Chemically Synthesized SDF-1α Analogue, N33A, Is a Potent Chemotactic Agent for CXCR4/Fusin/LESTR-expressing Human Leukocytes
1997; Elsevier BV; Volume: 272; Issue: 40 Linguagem: Inglês
10.1074/jbc.272.40.24966
ISSN1083-351X
AutoresHirotsugu Ueda, Michael A. Siani, Wanghua Gong, Darren A. Thompson, Garth G. Brown, Jiming Wang,
Tópico(s)Chemokine receptors and signaling
ResumoStromal cell-derived factor (SDF) 1 is a potent chemoattractant for leukocytes through activation of the receptor CXCR4/Fusin/LESTR, which is a fusion co-factor for the entry of T lymphocytotropic human immunodeficiency virus type 1 (HIV-1). This CXCR4-mediated HIV-1 fusion can be inhibited by SDF-1. Because of its importance in the study of immunity and AIDS, large scale production of SDF-1 is desirable. In addition to recombinant technology, chemical synthesis provides means by which biologically active proteins can be produced not only in large quantity but also with a variety of designed modifications. In this study, we investigated the binding and function of an SDF-1α analogue, N33A, synthesized by a newly developed native chemical ligation approach. Radioiodinated N33A showed high affinity binding to human monocytes, T lymphocytes, as well as neutrophils, and competed equally well with native recombinant SDF-1α for binding sites on leukocytes. N33A also showed equally potent chemoattractant activity as native recombinant SDF-1α for human leukocytes. Further study with CXCR4/Fusin/LESTR transfected HEK 293 cells showed that N33A binds and induces directional migration of these cells in vitro. These results demonstrate that the chemically synthesized SDF-1α analogue, N33A, which can be produced rapidly in large quantity, possesses the same capacity as native SDF-1α to activate CXCR4-expressing cells and will provide a valuable agent for research on the host immune response and AIDS. Stromal cell-derived factor (SDF) 1 is a potent chemoattractant for leukocytes through activation of the receptor CXCR4/Fusin/LESTR, which is a fusion co-factor for the entry of T lymphocytotropic human immunodeficiency virus type 1 (HIV-1). This CXCR4-mediated HIV-1 fusion can be inhibited by SDF-1. Because of its importance in the study of immunity and AIDS, large scale production of SDF-1 is desirable. In addition to recombinant technology, chemical synthesis provides means by which biologically active proteins can be produced not only in large quantity but also with a variety of designed modifications. In this study, we investigated the binding and function of an SDF-1α analogue, N33A, synthesized by a newly developed native chemical ligation approach. Radioiodinated N33A showed high affinity binding to human monocytes, T lymphocytes, as well as neutrophils, and competed equally well with native recombinant SDF-1α for binding sites on leukocytes. N33A also showed equally potent chemoattractant activity as native recombinant SDF-1α for human leukocytes. Further study with CXCR4/Fusin/LESTR transfected HEK 293 cells showed that N33A binds and induces directional migration of these cells in vitro. These results demonstrate that the chemically synthesized SDF-1α analogue, N33A, which can be produced rapidly in large quantity, possesses the same capacity as native SDF-1α to activate CXCR4-expressing cells and will provide a valuable agent for research on the host immune response and AIDS. Stromal cell-derived factor (SDF) 1The abbreviations used are: SDF, stromal cell-derived factor; HIV-1, human immunodeficiency virus type 1; HPLC, high pressure liquid chromatography; Boc, t-butoxycarbonyl; CI, chemotaxis index; rh, recombinant human.1The abbreviations used are: SDF, stromal cell-derived factor; HIV-1, human immunodeficiency virus type 1; HPLC, high pressure liquid chromatography; Boc, t-butoxycarbonyl; CI, chemotaxis index; rh, recombinant human. 