Hyaluronan and the functional organization of the interphotoreceptor matrix.

The interphotoreceptor matrix (IPM) fills the part of the eye referred to by ophthalmologists as the subretinal space. Located between the outer limiting membrane of the retina and the apical border of the retinal pigment epithelium (RPE), this unique matrix surrounds photoreceptor inner and outer segments projecting from the outer retinal surface. Structure–function activities of fundamental importance to vision occur within this matrix, including the trafficking of retinoids and other metabolites between photoreceptors and the RPE; retinal attachment; maintenance of photoreceptor specific microenvironments; photoreceptor alignment; and cellcell interactions involved in outer segment shedding and RPE phagocytosis. The molecular interactions responsible for these activities are not known. New evidence implicates hyaluronan (HA) and several HA-binding proteins as key participants in the organization of the IPM and in retinal attachment. In the space allowed for this short report it will not be possible to review all the important studies that have advanced our understanding of the IPM. For more detailed coverage and an extensive bibliography the reader is directed to comprehensive reviews published elsewhere. This discussion will be limited to recent advances in understanding novel molecules present in the IPM; to HA and its properties that permit selfassembly into a complex matrix; and to a unifying organizational concept based on interactions between HA and HAbinding proteins located in and around the IPM. In early attempts to study the IPM, gentle saline rinses of the outer retinal surface were used to remove soluble matrix molecules for subsequent biochemical analyses. Using such procedures interphotoreceptor matrix retinoid binding protein (IRBP), a variety of enzymes, mucins, and immunoglobulins were successfully isolated from the IPM. Later, the presence of a relatively insoluble IPM complex was documented in studies of isolated Xenopus and rat retina. We now know that an aqueous insoluble IPM survives saline rinses and can be removed with water as an intact unit. Distilled water detaches this matrix from the outer retina because polyanions, present in high density in the insoluble IPM, hydrate in the absence of salts, swelling the matrix to over twice its original diameter. Rinses of the outer retina with high pH buffer can also disrupt the interactions that stabilize the insoluble IPM. A novel glycoprotein named SPACR (an acronym for “SialoProtein Associated with Cones and Rods”) was recently identified in the insoluble human IPM. Sequence analysis of peptides from purified SPACR revealed 100% identity to the deduced sequence of IMPG1 cDNA (also called IPM150). The gene product of IMPG1 was initially thought to be a chondroitin sulfate proteoglycan core protein localized to the human IPM (Gene Bank accession number AF047492). However, carbohydrate analyses demonstrate that this molecule is a glycoprotein, not a proteoglycan. A polyclonal antibody prepared against SPACR intensely labels the rod-associated matrix with weaker labeling of the cone matrix. Another novel protein named SPACRCAN (also called IPM200) was recently identified in the insoluble IPM. SPACRCAN is clearly a chondroitin sulfate proteoglycan. It will only enter a 7% polyacrylamide gel after digestion with chondroitinase ABCand then will also exhibit intense immunoreactivity in western blot analysis to a chondroitin DDi6S monoclonal antibody. Analysis of the N-terminal sequence of human SPACRCAN led us to PG10.2, a gene coding for a proteoglycan core protein expressed by rat photoreceptors and pinealocytes. The human SPACRCAN gene has now been cloned (Gene Bank Accession No. AF157624). Immunohistochemistry shows intense SPACRCAN immunoreactivity in the IPM around cones with weaker labeling around rods. Although SPACR in human and macaque (author’s unpublished observations, 1999) is a glycoprotein and SPACRCAN is a chondroitin sulfate proteoglycan, in nonprimate retinas (bovine, mouse, and rat) both SPACR and SPACRCAN are chondroitin sulfate proteoglycans. The functional role of these highly conserved IPM molecules remains to be determined, however, the absence of the chondroitin sulfate chains on SPACR in species with foveate retinas may be fundamentally related to foveal specialization. Because glycosaminoglycans (GAGs) occupy a large extracellular volume, one obvious consequence of the absence of the chondroitin sulfate from SPACR may be related to the need for a smaller IPM volume in foveate retinas, thereby permitting the high packing density of foveal cones. Knowledge of the carbohydrate structures of SPACR and SPACRCAN in the IPM of other primates will be important for unraveling the functional roles of these novel molecules in this critical area of primate vision. The IPM also contains HA. HA is an extremely large (with molecular weights between 1 and 10 million Da and containing between 2,500 and 25,000 disaccharides), polyanionic GAG composed of repeating disaccharide units of b-(1,4)-D-glucuronic acidb-(1,3)-N-acetyl-D-glucosamine. A 10 million Da molecule of HA From the Cole Eye Institute, The Cleveland Clinic Foundation, Cleveland, Ohio. The research that led to the model presented in this report was supported by the National Eye Institute, National Institutes of Health, Bethesda, MD; The Foundation Fighting Blindness, Hunt Valley, MD; The Retina Research Foundation, Houston, TX; and The Alcon Research Institute, Ft. Worth, TX. Submitted for publication July 26, 1999; accepted July 26, 1999. Commercial relationships policy: N. Corresponding author: Joe G. Hollyfield, Cole Eye Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195. E-mail: [email protected]