Krishna Mohan Sepuru

Chemokine CXCL1 mediated neutrophil recruitment: Role of glycosaminoglycan interactions

The chemokine CXCL1/MGSA plays a pivotal role in the host immune response by recruiting and activating neutrophils for microbial killing at the tissue site. CXCL1 exists reversibly as monomers and dimers, and mediates its function by binding glycosaminoglycans (GAG) and CXCR2 receptor. We recently showed that both monomers and dimers are potent CXCR2 agonists, the dimer is the high-affinity GAG ligand, lysine and arginine residues located in two non-overlapping domains mediate GAG interactions, and there is extensive overlap between GAG and receptor-binding domains. To understand how these structural properties influence in vivo function, we characterized peritoneal neutrophil recruitment of a trapped monomer and trapped dimer and a panel of WT lysine/arginine to alanine mutants. Monomers and dimers were active, but WT was more active indicating synergistic interactions promote recruitment. Mutants from both domains showed reduced GAG heparin binding affinities and reduced neutrophil recruitment, providing compelling evidence that both GAG-binding domains mediate in vivo trafficking. Further, mutant of a residue that is involved in both GAG binding and receptor signaling showed the highest reduction in recruitment. We conclude that GAG interactions and receptor activity of CXCL1 monomers and dimers are fine-tuned to regulate neutrophil trafficking for successful resolution of tissue injury.

CXCL1/MGSA is a novel glycosaminoglycan (GAG)-binding chemokine: structural evidence for two distinct non-overlapping binding domains

In humans, the chemokine CXCL1/MGSA (hCXCL1) plays fundamental and diverse roles in pathophysiology, from microbial killing to cancer progression, by orchestrating the directed migration of immune and non-immune cells. Cellular trafficking is highly regulated and requires concentration gradients that are achieved by interactions with sulfated glycosaminoglycans (GAGs). However, very little is known regarding the structural basis underlying hCXCL1-GAG interactions. We addressed this by characterizing the binding of GAG heparin oligosaccharides to hCXCL1 using NMR spectroscopy. Binding experiments under conditions at which hCXCL1 exists as monomers and dimers indicate that the dimer is the high-affinity GAG ligand. NMR experiments and modeling studies indicate that lysine and arginine residues mediate binding and that they are located in two non-overlapping domains. One domain, consisting of N-loop and C-helical residues (defined as α-domain) has also been identified previously as the GAG-binding domain for the related chemokine CXCL8/IL-8. The second domain, consisting of residues from the N terminus, 40s turn, and third β-strand (defined as β-domain) is novel. Eliminating β-domain binding by mutagenesis does not perturb α-domain binding, indicating two independent GAG-binding sites. It is known that N-loop and N-terminal residues mediate receptor activation, and we show that these residues are also involved in extensive GAG interactions. We also show that the GAG-bound hCXCL1 completely occlude receptor binding. We conclude that hCXCL1-GAG interactions provide stringent control over regulating chemokine levels and receptor accessibility and activation, and that chemotactic gradients mediate cellular trafficking to the target site.

Molecular Basis of Chemokine CXCL5-Glycosaminoglycan Interactions.

Chemokines, a large family of highly versatile small soluble proteins, play crucial roles in defining innate and adaptive immune responses by regulating the trafficking of leukocytes, and also play a key role in various aspects of human physiology. Chemokines share the characteristic feature of reversibly existing as monomers and dimers, and their functional response is intimately coupled to interaction with glycosaminoglycans (GAGs). Currently, nothing is known regarding the structural basis or molecular mechanisms underlying CXCL5-GAG interactions. To address this missing knowledge, we characterized the interaction of a panel of heparin oligosaccharides to CXCL5 using solution NMR, isothermal titration calorimetry, and molecular dynamics simulations. NMR studies indicated that the dimer is the high-affinity GAG binding ligand and that lysine residues from the N-loop, 40s turn, β3 strand, and C-terminal helix mediate binding. Isothermal titration calorimetry indicated a stoichiometry of two oligosaccharides per CXCL5 dimer. NMR-based structural models reveal that these residues form a contiguous surface within a monomer and, interestingly, that the GAG-binding domain overlaps with the receptor-binding domain, indicating that a GAG-bound chemokine cannot activate the receptor. Molecular dynamics simulations indicate that the roles of the individual lysines are not equivalent and that helical lysines play a more prominent role in determining binding geometry and affinity. Further, binding interactions and GAG geometry in CXCL5 are novel and distinctly different compared with the related chemokines CXCL1 and CXCL8. We conclude that a finely tuned balance between the GAG-bound dimer and free soluble monomer regulates CXCL5-mediated receptor signaling and function.

