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• W1593400773 abstract "Distinct from the noncovalently linked recombinant human stem cell factor (rhSCF) dimer, we report here the isolation and identification of an SDS-nondissociable dimer produced during folding/oxidation of rhSCF. Experimental evidence using various cleavage strategies and analyses shows that the isolated dimer is composed of two rhSCF monomers covalently linked by four disulfide bonds. The cysteines are paired as in the noncovalently associated dimer except that all pairings are intermolecular rather than intramolecular. Other structural models, involving intertwining of intramolecular disulfide loops, are ruled out. The molecule behaves similarly to the noncovalently associated dimer during ion-exchange or gel permeation chromatography. However, the disulfide-linked dimer exhibits increased hydrophobicity in reverse-phase columns and in the native state does not undergo spontaneous dimer dissociation-association as seen for the noncovalent dimer. Spectroscopic analyses indicate that the disulfide-linked and noncovalently associated rhSCF dimers have grossly similar secondary and tertiary structures. In vitro, the disulfide-linked dimer exhibits approximately 3-fold higher biological activity in supporting growth of a hematopoietic cell line and stimulating hematopoietic cell colony formation from enriched human CD34+ cells. The molecule binds to the rhSCF receptor, Kit, with an efficiency only half that of the noncovalently associated dimer. Formation of intermolecular disulfides in the disulfide-linked dimer with retention of biological activity has implications for the three-dimensional structure of noncovalently held dimer and disulfide-linked dimer. Distinct from the noncovalently linked recombinant human stem cell factor (rhSCF) dimer, we report here the isolation and identification of an SDS-nondissociable dimer produced during folding/oxidation of rhSCF. Experimental evidence using various cleavage strategies and analyses shows that the isolated dimer is composed of two rhSCF monomers covalently linked by four disulfide bonds. The cysteines are paired as in the noncovalently associated dimer except that all pairings are intermolecular rather than intramolecular. Other structural models, involving intertwining of intramolecular disulfide loops, are ruled out. The molecule behaves similarly to the noncovalently associated dimer during ion-exchange or gel permeation chromatography. However, the disulfide-linked dimer exhibits increased hydrophobicity in reverse-phase columns and in the native state does not undergo spontaneous dimer dissociation-association as seen for the noncovalent dimer. Spectroscopic analyses indicate that the disulfide-linked and noncovalently associated rhSCF dimers have grossly similar secondary and tertiary structures. In vitro, the disulfide-linked dimer exhibits approximately 3-fold higher biological activity in supporting growth of a hematopoietic cell line and stimulating hematopoietic cell colony formation from enriched human CD34+ cells. The molecule binds to the rhSCF receptor, Kit, with an efficiency only half that of the noncovalently associated dimer. Formation of intermolecular disulfides in the disulfide-linked dimer with retention of biological activity has implications for the three-dimensional structure of noncovalently held dimer and disulfide-linked dimer. Stem cell factor (SCF), 1The abbreviations used are: SCFstem cell factorrhSCFrecombinant human SCFM-CSFmacrophage colony-stimulating factorPDGFplatelet-derived growth factorCFU-GMcolony forming units-granulocyte macrophageBFU-Eburst forming unit-erythroidrhG-CSFrecombinant human granulocyte colony-stimulating factorGM-CFCgranulocyte