Heparan sulfate proteoglycans are ligands for receptor protein tyrosine phosphatase sigma

Abstract

RPTPsigma is a cell adhesion molecule-like receptor protein tyrosine phosphatase involved in nervous system development. Its avian orthologue, known as cPTPsigma or CRYPalpha, promotes intraretinal axon growth and controls the morphology of growth cones. The molecular mechanisms underlying the functions of cPTPsigma are still to be determined, since neither its physiological ligand(s) nor its substrates have been described. Nevertheless, a major class of ligand(s) is present in the retinal basal lamina and glial endfeet, the potent native growth substrate for retinal axons. We demonstrate here that cPTPsigma is a heparin-binding protein and that its basal lamina ligands include the heparan sulfate proteoglycans (HSPGs) agrin and collagen XVIII. These molecules interact with high affinity with cPTPsigma in vitro, and this binding is totally dependent upon their heparan sulfate chains. Using molecular modelling and site-directed mutagenesis, a binding site for heparin and heparan sulfate was identified in the first immunoglobulin-like domain of cPTPsigma. HSPGs are therefore a novel class of heterotypic ligand for cPTPsigma, suggesting that cPTPsigma signaling in axons and growth cones is directly responsive to matrix-associated cues.

FIG. 1.

Binding of cPTPσ to the E6 chick retinal basal lamina in vivo. (A) Time course of cPTPσ1-VSV production by chicken embryo fibroblasts infected with RCAS-α1VST retrovirus. Samples of culture medium conditioned for 1 (lane 2), 2 (lane 3) or 3 (lane 4) days after transfection were analyzed by immunoblotting using antibody specific for the VSV epitope. Lane 1 contains conditioned medium from untransfected cells. (B and D) RCAS-α1VSV retrovirus-infected retinas at E6, probed with a digoxigenin-labeled viral probe by in situ hybridization (B) or with VSV antibody (D). Noninfected tissues were probed by in situ hybridization (C) or with VSV antibody (E). Arrowheads indicate the retinal BL. pe, pigmented epithelium. Scale bar, 0.1 mm.

FIG. 2.

Saturation curves and Scatchard plots for the binding of cPTPσ to heparin, agrin, and collagen XVIII. Microtiter plates were coated with heparin-albumin (A), agrin (B), or collagen XVIII (C) and incubated with a range of cPTPσ1-AP concentrations. Saturation curves were fitted by nonlinear regression analysis as described in Materials and Methods. Each value represents the mean ± standard deviation of three measurements.

FIG. 3.

Binding of cPTPσ to the E6 chick retinal BL is mediated by HS chains. Results of receptor affinity probe assays using the extracellular region of cPTPσ1 fused to alkaline phosphatase are shown. Retina sections were untreated (A to D) or pretreated with heparinase III (E) or chondroitinase ABC (F). cPTPσ1-AP conditioned medium was used alone (A) or preincubated with heparin (B), HS (C), or chondroitin sulfate (D). The BL staining indicates cPTPσ1-AP binding. Arrowheads indicate the retinal BL. pe, pigmented epithelium. Scale bar, 0.1 mm.

FIG. 4.

cPTPσ binds agrin and collagen XVIII via their HS chains. An HSPG-enriched fraction from chicken embryo vitreous bodies (A), purified agrin (B), and purified collagen XVIII (C) samples, separated by SDS-6% polyacrylamide gel electrophoresis, were transferred to nitrocellulose and probed with antibodies specific for agrin or collagen XVIII or were incubated with cPTPσ1-VSV conditioned medium. cPTPσ1-VSV was detected using antibody to VSV. Samples were either not treated with enzymes (lanes a, e, and i) or predigested with heparinase III (lanes b, f, and j), collagenase (lanes c, g, and k), or chondroitinase ABC (lanes d, h, and l). Arrows in panel A indicate the two bands corresponding in molecular mass to agrin (upper arrow) and collagen XVIII (lower arrow) observed in the blot overlay assay.

