Identification and partial characterization of the secretory glycoproteins of the bovine subcommissural organ-Reissner's fiber complex. Evidence for the existence of two precursor forms.
The subcommissural organ (SCO) is a brain gland whose secretory material is released into the cerebrospinal fluid where it condenses into a thread-like structure known as Reissner's fiber (RF). This fiber extends along the aqueduct, fourth ventricle and central canal of the spinal cord. The present investigation was designed to identify and partially characterize the secretory products of the bovine SCO in their intracellular location and after they have been released and packed into RF form. 5,000 SCOs were dissected out under a microscope, whereas RF of 30,000 cows were collected by perfusing the central canal of the spinal cord with artificial cerebrospinal fluid. Extracts of SCO and RF were used for (i) raising polyclonal antibodies; (ii) immunoblotting; (iii) lectin binding on electrotransfers: concanavalin A (affinity = mannose, glucose) and Limax flavus agglutinin (affinity = sialic acid); (iv) immunoaffinity chromatography; (v) preparative SDS-PAGE and raising of polyclonal antibodies against each of the secretory glycoproteins identified in the immunoblots. All antibodies and the two lectins were also applied to tissue sections of the SCO and RF of several species. The immunocytochemical study of the bovine SCO using an anti-RF serum showed that the secretory material present in the rough endoplasmic reticulum (RER), secretory granules and in RF is strongly immunoreactive. Con A binding sites were only found in the endoplasmic reticulum, whereas Limax flavus agglutinin revealed secretory granules and RF, only. In the blots the immunostaining was used to identify secretory polypeptides. The glycosylated nature of the latter was established by their affinity for Con A and/or Limax flavus agglutinin. Furthermore, this latter lectin allowed us to distinguish whether the intracellular source of a secretory glycoprotein is from a pre-Golgi (RER) or a post-Golgi (secretory granules) compartment. Four glycoproteins were identified in the SCO with apparent molecular weights of 540, 450, 320 and 190 kDa. The three former were also purified by immunoaffinity chromatography. The 540 and 320 kDa forms are present in the SCO but missing in RF, have affinity for Con A, but not for LFA. It is suggested that these two compounds correspond to two precursor forms. The 450 and 190 kDa glycoproteins are present in both, the SCO and RF, and have affinity for Con A and Limax flavus agglutinin. These most likely correspond to processed forms. The presence of more than one precursor was further substantiated by immunocytochemical findings using antisera against the 540, 450 and 320 kDa forms.(ABSTRACT TRUNCATED AT 400 WORDS)
Nualart
F
F
Instituto de Histología y Patología, Universidad Austral de Chile, Valdivia.
Hein
S
S
Rodríguez
E M
EM
Oksche
A
A
D000818:Animals; D000906:Antibodies; D002417:Cattle; D002846:Chromatography, Affinity; D003594:Cytoplasmic Granules / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D015151:Immunoblotting; D007124:Immunoenzyme Techniques; D007150:Immunohistochemistry; D037102:Lectins; D008970:Molecular Weight; D013116:Spinal Cord / Q000166:cytology / Q000502:physiology*; D013351:Subcommissural Organ / Q000166:cytology / Q000378:metabolism*
D000906:Antibodies; D006023:Glycoproteins; D037102:Lectins