CXCL17 is a proinflammatory chemokine and promotes neutrophil trafficking.

CXCL17, a novel member of the CXC chemokine class, has been implicated in several human pathologies, but its role in mediating immune response is not well understood. Characteristic features of immune response include resident macrophages orchestrating successive and structured recruitment of neutrophils and monocytes to the insult site. Here, we show that Cxcl17 knockout (KO) mice, compared with the littermate wild-type control mice, were significantly impaired in peritoneal neutrophil recruitment post-lipopolysaccharide (LPS) challenge. Further, the KO mice show dysregulated Cxcl1, Cxcr2, and interleukin-6 levels, all of which directly impact neutrophil recruitment. Importantly, the KO mice showed no difference in monocyte recruitment post-LPS challenge or in peritoneal macrophage levels in both unchallenged and LPS-challenged mice. We conclude that Cxcl17 is a proinflammatory chemokine and that it plays an important role in the early proinflammatory response by promoting neutrophil recruitment to the insult site.

CXCR2 chemokine receptor - a master regulator in cancer and physiology.

Recent findings have modified our understanding of the roles of chemokine receptor CXCR2 and its ligands in cancer, inflammation, and immunity. Studies in Cxcr2 tissue-specific knockout mice show that this receptor is involved in, among other things, cancer, central nervous system (CNS) function, metabolism, reproduction, COVID-19, and the response to circadian cycles. Moreover, CXCR2 involvement in neutrophil function has been revisited not only in physiology but also for its major contribution to cancers. The recent unfolding of the role of CXCR2 in numerous cancers has led to extensive evaluation of multiple CXCR2 antagonists in preclinical and clinical studies. In this review we discuss the potential of targeting CXCR2 for cancer treatment.

Cxcl1 monomer-dimer equilibrium controls neutrophil extravasation.

The chemokine Cxcl1 plays a crucial role in recruiting neutrophils in response to infection. The early events in chemokine-mediated neutrophil extravasation involve a sequence of highly orchestrated steps including rolling, adhesion, arrest, and diapedesis. Cxcl1 function is determined by its properties of reversible monomer-dimer equilibrium and binding to Cxcr2 and glycosaminoglycans. Here, we characterized how these properties orchestrate extravasation using intravital microscopy of the cremaster. Compared to WT Cxcl1, which exists as both a monomer and a dimer, the trapped dimer caused faster rolling, less adhesion, and less extravasation. Whole-mount immunofluorescence of the cremaster and arrest assays confirmed these data. Moreover, the Cxcl1 dimer showed impaired LFA-1-mediated neutrophil arrest that could be attributed to impaired Cxcr2-mediated ERK signaling. We conclude that Cxcl1 monomer-dimer equilibrium and potent Cxcr2 activity of the monomer together coordinate the early events in neutrophil recruitment.

Chemokine Cxcl1-Cxcl2 heterodimer is a potent neutrophil chemoattractant.

Microbial infection is characterized by release of multiple proinflammatory chemokines that direct neutrophils to the insult site. How collective function of these chemokines orchestrates neutrophil recruitment is not known. Here, we characterized the role for heterodimer and show that the Cxcl1-Cxcl2 heterodimer is a potent neutrophil chemoattractant in mice and can recruit more neutrophils than the individual chemokines. Chemokine-mediated neutrophil recruitment is determined by Cxcr2 receptor signaling, Cxcr2 endocytosis, and binding to glycosaminoglycans. We have now determined heterodimer's Cxcr2 activity using cellular assays and Cxcr2 density in blood and recruited neutrophils in heterodimer-treated mice. We have shown that the heterodimer binds glycosaminoglycans with higher affinity and more efficiently than Cxcl1 or Cxcl2. These data collectively indicate that optimal glycosaminoglycan interactions and dampened receptor activity acting in concert in a dynamic fashion promote heterodimer-mediated robust neutrophil recruitment. We propose that this could play a critical role in combating infection.

Structural basis of a chemokine heterodimer binding to glycosaminoglycans.

