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 SPACR expression is restricted to retinal photoreceptors. The SPACR core protein was identified with Western blotting following SDS-PAGE with a SPACR C-terminal peptide polyclonal antibody and a chondroitin-6-sulfate Deltadisaccharide monoclonal antibody. The 150 kD immunopositive band was isolated, digested with trypsin and the peptides analysed by MALDI mass spectroscopy. Peptide mass mapping confirmed the identity of the 150 kD immunopositive band to be mouse SPACR core protein. Alignment comparisons of the deduced amino 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 oligosaccharide attachment, one EGF-like domain and four hyaluronan-binding motifs. Unlike human SPACR, which contains no conventional consensus sites for glycosaminoglycan attachment, mouse SPACR contains three. Recent biochemical studies of human and mouse SPACR protein indicate that this novel interphotoreceptor matrix molecule is a glycoprotein in human and a proteoglycan in the mouse. The presence of consensus sites for glycosaminoglycan attachment in the deduced sequence of mouse SPACR and the absence of these sites in human SPACR provide molecular verification of our biochemical results, suggesting that differences in post-translational modifications of SPACR may be important in SPACR function in foveate and non-foveate retinas.
Lee
J W
JW
Cole Eye Institute, The Cleveland Clinic Foundation, Cleveland, OH 44195, USA.
Chen
Q
Q
Rayborn
M E
ME
Shadrach
K G
KG
Crabb
J W
JW
Rodriguez
I R
IR
Hollyfield
J G
JG
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D002096:C-Peptide / Q000276:immunology; D002809:Chondroitin Sulfates / Q000276:immunology; D018076:DNA, Complementary / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D017403:In Situ Hybridization; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D015676:Osteonectin / Q000235:genetics* / Q000378:metabolism; D010449:Peptide Mapping; D020419:Photoreceptor Cells, Vertebrate / Q000378:metabolism*; D016133:Polymerase Chain Reaction
D000911:Antibodies, Monoclonal; D002096:C-Peptide; D018076:DNA, Complementary; D015676:Osteonectin; D002809:Chondroitin Sulfates