SPACR, a Novel Interphotoreceptor Matrix Glycoprotein in Human Retina That Interacts with Hyaluronan

Abstract

SPACR (sialoprotein associated with cones and rods), is the major 147-150-kDa glycoprotein present in the insoluble interphotoreceptor matrix of the human retina. Immunocytochemistry localizes SPACR to the matrix surrounding rods and cones (Acharya, S., Rayborn, M. E., and Hollyfield, J. G. (1998) Glycobiology 8, 997-1006). From affinity-purified SPACR, we obtained seven peptide sequences showing 100% identity to the deduced sequence of IMPG1, a purported chondroitin 6-sulfate proteoglycan core protein, which binds peanut agglutinin and is localized to the interphotoreceptor matrix. We show here that SPACR is the most prominent 147-150-kDa band present in the interphotoreceptor matrix and is the gene product of IMPG1. SPACR is not a chondroitin sulfate proteoglycan, since it is not a product of chondroitinase ABC digestion and does not react to a specific antibody for chondroitin 6-sulfate proteoglycan. Moreover, the deduced amino acid sequence reveals no established glycosaminoglycan attachment site. One hyaluronan binding motif is present in the predicted sequence of SPACR. We present evidence that SPACR has a functional hyaluronan binding domain, suggesting that interactions between SPACR and hyaluronan may serve to form the basic macromolecular scaffold, which comprises the insoluble interphotoreceptor matrix.The IPM 1 resides within the outer eye wall, at the interface between the neural retina and the retinal pigment epithelium. Projecting from the neural retina outer surface, the elongate photoreceptor inner and outer segments penetrate into and are surrounded by the IPM (1). A number of activities of fundamental importance to vision are thought to be mediated by the IPM, including retinal adhesion, visual pigment chromophore exchange, metabolite trafficking, photoreceptor alignment, and membrane turnover (2). The specific IPM molecules that participate in these activities are not well defined.

SPACRCAN, a Novel Human Interphotoreceptor Matrix Hyaluronan-binding Proteoglycan Synthesized by Photoreceptors and Pinealocytes

“…In contrast, when PNA was used in lectin blotting studies, the 150-kDa SPACR band was the only region of the blot stained in the undigested IPM sample (Fig. 5B, lane 5) (21). Following chondroitinase ABC digestion, in addition to the 150-kDa SPACR band, other proteins have entered the gel, which bind PNA (Fig.…”

“…CPC Precipitation-To demonstrate HA binding to SPACRCAN we used a protocol similar to that described (21). Briefly, PNA-agarosepurified SPACRCAN core protein samples (1 g) in 50 mM Tris acetate buffer (pH 7.3) containing 0.5 M NaCl were incubated for 1 h at room temperature with and without Healon (50 g).…”

“…IPM Isolation-After bisecting the eye, retinas were removed from the posterior pole and washed extensively with PBS containing protease inhibitors to remove soluble molecules followed by detachment of the insoluble IPM with distilled water (21,31). The insoluble IPM was collected (20) after centrifugation, and the pellet was solubilized in 0.1 M Tris-buffered saline, pH 8.0, containing 5 mM dithiothreitol.…”

“…After incubation with avidin-conjugated horseradish peroxidase (1:5000 in PBS), the protein bands were visualized with the peroxidase color reaction as described previously. BSA was included in the CPC precipitation studies as a negative control (21).…”

“…A polyclonal antibody prepared against SPACR intensely labels the rod-associated matrix with weaker labeling of the cone matrix (20). Sequence analysis of peptides from purified SPACR revealed 100% identity to the deduced sequence of interphotoreceptor matrix proteoglycan 1 (GenBank TM accession number AF047492) cDNA (also called IPM150) (21). The gene product of interphotoreceptor matrix proteoglycan 1 is listed in GenBank TM as a chondroitin sulfate proteoglycan core protein, but our analysis indicates that it is a glycoprotein and not a proteoglycan (21).…”