for 1 h at 37 °C (n = 12–18). Data were normalized to the quantity of internalized ExtHD in control CHO-K1 cells. CHO-K1 cells were treated with enzymes degrading HS (heparinases I, II, III) or CS (chondroitinase ABC). c, d Quantity of internalized ExtHD in CHO-K1 cells in the absence and presence of increasing amounts of extracellular heparin (n = 2-6). One million cells were incubated with 7 μM protein in DMEM-F12 medium for 1 hr either at 37 °C (c) or 4 °C (d). Data were normalized to the intracellular quantity of ExtHD in control CHO-K1 cells at 37 °C in the absence of heparin. The ExtHD/heparin molar ratio is indicated for each heparin concentration. Data are presented as mean values + /- SEM. One-way ANOVA and unpaired t tests were used in a, b to determine if differences between values were significant: ****P < 0.0001, ***P = 0.0005, not significant (ns). The list of P values is provided in Supplementary Table 3.

Fig. 3. NMR characterization of ExtHD interaction with heparin dp12 at the amino acid level.

Interaction of ExtHD with heparin dp12 is probed in 50 mM NaH₂PO₄ (pH 7.4) and 100 mM NaCl. a Overlay of ¹H-¹⁵N HSQC spectra (500 MHz) obtained for ¹⁵N-ExtHD in the absence and presence of increasing amounts of heparin dp12. Stoichiometry between ExtHD and heparin dp12 is: 1:0 (red), 1:0.3 (dark blue), 1:0.7 (orange), 1:1 (pink), 1:1.4 (green), 1:2 (light blue) and 1:2.5 (black). Amino acid residues exhibiting the highest perturbations (arrows) are indicated in the spectra. b Amide CSP of ExtHD induced by the addition of 1.0 and 2.5 molar equivalents of heparin dp12. c Saturation curves corresponding to the binding to heparin dp12 are shown for residues displaying the highest perturbations with experimental data represented by points and non-linear curve fitting by lines. K_d^app values are given in micromolar.

Fig. 4. The GAG-binding region 189-204 of Engrailed2 remains disordered upon binding to heparin or heparin fragments.

a CD spectra of ExtHD in the absence (blue) and presence (red) of 1 molar equivalent 12 kDa heparin. Chemical shift deviations (CSD) and {¹H}-¹⁵N NOEs of ExtHD recorded at 500 MHz in the absence (blue) and presence (red) of 2.4 molar equivalents heparin dp8 are shown in b, c, respectively. Error bars in c indicate the standard error for each value based on peak intensity relative to background noise (see the “Methods” section).

Fig. 5. Hypothetical model of GAG-dependent bidirectional transfert of HPs across the plasma membrane.

The role of highly sulfated HS in cell internalization of En2 through translocation and endocytosis pathways is illustrated in panels a and b, respectively.