Biosynthesis and molecular biology of the secretory proteins of the subcommissural organ.

Ependymal cells are specialized in the synthesis and release of different factors into the cerebrospinal fluid (CSF). The subcommissural organ (SCO) is one of the most active areas of the ventricular walls secreting into the CSF. This gland is localized in the roof of the third ventricle covering the posterior commissure. Glycoproteins synthesized in SCO cells are released into the ventricular CSF where they aggregate, in a highly ordered fashion, forming an elongated supramacromolecular structure known as the Reissner's fiber (RF). RF grows caudally and extends along the brain aqueduct, the fourth ventricle, and the whole length of the central canal of the spinal cord. The SCO cells synthesize glycoproteins of high molecular weight. A precursor form of 540 kDa is synthesized in bovine and chick SCO cells, and a transcript of 10--14 kb is expressed selectively in the bovine SCO cells. The processing of this molecule generates at least one protein of about 450 kDa (RF-Gly-I), which, after being released, is involved in the formation of RF. Additionally, biochemical data indicate that bovine SCO cells synthesize a second precursor compound of 320 kDa, which is also detected in rat, rabbit, and dog. We postulate that RF is formed by two different complexes, one of which has a very high molecular mass (700 kDa or more) and is made up of at least six polypeptides, with the polypeptide of 450 kDa being its main component. The molecules that form RF in different species have different primary structures but they express common epitopes associated to the existence of cysteine bridges, which are probably crucial for polymerization of RF. Molecular procedures involving the use of anti-RF antibodies have led to the isolation of cDNA clones encoding two proteins known as RF-GLY-I and SCO-spondin. In the last 3 years, five partial cDNA sequences encoding SCO-spondin-like proteins have been obtained (Y08560, Y08561, AJ132107, AJ132106, AJ133488). These clones along with RF-GLY-I and SCO-spondin were computer-assembled generating a cDNA consensus sequence of 14.4 kb. Analyses of the long consensus sequence revealed an extended open reading frame (ORF-1) spanning from base 1,634 to 14,400 that encodes for a putative protein of 4,256 amino acids (approximately 450 kDa). The Mr of the predicted protein is consistent with the observed Mr of the largest protein recognized with anti-RF antibodies in SCO and RF extracts. However, the absence of consensus sequences typically present near the 5J'-end of the translation initiation site suggests the existence of a second open reading frame (ORF-2) extending from base 1 to base 14,400 in frame with the ORF-1 and probably encoding for the largest protein precursor (540 kDa). An antibody raised against a peptide sequence, deduced from the open reading frame encoded by a SCO cDNA, reacted specifically with the bovine and rat SCO-RF complex, thus indicating that the protein encoded by the cloned cDNA is part of RF. Immunoblots of bovine SCO extracts using the anti-peptide serum revealed bands of 540 kDa and 450 kDa, but it did not react with the proteins of 320 and 190 kDa. These data support the existence of two precursors for the bovine RF-glycoproteins (540 and 320 kDa) with the 450-kDa protein being a processed form of the 540-kDa precursor. We postulate that the cloned cDNAs encode for a protein that corresponds to the 540-kDa precursor and that at least part of this sequence is present in the processed form of 450 kDa that is secreted to form the RF.