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 chromat ography. Three major labeled proteins were detected (apparent molecular wei ghts 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 appar ent molecular weight of the bands. Also, label incorporation into glycoprot ein species was blocked by tunicamycin. Thus, the three prominent polypepti des and most of the minor components of this fraction corresponded to bona fide N-glycoproteins. Incubation of synaptodendrosomes with cycloheximide a lso inhibited label incorporation into the isolated glycoproteins. indicati ng that the labeling resulted from local de novo synthesis. Subcellular fra ctionation revealed that the labeled glycoproteins were present in soluble and particulate fractions, mainly microsomes and synaptic membranes, and on e of the species (42 kDa) appeared in the incubation medium, indicating sec retion. In addition, these glycoproteins were dissimilarly distributed in s everal brain regions, and were expressed differentially during development, reaching their highest level of synthesis during the period of synaptogene sis. These results provide evidence for local dendritic synthesis of partic ular glycoprotein components of the synapse. (C) 2001 Elsevier Science B.V. All rights reserved.