macrophage-colony forming cellsEPOerythropoietinCDcircular dichroismPVDFpolyvinylene difluorideTFAtrifluoroacetic acidDTTdithiothreitolHPLChigh performance liquid chromatographyPAGEpolyacrylamide gel electrophoresisCHOChinese hamster ovaryBNPS-skatole3-bromo-3-methyl-2-(2-nitrophenylmercapto-3H-indole. also termed “kit ligand” or “mast cell growth factor”(1.Zsebo K.M. Wypych J. McNiece I.K. Lu H.S. Smith K.A. Karkare S.B. Sachdev R.K. Yuschenkoff V.N. Birkett N.C. Williams L.R. Satyagal V.N. Tung W. Bosselman R.A. Mendiaz E.A. Langley K.E. Cell. 1990; 63: 195-201Abstract Full Text PDF PubMed Scopus (657) Google Scholar, 2.Martin F.H. Suggs S.V. Langley K.E. Lu H.S. Ting J. Okino K.H. Morris C.F. McNiece I.K. Jacobsen F.W. Mendiaz E.A. Birkett N.C. Smith K.C. Johnson M.J. Parker V.P. Flores J.C. Patel A.C. Fisher E.F. Erjavec H.O. Herrera C.J. Wypych J. Sachdev R.K. Pope J.A. Leslie I. Wen D. Lin C.W. Cupples R.L. Zsebo K.M. Cell. 1990; 63: 203-211Abstract Full Text PDF PubMed Scopus (601) Google Scholar, 3.Zsebo K.M. Williams D.A. Geissler E.N. Broudy V.C. Martin F.H. Atkins H.L. Hsu R.-Y. Birkett N.C. Okino K.H. Murdock D.C. Jacobsen F.W. Langley K.E. Smith K.A. Takeishi T. Cattanach B.M. Galli S.J. Suggs S.V. Cell. 1990; 63: 213-224Abstract Full Text PDF PubMed Scopus (1219) Google Scholar, 4.Williams D.E. Eisenman J. Baird A. Rauch C. Van Ness K. March C.J. Park L.S. Martin U. Mochizuki D.Y. Boswell H.S. Burgess G.S. Cosman D. Lyman S.D. Cell. 1990; 63: 167-174Abstract Full Text PDF PubMed Scopus (774) Google Scholar, 5.Copeland N.G. Gilbert D.J. Cho B.C. Donovan P.J. Jenkins N.A. Cosman D. Anderson D. Lyman S.D. Williams D.E. Cell. 1990; 63: 175-183Abstract Full Text PDF PubMed Scopus (487) Google Scholar, 6.Huang E. Nocka K. Beier D.R. Chu T.-Y. Buck J. Lahm H.-W. Wellner D. Leder P. Besmer P. Cell. 1990; 63: 225-233Abstract Full Text PDF PubMed Scopus (941) Google Scholar) functions in the early stages of hematopoiesis and is also involved in the development and function of other cell lineages, including melanocytes and germ cells(7.Russell E.S. Adv. Genet. 1979; 20: 357-459Crossref PubMed Scopus (844) Google Scholar, 8.Silvers W.K. The Coat Colors of Mice. A Model for Mammalian Gene Action and Interaction. Springer-Verlag, New York1979Crossref Google Scholar). SCF is initially synthesized as membrane-bound forms of 248 or 220 amino acids, depending on alternative splicing of exon 6. A soluble SCF form of 165 amino acids is biologically functional, apparently arising by proteolytic release from the extracellular domain of the membrane-bound 248-amino acid SCF(9.Flanagan J.G. Chan D.C. Leder P. Cell. 1991; 64: 1025-1035Abstract Full Text PDF PubMed Scopus (618) Google Scholar, 10.Toksoz D. Zsebo K.M. Smith K.A. Hu S. Brankow D. Suggs S.V. Martin F.H. Williams D.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7350-7354Crossref PubMed Scopus (253) Google Scholar, 11.Lu H.S. Clogston C.L. Parker V.P. Wypych J. Lee T.D. Swiderek K. Baltera Jr., R.F. Patel A.C. Brankow D.W. Liu X.-D. Ogden S.G. Karkare S.B. Hu S.S. Zsebo K.M. Langley K.E. Arch. Biochem. Biophys. 1992; 298: 150-158Crossref PubMed Scopus (38) Google Scholar). The naturally occurring soluble SCF is glycosylated at both N-linked and O-linked sites(12.Lu H.S. Clogston C.L. Wypych J. Fausset P.R. Lauren S. Mendiaz E.A. Zsebo K.M. Langley K.E. J. Biol. Chem. 1991; 266: 8102-8107Abstract Full Text PDF PubMed Google Scholar, 13.Langley K.E. Bennett L.G. Wypych J. Yancik S.A. Liu X.-D. Westcott K.R. Chang D.G. Smith K.A. Zsebo K.M. Blood. 