FIGURE 5.

Binding of two gangliosides enhances HCR/C entry into N2A cells. Assessment of HCR/C, HCR/C(Y1119A), and HCR/C(A1126K) trafficking in N2A cells enriched with exogenous ganglioside. N2A cells were enriched with 50 μg of GD1b/well for 4 h. The indicated HCR (40 nm) was incubated in N2A cells with or without exogenous ganglioside for 30 min. Unbound HCR was washed away, and cells were processed for microscopy. Instrument settings and exposure times were consistent between treatments. Images were prepared for publication and set to equivalent brightness and contrast settings using NIS-Elements (Nikon Instruments). Representative micrographs are shown. Magnification is similar to that described in the legend for Fig. 1, D and E. A Sia-1 site-competent derivative of HCR/C (HCR/C(Y1119A)) is able to enter N2A cells with ganglioside. These results indicate that two intact ganglioside binding sites are required for efficient trafficking of HCR/C into cells. However, binding GD1b through the Sia-1 site appears to support entry.