1 has been reported to be a primordial chemokine of the CXC subfamily and has multiple biological activities on a variety of cell types (1Bleul C.C. Fuhlbrigge R.C. Casasnovas J.M. Aiuti A. Springer T.A. J. Exp. Med. 1996; 184: 1101-1109Crossref PubMed Scopus (1274) Google Scholar, 2Nagasawa T. Hirota S. Tachibana K. Takakura N. Nishikawa S. Kitamura Y. Yoshida N. Kikutani H. Kishimoto T. Nature. 1996; 382: 635-638Crossref PubMed Scopus (1999) Google Scholar, 3Bleul C.C. Fazzan M. Choe H. Parolin C. Clark-Lewis I. Sodroski J. Springer T.A. Nature. 1996; 382: 829-833Crossref PubMed Scopus (1749) Google Scholar, 4Oberlin E. Amara A. Bachelerie F. Bessia C. Virelizier J-L. Arenzana-Seisdedos F. Schwartz O. Heard J.-M. Clark-Lewis I. Legler D.F. Loetscher M. Baggiolini M. Moser B. Nature. 1996; 382: 833-835Crossref PubMed Scopus (1478) Google Scholar). SDF-1 was initially isolated as a T lymphocyte chemoattractant and was found to be active also on monocytes but not on neutrophils (1Bleul C.C. Fuhlbrigge R.C. Casasnovas J.M. Aiuti A. Springer T.A. J. Exp. Med. 1996; 184: 1101-1109Crossref PubMed Scopus (1274) Google Scholar, 3Bleul C.C. Fazzan M. Choe H. Parolin C. Clark-Lewis I. Sodroski J. Springer T.A. Nature. 1996; 382: 829-833Crossref PubMed Scopus (1749) Google Scholar). However, its activity on neutrophils was subsequently shown by using Ca2+ mobilization experiments (4Oberlin E. Amara A. Bachelerie F. Bessia C. Virelizier J-L. Arenzana-Seisdedos F. Schwartz O. Heard J.-M. Clark-Lewis I. Legler D.F. Loetscher M. Baggiolini M. Moser B. Nature. 1996; 382: 833-835Crossref PubMed Scopus (1478) Google Scholar). The SDF-1 gene is located on chromosome 10 (5Shirozu M. Nakano T. Inazawa J. Tashiro K. Tada H. Shinohara T. Nonjo T. Genomics. 1995; 28: 495-500Crossref PubMed Scopus (534) Google Scholar), while the genes for other known CXC chemokines are located chromosome 4 (6Oppenheim J.J. Wang J.M. Chertov O. Taub D.D. Ben-Baruch A. Tilney L.N. Strom T.B. Paul L.C. Transplantation Biology: Cellular and Molecular Aspects. Lippincott-Raven, Philadelphia1996: 187-200Google Scholar, 7Baggiolini M. Dewald B. Moser B. Adv. Immunol. 1994; 55: 97-179Crossref PubMed Scopus (2259) Google Scholar). SDF-1 is well conserved with only a single amino acid substitution between human and murirne molecules (1Bleul C.C. Fuhlbrigge R.C. Casasnovas J.M. Aiuti A. Springer T.A. J. Exp. Med. 1996; 184: 1101-1109Crossref PubMed Scopus (1274) Google Scholar, 2Nagasawa T. Hirota S. Tachibana K. Takakura N. Nishikawa S. Kitamura Y. Yoshida N. Kikutani H. Kishimoto T. Nature. 1996; 382: 635-638Crossref PubMed Scopus (1999) Google Scholar). SDF-1α and SDF-1β were believed to be the result of differential splicing of a single gene, with SDF-1α missing four amino acids in the carboxyl terminus. The CXCR4/Fusin/LESTR has been identified as one of the functional receptors for SDF-1 (3Bleul C.C. Fazzan M. Choe H. Parolin C. Clark-Lewis I. Sodroski J. Springer T.A. Nature. 1996; 382: 829-833Crossref PubMed Scopus (1749) Google Scholar, 4Oberlin E. Amara A. Bachelerie F. Bessia C. Virelizier J-L. Arenzana-Seisdedos F. Schwartz O. Heard J.-M. Clark-Lewis I. Legler D.F. Loetscher M. Baggiolini M. Moser B. Nature. 