1993; 81: 656-660Crossref PubMed Google Scholar). stem cell factor recombinant human SCF macrophage colony-stimulating factor platelet-derived growth factor colony forming units-granulocyte macrophage burst forming unit-erythroid recombinant human granulocyte colony-stimulating factor granulocyte macrophage-colony forming cells erythropoietin circular dichroism polyvinylene difluoride trifluoroacetic acid dithiothreitol high performance liquid chromatography polyacrylamide gel electrophoresis Chinese hamster ovary 3-bromo-3-methyl-2-(2-nitrophenylmercapto-3H-indole. SCF binds to its receptor, Kit, to elicit its specific biological functions(1.Zsebo K.M. Wypych J. McNiece I.K. Lu H.S. Smith K.A. Karkare S.B. Sachdev R.K. Yuschenkoff V.N. Birkett N.C. Williams L.R. Satyagal V.N. Tung W. Bosselman R.A. Mendiaz E.A. Langley K.E. Cell. 1990; 63: 195-201Abstract Full Text PDF PubMed Scopus (657) Google Scholar, 2.Martin F.H. Suggs S.V. Langley K.E. Lu H.S. Ting J. Okino K.H. Morris C.F. McNiece I.K. Jacobsen F.W. Mendiaz E.A. Birkett N.C. Smith K.C. Johnson M.J. Parker V.P. Flores J.C. Patel A.C. Fisher E.F. Erjavec H.O. Herrera C.J. Wypych J. Sachdev R.K. Pope J.A. Leslie I. Wen D. Lin C.W. Cupples R.L. Zsebo K.M. Cell. 1990; 63: 203-211Abstract Full Text PDF PubMed Scopus (601) Google Scholar, 3.Zsebo K.M. Williams D.A. Geissler E.N. Broudy V.C. Martin F.H. Atkins H.L. Hsu R.-Y. Birkett N.C. Okino K.H. Murdock D.C. Jacobsen F.W. Langley K.E. Smith K.A. Takeishi T. Cattanach B.M. Galli S.J. Suggs S.V. Cell. 1990; 63: 213-224Abstract Full Text PDF PubMed Scopus (1219) Google Scholar). The Kit receptor belongs to the type III tyrosine kinase family whose members include receptors for M-CSF and PDGF (14.Yarden Y. Kuang W.-J. Yang-Feng T. Coussens L. Munemitsu S. Dulli T.J. Chen E. Schlessinger J. Francke U. Ullrich A. EMBO J. 1987; 6: 3341-3351Crossref PubMed Scopus (1330) Google Scholar, 15.Ullrich A. Schlessinger J. Cell. 1990; 61: 203-212Abstract Full Text PDF PubMed Scopus (4619) Google Scholar, 16.Miyajima A. Kitamura T. Harada N. Yokota T. Arai K.-I. Annu. Rev. Immunol. 1992; 10: 295-331Crossref PubMed Scopus (540) Google Scholar). SCF, M-CSF, and PDGF are all dimeric ligands (17.Arakawa T. Yphantis D.A. Lary J.W. Narhi L.O. Lu H.S. Prestrelski S.J. Clogston C.L. Zsebo K.M. Mendiaz E.A. Wypych J. Langley K.E. J. Biol. Chem. 1991; 266: 18942-18948Abstract Full Text PDF PubMed Google Scholar, 18.Langley K.E. Wypych J. Mendiaz E.A. Clogston C.L. Parker V.P. Farrar D.H. Brothers M.O. Satyagal V.N. Leslie I. Birkett N.C. Smith K.A. Baltera Jr., R.F. Lyons D.E. Hogan J.M. Crandall C. Boone T.C. Pope J.A. Karkare S.B. Zsebo K.M. Sachdev R.K. Lu H.S. Arch. Biochem. Biophys. 1992; 295: 21-28Crossref PubMed Scopus (53) Google Scholar, 19.Johnsson A. Heldin C.-H. Westermark B. Wasteson A. Biochem. Biophys. Res. Commun. 1982; 104: 66-74Crossref PubMed Scopus (163) Google Scholar, 20.Das S.K. Stanley E.R. J. Biol. Chem. 1982; 257: 13679-13684Abstract Full Text PDF PubMed Google Scholar) that mediate receptor dimerization(17.Arakawa T. Yphantis D.A. Lary J.W. Narhi L.O. Lu H.S. Prestrelski S.J. Clogston C.L. Zsebo K.M. Mendiaz E.A. Wypych J. Langley K.E. J. Biol. Chem. 1991; 266: 18942-18948Abstract Full Text PDF PubMed Google Scholar, 18.Langley K.E. Wypych J. Mendiaz E.A. Clogston C.L. Parker V.P. Farrar D.H. Brothers M.O. Satyagal V.N. Leslie I. Birkett N.C. Smith K.A. Baltera Jr., R.F. Lyons D.E. Hogan J.M. Crandall C. Boone T.C. Pope J.A. Karkare S.B. Zsebo K.M. Sachdev R.K. Lu H.S. Arch. Biochem. Biophys. 1992; 295: 21-28Crossref PubMed Scopus (53) Google Scholar, 19.Johnsson A. Heldin C.-H. Westermark B. Wasteson A. Biochem. Biophys. Res. Commun. 1982; 104: 66-74Crossref PubMed Scopus (163) Google Scholar, 20.Das S.K. Stanley E.R. J. Biol. Chem. 1982; 257: 13679-13684Abstract Full Text PDF PubMed Google Scholar). We have previously described the isolation and characterization of soluble SCF1-165 recombinantly expressed in Escherichia coli in a nonglycosylated form (rhSCF), and by CHO cells in a glycosylated form (11.Lu H.S. Clogston C.L. Parker V.P. Wypych J. Lee T.D. Swiderek K. Baltera Jr., R.F. Patel A.C. Brankow D.W. Liu X.