Solution Structure of CXCL5 - A Novel Chemokine and Adipokine Implicated in Inflammation and Obesity.

The chemokine CXCL5 is selectively expressed in highly specialized cells such as epithelial type II cells in the lung and white adipose tissue macrophages in muscle, where it mediates diverse functions from combating microbial infections by regulating neutrophil trafficking to promoting obesity by inhibiting insulin signaling. Currently very little is known regarding the structural basis of how CXCL5 mediates its novel functions. Towards this missing knowledge, we have solved the solution structure of the CXCL5 dimer by NMR spectroscopy. CXCL5 is a member of a subset of seven CXCR2-activating chemokines (CAC) that are characterized by the highly conserved ELR motif in the N-terminal tail. The structure shows that CXCL5 adopts the typical chemokine fold, but also reveals several distinct differences in the 30 s loop and N-terminal residues; not surprisingly, crosstalk between N-terminal and 30 s loop residues have been implicated as a major determinant of receptor activity. CAC function also involves binding to highly sulfated glycosaminoglycans (GAG), and the CXCL5 structure reveals a distinct distribution of positively charged residues, suggesting that differences in GAG interactions also influence function. The availability of the structure should now facilitate the design of experiments to better understand the molecular basis of various CXCL5 functions, and also serve as a template for the design of inhibitors for use in a clinical setting.

Structural Basis of Native CXCL7 Monomer Binding to CXCR2 Receptor N-Domain and Glycosaminoglycan Heparin

CXCL7, a chemokine highly expressed in platelets, orchestrates neutrophil recruitment during thrombosis and related pathophysiological processes by interacting with CXCR2 receptor and sulfated glycosaminoglycans (GAG). CXCL7 exists as monomers and dimers, and dimerization (~50 μM) and CXCR2 binding (~10 nM) constants indicate that CXCL7 is a potent agonist as a monomer. Currently, nothing is known regarding the structural basis by which receptor and GAG interactions mediate CXCL7 function. Using solution nuclear magnetic resonance (NMR) spectroscopy, we characterized the binding of CXCL7 monomer to the CXCR2 N-terminal domain (CXCR2Nd) that constitutes a critical docking site and to GAG heparin. We found that CXCR2Nd binds a hydrophobic groove and that ionic interactions also play a role in mediating binding. Heparin binds a set of contiguous basic residues indicating a prominent role for ionic interactions. Modeling studies reveal that the binding interface is dynamic and that GAG adopts different binding geometries. Most importantly, several residues involved in GAG binding are also involved in receptor interactions, suggesting that GAG-bound monomer cannot activate the receptor. Further, this is the first study that describes the structural basis of receptor and GAG interactions of a native monomer of the neutrophil-activating chemokine family.

Characterizing Protein–Glycosaminoglycan Interactions Using Solution NMR Spectroscopy

Solution nuclear magnetic resonance (NMR) spectroscopy and, in particular, chemical shift perturbation (CSP) titration experiments are ideally suited for characterizing the binding interface of macromolecular complexes. (1)H-(15) N-HSQC-based CSP studies have become the method of choice due to their simplicity, short time requirements, and not requiring high-level NMR expertise. Nevertheless, CSP studies for characterizing protein-glycosaminoglycan (GAG) interactions have been challenging due to binding-induced aggregation/precipitation and/or poor quality data. In this chapter, we discuss how optimizing experimental variables such as protein concentration, GAG size, and sensitivity of NMR instrumentation can overcome these roadblocks to obtain meaningful structural insights into protein-GAG interactions.

