Abstract
The fly morphogen Hedgehog (Hh) and its mammalian orthologs, Sonic, Indian, and Desert hedgehog, are secreted signaling molecules that mediate tissue patterning during embryogenesis and function in tissue homeostasis and regeneration in the adult. The function of all Hh family members is regulated at the levels of morphogen multimerization on the surface of producing cells, multimer release, multimer diffusion to target cells, and signal reception. These mechanisms are all known to depend on interactions of positively charged Hh amino acids (the Cardin-Weintraub (CW) motif) with negatively charged heparan sulfate (HS) glycosaminoglycan chains. However, a precise mechanistic understanding of these interactions is still lacking. In this work, we characterized ionic HS interactions of multimeric Sonic hedgehog (called ShhNp) as well as mutant forms lacking one or more CW residues. We found that deletion of all five CW residues as well as site-directed mutagenesis of CW residues Lys(33), Arg(35), and Lys(39) (mouse nomenclature) abolished HS binding. In contrast, CW residues Arg(34) and Lys(38) did not contribute to HS binding. Analysis and validation of Shh crystal lattice contacts provided an explanation for this finding. We demonstrate that CW residues Arg(34) and Lys(38) make contact with an acidic groove on the adjacent molecule in the multimer, suggesting a new function of these residues in ShhNp multimerization rather than HS binding. Therefore, the recombinant monomeric morphogen (called ShhN) differs in CW-dependent HS binding and biological activity from physiologically relevant ShhNp multimers, providing new explanations for functional differences observed between ShhN and ShhNp.
Highlights
The proteins of the Hh family are powerful morphogens that control growth and patterning of developing embryos
heparan sulfate proteoglycan (HSPG) have been implicated in Hh release, spreading, and reception, and Shh binds to heparan sulfate (HS) via its CW motif
We demonstrate that monomeric and multimeric morphogens differ in their capacity to bind HS
Summary
Cloning and Expression of Recombinant Shh—Shh constructs were generated from murine cDNA (NM 009170) by PCR. To determine heparin binding properties of ShhN and ShhNp, the supernatant of transfected Bosc cells was subjected to heparin affinity chromatography (Akta Protein Purifier) using heparin-Sepharose columns (Amersham Biosciences) at 4 °C. Proteins were applied to the columns in the absence of salt, and bound material was eluted with a linear NaCl gradient from 0 to 1.5 M in 0.1 M sodium acetate buffer (pH 6.0). Cleared supernatants of transfected cells, containing the various morphogens, were subjected to automated Akta affinity purification using a linear salt gradient from 0 to 1 M NaCl in 0.1 M sodium acetate buffer (pH 6.0) for protein elution. SAP-ShhN-containing medium was adjusted to 0.6 M NaCl to increase binding specificity and was applied to 4% paraformaldehyde-fixed C3H10T1/2 osteoblast precursor cells and frozen embryo sections overnight, followed by three washing steps with PBS. Crystal contacts were calculated employing CryCo (available on the World Wide Web)
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