-D. Ogden S.G. Karkare S.B. Hu S.S. Zsebo K.M. Langley K.E. Arch. Biochem. Biophys. 1992; 298: 150-158Crossref PubMed Scopus (38) Google Scholar, 17.Arakawa T. Yphantis D.A. Lary J.W. Narhi L.O. Lu H.S. Prestrelski S.J. Clogston C.L. Zsebo K.M. Mendiaz E.A. Wypych J. Langley K.E. J. Biol. Chem. 1991; 266: 18942-18948Abstract Full Text PDF PubMed Google Scholar, 18.Langley K.E. Wypych J. Mendiaz E.A. Clogston C.L. Parker V.P. Farrar D.H. Brothers M.O. Satyagal V.N. Leslie I. Birkett N.C. Smith K.A. Baltera Jr., R.F. Lyons D.E. Hogan J.M. Crandall C. Boone T.C. Pope J.A. Karkare S.B. Zsebo K.M. Sachdev R.K. Lu H.S. Arch. Biochem. Biophys. 1992; 295: 21-28Crossref PubMed Scopus (53) Google Scholar). These SCFs are fully native and biologically functional. In contrast with the M-CSF and PDGF dimers whose monomers are disulfide-linked(19.Johnsson A. Heldin C.-H. Westermark B. Wasteson A. Biochem. Biophys. Res. Commun. 1982; 104: 66-74Crossref PubMed Scopus (163) Google Scholar, 20.Das S.K. Stanley E.R. J. Biol. Chem. 1982; 257: 13679-13684Abstract Full Text PDF PubMed Google Scholar, 21.Glocker M.O. Arbogast B. Schreurs J. Deinzer M.L. Biochemistry. 1993; 32: 482-488Crossref PubMed Scopus (54) Google Scholar), both glycosylated and nonglycosylated SCF dimers contain noncovalently linked monomers(12.Lu H.S. Clogston C.L. Wypych J. Fausset P.R. Lauren S. Mendiaz E.A. Zsebo K.M. Langley K.E. J. Biol. Chem. 1991; 266: 8102-8107Abstract Full Text PDF PubMed Google Scholar, 17.Arakawa T. Yphantis D.A. Lary J.W. Narhi L.O. Lu H.S. Prestrelski S.J. Clogston C.L. Zsebo K.M. Mendiaz E.A. Wypych J. Langley K.E. J. Biol. Chem. 1991; 266: 18942-18948Abstract Full Text PDF PubMed Google Scholar). The SCF noncovalently associated dimer was observed to undergo spontaneous dissociation-reassociation of monomers in its native state(22.Lu H.S. Chang W.-C. Mendiaz E.A. Mann M.B. Langley K.E. Hsu Y.-R. Biochem. J. 1995; 305: 563-568Crossref PubMed Scopus (27) Google Scholar). In a companion paper(23.Jones M.D. Narhi L.O. Chang W.-C. Lu H.S. J. Biol. Chem. 1996; 271: xxxx-xxxxGoogle Scholar), we described the isolation and characterization of intermediates derived during folding and oxidation of the reduced and denatured rhSCF and the assignment of a predominant in vitro folding pathway. The major folded SCF is the noncovalently linked dimer (SDS-dissociable) and a small fraction is SDS-nondissociable dimer. In the present study, we isolate the nondissociable dimer to apparent purity and demonstrate that it is biologically functional and is covalently linked by four intermolecular disulfide bonds involving all cysteinyl residues. The biological, biochemical, biophysical, and structural properties of the noncovalently and covalently linked dimers are compared, and the results provide some insights to the structure and function of SCF. E. coli-derived rhSCF (SDS-dissociable dimer) was purified according to methods described previously(17.Arakawa T. Yphantis D.A. Lary J.W. Narhi L.O. Lu H.S. Prestrelski S.J. Clogston C.L. Zsebo K.M. Mendiaz E.A. Wypych J. Langley K.E. J. Biol. Chem. 1991; 266: 18942-18948Abstract Full Text PDF PubMed Google Scholar, 18.Langley K.E. Wypych J. Mendiaz E.A. Clogston C.L. Parker V.P. Farrar D.H. Brothers M.O. Satyagal V.N. Leslie I. Birkett N.C. Smith K.A. Baltera Jr., R.F. Lyons D.E. Hogan J.M. Crandall C. Boone T.C. Pope J.A. Karkare S.B. Zsebo K.M. Sachdev R.K. Lu H.S. Arch. Biochem. Biophys. 