Regulation of chemokine function: The roles of GAG-binding and post-translational nitration

The primary function of chemokines is to direct the migration of leukocytes to the site of injury during inflammation. The effects of chemokines are modulated by several means, including binding to G-protein coupled receptors (GPCRs), binding to glycosaminoglycans (GAGs), and through post-translational modifications (PTMs). GAGs, present on cell surfaces, bind chemokines released in response to injury. Chemokines bind leukocytes via their GPCRs, which directs migration and contributes to local inflammation. Studies have shown that GAGs or GAG-binding peptides can be used to interfere with chemokine binding and reduce leukocyte recruitment. Post-translational modifications of chemokines, such as nitration, which occurs due to the production of reactive species during oxidative stress, can also alter their biological activity. This review describes the regulation of chemokine function by GAG-binding ability and by post-translational nitration. These are both aspects of chemokine biology that could be targeted if the therapeutic potential of chemokines, like CXCL8, to modulate inflammation is to be realised.

Platelet-Derived Chemokine CXCL7 Dimer Preferentially Exists in the Glycosaminoglycan-Bound Form: Implications for Neutrophil–Platelet Crosstalk

Platelet-derived chemokine CXCL7 (also known as NAP-2) plays a crucial role in orchestrating neutrophil recruitment in response to vascular injury. CXCL7 exerts its function by activating the CXC chemokine receptor 2 (CXCR2) receptor and binding sulfated glycosaminoglycans (GAGs) that regulate receptor activity. CXCL7 exists as monomers, dimers, and tetramers, and previous studies have shown that the monomer dominates at lower and the tetramer at higher concentrations. These observations then raise the question: what, if any, is the role of the dimer? In this study, we make a compelling observation that the dimer is actually the favored form in the GAG-bound state. Further, we successfully characterized the structural basis of dimer binding to GAG heparin using solution nuclear magnetic resonance (NMR) spectroscopy. The chemical shift assignments were obtained by exploiting heparin binding-induced NMR spectral changes in the WT monomer and dimer and also using a disulfide-linked obligate dimer. We observe that the receptor interactions of the dimer are similar to the monomer and that heparin-bound dimer is occluded from receptor interactions. Cellular assays also show that the heparin-bound CXCL7 is impaired for CXCR2 activity. We conclude that the dimer–GAG interactions play an important role in neutrophil–platelet crosstalk, and that these interactions regulate gradient formation and the availability of the free monomer for CXCR2 activation and intrathrombus neutrophil migration to the injury site.

Interleukin-8 mediates neutrophil-endothelial interactions in pig-to-human xenogeneic models

Human neutrophils are sequestered by pig lung xenografts within minutes during ex vivo perfusion. This phenomenon is not prevented by pig genetic modifications that remove xeno‐antigens or added human regulatory molecules intended to down‐regulate activation of complement and coagulation pathways. This study investigated whether recipient and donor interleukin‐8 (IL‐8), a chemokine known to attract and activate neutrophils during inflammation, is elaborated in the context of xenogeneic injury, and whether human or pig IL‐8 promote the adhesion of human neutrophils in in vitro xenograft models.

Glycosaminoglycan Interactions Fine-Tune Chemokine-Mediated Neutrophil Trafficking: Structural Insights and Molecular Mechanisms

Circulating neutrophils, rapidly recruited in response to microbial infection, form the first line in host defense. Humans express ~50 chemokines, of which a subset of seven chemokines, characterized by the conserved “Glu-Leu-Arg” motif, mediate neutrophil recruitment. Neutrophil-activating chemokines (NACs) share similar structures, exist as monomers and dimers, activate the CXCR2 receptor on neutrophils, and interact with tissue glycosaminoglycans (GAGs). Considering cellular assays have shown that NACs have similar CXCR2 activity, the question has been and remains, why do humans express so many NACs? In this review, we make the case that NACs are not redundant and that distinct GAG interactions determine chemokine-specific in vivo functions. Structural studies have shown that the GAG-binding interactions of NACs are distinctly different, and that conserved and specific residues in the context of structure determine geometries that could not have been predicted from sequences alone. Animal studies indicate recruitment profiles of monomers and dimers are distinctly different, monomer–dimer equilibrium regulates recruitment, and that recruitment profiles vary between chemokines and between tissues, providing evidence that GAG interactions orchestrate neutrophil recruitment. We propose in vivo GAG interactions impact several chemokine properties including gradients and lifetime, and that these interactions fine-tune and define the functional response of each chemokine that can vary between different cell and tissue types for successful resolution of inflammation.