Copper staining method for extracting biologically active proteins from native gels.

An attempt was made to make protein bands visible on native gel using copper staining, since such a mild staining procedure would make the entire native gel electrophoresis process non-denaturing. Copper staining not only was able to detect various proteins on native gel with reasonable sensitivity, but also made extraction and recovery of active proteins possible from the gel using a gentle procedure.

Stem cell factor and IL-2 act synergistically in inducing intraepithelial lymphocyte proliferation and cytokine production: upregulation of the IL-2 receptor gamma-chain and signaling via JAK-3.

Murine intraepithelial lymphocytes (IEL) that express the gamma/delta form of the T cell receptor for antigen (TCRgammadelta) also express c-kit, the receptor for stem cell factor (SCF). We show here that SCF upregulates the expression of gammadelta TCR on IEL. More importantly, SCF induces upregulation in the expression of the common gamma-chain (gammac), which is a shared subunit of the receptor complexes for IL-2, -4, -7, -9, and -15. SCF was shown to act synergistically with IL-2 in inducing IEL proliferation, IFNgamma production, non-MHC-restricted cytotoxic activity, and upregulation of the expression of the gammac. SCF also acted synergistically with IL-7 and IL-15 in inducing IEL proliferation. IEL exposed to SCF were shown to have enhanced phosphorylation of JAK-3, and when SCF was combined with IL-2, there was an enhancement in the phosphorylation of JAK-3. These results suggest that SCF may play a more important role in regulating mucosal immune responses than previously appreciated.

Structure of the active core of human stem cell factor and analysis of binding to its receptor kit.

Stem cell factor (SCF) is an early-acting hematopoietic cytokine that elicits multiple biological effects. SCF is dimeric and occurs in soluble and membrane-bound forms. It transduces signals by ligand- mediated dimerization of its receptor, Kit, which is a receptor tyrosine kinase related to the receptors for platelet-derived growth factor (PDGF), macrophage colony-stimulating factor, Flt-3 ligand and vascular endothelial growth factor (VEGF). All of these have extracellular ligand-binding portions composed of immunoglobulin-like repeats. We have determined the crystal structure of selenomethionyl soluble human SCF at 2.2 A resolution by multiwavelength anomalous diffraction phasing. SCF has the characteristic helical cytokine topology, but the structure is unique apart from core portions. The SCF dimer has a symmetric 'head-to-head' association. Using various prior observations, we have located potential Kit-binding sites on the SCF dimer. A superimposition of this dimer onto VEGF in its complex with the receptor Flt-1 places the binding sites on SCF in positions of topographical and electrostatic complementarity with the Kit counterparts of Flt-1, and a similar model can be made for the complex of PDGF with its receptor.

Stem cell factor (SCF) can regulate the activation and expansion of murine intraepithelial lymphocytes.

Murine intraepithelial lymphocytes (IEL) express c-kit, the receptor for stem cell factor (SCF). SCF induced a low but significant proliferative response in IEL, but not in splenic T cells. SCF stimulation of IEL resulted in an expansion of the c-kit(+), TCRgammadelta(+)cell population. SCF-induced proliferation was dependent upon SCF-c-kit interactions, since antibody to c-kit blocked this response, and IEL obtained from c-kit mutant (W/W(v)) mice failed to respond to SCF. SCF acted synergistically with anti-TCRgammadelta and with concavalin A (Con A) to induce proliferation and interferon gamma (IFN-gamma) production in IEL. Finally, mice injected with SCF had a significant increase in the number of IEL in the small intestine. SCF-treated mice had increased numbers of TCRalphabeta(+)and TCRgammadelta(+)cell populations, as well as increased numbers of c-kit(+)and c-kit(-)IEL. These data suggest that SCF-c-kit interactions play an important role in regulating IEL expansion and activation.

The c-kit ligand, stem cell factor, can enhance innate immunity through effects on mast cells.