1992; 295: 21-28Crossref PubMed Scopus (53) Google Scholar). The recombinant molecule contains 165 amino acids plus an N-terminal methionine at position −1. Iodoacetic acid was purchased from Sigma. HPLC solvents and water were purchased from Burdick and Jackson. Sequencing reagents and solvents were supplied by Applied Biosystems (Foster City, CA) and Hewlett Packard (Mountain View, CA). All other reagents were of the highest quality available. SeaPlaque Agarose low gelling temperature was obtained from FMC BioProducts (Rockland, ME). X VIVO-15 medium and fetal calf serum were purchased from BioWhittaker Inc. (Walkerville, MD) and Hyclone Labs (Logan, UT), respectively. Recovery of rhSCF expressed in E. coli includes solubilization of rhSCF-containing inclusion bodies, oxidation, and folding and subsequent chromatographic steps(18.Langley K.E. Wypych J. Mendiaz E.A. Clogston C.L. Parker V.P. Farrar D.H. Brothers M.O. Satyagal V.N. Leslie I. Birkett N.C. Smith K.A. Baltera Jr., R.F. Lyons D.E. Hogan J.M. Crandall C. Boone T.C. Pope J.A. Karkare S.B. Zsebo K.M. Sachdev R.K. Lu H.S. Arch. Biochem. Biophys. 1992; 295: 21-28Crossref PubMed Scopus (53) Google Scholar). After cation-exchange chromatography using an S Sepharose column, pooled SCF (approximately 1 liter containing 600 mg of rhSCF) was further subjected to C-4 reverse-phase chromatography performed with a BioCat liquid chromatographic system (Perceptive Inc., NJ). Sample in 10 mM sodium acetate buffer, pH 4.5 was loaded onto a column (2.6 × 7.8 cm) packed with C4 silica gel (100 Å, wide pore; Vydac, San Jose, CA) preequilibrated with 12 mM HCl. After loading, the column was washed with mobile phase A (30% ethanol in 12 mM HCl). The separation was accomplished by a linear gradient from 0% mobile phase A to 100% mobile phase B (80% ethanol in 12 mM HCl) at a flow rate of 20 ml/min for 10 h. The column effluent was monitored continuously by a UV detector set at 280 nm for protein detection. SDS nondissociable SCF dimer elutes later than the bulk of the SCF (18.Langley K.E. Wypych J. Mendiaz E.A. Clogston C.L. Parker V.P. Farrar D.H. Brothers M.O. Satyagal V.N. Leslie I. Birkett N.C. Smith K.A. Baltera Jr., R.F. Lyons D.E. Hogan J.M. Crandall C. Boone T.C. Pope J.A. Karkare S.B. Zsebo K.M. Sachdev R.K. Lu H.S. Arch. Biochem. Biophys. 1992; 295: 21-28Crossref PubMed Scopus (53) Google Scholar) (see “Results”); it was pooled separately and immediately diluted into 10 volumes of 10 mM sodium acetate buffer, pH 5.0, concentrated by ultrafiltration, and buffer-exchanged by diafiltration using the same sodium acetate buffer. All purification steps were carried out at 5°C. The final material at 7 mg/ml was stored at −80°C. Reverse-phase HPLC was performed using TFA-acetonitrile gradient elution. A Vydac C4 column (4.6 mm × 25 cm; 300 Å) was equilibrated with 97% solvent A (0.1% TFA), 3% solvent B (0.1% TFA in 90% acetonitrile) with 215 and 280-nm UV detection at a flow rate of 0.7 ml/min. After samples were injected into the column, the following elution program was used: a linear gradient to 20% solvent B in 5 min and to 70% B in 60 min, then isocratic elution at 70% B for 20 min. Endoproteinase Asp-N digestion of SCF samples and peptide mapping procedures were essentially identical to those described earlier(18.Langley K.E. Wypych J. Mendiaz E.A. Clogston C.L. Parker V.P. Farrar D.H. Brothers M.O. Satyagal V.N. Leslie I. Birkett N.C. Smith K.A. Baltera Jr., R.F. Lyons D.E. Hogan J.M. Crandall C. Boone T.C. Pope J.A. Karkare S.B. Zsebo K.M. Sachdev R.K. Lu H.S. Arch. Biochem. Biophys. 