The Novel CXCL12γ Isoform Encodes an Unstructured Cationic Domain Which Regulates Bioactivity and Interaction with Both Glycosaminoglycans and CXCR4

International audienceBACKGROUND: CXCL12alpha, a chemokine that importantly promotes the oriented cell migration and tissue homing of many cell types, regulates key homeostatic functions and pathological processes through interactions with its cognate receptor (CXCR4) and heparan sulfate (HS). The alternative splicing of the cxcl12 gene generates a recently identified isoform, CXCL12gamma, which structure/function relationships remain unexplored. The high occurrence of basic residues that characterize this isoform suggests however that it could feature specific regulation by HS. METHODOLOGY/PRINCIPAL FINDINGS: Using surface plasmon resonance and NMR spectroscopy, as well as chemically and recombinantly produced chemokines, we show here that CXCL12gamma first 68 amino acids adopt a structure closely related to the well described alpha isoform, followed by an unfolded C-terminal extension of 30 amino acids. Remarkably, 60 % of these residues are either lysine or arginine, and most of them are clustered in typical HS binding sites. This provides the chemokine with the highest affinity for HP ever observed (Kd = 0.9 nM), and ensures a strong retention of the chemokine at the cell surface. This was due to the unique combination of two cooperative binding sites, one strictly required, found in the structured domain of the protein, the other one being the C-terminus which essentially functions by enhancing the half life of the complexes. Importantly, this peculiar C-terminus also regulates the balance between HS and CXCR4 binding, and consequently the biological activity of the chemokine. CONCLUSIONS/SIGNIFICANCE: Together these data describe an unusual binding process that gives rise to an unprecedented high affinity between a chemokine and HS. This shows that the gamma isoform of CXCL12, which features unique structural and functional properties, is optimized to ensure its strong retention at the cell surface. Thus, depending on the chemokine isoform to which it binds, HS could differentially orchestrate the CXCL12 mediated directional cell kinesis

CXCL12 is displayed by rheumatoid endothelial cells through its basic amino-terminal motif on heparan sulfate proteoglycans

The chemokine CXCL12 (also known as stromal cell-derived factor, SDF-1) is constitutively expressed by stromal resident cells and is involved in the homeostatic and inflammatory traffic of leukocytes. Binding of CXCL12 to glycosaminoglycans on endothelial cells (ECs) is supposed to be relevant to the regulation of leukocyte diapedesis and neoangiogenesis during inflammatory responses. To improve our understanding of the relevance of this process to rheumatoid arthritis (RA), we have studied the mechanisms of presentation of exogenous CXCL12 by cultured RA ECs. RA synovial tissues had higher levels of CXCL12 on the endothelium than osteoarthritis (OA) tissues; in both, CXCL12 colocalized to heparan sulfate proteoglycans (HSPGs) and high endothelial venules. In cultured RA ECs, exogenous CXCL12α was able to bind in a CXCR4-independent manner to surface HSPGs. Desulfation of RA EC HSPGs by pretreatment with sodium chlorate, or by replacing in a synthetic CXCL12α the residues Lys24 and Lys27 by Ser (CXCL12α-K2427S), decreased or abrogated the ability of the chemokine to bind to RA ECs. Ex vivo, synovial ECs from patients with either OA or RA displayed a higher CXCL12-binding capacity than human umbilical vein ECs (HUVECs), and in HUVECs the binding of CXCL12 was increased on exposure to tumor necrosis factor-α or lymphotoxin-α(1)β(2). Our findings indicate that CXCL12 binds to HSPGs on ECs of RA synovium. The phenomenon relates to the interaction of HSPGs with a CXCL12 domain with net positive surface charge located in the first β strand, which encompasses a canonical BXBB HSPG-binding motif. Furthermore, we show that the attachment of CXCL12 to HSPGs is upregulated by inflammatory cytokines. Both the upregulation of a constitutive chemokine during chronic inflammation and the HSPG-dependent immobilization of CXCL12 in EC surfaces are potential sites for therapeutic intervention

Homeostatic and Tissue Reparation Defaults in Mice Carrying Selective Genetic Invalidation of CXCL12/Proteoglycan Interactions.

