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.
References
[1]
Tashiro K, Tada H, Heilker R, Shirozu M, Nakano T, et al. (1993) Signal sequence trap: a cloning strategy for secreted proteins and type I membrane proteins. Science 261: 600–603.
[2]
Bleul CC, Farzan M, Choe H, Parolin C, Clark-Lewis I, et al. (1996) The lymphocyte chemoattractant SDF-1 is a ligand for LESTR/fusin and blocks HIV-1 entry. Nature 382: 829–833.
[3]
Balabanian K, Lagane B, Infantino S, Chow KY, Harriague J, et al. (2005) The chemokine SDF-1/CXCL12 binds to and signals through the orphan receptor RDC1 in T lymphocytes. J Biol Chem 280: 35760–35766.
[4]
Burns JM, Summers BC, Wang Y, Melikian A, Berahovich R, et al. (2006) A novel chemokine receptor for SDF-1 and I-TAC involved in cell survival, cell adhesion, and tumor development. J Exp Med 203: 2201–2213.
[5]
Miao Z, Luker KE, Summers BC, Berahovich R, Bhojani MS, et al. (2007) CXCR7 (RDC1) promotes breast and lung tumor growth in vivo and is expressed on tumor-associated vasculature. Proc Natl Acad Sci U S A 104: 15735–15740.
[6]
Shirozu M, Nakano T, Inazawa J, Tashiro K, Tada H, et al. (1995) Structure and chromosomal localization of the human stromal cell-derived factor 1 (SDF1) gene. Genomics 28: 495–500.
[7]
Nagasawa T, Tachibana K, Kishimoto T (1998) A novel CXC chemokine PBSF/SDF-1 and its receptor CXCR4: their functions in development, hematopoiesis and HIV infection. Semin Immunol 10: 179–185.
[8]
Zou YR, Kottmann AH, Kuroda M, Taniuchi I, Littman DR (1998) Function of the chemokine receptor CXCR4 in haematopoiesis and in cerebellar development. Nature 393: 595–599.
[9]
Klein RS, Rubin JB, Gibson HD, DeHaan EN, Alvarez-Hernandez X, et al. (2001) SDF-1 alpha induces chemotaxis and enhances Sonic hedgehog-induced proliferation of cerebellar granule cells. Development 128: 1971–1981.
[10]
Ma Q, Jones D, Borghesani PR, Segal RA, Nagasawa T, et al. (1998) Impaired B-lymphopoiesis, myelopoiesis, and derailed cerebellar neuron migration in CXCR4- and SDF-1-deficient mice. Proc Natl Acad Sci U S A 95: 9448–9453.
[11]
Ara T, Nakamura Y, Egawa T, Sugiyama T, Abe K, et al. (2003) Impaired colonization of the gonads by primordial germ cells in mice lacking a chemokine, stromal cell-derived factor-1 (SDF-1). Proc Natl Acad Sci U S A 100: 5319–5323.
[12]
Shamri R, Grabovsky V, Gauguet JM, Feigelson S, Manevich E, et al. (2005) Lymphocyte arrest requires instantaneous induction of an extended LFA-1 conformation mediated by endothelium-bound chemokines. Nat Immunol 6: 497–506.
[13]
Kantele JM, Kurk S, Jutila MA (2000) Effects of continuous exposure to stromal cell-derived factor-1 alpha on T cell rolling and tight adhesion to monolayers of activated endothelial cells. J Immunol 164: 5035–5040.
[14]
Grabovsky V, Feigelson S, Chen C, Bleijs DA, Peled A, et al. (2000) Subsecond induction of alpha4 integrin clustering by immobilized chemokines stimulates leukocyte tethering and rolling on endothelial vascular cell adhesion molecule 1 under flow conditions. J Exp Med 192: 495–506.
[15]
Campbell JJ, Hedrick J, Zlotnik A, Siani MA, Thompson DA, et al. (1998) Chemokines and the arrest of lymphocytes rolling under flow conditions. Science 279: 381–384.
[16]
Aiuti A, Webb IJ, Bleul C, Springer T, Gutierrez-Ramos JC (1997) The chemokine SDF-1 is a chemoattractant for human CD34+ hematopoietic progenitor cells and provides a new mechanism to explain the mobilization of CD34+ progenitors to peripheral blood. J Exp Med 185: 111–120.
[17]
Nanki T, Hayashida K, El-Gabalawy HS, Suson S, Shi K, et al. (2000) Stromal cell-derived factor-1-CXC chemokine receptor 4 interactions play a central role in CD4+ T cell accumulation in rheumatoid arthritis synovium. J Immunol 165: 6590–6598.