1992; 295: 21-28Crossref PubMed Scopus (53) Google Scholar). N-terminal amino acid sequence analysis of peptides was performed on an automatic protein sequencer (Applied Biosystems models 477A and 470 or Hewlett Packard HPG1000A) as described elsewhere(12.Lu H.S. Clogston C.L. Wypych J. Fausset P.R. Lauren S. Mendiaz E.A. Zsebo K.M. Langley K.E. J. Biol. Chem. 1991; 266: 8102-8107Abstract Full Text PDF PubMed Google Scholar). Procedures used to sequence peptides recovered from gel bands electroblotted onto PVDF membranes were described in a previous report(24.Fausset P.R. Lu H.S. Electrophoresis. 1991; 12: 22-27Crossref PubMed Scopus (10) Google Scholar). The conditions used were found to completely oxidize all Met residues except Met48. SDS-nondissociable rhSCF dimer at 1 mg/ml in 10 mM sodium acetate, pH 5.0, was incubated with 0.5% (w/v) H2O2 at 25°C for 3 h. After reaction, the mixture was purified by analytical reverse-phase HPLC as described above. A major oxidized peak eluting earlier than the unoxidized SCF dimer was obtained. Peptide mapping using Asp-N endoproteinase digestion (below) showed that all Met residues but Met48 were completely converted to the sulfoxide derivatives. Only a small fraction (about 10%) of Met48 was oxidized. The HPLC purified sample was subjected to CNBr cleavage as described below. Cleavage of rhSCF dimer species by BNPS-skatole (Pierce) was carried out in 50% acetic acid as described elsewhere(12.Lu H.S. Clogston C.L. Wypych J. Fausset P.R. Lauren S. Mendiaz E.A. Zsebo K.M. Langley K.E. J. Biol. Chem. 1991; 266: 8102-8107Abstract Full Text PDF PubMed Google Scholar). This reaction allows the cleavage of the sample at Trp44, which is the lone Trp in each monomer. A complete CNBr cleavage at the Met residues of SCF dimer species or H2O2-oxidized SCF dimer species was performed as follows. Vacuum-dried samples were redissolved in 70% formic acid (0.2 mg in 150 μl) and then incubated with freshly prepared CNBr (400 molar ratio to SCF) at 25°C for 24 h in the dark. For partial CNBr cleavages, 50-fold molar excess of CNBr was used and the incubation times were shortened to 2-8 h. All the cleaved samples were immediately vacuum dried for further analysis. Aliquots of dried samples (5-20 μg) were loaded onto individual lanes of precast 16% Laemmli polyacrylamide gels (10 wells; Novex Inc., San Diego, CA) and electrophoresed (25.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) under nonreducing and reducing conditions. After Coomassie Blue staining and destaining, protein band intensity in each gel lane was measured using an image scanner (PDI Inc., New York); images were integrated using PDQuest software (PDI Inc.). In separate analyses, gel bands were also electrophoretically transferred onto PVDF membrane and the Coomassie Blue-stained bands were excised for N-terminal sequence analysis (24.Fausset P.R. Lu H.S. Electrophoresis. 1991; 12: 22-27Crossref PubMed Scopus (10) Google Scholar). A sample (1 mg/ml) was reconstituted in 20 mM Tris-HCl buffer and digested with endoproteinase Lys-C (enzyme-to-substrate ratio = 1:100) at 25°C. At 15 min and 2 h, sample aliquots (100 μl each) were taken, and digestion was stopped by adding 5 μl of 20% TFA. Samples of 5-20 μg were dried completely and subjected to SDS-PAGE as described. One mg/ml solutions of SCF dimer species were incubated in the presence of 1.24 mg/ml DTT in 0.1 M Tris-HCl buffer (pH 8.5) containing 2.5 M urea, 60 mM NaCl, 2 mM EDTA. Aliquots of the reaction mixture were removed at selected time intervals, and unreacted thiols were blocked by the addition of 1 M iodoacetic acid (10:1 molar ratio to the thiol) in 0.3 M Tris, pH 8.0, for 2 min at room temperature. Samples were then quickly frozen in a methanol/dry ice bath and subsequently analyzed by reverse phase-HPLC using conditions described previously(23.Jones M.D. Narhi L.O. Chang W.-C. Lu H.S. J. Biol. Chem. 1996; 271: xxxx-xxxxGoogle Scholar). Circular dichroism (CD) and fluorescence spectroscopic studies and thermostability analysis of SCF forms were performed as described in a companion study(23.Jones M.D. Narhi L.O. Chang W.