Chemokines Cxcl1/KC and Cxcl2/MIP2 play a crucial role in coordinating neutrophil migration to the insult site. Chemokines' recruitment activity is regulated by monomer-dimer equilibrium and binding to glycosaminoglycans (GAGs). GAG chains exist as covalently linked to core proteins of proteoglycans (PGs) and also as free chains due to cleavage by heparanases during the inflammatory response. Compared with free GAGs, binding to GAGs in a PG is influenced by their fixed directionality due to covalent linkage and restricted mobility. GAG interactions impact chemokine monomer/dimer levels, chemotactic and haptotactic gradients, life time, and presentation for receptor binding. Here, we show that Cxcl1 and Cxcl2 also form heterodimers. Using a disulfide-trapped Cxcl1-Cxcl2 heterodimer, we characterized its binding to free heparin using nuclear magnetic resonance and isothermal titration calorimetry, and to immobilized heparin and heparan sulfate using surface plasmon resonance. These data, in conjunction with molecular docking, indicate that the binding characteristics such as geometry and stoichiometry of the heterodimer are different between free and immobilized GAGs and are also distinctly different from those of the homodimers. We propose that the intrinsic asymmetry of the heterodimer structure, along with differences in its binding to PG GAGs and free GAGs, regulate chemokine function.

Neutrophil recruitment by chemokines Cxcl1/KC and Cxcl2/MIP2: Role of Cxcr2 activation and glycosaminoglycan interactions.

Chemokines play a crucial role in combating microbial infection by recruiting blood neutrophils to infected tissue. In mice, the chemokines Cxcl1/KC and Cxcl2/MIP2 fulfill this role. Cxcl1 and Cxcl2 exist as monomers and dimers, and exert their function by activating the Cxcr2 receptor and binding glycosaminoglycans (GAGs). Here, we characterized Cxcr2 G protein and ß-arrestin activities, and GAG heparan sulfate (HS) interactions of Cxcl1 and Cxcl2 and of the trapped dimeric variants. To understand how Cxcr2 and GAG interactions impact in vivo function, we characterized their neutrophil recruitment activity to the peritoneum, Cxcr2 and CD11b levels on peritoneal and blood neutrophils, and transport profiles out of the peritoneum. Cxcl2 variants compared with Cxcl1 variants were more potent for Cxcr2 activity. Native Cxcl1 compared with native Cxcl2 and dimers compared with native proteins bound HS with higher affinity. Interestingly, recruitment activity between native Cxcl1 and Cxcl2, between dimers, and between the native protein and the dimer could be similar or very different depending on the dose or the time point. These data indicate that peritoneal neutrophil recruitment cannot be solely attributed to Cxcr2 or GAG interactions, and that the relationship between recruited neutrophils, Cxcr2 activation, GAG interactions, and chemokine levels is complex and highly context dependent. We propose that the ability of Cxcl1 and Cxcl2 to reversibly exist as monomers and dimers and differences in their Cxcr2 activity and GAG interactions coordinate neutrophil recruitment and activation, which play a critical role for successful resolution of inflammation.

Structural Insights Into How Proteoglycans Determine Chemokine-CXCR1/CXCR2 Interactions: Progress and Challenges.

Proteoglycans (PGs), present in diverse environments, such as the cell membrane surface, extracellular milieu, and intracellular granules, are fundamental to life. Sulfated glycosaminoglycans (GAGs) are covalently attached to the core protein of proteoglycans. PGs are complex structures, and are diverse in terms of amino acid sequence, size, shape, and in the nature and number of attached GAG chains, and this diversity is further compounded by the phenomenal diversity in GAG structures. Chemokines play vital roles in human pathophysiology, from combating infection and cancer to leukocyte trafficking, immune surveillance, and neurobiology. Chemokines mediate their function by activating receptors that belong to the GPCR class, and receptor interactions are regulated by how, when, and where chemokines bind GAGs. GAGs fine-tune chemokine function by regulating monomer/dimer levels and chemotactic/haptotactic gradients, which are also coupled to how they are presented to their receptors. Despite their small size and similar structures, chemokines show a range of GAG-binding geometries, affinities, and specificities, indicating that chemokines have evolved to exploit the repertoire of chemical and structural features of GAGs. In this review, we summarize the current status of research on how GAG interactions regulate ELR-chemokine activation of CXCR1 and CXCR2 receptors, and discuss knowledge gaps that must be overcome to establish causal relationships governing the impact of GAG interactions on chemokine function in human health and disease.

Lyophilization of human amniotic fluid is feasible without affecting biological activity.

BACKGROUND: Fetal swallowing of human amniotic fluid (hAF) containing trophic factors (TFs) promotes gastrointestinal tract (GIT) development. Preterm birth interrupts hAF swallowing, which may increase the risk of necrotizing enterocolitis (NEC). Postnatally, it is difficult to replicate fetal swallowing of hAF due to volume. We aimed to evaluate whether hAF lyophilization is feasible and its effect on hAF-borne TFs. METHODS: We collected hAF (n = 16) from uncomplicated pregnancies. hAF was divided into three groups: unprocessed control (C), concentration by microfiltration (F), and by dialysis and lyophilization (L). EGF, HGF, GM-CSF, and TGF-α were measured in each group by multiplex assay. Bioavailability of TFs was measured by proliferation and LPS-induced IL-8 production by intestinal epithelial cells FHs74. RESULTS: After dialysis/lyophilization, GM-CSF and TGF-α were preserved with partial loss of EGF and HGF. hAF increased cell proliferation and reduced LPS-induced IL-8 production compared to medium alone. Compared to control, dialysis/lyophilization and filtration of hAF increased FHs74 cell proliferation (p < 0.001) and decreased LPS-induced IL-8 production (p < 0.01). CONCLUSIONS: Lyophilization and filtration of hAF is feasible with partial loss of TFs but maintains and even improves bioavailability of TFs measured by proliferation and LPS-induced IL-8 production by FHs74.