[18]
Gallagher KA, Liu ZJ, Xiao M, Chen H, Goldstein LJ, et al. (2007) Diabetic impairments in NO-mediated endothelial progenitor cell mobilization and homing are reversed by hyperoxia and SDF-1 alpha. J Clin Invest 117: 1249–1259.
[19]
Ceradini DJ, Kulkarni AR, Callaghan MJ, Tepper OM, Bastidas N, et al. (2004) Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1 induction of SDF-1. Nat Med 10: 858–864.
[20]
Burger JA, Kipps TJ (2006) CXCR4: a key receptor in the crosstalk between tumor cells and their microenvironment. Blood 107: 1761–1767.
[21]
Esko JD, Selleck SB (2002) Order out of chaos: assembly of ligand binding sites in heparan sulfate. Annu Rev Biochem 71: 435–471.
[22]
Proudfoot AE, Handel TM, Johnson Z, Lau EK, LiWang P, et al. (2003) Glycosaminoglycan binding and oligomerization are essential for the in vivo activity of certain chemokines. Proc Natl Acad Sci U S A 100: 1885–1890.
[23]
Sadir R, Imberty A, Baleux F, Lortat-Jacob H (2004) Heparan sulfate/heparin oligosaccharides protect stromal cell-derived factor-1 (SDF-1)/CXCL12 against proteolysis induced by CD26/dipeptidyl peptidase IV. J Biol Chem 279: 43854–43860.
[24]
Sweeney EA, Lortat-Jacob H, Priestley GV, Nakamoto B, Papayannopoulou T (2002) Sulfated polysaccharides increase plasma levels of SDF-1 in monkeys and mice: involvement in mobilization of stem/progenitor cells. Blood 99: 44–51.
[25]
Valenzuela-Fernandez A, Palanche T, Amara A, Magerus A, Altmeyer R, et al. (2001) Optimal inhibition of ×4 HIV isolates by the CXC chemokine stromal cell-derived factor 1 alpha requires interaction with cell surface heparan sulfate proteoglycans. J Biol Chem 276: 26550–26558.
[26]
Gleichmann M, Gillen C, Czardybon M, Bosse F, Greiner-Petter R, et al. (2000) Cloning and characterization of SDF-1gamma, a novel SDF-1 chemokine transcript with developmentally regulated expression in the nervous system. Eur J Neurosci 12: 1857–1866.
[27]
Yu L, Cecil J, Peng SB, Schrementi J, Kovacevic S, et al. (2006) Identification and expression of novel isoforms of human stromal cell-derived factor 1. Gene 374: 174–179.
[28]
Laguri C, Sadir R, Rueda P, Baleux F, Gans P, et al. (2007) The Novel CXCL12gamma Isoform Encodes an Unstructured Cationic Domain Which Regulates Bioactivity and Interaction with Both Glycosaminoglycans and CXCR4. PLoS ONE 2: e1110.
[29]
Allen CD, Ansel KM, Low C, Lesley R, Tamamura H, et al. (2004) Germinal center dark and light zone organization is mediated by CXCR4 and CXCR5. Nat Immunol 5: 943–952.
[30]
Coulomb-L'Hermin A, Amara A, Schiff C, Durand-Gasselin I, Foussat A, et al. (1999) Stromal cell-derived factor 1 (SDF-1) and antenatal human B cell lymphopoiesis: expression of SDF-1 by mesothelial cells and biliary ductal plate epithelial cells. Proc Natl Acad Sci U S A 96: 8585–8590.
[31]
Amara A, Lorthioir O, Valenzuela A, Magerus A, Thelen M, et al. (1999) Stromal cell-derived factor-1alpha associates with heparan sulfates through the first beta-strand of the chemokine. J Biol Chem 274: 23916–23925.
[32]
Sandhoff R, Grieshaber H, Djafarzadeh R, Sijmonsma TP, Proudfoot AE, et al. (2005) Chemokines bind to sulfatides as revealed by surface plasmon resonance. Biochim Biophys Acta 1687: 52–63.
[33]
Altenburg JD, Broxmeyer HE, Jin Q, Cooper S, Basu S, et al. (2007) A naturally occurring splice variant of CXCL12/stromal cell-derived factor 1 is a potent human immunodeficiency virus type 1 inhibitor with weak chemotaxis and cell survival activities. J Virol 81: 8140–8148.
[34]
Jones KS, Petrow-Sadowski C, Bertolette DC, Huang Y, Ruscetti FW (2005) Heparan sulfate proteoglycans mediate attachment and entry of human T-cell leukemia virus type 1 virions into CD4+ T cells. J Virol 79: 12692–12702.
[35]
Orimo A, Gupta PB, Sgroi DC, Arenzana-Seisdedos F, Delaunay T, et al. (2005) Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion. Cell 121: 335–348.
[36]
Passaniti A, Taylor RM, Pili R, Guo Y, Long PV, et al. (1992) A simple, quantitative method for assessing angiogenesis and antiangiogenic agents using reconstituted basement membrane, heparin, and fibroblast growth factor. Lab Invest 67: 519–528.
[37]
Segret A, Rucker-Martin C, Pavoine C, Flavigny J, Deroubaix E, et al. (2007) Structural localization and expression of CXCL12 and CXCR4 in rat heart and isolated cardiac myocytes. J Histochem Cytochem 55: 141–150.
[38]
Stumm RK, Rummel J, Junker V, Culmsee C, Pfeiffer M, et al. (2002) A dual role for the SDF-1/CXCR4 chemokine receptor system in adult brain: isoform-selective regulation of SDF-1 expression modulates CXCR4-dependent neuronal plasticity and cerebral leukocyte recruitment after focal ischemia. J Neurosci 22: 5865–5878.
[39]
Nickel W (2007) Unconventional secretion: an extracellular trap for export of fibroblast growth factor 2. J Cell Sci 120: 2295–2299.
[40]
Brelot A, Heveker N, Adema K, Hosie MJ, Willett B, et al. (1999) Effect of mutations in the second extracellular loop of CXCR4 on its utilization by human and feline immunodeficiency viruses. J Virol 73: 2576–2586.
[41]
Soriano SF, Hernanz-Falcon P, Rodriguez-Frade JM, De Ana AM, Garzon R, et al. (2002) Functional inactivation of CXC chemokine receptor 4-mediated responses through SOCS3 up-regulation. J Exp Med 196: 311–321.
[42]
Ferrara N, Alitalo K (1999) Clinical applications of angiogenic growth factors and their inhibitors. Nat Med 5: 1359–1364.
[43]
Tischer E, Mitchell R, Hartman T, Silva M, Gospodarowicz D, et al. (1991) The human gene for vascular endothelial growth factor. Multiple protein forms are encoded through alternative exon splicing. J Biol Chem 266: 11947–11954.
[44]
Neufeld G, Cohen T, Gengrinovitch S, Poltorak Z (1999) Vascular endothelial growth factor (VEGF) and its receptors. Faseb J 13: 9–22.
[45]
Houck KA, Leung DW, Rowland AM, Winer J, Ferrara N (1992) Dual regulation of vascular endothelial growth factor bioavailability by genetic and proteolytic mechanisms. J Biol Chem 267: 26031–26037.
[46]
Ruhrberg C, Gerhardt H, Golding M, Watson R, Ioannidou S, et al. (2002) Spatially restricted patterning cues provided by heparin-binding VEGF-A control blood vessel branching morphogenesis. Genes Dev 16: 2684–2698.
[47]
Ferrara N (1999) Vascular endothelial growth factor: molecular and biological aspects. Curr Top Microbiol Immunol 237: 1–30.
[48]
Sadir R, Baleux F, Grosdidier A, Imberty A, Lortat-Jacob H (2001) Characterization of the stromal cell-derived factor-1alpha-heparin complex. J Biol Chem 276: 8288–8296.
[49]
Staropoli I, Chanel C, Girard M, Altmeyer R (2000) Processing, stability, and receptor binding properties of oligomeric envelope glycoprotein from a primary HIV-1 isolate. J Biol Chem 275: 35137–35145.
[50]
Lagane B, Ballet S, Planchenault T, Balabanian K, Le Poul E, et al. (2005) Mutation of the DRY motif reveals different structural requirements for the CC chemokine receptor 5-mediated signaling and receptor endocytosis. Mol Pharmacol 67: 1966–1976.
[51]
Balabanian K, Harriague J, Decrion C, Lagane B, Shorte S, et al. (2004) CXCR4-tropic HIV-1 envelope glycoprotein functions as a viral chemokine in unstimulated primary CD4+ T lymphocytes. J Immunol 173: 7150–7160.
[52]
Balabanian K, Lagane B, Pablos JL, Laurent L, Planchenault T, et al. (2005) WHIM syndromes with different genetic anomalies are accounted for by impaired CXCR4 desensitization to CXCL12. Blood 105: 2449–2457.
[53]
Pablos JL, Santiago B, Galindo M, Torres C, Brehmer MT, et al. (2003) Synoviocyte-derived CXCL12 is displayed on endothelium and induces angiogenesis in rheumatoid arthritis. J Immunol 170: 2147–2152.
[54]
Zimrin AB, Villeponteau B, Maciag T (1995) Models of in vitro angiogenesis: endothelial cell differentiation on fibrin but not matrigel is transcriptionally dependent. Biochem Biophys Res Commun 213: 630–638.
