Fig 2: Interactions between RPTP? and the homologous TNC and TNR.A, overview of the domain organization of RPTP?, TNR, and TNC. RPTP? includes a carbonic anhydrase-like (CA) domain, a single fibronectin type III domain (FN), a spacer region, and two cytoplasmic tyrosine phosphatase domains. The splice form of RPTP? called phosphacan does not include the transmembrane or cytoplasmic region but include a large segment featuring multiple glycosaminoglycan attachment sites in addition to the spacer region. TNR includes a cysteine-rich domain (CRD), four EGF repeats, nine FN domains, and a C-terminal fibrinogen-like (Fg) domain. A segment akin to “half” of an EGF repeat is inserted between the CRD and the first full EGF repeat. TNC includes a CRD and 14 EGF repeats separated by a segment that is akin to “half” of an EGF repeat. TNC also features eight FN domains and a C-terminal Fg domain. Multiple splice forms of TNC exist that include manifold combinations of FN repeats between FN domains 5 and 6. Here, repeats A1, A2, A4, B, and D are shown. B, design of constructs used in the binding assay using RPTP? and TNR as examples. The ectodomain of RPTP? and regions of TNR (here, the FN1-Fg domains) are fused to the Fc domain of human IgG1 or a protein designated FcYTS that includes the following: (1) the C?3 and C?4 domains of chicken IgY and (2) a Twin-Strep tag (TS) for detection with streptactin. Proteins are expressed in HEK293 cells and conditioned media is used for experiments. Potential interactions between the Fc and FcYTS candidate proteins are tested in an Alpha-binding assay by mixing conditioned media including Fc and FcYTS fusion proteins along with streptactin donor beads and protein A-acceptor beads. In AlphaScreen assays, excitation of donor beads at 680 nm triggers the release of a singlet oxygen. An acceptor bead hit by this highly reactive molecule emits a signal between 520 and 620 nm. Because the half-life of the singlet oxygen is limited, a luminescent signal is only obtained when the donor beads and acceptor beads are within 200 nm. Here, the binding of the FcYTS fusion protein of RPTP? immobilized on donor beads to the Fc fusion protein of TNR(FN1-Fg) bound to acceptor beads brings the two beads in proximity. C, results of the binding assay shown in a heat map representation. The scale indicates the value of the signal for the protein pair protein 1-FcYTS/protein 2-Fc divided by the signal measured for the protein 1-FcYTS/Fc only pair. Accordingly, these calculated values are unitless. Raw signals are included in Table S1. CA, carbonic anhydrase-like; CRD, cysteine-rich domain; EGF, epidermal growth factor-like; Fg, fibrinogen-like; FN, fibronectin type III; FN1, first FN repeat; RPTP?, protein tyrosine phosphatase receptor type G; RPTP?, protein tyrosine phosphatase receptor type Z; TNC, tenascin-C; TNR, tenascin-R.

Fig 3: Alanine substitutions at the RPTP?–TNR interface prevent interactions between RPTP? and TNR.A, a fusion protein of human IgG1 Fc and the FN1-Fg domains of human TNR binds to HEK293 cells transfected with an Emerald-RPTP? fusion protein. RPTP? was detected on the surface of cells using its N-terminal Emerald fluorescent tag, while TNR is bound to a goat anti-human IgG antibody conjugated to Alexa Fluor 568. The nuclei were visualized using DAPI. The experiment was performed three times, one representative set of images is shown. Scale bar represents 25 µM. B, a fusion protein of human IgG1 Fc and the FN1-Fg domains of human TNR does not bind to HEK293 cells transfected with an Emerald-RPTP? fusion protein in which residues Y368, Q369, and D370 are changed to alanine. RPTP? and bound TNR were detected as described in panel A. The nuclei were visualized using DAPI. The experiment was performed three times, one representative set of images is shown. C, fold differences of the fluorescent intensity of cell surface staining for TNR(FN1-Fg) between Emerald-RPTP?-WT–expressing (A) and Emerald-RPTP?-WT-AAA–expressing (B) cells normalized to the Emerald-RPTP?-WT-AAA–expressing cells. ***p = 0.0004. There was no statistical difference in the fluorescent intensity of Emerald-RPTP?-WT and Emerald-RPTP?-WT-AAA between cells. Individual data points are represented by black squares and triangles. Error bars represent the SD of the mean. DAPI, 4',6-diamidino-2-phenylindole; Fg, fibrinogen-like; FN, fibronectin type III; FN1, first FN repeat; RPTP?, protein tyrosine phosphatase receptor type Z; TNR, tenascin-R.

