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 cell-cell interactions involved in outer segment shedding and RPE phagocytosis. ^{1 2 3 4 5 6 7 8 9 10 11} 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. ^{12 13} This discussion will be limited to recent advances in understanding novel molecules present in the IPM; to HA and its properties that permit self-assembly into a complex matrix; and to a unifying organizational concept based on interactions between HA and HA-binding 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. ^{14 15 16 17} 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 ¹⁸ and rat retina. ¹⁹ We now know that an aqueous insoluble IPM survives saline rinses and can be removed with water as an intact unit. ²⁰ 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. ^{21 22} Rinses of the outer retina with high pH buffer can also disrupt the interactions that stabilize the insoluble IPM. ²³  

A novel glycoprotein named SPACR (an acronym for “SialoProtein Associated with Cones and Rods”) was recently identified in the insoluble human IPM. ²³ Sequence analysis of peptides from purified SPACR revealed 100% identity to the deduced sequence of IMPG1 cDNA ²⁴ (also called IPM150 ^{12 13} ). 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. ^{23 24} A polyclonal antibody prepared against SPACR intensely labels the rod-associated matrix with weaker labeling of the cone matrix. ²⁴  

Another novel protein named SPACRCAN (also called IPM200 ^{12 13} ) 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 ABC ²⁵ and then will also exhibit intense immunoreactivity in western blot analysis to a chondroitin ΔDi6S 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. ²⁶ 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. ²⁵  

Although SPACR in human ²⁵ 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. ²⁵ 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. ¹⁴ 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 β-(1,4)-D-glucuronic acid-β-(1,3)-N-acetyl-D-glucosamine. A 10 million Da molecule of HA would extend linearly to 25 μm. ^{32 33} Considering that the IPM is approximately 50-μm thick (the distance between the outer limiting membrane of the retina and the apical surface of the RPE), the larger HA molecules could theoretically bridge half this distance. Moreover, studies of its secondary structure indicate that the otherwise hydrophilic HA molecule contains repeating hydrophobic patches. ³⁴ In an aqueous environment, the hydrophobic patches on adjacent HA molecules can align to form associations that limit their aqueous exposure. ³⁵ The optimum alignment for maximum hydrophobic interaction occurs when adjacent molecules are in antiparallel orientation. Such hydrophobic interactions between HA molecules result in the formation of a continuous three-dimensional HA network, with each molecule interconnected with all the rest via this highly organized viscoelastic matrix. ³⁶  

RHAMM-type hyaluronan binding motifs (Receptor for HA Mediated Motility) ^{29 30} are present in the protein sequence of both SPACR and SPACRCAN, suggesting that these molecules may be retained in the insoluble IPM through binding to HA. RHAMM-type motifs are represented by the linear sequence B(X₇)B, where B is a basic amino acid residue and X is any nonacidic amino acid. Coprecipitation studies with detergents that precipitate GAGs, in conjunction with digests using an HA-specific hyaluronidase, indicate that in the IPM, both SPACR and SPACRCAN associate directly with HA. ²³ PEDF (Pigment Epithelium-Derived Factor), present in the IPM and vitreous, also binds to HA, presumably through the RHAMM-type motifs present in its primary sequence. ³¹  

Because HA is present in the IPM of most species studied, ³⁷ and because HA is recovered with SPACR and SPACRCAN when the insoluble IPM is isolated from the human retina, we propose that a complex of interacting HA molecules forms the primary scaffold of the IPM (Fig. 1) . Such a scaffold would allow secreted proteins, like SPACR and SPACRCAN, to bind through HA-binding motifs in their polypeptide. The binding of SPACR and SPACRCAN to HA may further stabilize and/or modulate interactions within the scaffold. It is of interest that SPACRCAN contains two potential HA-binding motifs, which may permit it to form a bridge between adjacent HA molecules. Because hydrophobic patches are also present along chondroitin sulfate GAG chains, chondroitin–HA interactions are thought to occur in a manner similar to that of HA–HA associations. ^{34 35} Thus, the chondroitin sulfate chains covalently linked to SPACRCAN provide yet another mechanism for this proteoglycan to associate with the HA scaffold. 

