Molecular Cloning and Characterization of Chick SPACRCAN

Abstract

Abstract: : Purpose: Previously we showed that a monoclonal antibody MY174 recognized a specific sialylated O-glycoconjugates of chick SPACR, an interphotoreceptor matrix (Zako et al., 2002). To perform screening of similar molecules with SPACR by hybridization. Methods: The 32P-labeled probe was prepared based on a known DNA sequence of chick SPACR. We used chick retina cDNA library for screening by hybridization. Obtained clones were sequenced and only clones except SPACR proceeded to polymerase chain reactions to analyze full sequence of the molecules. We newly prepared a polyclonal antibody against the molecule and used for western blot studies. We examined whether MY174 recognized the molecule. The modifications by glycosylations were also examined by a variety of glycosidase digestions. Results: The new sequences obtained from hybridization showed we had sequenced a full-length cDNA with an open reading frame of 3759 base pairs, encoding a polypeptide of 1252 amino acids. BLAST analysis of public databases revealed human SPACRCAN to be its most homologous relatively. Chondroitinase digestion showed that chick SPACRCAN has chondroitin sulfate chains like human SPACRCAN, but interestingly there were two kinds of isoforms in chick. Chick SPACRCAN was recognized by MY174 and the signal was abolished after a combination of neuraminidase and O-glycanase treatments like chick SPACR. Conclusions: Newly cloned molecule was chick SPACRCAN which consists of two isoforms. Chick SPACRCAN and SPACR has identical sialylated O-glycoconjugates as the epitope for MY174 suggesting this specific glycoconjugates may play an important roles for retinal functions.

Keywords: extracellular matrix • glycoconjugates/glycoproteins • photoreceptors