1996; 382: 833-835Crossref PubMed Scopus (1478) Google Scholar), a member of the seven transmembrane spanning receptor superfamily (8Kelvin D.J. Michiel D.F. Johnston J.A. Lloyd A.R. Sprenger H. Oppenheim J.J. Wang J.M. J. Leukocyte Biol. 1993; 54: 604-612Crossref PubMed Scopus (145) Google Scholar, 9Murphy P.M. Cytokine Growth Factor Rev. 1996; 7: 47-64Crossref PubMed Scopus (281) Google Scholar). CXCR4 was initially cloned as an orphan receptor (10Loetscher M. Geiser T. O'Reilly T. Zwahlen R. Baggiolini M. Moser B. J. Biol. Chem. 1994; 269: 232-237Abstract Full Text PDF PubMed Google Scholar, 11Federsppiel B. Melhado I.G. Duncan A.M.V. Delaney A. Schappert K. Clark-Lewis I. Jirik F.R. Genomics. 1993; 16: 707-712Crossref PubMed Scopus (162) Google Scholar, 12Nomura H. Nielsen B.W. Matsushima K. Int. Immunol. 1993; 5: 1239-1249Crossref PubMed Scopus (141) Google Scholar) and was later identified as a fusion co-factor for the entry of HIV-1 of the T lymphotropic strain (13Feng Y. Broder C.C. Kennedy P.E. Berger E.A. Science. 1996; 272: 872-877Crossref PubMed Scopus (3624) Google Scholar). In addition to its activities on human leukocytes, SDF-1 has been shown to inhibit the fusion and replication of T lymphotropic HIV-1 in host cells bearing CD4 and CXCR4 (3Bleul C.C. Fazzan M. Choe H. Parolin C. Clark-Lewis I. Sodroski J. Springer T.A. Nature. 1996; 382: 829-833Crossref PubMed Scopus (1749) Google Scholar, 4Oberlin E. Amara A. Bachelerie F. Bessia C. Virelizier J-L. Arenzana-Seisdedos F. Schwartz O. Heard J.-M. Clark-Lewis I. Legler D.F. Loetscher M. Baggiolini M. Moser B. Nature. 1996; 382: 833-835Crossref PubMed Scopus (1478) Google Scholar). Therefore, SDF-1 plays a pivotal role in host immune system and its defense against infection. Rapid production of cytokines and chemokines is essential for structure-function studies and design of molecules with agonist or antagonist activities. The turbocharged peptide synthesis technology (14Schnolzer M. Alewood P. Jones A Alwood D. Kent S.B.H. Int. J. Pept. Protein Res. 1992; 40: 180-193Crossref PubMed Scopus (938) Google Scholar) and chemical ligation of peptide segments (15Dawson P.E. Muir T.W. Clark-Lewis I. Kent S.B.H. Science. 1994; 266: 776-779Crossref PubMed Scopus (3139) Google Scholar) permit the synthesis of chemokines in large quantity. This approach was therefore utilized to synthesize SDF-1α based on its great utility in studies of immune responses and infectious diseases. However, the thioacid peptide (SDF-1α(1–33)-αCOSH) could not be readily generated on the standard α-thiocarboxylate resin (16Canne L.E. Walker S.M. Kent S.B.H. Tetrahedron Lett. 1995; 36: 1217-1220Crossref Scopus (74) Google Scholar) due to the presence of an Asn33 residue at the ligation site in SDF-1α. Instead, alanine was coupled to the α-thiocarboxylate resin, then the mutant SDF-1α(1–33, N33A) was synthesized. In this study, we report that this SDF-1α analogue, N33A, displays full biological activity through the activation of the receptor CXCR4. This analogue, which can be rapidly produced in large quantity in the absence of contaminating peptides, will prove to be an important tool in the study of host immunity and AIDS. Human recombinant SDF-1α was purchased from PeproTech Inc. (Rocky Hill, NJ). Radioiodination of the SDF-1α and the analogue N33A was performed by a lactoperoxidase-labeling procedure. The radioactive ligands were further purified by reversed phase HPLC. The specific activity of the radioiodinated chemokines was 2200 Ci/mmol. Boc protected amino acids were obtained from the following sources: AnaSpec (San Jose, CA), Bachem (Philadelphia, PA), NovaBiochem (San