Glycoprotein synthesis at the synapse: fractionation of polypeptides synthesized within isolated dendritic fragments by concanavalin A affinity chromatography.
The synthesis of glycosylated proteins at postsynaptic sites was evaluated by combining metabolic labeling of isolated pinched-off dendritic fragments (synaptodendrosomes) with glycoprotein isolation by Con A affinity chromatography. Three major labeled proteins were detected (apparent molecular weights of 128, 42 and 19 kDa) along with seven minor polypeptides. Treatment of the glycoprotein fraction with N-glycosidase F led to shift in the apparent molecular weight of the bands. Also, label incorporation into glycoprotein species was blocked by tunicamycin. Thus, the three prominent polypeptides and most of the minor components of this fraction corresponded to bona fide N-glycoproteins. Incubation of synaptodendrosomes with cycloheximide also inhibited label incorporation into the isolated glycoproteins, indicating that the labeling resulted from local de novo synthesis. Subcellular fractionation revealed that the labeled glycoproteins were present in soluble and particulate fractions, mainly microsomes and synaptic membranes, and one of the species (42 kDa) appeared in the incubation medium, indicating secretion. In addition, these glycoproteins were dissimilarly distributed in several brain regions, and were expressed differentially during development, reaching their highest level of synthesis during the period of synaptogenesis. These results provide evidence for local dendritic synthesis of particular glycoprotein components of the synapse.
Villanueva
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S
Facultad de Medicina, Instituto de Ciencias Biomedicas, Universidad de Chile, Casilla 70005-7, 6530499, Santiago, Chile.
Steward
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O
D000581:Amidohydrolases; D000818:Animals; D000900:Anti-Bacterial Agents; D001921:Brain / Q000166:cytology / Q000254:growth & development / Q000502:physiology; D002846:Chromatography, Affinity / Q000379:methods*; D003208:Concanavalin A; D003513:Cycloheximide; D003712:Dendrites / Q000737:chemistry / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D018507:Gene Expression Regulation, Developmental; D006023:Glycoproteins / Q000032:analysis* / Q000235:genetics* / Q000302:isolation & purification; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D013347:Subcellular Fractions / Q000737:chemistry; D013569:Synapses / Q000737:chemistry / Q000502:physiology*; D014415:Tunicamycin
D000900:Anti-Bacterial Agents; D006023:Glycoproteins; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D003208:Concanavalin A; D014415:Tunicamycin; D003513:Cycloheximide; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase