Regulation of eye size by the retinal basement membrane and vitreous body.

PURPOSE: Congenital high myopia is an early-onset enlargement of the eye globes that carries a high risk for retinal detachment. The genetic basis for congenital high myopia has frequently been connected to mutations in genes encoding extracellular matrix proteins of the vitreous body (VB) and the inner limiting membrane (ILM). Why defective or missing VB and ILM proteins lead to an increase in eye size is unknown. The present study introduces the chick embryo as a model to study the role of ILM and VB in regulating eye size. METHODS: The ILM and VB were disrupted by injecting collagenase into the eyes of E5 chick embryos. The digestion of VB and ILM proteins was monitored by Western blot and immunocytochemistry. Eye size was assessed up to 9 days after the enzyme injections. RESULTS: Intraocular injection of collagenase led to the disruption of the ILM and the VB by digesting their collagen constituents. Once disrupted, the ILM and the collagen II fibrillar network failed to regenerate despite continued synthesis of VB and ILM proteins. ILM and VB disruption resulted in eye enlargement of 50% within 4 days. The increase in eye size was greatly reduced by reconstituting the ILM. CONCLUSIONS: The present data show that the ILM and the VB play major roles in the early regulation of eye size. The authors speculate that the integrity of the vitreoretinal border is an important factor in preventing congenital high myopia.

Glycosaminoglycan-dependent and -independent inhibition of neurite outgrowth by agrin.

Agrin is a proteoglycan that can inhibit neurite outgrowth from multiple neuronal types when present as a substrate. Agrin's neurite inhibitory activity is confined to the N-terminal segment of the protein (agrin N150), which contains heparan sulfate (HS) and chondroitin sulfate (CS) side chains. We have examined the activities of various purified recombinant agrin fragments and their glycosaminoglycan (GAG) side chains in neurite outgrowth inhibition. Inhibitory activity was tested using dissociated chick ciliary ganglion neurons or dorsal root ganglion explants growing on laminin or N-cadherin. Initial experiments demonstrated that agrin N150 lacking GAG chains inhibited neurite outgrowth. Both halves of N150, each containing HS and/or CS side chains, could also inhibit neurite growth. Experiments using agrin fragments in which the GAG acceptor residues were mutated, or using agrin fragments purified from cells deficient in GAG synthesis, demonstrated that inhibition by the N-terminal portion of N150 requires GAGs, but that inhibition from the C-terminal part of N150 does not. Thus, the core protein or other types of glycosylation are important for inhibition from the more C-terminal region. Our results suggest that there are two distinct mechanisms for neurite outgrowth inhibition by agrin, one that is GAG-dependent and one that is GAG-independent.

Agrin is a chimeric proteoglycan with the attachment sites for heparan sulfate/chondroitin sulfate located in two multiple serine-glycine clusters.

Agrin is a large extracellular matrix protein that plays a key role in the formation and maintenance of the vertebrate neuromuscular junction. The amino acid sequence of agrin encodes a protein with a molecular size of 220 kDa, whereas SDS-PAGE shows a diffuse band around 400 kDa. Further studies showed that agrin is highly glycosylated and belongs to the family of heparan sulfate proteoglycans. By expressing different protein fragments, we localized the glycosaminoglycan (GAG) attachment sites to two locations within the agrin molecule. One site that is located between the seventh and eight follistatin-like domain includes 3 closely spaced serine-glycine (SG) consensus sequences and carries exclusively heparan sulfate side chains. The second site is located further downstream in the centrally located serine-threonine-rich domain and contains a cluster of 4 closely packed SG consensus sequences. This site predominantly carries chondroitin sulfate side chains. Investigating the contribution of individual serines in GAG priming by site-directed mutagenesis showed that each serine of the two SG clusters has the potential to carry GAGs. In accordance with the mixed GAG glycosylation of agrin peptide fragments, it was found that recombinant and in vivo-derived full-length agrin are not exclusively heparan sulfate proteoglycans but also carry chondroitin sulfate side chains.

Mapping of the laminin-binding site of the N-terminal agrin domain (NtA).

Agrin is a key organizer of acetylcholine receptor (AChR) clustering at the neuromuscular junction. The binding of agrin to laminin is required for its localization to synaptic basal lamina and other basement membranes. The high-affinity interaction with the coiled-coil domain of laminin is mediated by the N-terminal domain of agrin. We have adopted a structurally guided site-directed mutagenesis approach to map the laminin-binding site of NtA. Mutations of L117 and V124 in the C-terminal helix 3 showed that they are crucial for binding. Both residues are located in helix 3 and face the groove between the beta-barrel and the C-terminal helical segment of NtA. Remarkably, the distance between both residues matches a heptad repeat distance of two aliphatic residues which are solvent exposed in the coiled-coil domain of laminin. A lower but significant contribution originates from R43 and a charged cluster (E23, E24 and R40) at the open face of the beta-barrel structure. We propose that surface-exposed, conserved residues of the laminin gamma1 chain interact with NtA via hydrophobic and ionic interactions.