Diego, CA), and Peptides International (Louisville, KY). Peptides were synthesized on a modified ABI430A instrument using in situ neutralization Boc chemistry protocols (17Canne L.E. Ferre-D'Amare A.R. Burley S.K. Kent S.B.H.J. J. Am. Chem. Soc. 1995; 117: 2998-3007Crossref Scopus (179) Google Scholar). C-terminal segments were prepared on –OCH2Pam resins (ABI, Foster City, CA). N-terminal segments were prepared on α-thiocarboxylate resin (16Canne L.E. Walker S.M. Kent S.B.H. Tetrahedron Lett. 1995; 36: 1217-1220Crossref Scopus (74) Google Scholar). Standard HF cleavage protocols were employed following N-terminal Boc removal and drying of the resin. HPLC purification was performed on Rainin HPLCs (Woburn, MA) using Vydac C4 or Dynamax C4 columns with gradient elution (A, H2O, 0.1% trifluoroacetic acid; B, acetonitrile, 0.1% trifluoroacetic acid). Electrospray mass spectrometry was performed on a Sciex API1(PE-Sciex). Ligation of the peptide segments was performed at 4 mmpeptide concentration in 6 m guanidine, 0.1 mTris, pH 7, in the presence of 33 mm thiophenol (Fluka, Switzerland) at room temperature. Ligation was monitored by HPLC and was typically complete within 24 h. Ligation was followed by HPLC purification and lyophilization. After purification, the full-length peptide was reduced at 1 mg/ml in 8 m urea (Fluka), 0.1m Tris (Fluka), 5.37 mm EDTA ((Fluka), pH 8.6, in the presence of 100 mm 2-mercaptoethanol (Fluka). Reduction occurs under a nitrogen atmosphere at 40 °C for 1 h. After complete reduction, the mixture was reconstituted with the same buffer at 0.2 mg/ml with 18.7 mm oxidized glutathione (Sigma). The solution was dispensed into a Spectrum Spectra/Por*7 dialysis membrane (Houston, TX) (M r cut-off, 3500) and the bag was placed in 1 liter of initial dialysis buffer of 8m urea, 0.1 m Tris, 1 mm EDTA, 3 mm 2-mercaptoethanol, 1.3 mm oxidized glutathione, pH 8.6. Over a period of 2 days, 4 liters of 2m urea, 0.1 m Tris, pH 8.6, was pumped into the vessel containing the dialysis bag (18Maeda Y. Ueda T. Imoto T. Protein Eng. 1996; 9: 95-100Crossref PubMed Scopus (28) Google Scholar). After lyophilization, the full-length peptide was oxidized at 1 mg/ml in 2 mguanidine HCl (Fluka), 0.1 m Tris (Fluka), pH 8.6, at room temperature in the presence of air. Folding was complete after stirring overnight and was monitored by HPLC and mass spectrometry. Human peripheral blood leukocytes were isolated from normal donors (National Institutes of Health Clinical Center Transfusion Department, Bethesda, MD) according to the established protocols in this laboratory for purification of monocytes (purity was >90%) (19Xu L.L. MacVicar D.W. Ben-Baruch A. Kuhns D.B. Johnston J. Oppenheim J.J. Wang J.M. Eur. J. Immunol. 1995; 25: 2612-2617Crossref PubMed Scopus (76) Google Scholar), T lymphocytes (purity, >95% CD3+) (20Xu L. Kelvin D. Ye G.Q. Taub D.D. Ben-Baruch A. Oppenheim J.J. Wang J.M. J. Leukocyte Biol. 1995; 57: 335-342Crossref PubMed Scopus (65) Google Scholar) and neutrophils (purity, >98%) (19Xu L.L. MacVicar D.W. Ben-Baruch A. Kuhns D.B. Johnston J. Oppenheim J.J. Wang J.M. Eur. J. Immunol. 1995; 25: 2612-2617Crossref PubMed Scopus (76) Google Scholar). The chemokine receptor, CXCR4/fusin/LESTR cDNA was isolated in this laboratory and was stably transfected into human kidney embryonic epithelial 293 cells (CXCR4/293 cells) as described previously (21Ben-Baruch A. Xu L. Young P.R. Bengali K. Oppenheim J.J. Wang J.M. J. Biol. Chem. 1995; 270: 22123-22128Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Leukocyte migration was evaluated using a 48-well microchamber (Neuroprobe, Cabin John, MD) technique as described previously (19Xu L.L. MacVicar D.W. Ben-Baruch A. Kuhns D.B. Johnston J. Oppenheim J.J. Wang J.M. Eur. J. Immunol. 1995; 25: 2612-2617Crossref PubMed Scopus (76) Google Scholar, 20Xu L. Kelvin D. Ye G.Q. Taub D.D. Ben-Baruch A. Oppenheim J.J. Wang J.M. J. Leukocyte Biol. 1995; 57: 335-342Crossref PubMed Scopus (65) Google Scholar,22Falk W.R. Goodwin Jr., R.H. Leonard E.J. J. Immunol. Methods. 1980; 33: 239-247Crossref PubMed Scopus (251) Google Scholar). The migration of CXCR4/293 cells was also assessed by the 48-well microchamber technique with the polycarbonate filters (10-μm pore size) (21Ben-Baruch A. Xu L. Young P.R. Bengali K. Oppenheim J.J. Wang J.M. J. Biol. Chem. 1995; 270: 22123-22128Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar) precoated with collagen type I (Collaborative Biomedical Products, Bedford, MA). The results are expressed as the the chemotaxis index (CI) representing the fold increase in the cell migration induced by stimuli versus control medium. All experiments were performed at least two times, and results from one experiment are shown. The statistical significance of the difference between migration in response to stimuli and control was assessed by Student'st test. Binding assays were performed using a single concentration of125I-labeled chemokines in the presence of increasing concentrations of unlabeled ligands (19Xu L.L. MacVicar D.W. Ben-Baruch A. Kuhns D.B. Johnston J. Oppenheim J.J. Wang J.M. Eur. J. Immunol. 1995; 25: 2612-2617Crossref PubMed Scopus (76) Google Scholar, 21Ben-Baruch A. Xu L. Young P.R. Bengali K. Oppenheim J.J. Wang J.M. J. Biol. Chem. 1995; 270: 22123-22128Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). The binding data were analyzed with a Macintosh computer program LIGAND (P. Munson, Division of Computer Research and Technology, NIH, Bethesda, MD). In Scatchard plots, the binding data were analyzed with both "one-site" and "two-site" models, and only the one-site model better fit the curves obtained with either native leukocytes or CXCR4/293 cells. The rate of competition for binding by unlabeled ligands was calculated with the following formula:%inhibition=1−(binding in the presence of unlabeledEquation 1 chemokine/binding in the presence of medium alone)×100 We first examined whether the SDF-1α analogue N33A was able to bind and activate human peripheral blood monocytes and T cells. Fig.1 shows that 125I-N33A specifically bound to human peripheral blood monocytes (Fig.1 A) and T lymphocytes (Fig. 1 B) with high affinity (1.8 and 1.4 nm for monocytes and T cells, respectively). This level of binding to monocytes and T cells by125I-N33A was comparable to that of125I-rhSDF-1α as examined in parallel experiments (data not shown). Unlabeled N33A displaced 125I-rhSDF-1α binding to both monocytes and T lymphocytes (Fig. 1, C andD), and likewise, the binding of 125I-N33A to these cell types was also displaced by both unlabeled N33A and rhSDF-1α (not shown). Consistent and considerable migration of monocytes and T cells was induced by N33A (Fig.2). The potency and efficacy of N33A in the induction of mononuclear cell migration was comparable with rhSDF-1α, suggesting that chemically synthesized N33A retains the tertiary structure and functions as well as rhSDF-1α.Figure 2Chemotactic response of monocytes and T cells to N33A and rhSDF-1α. Different concentrations of N33A or rhSDF-1α were placed in the lower wells of the microchemotaxis chamber. The cells were placed in the upper wells, which were separated from the lower wells by polycarbonate filters. After incubation the filters were removed, stained, and the cells that migrated across the filters were counted. The results are expressed as the CI representing the fold increase of cell migration in response to chemokinesversus medium control. CI ≥ 2 are statistically significant in comparison with the spontaneous migration (in response to control medium alone, CI = 1).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The effect of SDF-1 on neutrophils is controversial (1Bleul C.C. Fuhlbrigge R.C. Casasnovas J.M. Aiuti A. Springer T.A. J. Exp. Med. 1996; 184: 1101-1109Crossref PubMed Scopus (1274) Google Scholar, 3Bleul C.C. Fazzan M. Choe H. Parolin C. Clark-Lewis I. Sodroski J. Springer T.A. Nature. 1996; 382: 829-833Crossref PubMed Scopus (1749) Google Scholar, 4Oberlin E. Amara A. Bachelerie F. Bessia C. Virelizier J-L. Arenzana-Seisdedos F. Schwartz O. Heard J.-M. Clark-Lewis I. Legler D.F. Loetscher M. Baggiolini M. Moser B. Nature. 1996; 382: 833-835Crossref PubMed Scopus (1478) Google Scholar). While some investigators failed to detect chemotactic activity of SDF-1 for neutrophils (1Bleul C.C. Fuhlbrigge R.C. Casasnovas J.M. Aiuti A. Springer T.A. J. Exp. Med. 1996; 184: 1101-1109Crossref PubMed Scopus (1274) Google Scholar), others were able to induce significant Ca2+ mobilization in neutrophils at physiologically relevant concentrations of SDF-1 (4Oberlin E. Amara A. Bachelerie F. Bessia C. Virelizier J-L. Arenzana-Seisdedos F. Schwartz O. Heard J.-M. Clark-Lewis I. Legler D.F. Loetscher M. Baggiolini M. Moser B. Nature. 1996; 382: 833-835Crossref PubMed Scopus (1478) Google Scholar). In an effort to clarify the activity of SDF-1 on neutrophils, we tested the binding and function of N33A on neutrophils in comparison rhSDF-1α. As shown in Fig.3, human peripheral blood neutrophils expressed a substantial number of specific binding sites for both N33A (Fig. 3 A) and native SDF-1α (Fig. 3 B). The binding is of high affinity with estimated K d values of about 5 nm. N33A and rhSDF-1α competed equally well for each other's binding as shown by the displacement curve (Fig.3 C and data not shown). Neutrophils also migrated in response to both N33A and rhSDF-1α, indicating that neutrophils are indeed among the target cell types for SDF-1. To further confirm that N33A utilizes CXCR4 as its functional receptor, HEK293 cells stably expressing CXCR4 (CXCR4/293 cells) were employed. Wild type HEK 293 cells exhibited a low level of specific binding (about 200 binding sites/cell) for both N33A and native SDF-1α. Both N33A and native SDF-1 also induced a weak but significant directional migration of wild type HEK293 cells (CI = 2.1 ± 0.2, at 120 nm ligand concentration). This is in agreement with the notion that a great variety of nonhematopoietic cells express CXCR4 mRNA. However, HEK293 cells overexpressing CXCR4 expressed markedly increased number of specific binding sites for radiolabeled N33A and rhSDF-1α (Fig. 4, A andB). N33A and rhSDF-1α mutually competed for binding to CXCR4/293 cells (Fig. 4, C and D). Both N33A and rhSDF-1α were able to induce a remarkable directional migration of CXCR4/293 cells (Fig. 5) with similar potency and efficacy. HEK293 cells transfected with known chemokine receptors, including CXCR1, CXCR2, and CCR1–5, did not show any increased binding or activation by N33A or rhSDF-1α over background levels, whereas these cells specifically bound and migrated in response to their ligands (data not shown). These results demonstrate that N33A, like rhSDF-1α, uses CXCR4 as a functional receptor.Figure 5Chemotactic response of CXCR4/293 cells induced by N33A and rhSDF-1α. CXCR4/293 cell migration was measured by 300 min incubation at 37 °C in microchemotaxis chambers as described previously (21Ben-Baruch A. Xu L. Young P.R. Bengali K. Oppenheim J.J. Wang J.M. J. Biol. Chem. 1995; 270: 22123-22128Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). CI ≥ 2 are statistically significant in comparison with the spontaneous migration.