Wheat germ agglutinin is selectively transported to multivesicular bodies.
Colloidal iron dextran particles bearing wheat germ agglutinin (WGA/FeDex) were bound by glycoconjugates expressed at the surface of HepG2 cells. Bound WGA/FeDex was internalized when cells were incubated at 37 degrees C and accumulated in intracellular structures which have the same buoyant density as the plasma membrane when examined on Percoll density gradients. The intracellular structures containing WGA/FeDex were identified as multivesicular bodies (MVB) by transmission electron microscopy. WGA/FeDex was not transported to lysosomes nor did it interfere with uptake and transport of GalBSA to lysosomes by the asialoglycoprotein receptor. WGA/FeDex was seen predominantly in non-coated invaginations at the cell surface, suggesting it may enter cells at a different site than GalBSA/FeDex. Highly enriched plasma membranes and MVBs containing superparamagnetic [125I]WGA/FeDex particles were prepared by high gradient magnetic affinity chromatography (HIMAC). Plasma membranes prepared by HIMAC were enriched 30-fold for [125I]WGA/FeDex, 15-fold for alkaline phosphodiesterase I, and 9-fold for galactosyltransferase relative to the crude post-nuclear homogenate and consisted entirely of plasmalemmal sheets. Intracellular structures containing WGA/FeDex were enriched 35-fold for [125I]WGA/FeDex, 10-fold for alkaline phosphodiesterase I, and 10-fold for galactosyltransferase but did not contain lysosomal beta-galactosidase. WGA/FeDex has a different ultimate destination in HepG2 cells than ligands internalized by the asialoglycoprotein receptor and can be used to obtain highly enriched plasma membranes and MVBs from cultured cells.
Becich
M J
MJ
Department of Pathology, Washington University School of Medicine, St. Louis, MO 63110.
Mahklouf
S
S
Baenziger
J U
JU
D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D002458:Cell Fractionation / Q000379:methods; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D011992:Endosomes / Q000378:metabolism*; D006001:Glycoconjugates / Q000378:metabolism*; D006801:Humans; D007505:Iron-Dextran Complex / Q000378:metabolism*; D008024:Ligands; D008247:Lysosomes / Q000378:metabolism; D008854:Microscopy, Electron; D013696:Temperature; D014407:Tumor Cells, Cultured; D014909:Wheat Germ Agglutinins / Q000378:metabolism*
D006001:Glycoconjugates; D008024:Ligands; D014909:Wheat Germ Agglutinins; D007505:Iron-Dextran Complex