FIGURE 8.

CW residues 33, 35, and 39 selectively interact with HS. A–C, ionic interactions of CW mutant forms ShhNp^{K33A/R35A/K39A}, ShhNp^K33A/R35A, and ShhNp^K39A with embryonic HS as determined by HS affinity chromatography. Bound proteins were eluted in a 0–1 m NaCl gradient; signal intensity is expressed relative to the signal intensity of the input set to 100%. ShhNp^{K33A/R35A/K39A} did not bind to any HS column, but ShhNp^K39A and ShhNp^K33A/R35A variably bound to immobilized HS. ShhNp^R34A/K38A always bound to all HS preparations. This indicates that residues Lys³³, Arg³⁵, and Lys³⁹ contribute to HS binding, Lys³⁹ being the most critical residue. Insets, representative immunoblotted fractions following HS affinity chromatography. D, C3H10T1/2 osteoblast precursor cell differentiation induced by mutant morphogens. C3H10T1/2 cells were stimulated with equivalent amounts of ShhNp, ShhNp^R34A/K38A, ShhNp^{K33A/R35A/K39A}, ShhNp^K33A/R35A, and ShhNp^K39A, and AP activity was quantified. ShhNp AP values were set to 100% in each experiment, and relative values for each mutant form were calculated. The observed increase in biological activity compared with ShhNp was always insignificant (ShhNp^{K33A/R35A/K39A}: 154 ± 25%, p = 0,055, n = 7; ShhNp^K33A/R35A: 134 ± 19%, p = 0.0891, n = 7; ShhNp^K39A: 127 ± 19%, p = 0.18, n = 7). The observed decrease in ShhNp^R34A/K38A biological activity was significant (72 ± 3%, p = 0,0001, n = 7).