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Chemokines are important mediators that participate in a variety of pathophysiological conditions including inflammation, infection, tissue injury and repair, immune responses, as well as malignancy (6Oppenheim J.J. Wang J.M. Chertov O. Taub D.D. Ben-Baruch A. Tilney L.N. Strom T.B. Paul L.C. Transplantation Biology: Cellular and Molecular Aspects. 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Thus, the role for SDF-1 as a primordial chemokine regulating primarily the tissue distribution of leukocytes needs further investigation. SDF-1 also has several essential functions in development (2Nagasawa T. Hirota S. Tachibana K. Takakura N. Nishikawa S. Kitamura Y. Yoshida N. Kikutani H. Kishimoto T. Nature. 1996; 382: 635-638Crossref PubMed Scopus (1999) Google Scholar). Mice lacking SDF-1 due to homologous recombination died perinatally and although the numbers of B cell progenitors in mutant embryos were severely reduced in fetal liver and bone marrow, myeloid progenitors were reduced only in the bone marrow not in the fetal liver, indicating that SDF-1 is responsible for B cell lymphopoiesis and bone marrow myelopoiesis. In addition, mice deprived of SDF-1 gene had a cardiac ventricular septal defect (2Nagasawa T. Hirota S. Tachibana K. Takakura N. Nishikawa S. Kitamura Y. Yoshida N. Kikutani H. Kishimoto T. Nature. 1996; 382: 635-638Crossref PubMed Scopus (1999) Google Scholar). The SDF-1α analogue N33A was chemically synthesized and was shown in this study to be equally as potent as native SDF-1α in binding and activating human leukocytes as well as CXCR4-transfected HEK293 cells. N33A has also been shown to induce human B lymphocyte migration and to effectively inhibit infection of PM-1 cells by HIV-1(LAV). 2M. A. Siani, D. A. Thompson, L. E. Canne, G. M. Figliozzi, S. B. H. Kent, R. Simon, J. Cyster, P. E. Kennedy, E. D. Smith, and E. A. Berger, unpublished observation. The preparation of analogues is greatly facilitated by molecular chemical synthesis, in which proteins can be produced either singly or by combinational methods. The analogues could include a full range of genetically encoded amino acids as well as unnatural backbone structures and unclonable residues such as d-amino acids, fluorescent or nuclear magnetic resonance-sensitive nuclei. The activities of analogues can also be tuned by fast cycles of synthesis-design-assay-resynthesis (33Robson B. Nat. Biotechnol. 1996; 14: 892-893Crossref PubMed Scopus (11) Google Scholar). The validity of this approach is demonstrated by the fact that it yields functional molecules such as the SDF-1α analogue N33A, a biological contamination-free agonist of SDF-1 that can be utilized in studies of the immune system and host defense against AIDS. We thank Kathleen Bengali and Nancy Dunlop for technical support, and Cheryl Fogle and Teresa Covell for secretarial assistance. The critical review of this manuscript by Dr. Joost J. Oppenheim is greatly appreciated.
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