Fig 4: The assembly of PNNs depends on interactions between RPTP? and TNR.A, addition of RPTP?-AAA mutant is unable to recover binding of PNN component ACAN in Ptprz1 KO neuronal cultures. Cortical cultures from E16 Ptprz1 KO mice were fixed at DIV 9. Binding of PNN component ACAN appeared disrupted with a broken and aggregated staining pattern in these cultures. Addition of recombinant RPTP?-WT (2 µg per well at DIV 3) to the cells was able to restore regular pattern of aggrecan binding to the cell surface. However, TNR-binding mutant RPTP?-AAA was unable to restore ACAN binding to the cell. Similar to untreated cultures, the ACAN staining pattern remained broken and discontinuous with an aggregated and punctate appearance. Mean prominence or isolation of PNN peaks was visualized using PNN marker ACAN and quantified using our PNN node/peak analysis. Analysis by ordinary one-way ANOVA showed that the average isolation or prominence of PNN peaks is significantly different between groups (F(2,46) = 3.232, p = 0.048). RPTP?-WT–treated cells (n = 12 cells, 3 cultures) showed a significantly lower mean prominence (*p = 0.037, Tukey’s post hoc testing) than untreated cells (n = 12 cells, 3 cultures) indicating more regular pattern of ACAN staining and a decrease in node/peak isolation. Mean prominence of peaks in RPTP?-AAA–treated PNNs (n = 25 cells, 3 cultures) was not significantly different from untreated cells (RPTP?-AAA p = 0.35, Tukey’s post hoc testing). B, addition of RPTP?-AAA disrupts binding of PNN component ACAN in WT neuronal cultures. Cortical cultures derived from E16 WT mice were fixed at DIV 9 and stained with PNN marker, ACAN. ACAN staining appeared regular and continuous in untreated group. Addition of RPTP?-AAA (2 µg per well at DIV 6) resulted in disrupted and aggregated aggrecan staining similar to pattern seen in Ptprz1 KO cultures. Interestingly, addition of RPTP?-WT (2 µg per well at DIV 6) did not result in disruption of ACAN binding to the cell surface. PNNs were visualized using PNN marker ACAN, and their mean prominence or isolation was calculated using our PNN node/peak analysis. There were significant differences in the average isolation index of PNN peaks among the various treatment groups (F(2,66) = 6.203, p = 0.0034, ordinary one-way ANOVA). Average isolation index or mean prominence of PNNs was significantly higher in RPTP?-AAA–treated cells (n = 24 PNNs, 6 cultures) as compared to untreated control cells (n = 31 PNNs, 6 cultures, *p = 0.0079) and RPTP?-WT–treated cells (n = 14 PNNs, 4 cultures, #, p = 0.013). Scale bar represents 10 µm. ACAN, aggrecan; ANOVA, analysis of variance; DIV, day in vitro; E, embryonic day; PNN, perineuronal net; RPTP?, protein tyrosine phosphatase receptor type Z; TNR, tenascin-R.

Fig 5: Crystal structure of the RPTP?–TNC complex and comparison with the RPTP?–TNR complex.A, the RPTP?–TNC complex is shown in ribbon diagram overlaid onto the RPTP?-TNR (colored white). The FN domain of RPTP? is colored green, while the Fg domain of TNC is shown in pink. The position of Ca2+ ions bound to TNC and TNR is indicated by gray and white transparent spheres, respectively. The letters N and C indicate the N- and C-termini, respectively. B, the complex interface can be divided into a major binding site (solid line square) and a minor binding site (dotted line circle). Contacting residues are represented as sticks, while transparent surfaces denote residues involved in van der Waals or packing interactions. Potential hydrogen bonds are represented as dashed lines. C, detailed view of the interactions in the major site. For clarity, only stacking interactions are shown on the two left panels while the hydrogen bonding residues are shown only in the two rightmost panels. In these panels, interacting atoms of side chains or main chain atoms are displayed only if they participate in the interactions. D, detailed view of the interactions at the minor site. The gray sphere denotes the Ca2+ ion bound to the Fg domain of TNC. E, comparison of the RPTP?-TNC– and the RPTP?-TNR–binding sites. The color coding for the RPTP?-TNC complex is identical to the one used in panel A, while residues in the RPTP?–TNR complex are shown in transparent white. For clarity, only the relevant main chain or side chain atoms are represented. Fg, fibrinogen-like; FN, fibronectin type III; RPTP?, protein tyrosine phosphatase receptor type Z; TNC, tenascin-C; TNR, tenascin-R.