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 into acarbohydrate-rich interphotoreceptor matrix (IPM). Unique IPMglycoconjugates are distributed around rods and cones. Wheat germagglutinin (WGA) strongly decorates the rod matrix domains and weaklydecorates the cone matrix domains. This study characterizes the majorWGA-binding glycoprotein in the human IPM, which we refer to as SPACR(sialoprotein associated with cones and rods). SPACR, which has a molecularweight of 147 kDa, was isolated and purified from the IPM by lectinaffinity chromatography. A polyclonal antibody to SPACR was prepared thatcolocalizes in tissue preparations with WGA-binding domains in the IPM.Sequential digestion of SPACR with N- and O- glycosidases results in asystematic increase in electrophorectic mobility, indicating the presenceof both N- and O-linked glycoconjugates. Complete deglycosylation resultsin a reduction in the relative molecular mass of SPACR by about 30%.Analysis of lectin binding allowed us to identify some of the structuralcharacteristics of SPACR glycoconjugates. Treatment with neuraminidaseexposes Galbeta1- 3GalNAc disaccharide as indicated by positive peanutagglutinin (PNA) staining, accompanied by the loss of WGA staining. Maackiaamurensis agglutinins (MAA-1 and MAA-2), specific for sialic acid inalpha2-3 linkage to Gal, bind SPACR, while Sambucus nigra agglutinin (SNA),specific for alpha2-6 linked sialic acid, does not, indicating that thedominant glycoconjugate determinant on SPACR is the O-linked carbohydrate,NeuAcalpha2-3Galbeta1-3GalNAc. The abundance of sialic acid in SPACRsuggests that this glycoprotein may contribute substantially to thepolyanionic nature of the IPM. The carbohydrate chains present on SPACRcould also provide sites for extensive crosslinking and participate in theformation of the ordered IPM lattice that surrounds the elongatephotoreceptors projecting from the outer retinal surface.