Mast cells are thought to contribute significantly to the pathology and mortality associated with anaphylaxis and other allergic disorders. However, studies using genetically mast cell-deficient WBB6F1-KitW/KitW-v and congenic wild-type (WBB6F1-+/+) mice indicate that mast cells can also promote health, by participating in natural immune responses to bacterial infection. We previously reported that repetitive administration of the c-kit ligand, stem cell factor (SCF), can increase mast cell numbers in normal mice in vivo. In vitro studies have indicated that SCF can also modulate mast cell effector function. We now report that treatment with SCF can significantly improve the survival of normal C57BL/6 mice in a model of acute bacterial peritonitis, cecal ligation and puncture (CLP). Experiments in mast cell-reconstituted WBB6F1-KitW/KitW-v mice indicate that this effect of SCF treatment reflects, at least in part, the actions of SCF on mast cells. Repetitive administration of SCF also can enhance survival in mice that genetically lack tumor necrosis factor (TNF)-alpha, demonstrating that the ability of SCF treatment to improve survival after CLP does not solely reflect effects of SCF on mast cell- dependent (or -independent) production of TNF-alpha. These findings identify c-kit and mast cells as potential therapeutic targets for enhancing innate immune responses.

Changes in conformation and stability upon SCF/sKit complex formation.

Stem cell factor (SCF) is thought to be a member of the four-helical bundle cytokine superfamily, and exists in solution as a noncovalent homodimer. It is the ligand for Kit, a tyrosine kinase type III receptor. The interaction of SCF and Kit affects early hematopoietic progenitors, as well as gametocytes, melanocytes, and mast cells. Upon binding of SCF the Kit undergoes dimerization and transphosphorylation. Circular dichroism (CD), intrinsic fluorescence, and Fourier transform infrared (FTIR) spectroscopy were used for conformational analyses of free SCF, soluble Kit (sKit), and the complex. The sKit consisted of the extracellular domain of Kit, contained five Ig-like domains, and was prepared from the conditioned media of transfected Chinese hamster ovary cells. With these techniques, a reproducible conformational change was seen upon ligand/receptor binding. The far-UV CD and FTIR spectroscopy indicated a slight increase in the alpha-helical content. The near-UV CD and fluorescence spectra showed changes in the environments of the aromatic amino acids. The thermal denaturation of SCF was not affected by complex formation, while the melting temperature of sKit increased only a few degrees when binding SCF. This indicates that binding is temperature dependent, consistent with titration calorimetry results published previously which demonstrated that there is a large enthalpy of binding. The conformational changes which accompany SCF/sKit binding could play a role in the receptor dimerization and signal transduction which follow.

Selective deamidation of recombinant human stem cell factor during in vitro aging: isolation and characterization of the aspartyl and isoaspartyl homodimers and heterodimers.

During in vitro aging, deamidation of recombinant human stem cell factor produced in Escherichia. coli was detected by HPLC analysis and by the release of soluble ammonia. The deamidation rate is very slow in buffers at low pH or at low temperatures; however, the rate is significantly accelerated in alkaline buffers such as sodium bicarbonate in combination with elevated temperatures. HPLC isolation of various deamidated forms followed by peptide mapping and mass spectrometric analyses revealed that the deamidation involves Asn10 in the sequence -T9NNV- near the N-terminus of the protein. Following peptide mapping analysis, significant amounts of aspartyl and isoaspartyl peptides were identified, indicating the conversion of asparagine into both aspartate and isoaspartate residues. As a result of spontaneous association-dissociation of stem cell factor dimer, a total of five deamidated forms, including two homodimers and three heterodimers, were detected and isolated. Cell proliferation assays showed that two rhSCF heterodimeric species, derived from dimerization between isoaspartyl and other stem cell factor monomers, retain only approximately half of the biological activity. The homodimer with isoaspartic acid in place of Asn10 is 50-fold less potent, while the aspartyl homodimer, either isolated during deamidation experiments or recombinantly prepared by site-directed mutagenesis (e.g., N10D and N10D/N11D variants), exhibits higher activity than the standard molecule. In comparison, synthetic N10A and N10E variants, though missing the deamidation site, are significantly less active. All these variants lacking the Asn10 deamidation site are relatively more stable than those containing the asparagine residue. The results indicate that the biological function and chemical stability of stem cell factor are influenced by the nature of the residue at position 10.

Tissue inhibitor of metalloproteinase-2 (TIMP-2) binds to the catalytic domain of the cell surface receptor, membrane type 1-matrix metalloproteinase 1 (MT1-MMP).

