Chapter 4. Interactions of chemokines with glycosaminoglycans.

Many proteins require interactions with cell surface glycosaminoglycans (GAGs) to exert their biologic activity. The effect of GAG binding on protein function ranges from essential roles in development, organogenesis, cell growth, cell adhesion, inflammation, tumorigenesis, and interactions with pathogens. A classic example is the role of GAGs in the interaction of fibroblast growth factors with their receptors, where GAGs play a role in specificity determination and control of receptor-ligand engagement. The other well-studied example involves the binding of antithrombin to heparin/heparan sulfate, which results in the inactivation of the coagulation cascade. In view of their specialized activity in cellular recruitment, chemokines interact with GAGs, minimally as a mechanism for localization of chemokines to specific anatomical spaces enabling them to act as directional signals for migrating cells. The biological relevance of these interactions has been recently demonstrated by functional characterization of mutants that are deficient in GAG binding. These mutants bind receptor normally in vitro but are unable to recruit cells in vivo. Observations like this have motivated investigations to identify GAG-binding epitopes on chemokines, the specificity and affinity of chemokines for different GAGs, the oligomerization of chemokines on GAGs, and the efficacy of GAG-binding mutants in the context of in vivo cell recruitment and animal models of disease. To this end, several techniques have been developed to measure the interactions of chemokines with GAGs. In this chapter we describe these various assays with particular reference to those that have been used to assess the binding of chemokines to GAGs and to define their epitopes. In the end, we believe both in vitro and in vivo characterization are absolutely necessary for understanding these interactions and their biologic relevance in the context of the whole organism.

Protein function microarrays based on self-immobilizing and self-labeling fusion proteins.

Protein microarrays are an attractive approach for the high-throughput analysis of protein function, but their impact on proteomics has been limited by the technical difficulties associated with their generation. Here we demonstrate that fusion proteins of O6-alkylguanine-DNA alkyltransferase (AGT) can be used for the simple and reliable generation of protein microarrays for the analysis of protein function. Important features of the approach are the selectivity of the covalent immobilization; this allows for direct immobilization of proteins out of cell extracts, and the option both to label and to immobilize AGT fusion proteins, which allows for direct screening for protein-protein interactions between different AGT fusion proteins. In addition to the identification of protein-protein interactions, AGT-based protein microarrays can be used for the characterization of small molecule-protein interactions or post-translational modifications. The potential of the approach was demonstrated by investigating the post-translational modification of acyl carrier protein (ACP) from E. coli by different phosphopantetheine transferases (PPTases), yielding insights into the role of selected ACP amino acids in the ACP-PPTase interaction.

Engineering substrate specificity of O6-alkylguanine-DNA alkyltransferase for specific protein labeling in living cells.

Fusion proteins of human O(6)-alkylguanine-DNA alkyltransferase (AGT) can be specifically labeled with a wide variety of synthetic probes in mammalian cells; this makes them an attractive tool for studying protein function. However, to avoid undesired labeling of endogenous wild-type AGT (wtAGT), the specific labeling of AGT fusion proteins has been restricted to AGT-deficient mammalian cell lines. We present here the synthesis of an inhibitor of wtAGT and the generation of AGT mutants that are resistant to this inhibitor. This enabled the inactivation of wtAGT and specific labeling of fusion proteins of the AGT mutant in vitro and in living cells. The ability to specifically label AGT fusion proteins in the presence of endogenous AGT, after brief incubation of the cells with a small-molecule inhibitor, should significantly broaden the scope of application of AGT fusion proteins for studying protein function in living cells.

Protein-functionalized polymer brushes.

A new strategy for the preparation of protein-functionalized polymer brushes is reported, which is based on a combination of surface-initiated atom transfer radical polymerization (ATRP), p-nitrophenyl chloroformate activation of the surface hydroxyl groups, and subsequent O(6)-benzylguanine (BG) functionalization. The BG-functionalized brushes are used to chemoselectively immobilize O(6)-alkylguanine-DNA-alkyltransferase (AGT) fusion proteins with a defined orientation and surface density. These protein-modified polymer brushes are attractive candidates for the development of protein microarrays.

Synthesis and characterization of bifunctional probes for the specific labeling of fusion proteins.

Labeling proteins with synthetic probes is important for studying and characterizing protein function. We have recently introduced a general method for the specific in vivo and in vitro labeling of fusion proteins that is based on the reaction of O6-alkylguanine-DNA alkyltransferase (AGT) with O6-benzylguanine derivatives. Here we report two complementary routes for the synthesis of O6-benzylguanine derivatives, which allow for the labeling of AGT fusion proteins with bifunctional synthetic probes and demonstrate the specific labeling of AGT fusion proteins with these probes. These molecules should become useful tools for various applications in functional proteomics.