N-Glycosylation of the Discoidin Domain Receptor Is Required for Axon Regeneration in Caenorhabditis elegans

Abstract

Axon regeneration following neuronal injury is an important repair mechanism that is not well understood at present. In Caenorhabditis elegans, axon regeneration is regulated by DDR-2, a receptor tyrosine kinase (RTK) that contains a discoidin domain and modulates the Met-like SVH-2 RTK-JNK MAP kinase signaling pathway. Here, we describe the svh-10/sqv-3 and svh-11 genes, which encode components of a conserved glycosylation pathway, and show that they modulate axon regeneration in C. elegans Overexpression of svh-2, but not of ddr-2, can suppress the axon regeneration defect observed in svh-11 mutants, suggesting that SVH-11 functions between DDR-2 and SVH-2 in this glycosylation pathway. Furthermore, we found that DDR-2 is N-glycosylated at the Asn-141 residue located in its discoidin domain, and mutation of this residue caused an axon regeneration defect. These findings indicate that N-linked glycosylation plays an important role in axon regeneration in C. elegans.

Keywords: Caenorhabditis elegans; DDR-2; N-glycosylation; axon regeneration.

Figure 1

Identification of svh-9/nstp-1, svh-10/sqv-3, and svh-11 genes. (A) EMB-9 collagen–DDR-2 discoidin domain RTK modulates the SVH-1 growth factor–SVH-2 RTK–JNK pathway in C. elegans. (B) Structures of SVH-9/NSTP-1, SVH-10/SQV-3, and SVH-11. Schematic domain diagrams of C. elegans SVH-9/NSTP-1, SVH-10/SQV-3, SVH-11 and their human counterparts (SLC35B4, GalT1, and FuT2) are shown. Domains are shown as follows: a transmembrane domain (TM; black), a Gal transferase domain (GalT; yellow), and a Fuc transferase domain (FuT; red). The sites of each mutation are indicated.

Figure 2

The sqv-3 and svh-11 genes are required for axon regeneration. (A) Representative D-type motor neurons in wild-type, sqv-3, and svh-11 mutant animals 24 hr after laser surgery. In wild-type animals, a severed axon has regenerated a growth cone (arrow). In mutants, the proximal ends of axons failed to regenerate (arrowheads). Bar, 10 μm. (B and C) Percentages of axons that initiated regeneration 24 hr after laser surgery. The numbers (n) of axons examined are shown. Error bars indicate 95% confidence intervals. * P < 0.05, ** P < 0.01, *** P < 0.001 as determined by Fisher’s exact test. NS, not significant.

Figure 3

The relationship between SVH-11 and components involved in the JNK pathway in axon regeneration. (A–C) Percentages of axons that initiated regeneration 24 hr after laser surgery are shown. svh-11(km86) mutants carrying Pmlk-1::mlk-1 or Punc-25::svh-2 were analyzed for axon regeneration in nontransgenic siblings of the extrachromosomal transgenic lines (-array) (C). The numbers (n) of axons examined are shown. Error bars indicate 95% confidence intervals. * P < 0.05, ** P < 0.01 as determined by Fisher’s exact test. NS, not significant.

Figure 4

DDR-2 is N-glycosylated at Asn-141 in animals. (A) N-glycosylation of DDR-2. Wild-type animals carrying Phsp::ddr-2::gfp and Phsp::ddr-2(N141A)::gfp were subjected to heat shock at 37° for 30 min and incubated at 20° for additional 4 hr. Animal lysates were prepared and immunoprecipitated with anti-GFP antibody. The DDR-2::GFP immunoprecipitate was treated with (+) or without (−) PNGase, and immunoblotted with anti-GFP antibody. (B) Schematic diagram of DDR-2 domains. Potential N-glycosylation sites are indicated (red). Domains are shown as follows: discoidin (DS) and DS-like domains (yellow), a transmembrane domain (TM; black box), and a kinase domain (orange). (C) Effect of the ddr-2(NA) mutations on axon regeneration. Percentages of axons that initiated regeneration 24 hr after laser surgery are shown. The numbers (n) of animals examined are shown. Error bars indicate 95% confidence intervals. * P < 0.05, ** P < 0.01, *** P < 0.001 as determined by Fisher’s exact test. NS, not significant. (D) Effect of the ddr-2(N141A; N167A) double mutations on DDR-2 N-glycosylation. ddr-2(tm797) mutants carrying Phsp::ddr-2(N141A)::gfp (N141A) or Phsp::ddr-2(N141A; N167A)::gfp (N141A; N167A) were subjected to heat shock at 37° for 30 min and incubated at 20° for additional 4 hr. Animal lysates were prepared and immunoprecipitated with anti-GFP antibody. The DDR-2::GFP immunoprecipitate was treated with (+) or without (−) PNGase, and immunoblotted with anti-GFP antibody.

Figure 5

Fucosylation and tyrosine kinase activity of DDR-2. (A) Effects of the ddr-2(N141A) mutation on fucosylation and tyrosine kinase activity of DDR-2. ddr-2(tm797) mutants carrying Phsp::ddr-2::gfp (WT), Phsp::ddr-2(N141A)::gfp (N141A), or Phsp::ddr-2(K554E)::gfp (KN) were subjected to heat shock (+HS) at 37° for 30 min and incubated at 20° for additional 4 hr. Animal lysates were prepared and immunoprecipitated with anti-GFP antibody. The DDR-2::GFP immunoprecipitate was blotted with a lectin (AOL) specific for L-fucose and immunoblotted with anti–phospho-tyrosine (pY) and anti-GFP antibodies. Experiments were performed three times with similar results. (B) Effect of the svh-11(gk819558) mutation on DDR-2 N-glycosylation. ddr-2(tm797) (WT) and svh-11(gk819558); ddr-2(tm797) (svh-11) mutants carrying Phsp::ddr-2::gfp were subjected to heat shock at 37° for 30 min and incubated at 20° for additional 4 hr. Animal lysates were prepared and immunoprecipitated with anti-GFP antibody. The DDR-2::GFP immunoprecipitate was treated with (+) or without (−) PNGase, and immunoblotted with anti-GFP antibody. (C) Effect of the svh-11(km86) mutation on DDR-2 fucosylation. ddr-2(tm797) (WT) and svh-11(km86); ddr-2(tm797) (svh-11) mutants carrying Phsp::ddr-2::gfp were subjected to heat shock at 37° for 30 min and incubated at 20° for additional 4 hr. Animal lysates were prepared and immunoprecipitated with anti-GFP antibody. The DDR-2::GFP immunoprecipitate was blotted with a lectin (AOL) specific for L-fucose and immunoblotted with anti-GFP antibody.