Soluble Escherichia coli-derived recombinant human stem cell factor (rhSCF) forms a non-covalently associated dimer. We have determined a dimer association constant (K a ) of 2-4 ؋ 10 8 M ؊1 , using sedimentation equilibrium and size exclusion chromatography. SCF has been shown previously to be present at concentrations of approximately 3.3 ng/ml in human serum. Based on the dimerization K a , greater than 90% of the circulating SCF would be in the monomeric form. When 125 I-rhSCF was added to human serum and the serum analyzed by size exclusion chromatography, 72-49% of rhSCF was monomer when the total SCF concentration was in the range of 10 -100 ng/ml, consistent with the K a determination. Three SCF variants, SCF(F63C), SCF (V49L,F63L), and SCF(A165C), were recombinantly expressed in Escherichia coli, purified, and characterized. The dimer K a values, biophysical properties, and biological activities of these variants were studied. Dimerization-defective variants SCF(F63C)S-CH 2 CONH 2 and SCF(V49L,F63L) showed substantially reduced mitogenic activity, while the activity of the Cys 165 -Cys 165 disulfide-linked SCF(A165C) dimer was 10-fold higher than that of wild type rhSCF. The results suggest a correlation between dimerization affinity and biological activity, consistent with a model in which SCF dimerization mediates dimerization of its receptor, Kit, and subsequent signal transduction.Stem cell factor is a cytokine that is active toward early hematopoietic cells and also plays roles in gametogenesis, melanogenesis, and mast cell function. Its biological and other properties have been extensively reviewed (1, 2). It is found in both membrane-bound and soluble forms, with the latter being derived from a membrane-bound form by proteolytic cleavage. The soluble SCF has 165 amino acids.Both soluble Escherichia coli-derived and CHO 1 cell-derived recombinant human SCF have been reported to be non-covalently associated dimers, as determined by sedimentation equilibrium and size exclusion chromatography at protein concentrations above 0.4 mg/ml (3). In a previous paper (4), we demonstrated that SCF dimer is dissociable under non-denaturing conditions and the dissociation rate constant (k d ) of E. coli-derived rhSCF dimer is approximately 1.35 ϫ 10 Ϫ4 s Ϫ1 at pH 4.8, 25°C. In the present work, we arrive at a value of 2-4 ϫ 10 8 M Ϫ1 for the dimer association constant (K a ) of E. coli-derived rhSCF, based on several approaches including ultracentrifugation and size exclusion chromatography at low SCF concentrations. Since the SCF concentration in human serum has been found previously to be a few nanograms/ml (5), the K a value suggests that the majority of SCF in serum may be monomeric. We use 125 I-SCF as a tracer added to serum to show that this does in fact appear to be the case.The binding of ligands to cell receptors, followed by receptor dimerization, is essential for signal transduction by the family of transmembrane receptor tyrosine kinases (6 -8). The receptor for SCF on target cells is Kit (see Refs....

Keratinocyte growth factor (KGF or FGF-7) is a member of the heparin binding fibroblast growth factor (FGF) family and is a paracrine mediator of proliferation and differentiation of a wide variety of epithelial cells. To examine the stoichiometry of complexes formed between KGF and its receptor, we have utilized a soluble variant of the extracellular region of the KGF receptor containing two tandem immunoglobulin-like loops, loops II and III (sKGFR). Ligand-receptor complexes were examined by size exclusion chromatography, light scattering, N-terminal protein sequencing, and sedimentation velocity. In the presence of low-molecular mass heparin ( approximately 3 kDa), we demonstrate the formation of complexes containing two molecules of sKGFR and one molecule of KGF. In the absence of heparin, we were unable to detect any KGF-sKGFR complexes using the above techniques, and additional studies in which sedimentation equilibrium was used show that the binding is very weak (Kd >/= 70 microM). Furthermore, using heparin fragments of defined size, we demonstrate that a heparin octamer or decamer can promote formation of a 2:1 complex, while a hexamer does not. Utilizing the highly purified proteins and defined conditions described in this study, we find that heparin is obligatory for formation of a KGF-sKGFR complex. Finally, 32D cells, which appear to lack low-affinity FGF binding sites, were transfected with a KGFR-erythropoeitin receptor chimera and were found to require heparin to achieve maximal KGF stimulation. Our data are consistent with the previously described concept that cell- or matrix-associated heparan sulfate proteoglycans (HSPGs) and FGF ligands participate in a concerted mechanism that facilitates FGFR dimerization and signal transduction in vivo.

The effect of oxidation of the methionine residues of Escherichia coli-derived recombinant human stem cell factor (huSCF) to methionine sulfoxide on the structure and activity of SCF was examined. Oxidation was performed using hydrogen peroxide under acidic conditions (pH 5.0). The kinetics of oxidation of the individual methionine residues was determined by quantitation of oxidized and unoxidized methionine-containing peptides, using RP-HPLC of Asp-N endoproteinase digests. The initial oxidation rates for Met"', Met", Met2', Met", and Met4' were 0.1 1 min", 0.098 min", 0.033 min", 0.0063 min-l, and 0.00035 min-I, respectively, when SCF was incubated in 0.5% H,Oz at room temperature. Although oxidation of these methionines does not affect the secondary structure of SCF, the oxidation of Met36 and Met48 affects the local structure as indicated by CD and fluorescence spectroscopy. The 295-nm Trp peak in the near-UV CD is decreased upon oxidation of Met3', and lost completely following the oxidation of Met4', indicating that the Trp44 environment is becoming significantly less rigid than it is in native SCF. Consistent with this result, the fluorescence spectra revealed that Trp44 becomes more solvent exposed as the methionines are oxidized, with the hydrophobicity of the Trp44 environment decreasing significantly. The oxidations of Met36 and Met4' decrease biological activity by 40% and 6070, respectively, while increasing the dissociation rate constant of SCF dimer by two-and threefold. These results imply that the oxidation of Met36 and Met4* affects SCF dimerization and tertiary structure, and decreases biological activity.

In its native state, recombinant human-stem-cell-factor (SCF) dimer can spontaneously and rapidly undergo hybridization when two different SCF dimer species are incubated together. SCF species differing in molecular charge, e.g., a wild-type SCF form and a variant with Asp at position 10 instead of Asn, were used in the hybridization studies; the original species and newly formed dimer hybrid can be separated and quantified by cationic-exchange h.p.l.c. The hybridization reaches an equilibrium where the ratio of hybrid dimer to each of the original species is 2. Kinetic studies of the initial rate of hybridization enable a rate constant for monomer dissociation to be determined. This rate constant is influenced by pH, temperature and salt concentration. The pH and salt effects suggest that salt bridges between charged amino acids at the monomer-monomer interface may be present. From the temperature effects, the activation energy for monomer dissociation was determined to be 85.6 kJ/mol, which is typical for oligomeric proteins. Heavily glycosylated recombinant SCF from Chinese-hamster ovary cells exchanged equally well with the bacterially derived non-glycosylated SCF, indicating that the attached carbohydrate moieties had no effect on monomer exchange.

No abstract