The purification and immunocytochemical localization of the major iodinatable cell surface glycoproteins of chinese hamster ovary cells

A group of high molecular weight, acidic glycoproteins (HMWAG) are enriched in a plasma membrane subfraction of low density that can be isolated from Chinese hamster ovary (CHO) cells. The HMWAG represent the major components labelled by either lactoperoxidase-catalysed iodination of whole cells or by 125I-labelled wheat germ agglutinin (WGA) overlays of the membrane fraction after it had been analysed by 2-dimensional electrophoresis. The HMWAG were solubilized from membrane in Triton X100/borate buffer and purified by affinity chromatography on WGA-Sepharose. Antisera prepared against these glycoproteins in mice bound to several CHO cell lines including the WGA-resistant line Pro-5 WGAB1. Indirect immunofluorescence experiments performed at 4 °C showed that the glycoproteins had a fairly uniform but punctate distribution over the cell edge and dorsal surface of substratum-attached cells. Filopodia projecting along the substratum were also intensely labelled. After permeabilizing the cells the HMWAG were also shown to be located in cytoplasmic vesicles and in a perinuclear zone, which is assumed to be Golgi-associated. Electron microscopic observations using indirect immunoferritin procedures confirmed that the cell surface HMWAG were heavily concentrated on microvilli. The distribution of label seen with fluorescein-conjugated WGA was similar to that noted with fluorescent antibody, except that WGA was more uniformly distributed on the cell surface and also bound to the nuclear envelope in permeabilized cells. However, if cells labelled at 4 °C were warmed to 37 °C for 30 min the patterns of internalization of WGA and antibody were distinct. With the lectin, label rapidly became concentrated in the perinuclear zone, whereas with antibody the microvilli became denuded of label, which was then internalized into cytoplasmic vacuoles just beneath the plasma membrane. These results confirm that the HMWAG are concentrated on regions of the cell surface that are likely to produce vesicles readily upon cell breakage. They also suggest that pattern of internalization of a cell surface glycoprotein may be influenced by the nature of the ligand that binds to it.