Ran-2, a glial lineage marker, is a GPI-anchored form of ceruloplasmin

Abstract

Cell interactions in the nervous system are frequently mediated by surface proteins that are attached to the membrane by a glycosyl phosphatidylinositol (GPI) anchor. In this study, we have characterized the expression of such proteins on glial cells. We have detected a major GPI-anchored protein on astrocytes and Schwann cells, with a molecular weight of 140 kD. When Schwann cells were treated with forskolin to promote a myelinating phenotype, expression of this 140-kD protein dramatically decreased, whereas another GPI-anchored protein of 80 kD was strongly induced; expression of other integral membrane proteins were likewise dramatically altered. The size and pattern of expression of the 140-kD protein suggested that it might correspond to the Ran-2 antigen, a glial lineage marker. This notion was confirmed by immunoprecipitating this 140-kD protein with the Ran-2 monoclonal antibody. The Ran-2 antigen is expressed over the entire Schwann cell surface in a punctate fashion; it is removed by phosphatidylinositol phospholipase C treatment, thereby confirming that it is GPI-anchored. When Schwann cells are cocultured with neurons, the Ran-2 antigen initially concentrates at sites of Schwann cell contact with neurons, suggesting that it may play a role in early Schwann cell-neuron interactions; it is then downregulated. Protein sequencing of the Ran-2 antigen immunopurified from rat brain membranes showed complete identity over two extended segments with the copper binding protein ceruloplasmin. These findings indicate that astrocytes and Schwann cells express a novel GPI-anchored form of ceruloplasmin and suggest that this GPI form plays a role in axonal-glial interactions.

Targeted gene disruption reveals an essential role for ceruloplasmin in cellular iron efflux

“…A similar ferroxidase mechanism has also been proposed for the function of FET3 in iron uptake in yeast (28,29) and hephaestin in murine iron absorption (11). These two multicopper oxidases are membrane proteins, and recent studies have revealed a glycosyl-phosphatidylinositol-linked form of ceruloplasmin (30,31); however, additional experiments are needed to determine whether ceruloplasmin functions in solution or bound to the plasma membrane. The normal iron absorption in Cp Ϫ/Ϫ mice suggests that hephaestin is required for this process, consistent with the earlier observation of impaired iron transfer in copper-deficient animals.…”

Biochemical Analysis of a Missense Mutation in Aceruloplasminemia

“…Consistent with this concept, in patients with Wilson disease, the absence or dysfunction of a copper-transporting ATPase abrogates copper transfer into the secretory pathway, resulting in marked diminution in the serum concentration of ceruloplasmin (4). Extrahepatic synthesis of human ceruloplasmin has been detected in several tissues, including the retina and brain (5,6), and recent studies in rodents suggest that in brain and testis ceruloplasmin is synthesized as a glycosylphosphatidylinositol (GPI) 1 -anchored form via alternative RNA splicing (7)(8)(9)(10).…”

Copper Transport and Metabolism Are Normal in Aceruloplasminemic Mice

“…This protein is synthesized and secreted by hepatocytes as a holoprotein with six atoms of copper tightly incorporated during its biosynthesis (2). In addition to the plasma form of ceruloplasmin, an alternative splice product yields a glycosyl phosphatidylinositol membrane-anchored form that is present in specific tissues (3)(4)(5)(6). Although copper has no effect on the rate of apoceruloplasmin synthesis or secretion (7), the plasma half-life of apoceruloplasmin is shorter than holoceruloplasmin and makes the serum concentration of this protein and the loss of oxidase activity a sensitive marker for copper deficiency (8,9).…”