International audienceBACKGROUND: Interaction with heparan sulfate proteoglycans is supposed to provide chemokines with the capacity to immobilize on cell surface and extracellular matrix for accomplishing both tissue homing and signaling of attracted cells. However, the consequences of the exclusive invalidation of such interaction on the roles played by endogenous chemokines in vivo remain unascertained. METHODS AND RESULTS: We engineered a mouse carrying a Cxcl12 gene (Cxcl12(Gagtm)) mutation that precludes interactions with heparan sulfate structures while not affecting CXCR4-dependent cell signaling of CXCL12 isoforms (α, β, γ). Cxcl12(Gagtm/Gagtm) mice develop normally, express normal levels of total and isoform-specific Cxcl12 mRNA, and show increased counting of circulating CD34(+) hematopoietic precursor cells. After induced acute ischemia, a marked impaired capacity to support revascularization was observed in Cxcl12(Gagtm/Gagtm) animals associated with a reduced number of infiltrating cells in the ischemic tissue despite the massive expression of CXCL12 isoforms. Importantly, exogenous administration of CXCL12γ, which binds heparan sulfate with the highest affinity ever reported for a cytokine, fully restores vascular growth, whereas heparan sulfate-binding CXCL12γ mutants failed to promote revascularization in Cxcl12(Gagtm/Gagtm) animals. CONCLUSION: These findings prove the role played by heparan sulfate interactions in the functions of CXCL12 in both homeostasis and physiopathological settings and document for the first time the paradigm of chemokine immobilization in vivo

Elastase Release by Transmigrating Neutrophils Deactivates Endothelial-bound SDF-1α and Attenuates Subsequent T Lymphocyte Transendothelial Migration

Leukocyte trafficking to sites of inflammation follows a defined temporal pattern, and evidence suggests that initial neutrophil transendothelial migration modifies endothelial cell phenotype. We tested the hypothesis that preconditioning of human umbilical vein endothelial cells (HUVEC) by neutrophils would also modify the subsequent transendothelial migration of T lymphocytes across cytokine-stimulated HUVEC in an in vitro flow assay. Using fluorescence microscopy, preconditioning of HUVEC by neutrophils was observed to significantly reduce the extent of subsequent stromal cell–derived factor-1α (SDF-1α [CXCL12])-mediated T lymphocyte transendothelial migration, without reducing accumulation. In contrast, recruitment of a second wave of neutrophils was unaltered. Conditioned medium harvested after transendothelial migration of neutrophils or supernatants from stimulated neutrophils mediated a similar blocking effect, which was negated using a specific neutrophil elastase inhibitor. Furthermore, T lymphocyte transendothelial migration was inhibited by treatment of HUVEC with purified neutrophil elastase, which selectively cleaved the amino terminus of HUVEC-bound SDF-1α, which is required for its chemotactic activity. The reduction in T lymphocyte transendothelial migration was not observed using a different chemokine, ELC (CCL19), and was not reversed by replenishment of SDF-1α, indicating endothelial retention of the inactivated chemokine. In summary, transmigrating neutrophils secrete localized elastase that is protected from plasma inhibitors, and thereby modulate trafficking of other leukocyte subsets by altering the endothelial-associated chemotactic activities

The CXCL12γ Chemokine Displays Unprecedented Structural and Functional Properties that Make It a Paradigm of Chemoattractant Proteins

The CXCL12γ chemokine arises by alternative splicing from Cxcl12, an essential gene during development. This protein binds CXCR4 and displays an exceptional degree of conservation (99%) in mammals. CXCL12γ is formed by a protein core shared by all CXCL12 isoforms, extended by a highly cationic carboxy-terminal (C-ter) domain that encompass four overlapped BBXB heparan sulfate (HS)-binding motifs. We hypothesize that this unusual domain could critically determine the biological properties of CXCL12γ through its interaction to, and regulation by extracellular glycosaminoglycans (GAG) and HS in particular. By both RT-PCR and immunohistochemistry, we mapped the localization of CXCL12γ both in mouse and human tissues, where it showed discrete differential expression. As an unprecedented feature among chemokines, the secreted CXCL12γ strongly interacted with cell membrane GAG, thus remaining mostly adsorbed on the plasmatic membrane upon secretion. Affinity chromatography and surface plasmon resonance allowed us to determine for CXCL12γ one of the higher affinity for HS (Kd = 0.9 nM) ever reported for a protein. This property relies in the presence of four canonical HS-binding sites located at the C-ter domain but requires the collaboration of a HS-binding site located in the core of the protein. Interestingly, and despite reduced agonist potency on CXCR4, the sustained binding of CXCL12γ to HS enabled it to promote in vivo intraperitoneal leukocyte accumulation and angiogenesis in matrigel plugs with much higher efficiency than CXCL12α. In good agreement, mutant CXCL12γ chemokines selectively devoid of HS-binding capacity failed to promote in vivo significant cell recruitment. We conclude that CXCL12γ features unique structural and functional properties among chemokines which rely on the presence of a distinctive C-ter domain. The unsurpassed capacity to bind to HS on the extracellular matrix would make CXCL12γ the paradigm of haptotactic proteins, which regulate essential homeostatic functions by promoting directional migration and selective tissue homing of cells

CXCR7 heterodimerizes with CXCR4 and regulates CXCL12-mediated G protein signaling.

