PARTIAL SEQUENCING OF REISSNERS FIBER GLYCOPROTEIN-I (RF-GLY-I)

The bulk of the secretion of the subcommissural organ is formed by gly coproteins that appear to be derived from two precursor forms of 540 a nd 320 kDa. Upon release into the ventricle, these glycoproteins aggre gate to form Reissner's fiber. We report the isolation of three cDNA c lones from a cDNA library prepared from bovine subcommissural organ RN A, by using an anti-Reissner's fiber serum for immunoscreening. Insert s of 0.7, 1.2, and 2.5 kb were amplified by the polymerase chain react ion, subcloned into pUC18 vector, and sequenced. Although restriction mapping of the three inserts initially suggested that all of them were derived from the same mRNA, sequence analysis showed that a shea nonh omologous region was present in the 0.7-kb insert when compared with t he 1.2-kb and 2.5-kb inserts, suggesting that they corresponded to two different, although highly homologous, mRNAs. Northern analyses showe d a single mRNA species of approximately 9.5 kb present in the subcomm issural organ and missing in the choroid plexus, brain cortex, and liv er. Tn situ hybridization confirmed that the expression of the RNA was restricted to cells of the bovine subcommissural organ. Polyclonal an tibodies raised against a synthetic peptide, whose amino-acid sequence was deduced from the 2.5-kb cDNA, reacted specifically with the bovin e and rat subcommissural organ-Reissner's fiber complex. In immunoblot s of bovine subcommissural organ, this antibody revealed the precursor 540-kDa form and its putative processed form of 450 kDa. It is conclu ded that the cloned cDNA encodes for the major constitutive glycoprote in of Reissner's fiber, here designated as RF-Gly I. The sequenced reg ion of RF-Gly I displays a high degree of homology with some regions o f the von Willebrand factor and certain mucins; it also displays two m otifs homologous with repeats present in proteins of the spondin famil y and other proteins. A core sequence of the RF-Gly I repeats suggests that this molecule displays protein-binding properties.