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.
Department of Pathology, Washington University School of Medicine, St. Louis, MO 63110.
M J
MJ
Becich
S
S
Mahklouf
J U
JU
Baenziger
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