AvGp50, a predominantly axonally expressed glycoprotein, is a member of the IgLON's subfamily of cell adhesion molecules (CAMs).

We have previously reported a 50 kDa glycoprotein (AvGp50) expressed specifically in the chick nervous system [Hancox, K.A., Sheppard, A.M. and Jeffrey, P.L., Characterisation of a novel glycoprotein (AVGP50) in the avian nervous system, with a monoclonal antibody, Dev. Brain Res., 70 (1992) 25-37], and we present its molecular characterization. A PCR fragment was generated following sequencing of peptide and N-terminal fragments derived from purified AvGp50. A 1.58 kb clone (pUEX762) containing the 5'-UTR, the entire coding sequence and a short 3'-UTR was then isolated from a chick embryonic day 18 forebrain library. The deduced amino acid sequence encodes a 338 amino acid peptide containing a 31 amino acid signal peptide at the N-terminal and a 19 amino acid phosphatidylinositol glycan linkage sequence at the C-terminal. The mature protein contains three C2-immunoglobulin-like domains and a glycosyl phosphatidylinositol anchor and shares significant homology to other members of the immunoglobulin superfamily, including neural cell adhesion molecule (N-CAM), myelin-associated glycoprotein (MAG) and the Drosophila protein Amalgam. AvGp50 exhibits highest sequence identity to a recently classified subgroup of the immunoglobulin superfamily (IgLONs - immunoglobulin LAMP, OBCAM and neurotrimin - classified by Pimenta et al. [Pimenta, A.F., Zhukareva, V., Barbe, M.F., Reinoso, B.S., Grimley, C., Henzel, W., Fischer, I. and Levitt, P., The limbic system-associated membrane protein is an Ig superfamily member that mediates selective neuronal growth and axon targeting, Neuron, 15 (1995) 287-297], comprising the opioid binding cell adhesion molecule (OBCAM), neurotrimin and the limbic system-associated membrane protein (LAMP) suggesting that AvGp50 is a member of this subgroup. AvGp50 is expressed predominantly on the cell surface of axons, in particular Purkinje cell and granule cell axons in the cerebellum. In cerebellar and forebrain neuronal cultures, protein expression is exclusively located at the cell surface. Despite its cell surface localization, AvGp50 does not directly influence the outgrowth of neurons from explant cultures from ED8 to ED10 chick forebrain, prompting the suggestion that AvGp50 may act in later maturational events.

Characterization of two distinct populations of detergent resistant membrane complexes isolated from chick brain tissues.

We report the isolation of two distinct populations of detergent resistant membrane complexes (DRMCs) from 1-day-old chick brain, utilizing a procedure involving Triton X-100 insolubility and sucrose density gradient centrifugation. The first population is abundant (1.8% of the total homogenate protein), highly enriched for two glycosylphosphatidylinositol (GPI)-anchored proteins (Thy-1 and AvGp50), and not enriched for caveolin. The second population is of relatively low abundance (0.2% of the total homogenate), contains relatively low levels of Thy-1 and AvGp50 enrichment, and is highly enriched in caveolin. Both populations of DRMCs are enriched for cholesterol, ganglioside GM1, total kinase and tyrosine kinase activities, and c-Src and c-Fyn. However, there are differences in the Coomassie-stained protein profiles, phosphoprotein components, tyrosine kinase activity, and electron microscopic morphology when the Thy-1 and AvGp50-enriched DRMCs are compared to the caveolin-rich DRMCs. In addition, the GPI-enriched DRMCs contain CaM kinase type II immunoreactivity, whereas this molecule was undectable in the caveolin-rich DRMCs. The isolation of two distinct DRMC fractions may be representative of unique plasma membrane signaling domains involved in GPI-anchored protein or other receptor-mediated signal transduction within the avian nervous system. Further, we have demonstrated for the first time that nervous system tissue, in particular the hatch chick cerebellum, contains caveolin immunoreactivity.

Characterisation of a novel glycoprotein (AvGp50) in the avian nervous system, with a monoclonal antibody.

A size fractionated lentil lectin-positive fraction derived from a deoxycholate extract of 1-day-old chick forebrain membranes was used to generate a series of monoclonal antibodies (Mabs) against neural antigens. One of these, MabSA1.7 recognises a glycoprotein which is enriched in synaptic plasma membranes, designated AvGp50. Polyacrylamide gel electrophoresis and Western blots show that AvGp50 is comprised of at least two glycoforms, with M(r)s of 53 kDa and 49 kDa respectively. AvGp50 is nervous system specific and most abundantly expressed in the forebrain, tecta and cerebellum where its pattern of expression is developmentally regulated. Immunohistochemical data localises AvGp50 to regions characterised by highly concentrated synapses, in particular, the molecular and granule cell layers of the cerebellum and in the inner and outer plexiform layers in the retina. Solubilization of the protein with the detergent Triton X-100 shows that AvGp50 is predominantly a cytoskeletally associated glycoprotein. However, when a synaptic plasma membrane fraction was treated with Triton X-114, AvGp50 partitioned into the detergent phase. Digestion of the protein with N-glycanase cleaved five N-linked carbohydrate side chains and reduced the molecular weight to approximately 34 and 31 kDa. Removal of the carbohydrate side chains led to an almost complete loss of recognition of the 34 kDa glycoform by the MabSA1.7, suggesting that the monoclonal antibody recognises a carbohydrate rather than peptide epitope.