Distinct Differences in Structural States of Conserved Histidines in Two Related Proteins: NMR Studies of the Chemokines CXCL1 and CXCL8 in the Free Form and Macromolecular Complexes.

Hydrogen-bonding and ionic interactions play fundamental roles in macromolecular recognition and function. In contrast to lysines and arginines, how histidines mediate these interactions is less well-understood due to the unique properties of its side chain imidazole that include an aromatic ring with two titratable nitrogens, a p Ka that can vary significantly, and the ability to exist in three distinct forms: protonated imidazolium and two tautomeric neutral (Nδ1 and Nε2) states. Here, we characterized the structural features of histidines in the chemokines CXCL8 and CXCL1 in the free, GAG heparin-bound, and CXCR2 receptor N-terminal domain-bound states using solution NMR spectroscopy. CXCL8 and CXCL1 share two conserved histidines, one in the N-loop and the other in the 30s loop. In CXCL8, both histidines exist in the Nε2 tautomeric state in the free, GAG-bound, and receptor-bound forms. On the other hand, in unliganded CXCL1, each of the two histidines exists in two states, as the neutral Nε2 tautomer and charged imidazolium. Further, both histidines exclusively exist as the imidazolium in the GAG-bound and as the Nε2 tautomer in the receptor-bound forms. The N-loop histidine alone in both chemokines is involved in direct GAG and receptor interactions, indicating the role of the 30s loop varies between the chemokines. Our observation that the structural features of conserved histidines and their functional role in two related proteins can be quite different is novel. We further propose that directly probing the imidazole structural features is essential to fully appreciate the molecular basis of histidine function.

Structural basis, stoichiometry, and thermodynamics of binding of the chemokines KC and MIP2 to the glycosaminoglycan heparin.

Keratinocyte-derived chemokine (KC or mCXCL1) and macrophage inflammatory protein 2 (MIP2 or mCXCL2) play nonredundant roles in trafficking blood neutrophils to sites of infection and injury. The functional responses of KC and MIP2 are intimately coupled to their interactions with glycosaminoglycans (GAGs). GAG interactions orchestrate chemokine concentration gradients and modulate receptor activity, which together regulate neutrophil trafficking. Here, using NMR, molecular dynamics (MD) simulations, and isothermal titration calorimetry (ITC), we characterized the molecular basis of KC and MIP2 binding to the GAG heparin. Both chemokines reversibly exist as monomers and dimers, and the NMR analysis indicates that the dimer binds heparin with higher affinity. The ITC experiments indicate a stoichiometry of two GAGs per KC or MIP2 dimer and that the enthalpic and entropic contributions vary significantly between the two chemokine-heparin complexes. NMR-based structural models of heparin-KC and heparin-MIP2 complexes reveal that different combinations of residues from the N-loop, 40s turn, ß3-strand, and C-terminal helix form a binding surface within a monomer and that both conserved residues and residues unique to a particular chemokine mediate the binding interactions. MD simulations indicate significant residue-specific differences in their contribution to binding and affinity for a given chemokine and between chemokines. On the basis of our observations that KC and MIP2 bind to GAG via distinct molecular interactions, we propose that the differences in these GAG interactions lead to differences in neutrophil recruitment and play nonoverlapping roles in resolution of inflammation.

Direct detection of lysine side chain NH3+ in protein-heparin complexes using NMR spectroscopy.

Two NMR observables, the NζH3+ peak in the HISQC spectrum and Nζ chemical shift difference between the free and heparin-bound forms, can identify binding-interface lysines in protein-heparin complexes. Unlike backbone chemical shifts, these direct probes are stringent and are less prone to either false positives or false negatives.

Chemokine CXCL7 Heterodimers: Structural Insights, CXCR2 Receptor Function, and Glycosaminoglycan Interactions.

