CHARACTERIZATION OF SPACR, A SIALOPROTEIN ASSOCIATED WITH CONES AND RODS PRESENT IN THE INTERPHOTORECEPTOR MATRIX OF THE HUMAN RETINA - IMMUNOLOGICAL AND LECTIN-BINDING ANALYSIS

Rod and cone photoreceptors project from the outer retinal surface int o a carbohydrate-rich interphotoreceptor matrix (IPM), Unique IPM glyc oconjugates are distributed around rods and cones, Wheat germ agglutin in (WGA) strongly decorates the rod matrix domains and weakly decorate s the cone matrix domains. This study characterizes the major WGA-bind ing glycoprotein in the human IPM, which me refer to as SPACR (sialopr otein associated with cones and rods). SPACR, which has a molecular we ight of 147 kDa, was isolated and purified from the IPM by lectin affi nity chromatography, A polyclonal antibody to SPACR was prepared that colocalizes in tissue preparations with WGA-binding domains in the IPM . Sequential digestion of SPACR with N- and O-glycosidases results in a systematic increase in electrophorectic mobility, indicating the pre sence of both N- and O-linked glycoconjugates. Complete deglycosylatio n results in a reduction in the relative molecular mass of SPACR by ab out 30 %. Analysis of lectin binding allowed us to identify some of th e structural characteristics of SPACR glycoconjugates. Treatment with neuraminidase exposes Gal beta 1-3GalNAc disaccharide as indicated by positive peanut agglutinin (PNA) staining, accompanied by the loss of WGA staining. Maackia amurensis agglutinins (MAA-1 and MAA-2), specifi c for sialic acid in alpha 2-3 linkage to Gal, bind SPACR, while Sambu cus nigra agglutinin (SNA), specific for alpha 2-6 linked sialic acid, does not, indicating that the dominant glycoconjugate determinant on SPACR is the O-linked carbohydrate, NeuAc alpha 2-3Gal beta 1-3 GalNAc , The abundance of sialic acid in SPACR suggests that this glycoprotei n may contribute substantially to the polyanionic nature of the IPM, T he carbohydrate chains present on SPACR could also provide sites for e xtensive crosslinking and participate in the formation of the ordered IPM lattice that surrounds the elongate photoreceptors projecting from the outer retinal surface.