It has been proposed that tissue inhibitor of metalloproteinase-2 (TIMP-2), in stoichiometric concentrations, serves as an intermediate in progelatinase A activation by binding to activated membrane type 1-matrix metalloproteinase 1 (MT1-MMP) on the plasma membrane. An MT1-MMP-independent cell surface receptor for TIMP-2 has also been postulated. To clarify TIMP-2 binding, we have performed 125I-TIMP-2 binding studies on transfected COS-1 cells and endothelial cells. Specific receptors for TIMP-2 were identified on COS-1 cells transfected with MT1-MMP cDNA, but not on vector-transfected cells. Treatment of MT1-MMP transfected COS-1 cells with a hydroxamic acid inhibitor of MMPs, CT-1746, but not an inactive stereoisomer, CT-1915, produced dose-dependent inhibition of specific TIMP-2 binding comparable with that noted with excess unlabeled TIMP-2. This result suggests that TIMP-2 binds to the zinc catalytic site of MT1-MMP. As demonstrated by the limited competition for binding of C-terminal deleted TIMP-2, the C-terminal domain of TIMP-2 participates in binding to MT1-MMP. Cross-linking studies followed by immunoprecipitation using antibodies to MT1-MMP were employed to identify 125I-TIMP-2.MT1-MMP complexes in MT1-MMP-transfected COS-1 cell membrane extracts. TIMP-2 receptors were also identified on concanavalin A-treated human umbilical vein endothelial cells; inhibition of TIMP-2 binding with CT-1746 was demonstrated.

The C-terminal domain of tissue inhibitor of metalloproteinases-2 is required for cell binding but not for antimetalloproteinase activity.

We have generated a C-terminally-truncated form of recombinant tissue inhibitor of metalloproteinases-2 (designated rTIMP-2 delta) in which the region of the inhibitor extending from residue 128 to 194 and including 3 of the 6 disulfide bonds is deleted. rTIMP-2 and rTIMP-2 delta had similar inhibitory activities toward interstitial collagenase and inhibited the activation of the precursor form of matrix metalloproteinase-2 (proMMP-2). rTIMP-2 also bound with high affinity (Kd 0.99 nM) to HT1080 human fibrosarcoma cells treated with 12-O-tetradecanoyl-phorbol-13-acetate. However deletion of the C-terminal domain of TIMP-2 significantly lowered the cell surface binding affinity, with competition experiments indicating a 2 order of magnitude difference between rTIMP-2 and rTIMP-2 delta in the concentrations needed to displace 125I-labeled rTIMP-2 binding. These data indicate that the C-terminal domain of TIMP-2 is not required for the antimetalloproteinase activity but plays a major role in the high affinity cell surface binding of the inhibitor.

The majority of stem cell factor exists as monomer under physiological conditions. Implications for dimerization mediating biological activity.

Soluble Escherichia coli-derived recombinant human stem cell factor (rhSCF) forms a non-covalently associated dimer. We have determined a dimer association constant (Ka) of 2-4 x 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 Ka, greater than 90% of the circulating SCF would be in the monomeric form. When 125I-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 Ka determination. Three SCF variants, SCF(F63C), SCF (V49L,F63L), and SCF(A165C), were recombinantly expressed in Escherichia coli, purified, and characterized. The dimer Ka values, biophysical properties, and biological activities of these variants were studied. Dimerization-defective variants SCF(F63C)S-CH2CONH2 and SCF(V49L,F63L) showed substantially reduced mitogenic activity, while the activity of the Cys165-Cys165 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.

Enforced c-KIT expression renders highly metastatic human melanoma cells susceptible to stem cell factor-induced apoptosis and inhibits their tumorigenic and metastatic potential.

