SPACR in the interphotoreceptor matrix of the mouse retina: Molecular, biochemical and immunohistochemical characterization

Mouse SPACR cDNA was cloned by screening a mouse retina cDNA library using a PCR probe derived from human SPACR cDNA, Mouse SPACR cDNA comprises 3675 bp containing an open reading frame coding for 742 amino acids. Multitissue Northern blot analysis and in situ hybridization studies indicate that SPA CR expression is restricted to retinal photoreceptors. The SPACR core prote in was identified with Western blotting following SDS-PAGE with a SPACR C-t erminal peptide polyclonal antibody and a chondroitin-6-sulfate Delta disac charide monoclonal antibody. The 150 kD immunopositive band was isolated, d igested with trypsin and the peptides analysed by MALDI mass spectroscopy. Peptide mass mapping confirmed the identity of the 150 kD immunopositive ba nd to be mouse SPACR core protein. Alignment comparisons of the deduced ami no acid sequence of mouse and human SPACR show 64 % homology. Like SPACR in the human interphotoreceptor matrix, the mouse orthologue contains a large central mucin-like domain flanked by consensus sites for N-linked oligosac charide attachment, one EGF-like domain and four hyaluronan-binding motifs. Unlike human SPACR, which contains no conventional consensus sites for gly cosaminoglycan attachment, mouse SPACR contains three. Recent biochemical s tudies of human and mouse SPACR protein indicate that this never interphoto receptor matrix molecule is a glycoprotein in human and a proteoglycan in t he mouse. The presence of consensus sites for glycosaminoglycan attachment in the deduced sequence of mouse SPACR and the absence of these sites in hu man SPACR provide molecular Verification of our biochemical results, sugges ting that differences in post-translational modifications of SPACR may be i mportant in SPACR function in foveate and non-foveate retinas. (C) 2000 Aca demic Press.