-C. Lu H.S. J. Biol. Chem. 1996; 271: xxxx-xxxxGoogle Scholar). Samples were in 20 mM Tris-HCl, pH 7.5, for all biophysical analyses. [3H]Thymidine incorporation by the human megakaryoblastic leukemia cell line UT-7 was monitored in the cell proliferation assay as described previously(26.Smith K.A. Zsebo K.M. Coligan J.E. Kruisbeek A.M. Margulies D.H. Shevach E.M. Strober W. Current Protocols in Immunology. John Wiley & Sons, New York1992: 6.17.1-6.17.11Google Scholar). Receptor binding assays were also performed using whole cell lysate containing membrane-associated Kit receptor as reported elsewhere(26.Smith K.A. Zsebo K.M. Coligan J.E. Kruisbeek A.M. Margulies D.H. Shevach E.M. Strober W. Current Protocols in Immunology. John Wiley & Sons, New York1992: 6.17.1-6.17.11Google Scholar). Human leukapheresis products were obtained from consenting healthy donors and patients with approval of the Institute review committee. Isolation of CD34+ cells from peripheral blood and bone marrow cells was carried out using Miltenyi MiniMacs columns as suggested by the manufacturer (Miltenyi Biotec. Inc., Sunnyvale, CA). Briefly, Ficoll-Paque medium (Pharmacia Biotech Inc.) was used to fractionate mononuclear cells. The mononuclear cells were subsequently stained with mouse anti-human CD34 antibody (QBEND/10) and colloidal magnetic microbeads recognizing mouse IgG. The stained cells were then applied to the MiniMacs column with magnetic separator and washed with 500 μl of buffer four times. After removal of the column from the magnet, the retained cells were eluted with 500 μl of phosphate-buffered saline with 0.5% bovine serum albumin. Approximately 80% pure CD34+ cells were routinely isolated by this method as judged by fluorescein-activated cell sorting(27.Briddell R. Shieh J.-H. Chen Y.-F. Schuster L. McNiece I.K. Blood. 1995; 84 (abstr.): 2918Google Scholar). CFU-GM assays were performed in 35-mm Petri dishes with double-layer agarose cultures, as described previously with minor modifications(28.Bradley T. Hodgson G. Rosendaal M. J. Cell. Physiol. 1978; 94: 517Crossref Scopus (186) Google Scholar). An underlayer (1 ml of 0.5% agarose) contained X VIVO-15 media supplemented with 20% FCS (EX-15, 20% FCS), rhG-CSF (100 ng/dish) and rhSCF (from 0.01 to 300 ng). An overlayer (1 ml of 0.36% agarose) contained EX-15, 20% FCS and CD34+ cells. Two cell concentrations, 1,000 and 5,000 cells/dish, were assayed in triplicate. Cultures were incubated at 37°C in a fully humidified atmosphere containing 5% CO2 for 14 days. GM-CFC were defined as colonies containing 50 cells or more. BFU-E assays were performed using procedures identical to GM-CFC assays except that EPO (4 units/ml) was added to the cultures instead of rhG-CSF. Expression of rhSCF in bacteria has resulted in the production of insoluble, inactive SCF accumulated in inclusion bodies. Solubilization and in vitro folding and oxidation are therefore necessary for the recovery and chromatographic purification of active SCF(17.Arakawa T. Yphantis D.A. Lary J.W. Narhi L.O. Lu H.S. Prestrelski S.J. Clogston C.L. Zsebo K.M. Mendiaz E.A. Wypych J. Langley K.E. J. Biol. Chem. 1991; 266: 18942-18948Abstract Full Text PDF PubMed Google Scholar, 18.Langley K.E. Wypych J. Mendiaz E.A. Clogston C.L. Parker V.P. Farrar D.H. Brothers M.O. Satyagal V.N. Leslie I. Birkett N.C. Smith K.A. Baltera Jr., R.F. Lyons D.E. Hogan J.M. Crandall C. Boone T.C. Pope J.A. Karkare S.B. Zsebo K.M. Sachdev R.K. Lu H.S. Arch. Biochem. Biophys. 1992; 295: 21-28Crossref PubMed Scopus (53) Google Scholar, 23.Jones M.D. Narhi L.O. Chang W.