Expression of the tyrosine-kinase receptor encoded by the c-KIT proto-oncogene progressively decreases during local tumor growth and invasion of human melanomas. To provide direct evidence that c-KIT plays a role in metastasis of human melanoma, we transfected the c-KIT gene into the c-KIT negative highly metastatic human melanoma cell line A375SM and subsequently analysed its tumorigenic and metastatic potential. A375SM parental cells, A375SM-NOT (neo, control), and A375SM-KIT-positive cells were injected s.c. and i.v. into nude mice. A375SM-KIT cells produced significantly slower growing s.c. tumors and fewer lung metastases than control cells. Exposure of c-KIT-positive melanoma cells in vitro and in vivo to stem cell factor (SCF), the ligand for c-KIT, triggered apoptosis of these cells but not of c-KIT-negative melanoma cells or normal melanocytes. Since SCF is produced by keratinocytes and other dermal cells in the skin, these results suggest that the loss of c-KIT receptor expression may allow malignant melanoma cells to escape SCF/c-KIT-mediated apoptosis, hence contributing to tumor growth and eventually metastasis. The antitumor and antimetastatic properties of SCF may be useful in treating human melanomas in early stages.

Epitope mapping and immunoneutralization of recombinant human stem-cell factor.

The epitope regions of three anti-[stem-cell factor (SCF)]g have been mapped by characterization of immunoreactivities against truncated forms of SCF in immunoblots and against synthetic peptides in solution-phase competition ELISA. Two of the antibodies, mAb 7H6 and mAb 8H7A, were raised against Escherichia coli-derived human SCF-(1-164) while the third, polyclonal antibody (pAb) 1337, was raised against a peptide corresponding to residues 3-31 of human SCF. The epitopes of mAbs 7H6 and 8H7A have been mapped to residues 61-95 and 95-110, respectively. The epitope of pAb 1337 has been mapped to residues 21-31. The ability of the anti-SCF Ig to recognize E. coli-derived human SCF presented in various formats, i.e. partially denatured (fixed in standard ELISA or on a western blot) or native (in solution), was studied, mAb 7H6 recognized its epitope in partially denatured or native SCF with equally high affinity, while mAb 8H7A and pAb 1337 recognized their epitopes only when SCF was at least partially denatured, mAb 7H6 was found to neutralize in vitro SCF-mediated cell proliferation and SCF binding to its receptor, when present in equimolar concentrations relative to the ligand, suggesting that the epitope region is functionally significant. Evidence that the mAb 7H6 epitope is represented by discontinuous regions (residues within sequences 61-65 and 91-95 are critically involved) is presented. The observation that the mAb 7H6 epitope is discontinuous has implications for the structure of SCF.

A role for stem cell factor (SCF): c-kit interaction(s) in the intestinal tract response to Salmonella typhimurium infection.

Cholera toxin (CT) has been shown to induce stem cell factor (SCF) production in mouse ligated intestinal loops. Further, SCF interaction(s) with its receptor (c-kit) was shown to be important for the intestinal tract secretory response after CT exposure. In this study, we have investigated whether SCF production is induced in the intestinal tract after exposure to Salmonella typhimurium and whether this production could be an important intestinal tract response to Salmonella infection. Using a mouse ligated intestinal loop model, increased levels of SCF mRNA were detected at 2-4 h post-Salmonella challenge. Intestinal fluid obtained from Salmonella-challenged loops contained high levels of SCF by ELISA. Human and murine intestinal epithelial cell lines were also shown to have increased levels of SCF mRNA after exposure to Salmonella. Inhibition of Salmonella invasion of epithelial cells was shown to be one potentially important role for SCF:c-kit interactions in host defense to Salmonella infection. Pretreatment of human or murine intestinal cell lines with SCF resulted in a cellular state that was resistant to Salmonella invasion. Finally, mice having mutations in the white spotting (W) locus, which encodes the SCF-receptor (c-kit), were significantly more susceptible to oral Salmonella challenge than their control littermates. Taken together, the above results suggest that an important intestinal tract response to Salmonella infection is an enhanced production of SCF and its subsequent interactions with c-kit.

In vitro methionine oxidation of Escherichia coli-derived human stem cell factor: effects on the molecular structure, biological activity, and dimerization.

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 Met159, Met-1, Met27, Met36, and Met48 were 0.11 min-1, 0.098 min-1, 0.033 min-1, 0.0063 min-1, and 0.00035 min-1, respectively, when SCF was incubated in 0.5% H2O2 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 Met36, and lost completely following the oxidation of Met48, 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 Met48 decrease biological activity by 40% and 60%, respectively, while increasing the dissociation rate constant of SCF dimer by two- and threefold. These results imply that the oxidation of Met36 and Met48 affects SCF dimerization and tertiary structure, and decreases biological activity.