FIG. 5.

Expression patterns of cPTPσ, agrin, and collagen XVIII in the developing chick retina. E6 (A, C, and E) and E10 (B, D, and F) chick retina sections were probed with antibodies to cPTPσ (A and B), agrin (C and D), and collagen XVIII (E and F). At E6, cPTPσ is strongly expressed in the retinal axons (outer fiber layer [OFL]), while both agrin and collagen XVIII show strong expression in the juxtaposed BL. At E10, cPTPσ shows strong expression, mainly in neurite layers: OFL and IPL (inner plexiform layer). Agrin expression overlaps in fiber layers and is also a major constituent of the BL along with collagen XVIII. RGC, retinal ganglion cell layer; INL, inner nuclear layer; OPL, outer plexiform layer; PE, pigmented epithelium. White arrowheads indicate the BL staining. Scale bar, 0.1 mm.

FIG. 6.

Structural model of the Ig-1 domain of cPTPσ and sequence alignment with the Ig domains of telokin, NCAM, and FGFR1. (A) Electrostatic surface representation of the Ig-1 domain of cPTPσ; blue and red represent positive and negative electrostatic potential, respectively. The large positive-potential patch represents the putative heparin binding site. Basic residues are labeled in yellow and numbered according to Stoker (69). (B) Ribbon view of the predicted folding with the same orientation as in panel A. N and C denote the amino and carboxyl termini. The β strands are labeled, from A to G, according to the telokin fold. (C) Structure-based sequence alignment between telokin (the template used for modelling) and the heparin-binding Ig domains of cPTPσ, NCAM, and FGFR1. The corresponding sequences of the mouse and human orthologues of cPTPσ (mPTPσ and hPTPσ) are included. The heparin-binding sites are highlighted in blue (proposed site for cPTPσ). The telokin key structural residues and their conserved equivalents in cPTPσ are highlighted in green. The β strands are underlined. The secondary structure definitions were reported in the following sources: for telokin, reference ; for NCAM-Ig2, reference ; and for FGFR1-Ig2, reference . This figure was made using the Swiss-PdbViewer v3.6b2 (30).

FIG. 7.

Identification of the cPTPσ heparin-binding site by site-directed mutagenesis. (A) Basic residues in the heparin-binding site in domain Ig-1 (mutations M1, M2, M3, and M4), another cluster in Ig-2 (mutation M5), or in the loop connecting Ig-2 and Ig-3 (mutation M6) were replaced with alanine. wt corresponds to the original cPTPσ sequence. (B) The mutated proteins were tested in solid-phase binding assays for binding to heparin-BSA. Bars represent means ± standard errors of three determinations.

FIG. 8.

The heparin/HS binding site in domain Ig-1 is essential for retinal BL binding. Receptor affinity probe assays were performed using cPTPσ1-AP (A), FN3Δ-AP (B), M1 (C), M2 (D), M3 (E), M4 (F), M5 (G), and M6 (H) conditioned media, respectively. The BL staining indicates fusion protein binding. Arrowheads indicate the retinal BL. pe, pigmented epithelium. Scale bar, 0.1 mm.

FIG. 9.

Müller glia endfeet express two classes of cPTPσ ligands. Retinal basal laminae with glial endfeet attached were flat mounted and probed by RAP in situ with conditioned media containing cPTPσ1-AP (A to E), FN3Δ-AP (F), M1 (G), M2 (H), M3 (I), M4 (J), M5 (K), and M6 (L). Heparin addition (B) or heparinase III pretreatment (C) reduces binding compared to control treatments (A and F) but does not abolish it. cPTPσ mutants with impaired heparin-binding properties also show reduced binding on glial endfeet (H to J). Chondroitin sulfate addition (D) or chondroitinase ABC pretreatment (E) does not affect cPTPσ binding significantly. Arrows indicate ringlike endfeet. Scale bar, 10 μm.