Other molecules containing specific HA-binding motifs have been identified in the plasma membranes of cells that border the IPM (Müller cells, photoreceptors, and RPE cells). These include CD44, a well-recognized HA-binding protein present in the apical microvillae of Müller cells, ¹¹ and RHAMM, recently localized to the apical region of the RPE. ³⁸ Neuroglycan C, a proteoglycan that is associated with the plasma membrane of photoreceptors, contains a RHAMM-type HA-binding motif in its extracellular domain as well as covalently linked chondroitin sulfate. ³⁹ The newly discovered gene expressed in photoreceptors and linked to the loci for retinitis pigmentosa-1 codes for oxygen-regulated photoreceptor protein-1 (ORP-1) ⁴⁰ . The deduced sequence of the ORP-1 polypeptide contains 14 putative HA-binding motifs and 11 putative GAG-attachment sites. If ORP-1 is associated with the plasma membrane of photoreceptors, as is predicted from its deduced sequence, the HA-binding motifs and GAGs on this molecule would also be positioned to interact with the HA scaffold. Thus, the HA-binding motifs and chondroitin sulfate on the cells that border the IPM provide the structural components through which these cells can interact with the HA scaffold. Attachment of the HA scaffold to cells on both sides of the IPM would allow the IPM to form the structural link between the retina and the RPE by way of these protein–carbohydrate and carbohydrate–carbohydrate interactions (Fig. 1) . 

Additional studies are needed to test several aspects of the hypothesis implicit in this new model of IPM organization. Important areas for consideration are as follows: understanding the differences in the composition and stability of the scaffold surrounding cones versus rods and the relative importance of each in retinal attachment; determining the pattern and rates of addition and loss (turnover) of the various molecules present in the IPM; identifying other molecules in the IPM that associate with the scaffold; defining the cellular sources of HA present in the scaffold; identifying other molecules that function to attach the scaffold to cells that border the IPM; defining the changes in the scaffold that precede retinal detachment; and understanding the processes that repair the scaffold after retinal reattachment. Further studies are also required to identify other functional roles of SPACR and SPACRCAN and to determine their possible involvement as targets for inherited retinal disease. 

 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.

Figure 1.

 

Cartoon depicting the levels of organization of hyaluronan (HA), which forms the basic IPM scaffold, drawn by David Schumick, Medical Illustrator at The Cleveland Clinic Foundation. Left panel shows the antiparallel alignment of linear HA molecules forming the basic matrix scaffold structure (adapted from Scott et al. ^{29 30 31} ). Center panel depicts the continuous three-dimensional scaffold complex (not to scale) in the extracellular compartment adapted from electron microscope images of the IPM. Right panel depicts the interaction of the scaffold (not to scale) with HA-binding motifs on cells that border the IPM (CD44 on apical microvillae of Müller cell and RHAMM on apical RPE processes) and secreted molecules within the IPM (SPACR, SPACRCAN; Pigment Epithelium-Derived Factor [PEDF], and IRBP). Because IRBP can be removed from the IPM with saline rinses, it is not considered part of the insoluble complex. However, the deduced amino acid sequence of human IRBP does contain two RHAMM-like motifs (K³²¹-R³²⁹ and K⁷⁷³-R⁷⁸¹, Accession No. J03912), suggesting the possibility of IRBP interaction with the HA scaffold. Furthermore, RHAMM-like motifs in IRBP are highly conserved as evidenced by their presence in the deduced sequence of IRBP in 32 mammalian species. Enzyme-linked immunosorbent analyses also demonstrate that IRBP can bind to some as yet unidentified molecules in the insoluble IPM. ³⁹

 

The author thanks Vincent C. Hascall, John W. Crabb, and Alan D. Marmorstein for their valuable comments on early drafts of this manuscript. The author also acknowledges the numerous discussions with Endre Balazs, MD, a pioneer in hyaluronan research and its application in medicine.