Chemokines mediate diverse fundamental biological processes, including combating infection. Multiple chemokines are expressed at the site of infection; thus chemokine synergy by heterodimer formation may play a role in determining function. Chemokine function involves interactions with G-protein-coupled receptors and sulfated glycosaminoglycans (GAG). However, very little is known regarding heterodimer structural features and receptor and GAG interactions. Solution nuclear magnetic resonance (NMR) and molecular dynamics characterization of platelet-derived chemokine CXCL7 heterodimerization with chemokines CXCL1, CXCL4, and CXCL8 indicated that packing interactions promote CXCL7-CXCL1 and CXCL7-CXCL4 heterodimers, and electrostatic repulsive interactions disfavor the CXCL7-CXCL8 heterodimer. As characterizing the native heterodimer is challenging due to interference from monomers and homodimers, we engineered a "trapped" disulfide-linked CXCL7-CXCL1 heterodimer. NMR and modeling studies indicated that GAG heparin binding to the heterodimer is distinctly different from the CXCL7 monomer and that the GAG-bound heterodimer is unlikely to bind the receptor. Interestingly, the trapped heterodimer was highly active in a Ca2+ release assay. These data collectively suggest that GAG interactions play a prominent role in determining heterodimer function in vivo. Further, this study provides proof-of-concept that the disulfide trapping strategy can serve as a valuable tool for characterizing the structural and functional features of a chemokine heterodimer.

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.

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, ß3 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.

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 ß-strand (defined as ß-domain) is novel. Eliminating ß-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.

Solution NMR characterization of chemokine CXCL8/IL-8 monomer and dimer binding to glycosaminoglycans: structural plasticity mediates differential binding interactions.

Chemokine CXCL8/interleukin-8 (IL-8) plays a crucial role in directing neutrophils and oligodendrocytes to combat infection/injury and tumour cells in metastasis development. CXCL8 exists as monomers and dimers and interaction of both forms with glycosaminoglycans (GAGs) mediate these diverse cellular processes. However, very little is known regarding the structural basis underlying CXCL8-GAG interactions. There are conflicting reports on the affinities, geometry and whether the monomer or dimer is the high-affinity GAG ligand. To resolve these issues, we characterized the binding of a series of heparin-derived oligosaccharides [heparin disaccharide (dp2), heparin tetrasaccharide (dp4), heparin octasaccharide (dp8) and heparin 14-mer (dp14)] to the wild-type (WT) dimer and a designed monomer using solution NMR spectroscopy. The pattern and extent of binding-induced chemical shift perturbation (CSP) varied between dimer and monomer and between longer and shorter oligosaccharides. NMR-based structural models show that different interaction modes coexist and that the nature of interactions varied between monomer and dimer and oligosaccharide length. MD simulations indicate that the binding interface is structurally plastic and provided residue-specific details of the dynamic nature of the binding interface. Binding studies carried out under conditions at which WT CXCL8 exists as monomers and dimers provide unambiguous evidence that the dimer is the high-affinity GAG ligand. Together, our data indicate that a set of core residues function as the major recognition/binding site, a set of peripheral residues define the various binding geometries and that the structural plasticity of the binding interface allows multiplicity of binding interactions. We conclude that structural plasticity most probably regulates in vivo CXCL8 monomer/dimer-GAG interactions and function.

Chemokine CXCL1-Mediated Neutrophil Trafficking in the Lung: Role of CXCR2 Activation.

The chemokine CXCL1 and its receptor CXCR2 play a crucial role in host immune response by recruiting and activating neutrophils for microbial killing at the tissue site. Dysregulation in this process has been implicated in collateral tissue damage causing disease. CXCL1 reversibly exists as monomers and dimers, and it has been proposed that distinct monomer and dimer activities and the monomer-dimer equilibrium regulate the neutrophil function. However, the molecular mechanisms linking the CXCL1/CXCR2 axis and the neutrophil 'beneficial' and 'destructive' phenotypes are not known. In this study, we characterized neutrophil trafficking and its consequence in the mouse lung by the CXCL1 wild type (WT), which exists as monomers and dimers, and by a nondissociating dimer. Whereas the WT, compared to the dimer, was more active at low doses, both the WT and the dimer elicited a large neutrophil efflux at high doses. Importantly, robust neutrophil recruitment elicited by the WT or dimer was not detrimental to lung tissue integrity and, further, could not be correlated to surface CXCR2 levels. We conclude that the CXCL1 monomer-dimer distribution and receptor interactions are highly coupled and regulate neutrophil trafficking and that injury in the context of disease is a consequence of inappropriate CXCR2 activation at the target tissue and not due to mechanical forces exerted by neutrophils during recruitment.

Solution NMR characterization of WT CXCL8 monomer and dimer binding to CXCR1 N-terminal domain.