International audienceThe stromal cell-derived factor-1/CXCL12 chemokine engages the CXCR4 and CXCR7 receptors and regulates homeostatic and pathologic processes, including organogenesis, leukocyte homeostasis, and tumorigenesis. Both receptors are widely expressed in mammalian cells, but how they cooperate to respond to CXCL12 is not well understood. Here, we show that CXCR7 per se does not trigger G(alphai) protein-dependent signaling, although energy transfer assays indicate that it constitutively interacts with G(alphai) proteins and undergoes CXCL12-mediated conformational changes. Moreover, when CXCR4 and CXCR7 are coexpressed, we show that receptor heterodimers form as efficiently as receptor homodimers, thus opening the possibility that CXCR4/CXCR7 heterodimer formation has consequences on CXCL12-mediated signals. Indeed, expression of CXCR7 induces conformational rearrangements within preassembled CXCR4/G(alphai) protein complexes and impairs CXCR4-promoted G(alphai)-protein activation and calcium responses. Varying CXCR7 expression levels and blocking CXCL12/CXCR7 interactions in primary T cells suggest that CXCR4/CXCR7 heterodimers form in primary lymphocytes and regulate CXCL12-promoted chemotaxis. Taken together, these results identify CXCR4/CXCR7 heterodimers as distinct functional units with novel properties, which can contribute to the functional plasticity of CXCL12

1H-NMR conformational analysis of a high-affinity antigenic 11-residue peptide from the tryptophan synthase beta 2 subunit.

International audienceTwo synthetic peptides from the beta 2 subunit of tryptophan synthase have been studied by 1H-NMR spectroscopy at 300 MHz. One peptide, His-Gly-Arg-Val-Gly-Ile-Tyr-Phe-Gly-Met-Lys (peptide 11; Ile, isoleucine) is antigenic and binds with a high affinity to a monoclonal antibody that recognizes the native beta 2 subunit. The second peptide, His-Gly-Arg-Val-Gly-Ile-Tyr-Phe (peptide 8) reacts very weakly with the antibody. The 1H-NMR spectra of the two peptides have been assigned from two-dimensional techniques in H2O, 2H2O and (2H6) dimethyl sulfoxide [(2H6)Me2SO]. The structure has been evaluated through analysis of nuclear Overhauser effects, coupling constants, amide-proton exchange rates and their temperature coefficients, and chemical shifts. In aqueous solvent, the C-terminal part of peptide 11 presents some structure centered around residues Phe-Gly-Met. The relationship between the structure found in peptide 11 and its antigenic nature is discussed

Preparation of synthetic glycoconjugates as potential vaccines against Shigella flexneri serotype 2a disease.

International audienceThe synthesis of three neoglycopeptides incorporating carbohydrate haptens, differing in length, covalently linked to a non natural universal T helper peptide is disclosed. They were synthesized according to a blockwise strategy based on the condensation of appropriate di-, tri-, and tetrasaccharide trichloroacetimidate donors onto an azidoethyl 2-acetamido-2-deoxybeta-D-glucopyranoside acceptor. Use of thiol-maleimide coupling chemistry allowed site-selective efficient conjugation

Glycosaminoglycans and syndecan-4 are involved in SDF-1/CXCL12-mediated invasion of human epitheloid carcinoma HeLa cells.

International audienceBACKGROUND: In addition to their physiologic effects in inflammation and angiogenesis, chemokines are involved in cancer pathology. The CXC-chemokine stromal cell-derived factor-1 (SDF-1)/CXCL12 mediates its biological activities through activation of G protein-coupled receptor CXCR4 and binds to glycosaminoglycans (GAGs). METHODS: Using Bio-coat cell migration chambers, specific antagonists, flow cytometry and RNA interference, we evaluate the involvement of heparan sulfate proteoglycans (HSPG) in the SDF-1/CXCL12-induced invasion of human cervix epitheloid carcinoma HeLa cells. RESULTS: The SDF-1/CXCL12-induced cell invasion is dependent on CXCR4. Furthermore, Protein Kinase C delta (PKC delta) and c-jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) are implicated in this event, but not extracellular signal-regulated kinase (ERK) 1/2. Moreover, the invasion of HeLa cells induced by SDF-1/CXCL12 was dependent on matrix metalloproteinase-9 (MMP-9). The pre-incubation of HeLa cells with heparin or with anti-heparan sulfate antibodies or with beta-d-xyloside inhibited SDF-1/CXCL12-mediated cell invasion. Furthermore, the down-regulation of syndecan-4, a heparan sulfate proteoglycan, decreased SDF-1/CXCL12-mediated HeLa cell invasion. CONCLUSION: GAGs, probably on syndecan-4, are involved in SDF-1/CXCL12-mediated cell chemotaxis. GENERAL SIGNIFICANCE: These data suggest that targeting the glycosaminoglycan/chemokine interaction could be a new therapeutic approach for carcinomas in which SDF-1/CXCL12 is involved