Multivalency of Sonic Hedgehog Conjugated to Linear Polymer Chains Modulates Protein Potency

Abstract

A potently active multivalent form of the protein Sonic hedgehog (Shh) was produced by bioconjugation of a modified recombinant form of Shh to the linear polymers poly(acrylic acid) (pAAc) and hyaluronic acid (HyA) via a two-step reaction exploiting carboimiide and maleimide chemistry. Efficiency of the conjugation was approximately 75% even at stoichiometric ratios of 30 Shh molecules per linear HyA chain (i.e., 30:1 Shh/HyA). Bioactivity of the conjugates was tested via a cellular assay across a range of stoichiometric ratios of Shh molecules to HyA linear chains, which was varied from 0.6:1 Shh/HyA to 22:1 Shh/HyA. Results indicate that low conjugation ratios decrease Shh bioactivity and high ratios increase this activity beyond the potency of monomeric Shh, with approximately equal activity between monomeric soluble Shh and conjugated Shh at 7:1 Shh/HyA. In addition, high-ratio constructs increased angiogenesis determined by the in vivo chick chorioallantoic membrane (CAM) assay. These results are captured by a kinetic model of multiple interactions between the Shh/HyA conjugates and cell surface receptors resulting in higher cell signaling at lower bulk Shh concentrations.

Multivalent Conjugates of Sonic Hedgehog Accelerate Diabetic Wound Healing

“…28 We have previously validated methods to effectively measure the valency of these nanoscale protein conjugates, 29 and we have demonstrated that multivalent conjugation of the growth factor Sonic hedgehog (Shh) to linear polymer chains of hyaluronic acid (HyA) yields increasing Shh-pathway activation with higher Shh valencies. 30,31 Furthermore, multivalent Shh conjugates (mvShh) induced enhanced angiogenic function relative to an equivalent dose of unconjugated Shh. 30 Taken together, mvShh conjugates are capable of increasing the per-molecule bioactivity of Shh to elicit therapeutic cellular responses of Shh at lower tissue-level concentrations.…”

“…30,31 Furthermore, multivalent Shh conjugates (mvShh) induced enhanced angiogenic function relative to an equivalent dose of unconjugated Shh. 30 Taken together, mvShh conjugates are capable of increasing the per-molecule bioactivity of Shh to elicit therapeutic cellular responses of Shh at lower tissue-level concentrations. Thus, more generally, we propose that although growth factors may be cleared from the target tissues, their therapeutic function can be achieved at lower tissue-level concentrations by employing multivalent conjugates.…”

Structure of the protein core of the glypican Dally-like and localization of a region important for hedgehog signaling

“…An additional element complicating interpretation of results of studies of receptor-ligand interactions in solution arises from the absence of membrane tethering and ligand multivalency. Restricting components to a membrane surface orients them and greatly enhances their local concentration (36), and a multivalent ligand greatly increases the avidity of binding (37)(38)(39). Interactions between a monovalent ligand and cell-surface components may thus not be strong enough to be observed with soluble components in solution.…”

Peptide immobilized on gold particles enhances cell growth

“…The immobilized TPO-P-Cys had higher activity than soluble TPO-P-Cys, although the activity of immobilized TPO-P-Cys was lower than that of rhTPO. The effects of multivalent ligands have been discussed by many researchers (Huskens 2006;Kiessling et al 2006;Ito 2008;Wall et al 2008). They govern many interactions between proteins and small molecules, between proteins or antibodies and cell membranes, between viruses and cells, etc.…”

“…Natural and synthetic multivalent ligands can function as potent inhibitors or effectors of biological processes (Huskens 2006;Kiessling et al 2006;Wall et al 2008). Because they present multiple copies of a receptor-binding element, multivalent ligands can bind to receptors with high avidity and specificity, thereby acting as powerful inhibitors.…”