Chemokine CXCL8 and its receptor CXCR1 are key mediators in combating infection and have also been implicated in the pathophysiology of various diseases including chronic obstructive pulmonary disease (COPD) and cancer. CXCL8 exists as monomers and dimers but monomer alone binds CXCR1 with high affinity. CXCL8 function involves binding two distinct CXCR1 sites - the N-terminal domain (Site-I) and the extracellular/transmembrane domain (Site-II). Therefore, higher monomer affinity could be due to stronger binding at Site-I or Site-II or both. We have now characterized the binding of a human CXCR1 N-terminal domain peptide (hCXCR1Ndp) to WT CXCL8 under conditions where it exists as both monomers and dimers. We show that the WT monomer binds the CXCR1 N-domain with much higher affinity and that binding is coupled to dimer dissociation. We also characterized the binding of two CXCL8 monomer variants and a trapped dimer to two different hCXCR1Ndp constructs, and observe that the monomer binds with ∼10- to 100-fold higher affinity than the dimer. Our studies also show that the binding constants of monomer and dimer to the receptor peptides, and the dimer dissociation constant, can vary significantly as a function of pH and buffer, and so the ability to observe WT monomer peaks is critically dependent on NMR experimental conditions. We conclude that the monomer is the high affinity CXCR1 agonist, that Site-I interactions play a dominant role in determining monomer vs. dimer affinity, and that the dimer plays an indirect role in regulating monomer function.

Molecular basis of glycosaminoglycan heparin binding to the chemokine CXCL1 dimer.

Glycosaminoglycan (GAG)-bound and soluble chemokine gradients in the vasculature and extracellular matrix mediate neutrophil recruitment to the site of microbial infection and sterile injury in the host tissue. However, the molecular principles by which chemokine-GAG interactions orchestrate these gradients are poorly understood. This, in part, can be directly attributed to the complex interrelationship between the chemokine monomer-dimer equilibrium and binding geometry and affinities that are also intimately linked to GAG length. To address some of this missing knowledge, we have characterized the structural basis of heparin binding to the murine CXCL1 dimer. CXCL1 is a neutrophil-activating chemokine and exists as both monomers and dimers (Kd = 36 µm). To avoid interference from monomer-GAG interactions, we designed a trapped dimer (dCXCL1) by introducing a disulfide bridge across the dimer interface. We characterized the binding of GAG heparin octasaccharide to dCXCL1 using solution NMR spectroscopy. Our studies show that octasaccharide binds orthogonally to the interhelical axis and spans the dimer interface and that heparin binding enhances the structural integrity of the C-terminal helical residues and stability of the dimer. We generated a quadruple mutant (H20A/K22A/K62A/K66A) on the basis of the binding data and observed that this mutant failed to bind heparin octasaccharide, validating our structural model. We propose that the stability enhancement of dimers upon GAG binding regulates in vivo neutrophil trafficking by increasing the lifetime of "active" chemokines, and that this structural knowledge could be exploited for designing inhibitors that disrupt chemokine-GAG interactions and neutrophil homing to the target tissue.

Real-time kinetics of high-mobility group box 1 (HMGB1) oxidation in extracellular fluids studied by in situ protein NMR spectroscopy.

Some extracellular proteins are initially secreted in reduced forms via a non-canonical pathway bypassing the endoplasmic reticulum and become oxidized in the extracellular space. One such protein is HMGB1 (high-mobility group box 1). Extracellular HMGB1 has different redox states that play distinct roles in inflammation. Using a unique NMR-based approach, we have investigated the kinetics of HMGB1 oxidation and the half-lives of all-thiol and disulfide HMGB1 species in serum, saliva, and cell culture medium. In this approach, salt-free lyophilized (15)N-labeled all-thiol HMGB1 was dissolved in actual extracellular fluids, and the oxidation and clearance kinetics were monitored in situ by recording a series of heteronuclear (1)H-(15)N correlation spectra. We found that the half-life depends significantly on the extracellular environment. For example, the half-life of all-thiol HMGB1 ranged from ~17 min (in human serum and saliva) to 3 h (in prostate cancer cell culture medium). Furthermore, the binding of ligands (glycyrrhizin and heparin) to HMGB1 significantly modulated the oxidation kinetics. Thus, the balance between the roles of all-thiol and disulfide HMGB1 proteins depends significantly on the extracellular environment and can also be artificially modulated by ligands. This is important because extracellular HMGB1 has been suggested as a therapeutic target for inflammatory diseases and cancer. Our work demonstrates that the in situ protein NMR approach is powerful for investigating the behavior of proteins in actual extracellular fluids containing an enormous number of different molecules.