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.
Hancox
K A
KA
Children's Medical Research Foundation, Westmead, NSW, Australia.
Sheppard
A M
AM
Jeffrey
P L
PL
D000818:Animals; D000911:Antibodies, Monoclonal; D001921:Brain / Q000166:cytology / Q000196:embryology*; D001923:Brain Chemistry; D002531:Cerebellum / Q000737:chemistry* / Q000166:cytology / Q000196:embryology; D002642:Chick Embryo; D002645:Chickens; D002846:Chromatography, Affinity; D003840:Deoxycholic Acid; D003902:Detergents; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000032:analysis* / Q000302:isolation & purification; D007150:Immunohistochemistry; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology; D008970:Molecular Weight; D016548:Prosencephalon / Q000737:chemistry / Q000196:embryology; D012160:Retina / Q000737:chemistry / Q000166:cytology / Q000196:embryology; D012584:Sciatic Nerve / Q000737:chemistry / Q000166:cytology / Q000196:embryology*; D013116:Spinal Cord / Q000737:chemistry / Q000166:cytology / Q000196:embryology*; D013570:Synaptic Membranes / Q000737:chemistry / Q000502:physiology
D000911:Antibodies, Monoclonal; D003902:Detergents; D006023:Glycoproteins; D003840:Deoxycholic Acid