Protein binding to galactosyl sulfatide. The indicated components
(single or equimolar pairs) were incubated at the indicated concentrations in
lipid-coated wells. Binding was detected with antibodies to laminin (FLAG
epitope-horseradish peroxidase), αLNNd (Myc), or mAgrin (agrin). The
average and S.D. values (n = 3) and fitted regressions for simple
binding (solid and dashed lines) are shown in each graph.
Panel A, binding of αLNNd to galactosyl-3-sulfate-ceramide.
Panel B, binding of mAgrin to galactosyl-3-sulfate-ceramide undiluted
(1/1) or diluted (1/4, 1/8) in galactosyl-ceramide. Panel C, binding
of (wt) laminin to galactosyl-3-sulfate-ceramide undiluted (1/1) or diluted
(1/8, 1/16) in galactosyl-ceramide. Panel D, Binding of Lm
(closed circles), LmΔαLN (closed triangles),
LmΔαLN + αLNNd (open triangles), and
LmΔβLN (closed inverted triangles). Panel E,
Binding of Lm (closed circles), LmΔLG (closed
diamonds), LmΔLG + mA (open diamonds), and
LmΔ(αLN&LG) (open circles). Panel F, Binding
of Lm (closed circles), LmΔαLN-L4b (closed
triangles), and LmΔαLN-L4b +αLNNd (open
triangles). Contributions fromαLNNd, mAgrin, and laminin LG and
αLN domains were detected. Dilution of the sulfated galactosyl ceramide
in galactosyl ceramide resulted in decreased binding for mAgrin and laminin.
Coupling of αLNNd to LmΔαLN, αLNNd to
LmΔαLN-L4b, and mA to LmΔLG increased the laminin
affinities.