-C. Lu H.S. J. Biol. Chem. 1996; 271: xxxx-xxxxGoogle Scholar). The rhSCF isolated in this way is a noncovalently linked, SDS-dissociable dimer (17.Arakawa T. Yphantis D.A. Lary J.W. Narhi L.O. Lu H.S. Prestrelski S.J. Clogston C.L. Zsebo K.M. Mendiaz E.A. Wypych J. Langley K.E. J. Biol. Chem. 1991; 266: 18942-18948Abstract Full Text PDF PubMed Google Scholar), like naturally occurring SCF(1.Zsebo K.M. Wypych J. McNiece I.K. Lu H.S. Smith K.A. Karkare S.B. Sachdev R.K. Yuschenkoff V.N. Birkett N.C. Williams L.R. Satyagal V.N. Tung W. Bosselman R.A. Mendiaz E.A. Langley K.E. Cell. 1990; 63: 195-201Abstract Full Text PDF PubMed Scopus (657) Google Scholar). However, during the cationic exchange column after folding and oxidation, we have noticed that rhSCF bands of 18.5 and 37 kDa co-elute, as analyzed by nonreducing SDS-PAGE analysis (Fig. 1A, lanes 1 and 2). Sequence analysis of these two bands electrophoretically transferred onto a PVDF membrane revealed that both have the expected N-terminal sequence of rhSCF. When SDS-PAGE was performed under reducing conditions, the 37-kDa SCF band disappeared and the 18.5-kDa SCF band intensity increased, suggesting that the 37-kDa band is SCF dimer dissociable upon reduction (data not shown). Preparative reverse-phase column chromatography resolved these two species (Fig. 1B). Peak 1 eluting earlier represents the rhSCF which is 18.5 kDa on nonreducing SDS-PAGE and on reducing SDS-PAGE (Fig. 1 A, lanes 4 and 6; note that the reduced protein migrates slightly faster than the nonreduced, as described previously (17.Arakawa T. Yphantis D.A. Lary J.W. Narhi L.O. Lu H.S. Prestrelski S.J. Clogston C.L. Zsebo K.M. Mendiaz E.A. Wypych J. Langley K.E. J. Biol. Chem. 1991; 266: 18942-18948Abstract Full Text PDF PubMed Google Scholar) and as is typical for proteins containing intramolecular disulfide bonds). This form corresponds to the active noncovalently associated, SDS-dissociable rhSCF dimer(17.Arakawa T. Yphantis D.A. Lary J.W. Narhi L.O. Lu H.S. Prestrelski S.J. Clogston C.L. Zsebo K.M. Mendiaz E.A. Wypych J. Langley K.E. J. Biol. Chem. 1991; 266: 18942-18948Abstract Full Text PDF PubMed Google Scholar). Peak 2 represents the SCF dimer which is 37 kDa on nonreducing SDS-PAGE and about 18.5 kDa upon reducing SDS-PAGE (Fig. 1A, lanes 7 and 5, respectively). Analytical reverse-phase HPLC using a TFA-acetonitrile gradient elution is shown in Fig. 1C (bottom chromatogram). This analysis provides a full resolution of the two species and accurately estimates that the disulfide-linked dimer form is 10-20% of the total (varying somewhat between preparations) (top chromatogram). The middle and bottom chromatograms demonstrate the purity of both peaks obtained from the preparative C4 chromatography. Fig. 2 shows peptide map analyses to compare the primary sequence and disulfide structure between the two forms. The two maps derived from Asp-N endoproteinase digestion are indistinguishable. Since peptides 1 and 2, which contain the disulfide bonds Cys4-Cys89 and Cys43-Cys138, respectively, are present in both maps and since the monomers of the SDS-nondissociable SCF dimer become dissociable in the presence of reducing agent, as shown above, it follows that there are two types of structural models to explain the lack of dissociation in SDS (Fig. 3).Figure 3Possible structures for SDS-nondissociable rhSCF dimer. A, disulfide-linked dimers (A1, A2, and A3); the numbers -1, 26, 37, 48, and 159 shown in A1 are Met residues, and 4, 43, 89, and 138 are Cys residues. B, concatenated dimers (B1, B2, B3, B4, and B5).View Large Image" @default.
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• W1593400773 title "Isolation and Characterization of a Disulfide-linked Human Stem Cell Factor Dimer" @default.
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