Isolation and characterization of a disulfide-linked human stem cell factor dimer. Biochemical, biophysical, and biological comparison to the noncovalently held dimer.

Distinct from the noncovalently linked recombinant human stem call 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.

Human stem cell factor dimer forms a complex with two molecules of the extracellular domain of its receptor, Kit.

Stem cell factor (SCF) is a cytokine that is active toward hematopoietic progenitor cells and other cell types, including germ cells, melanocytes, and mast cells, which express its receptor, the tyrosine kinase, Kit. SCF exists as noncovalently associated dimer at concentrations where it has been possible to study its quaternary structure; it stimulates dimerization and autophosphorylation of Kit at the cell surface. We have used recombinant versions of human SCF and human Kit extracellular domain (sKit) to study SCF-Kit interactions. By size exclusion chromatography, plus various physical chemical methods including light scattering, sedimentation equilibrium, and titration calorimetry, we demonstrate the formation of complexes containing a dimer of SCF (unglycosylated SCF1-165) plus two molecules of sKit. The concentrations of SCF and sKit in these studies were in the range of 0.35-16.2 microM. The data are analyzed and discussed in the context of several possible models for complex formation. In particular, the sedimentation data are not consistent with a model involving cooperative binding. The Kd estimate for SCF-sKit interaction, obtained by sedimentation equilibrium, is about 17 nm at 25 degrees C. With glycosylated SCF1-165, the Kd is considerably higher.

A role for stem cell factor and c-kit in the murine intestinal tract secretory response to cholera toxin.

The role of stem cell factor (SCF) and its receptor (c-kit) in the intestinal secretory response to cholera toxin (CT) was investigated using a ligated intestinal loop model in mice having mutations in the dominant white spotting (W) locus and the steel (Sl) locus. W/Wv mice, which express an aberrant form of the c-kit protein, failed to give an intestinal secretory response after luminal CT challenge. In contrast, W/Wv mice and their control littermates had equivalent intestinal secretory responses to Escherichia coli heat-stable enterotoxin (STa). Sl/Sld mice, which express only a soluble truncated form of SCF, also gave a significantly reduced intestinal secretory response to CT when compared to the secretory response of their littermate controls. The unresponsiveness of W/Wv mice to CT was restricted to the intestinal tract since these mice had foot pad swelling responses to CT challenge that were equivalent to their littermate controls. Restoration of mast cells in W/Wv mice by bone marrow transplantation of control littermate bone marrow did not reverse the CT-unresponsiveness of the intestinal tract. Histological evaluation of the gastrointestinal tract from W/Wv mice showed a normal distribution of enterochromaffin cells (ECC). CT challenge of either ligated intestinal loops from C57B1/6 mice or a mouse intestinal epithelial cell line (MODE-K) resulted in elevated levels of mRNA for SCF. MODE-K cells exposed to CT also had enhanced expression of c-kit. Finally, fluid obtained from CT-challenged ligated intestinal loops from C57B1/6 mice contained significant levels of SCF. Taken together, the above results suggest that CT-induced intestinal secretory responses are dependent upon SCF-c-kit interactions. These interactions appear to be induced as a consequence of CT stimulation of the intestinal tract and may also play a role in the development or functionality of the enteric nervous system.

Identification and characterization of a soluble c-kit receptor produced by human hematopoietic cell lines.

Stem cell factor (SCF) triggers cell growth by binding to cell surface c-kit receptors. Soluble forms of several cytokine receptors have been described and may play a role in the modulation of cytokine activity in vivo. For these reasons, we investigated whether human hematopoietic cells produce soluble c-kit receptors. The human leukemia cell lines OCIM1 and MO7e display approximately 80,000 and approximately 35,000 high-affinity cell surface c-kit receptors, respectively. Soluble c-kit receptors were detected by enzyme immunoassay in OCIM1 and MO7e culture supernatants. We determined the molecular weight and binding affinity of soluble c-kit receptor produced by OCIM1 cells, soluble c-kit receptor purified from human serum, and recombinant soluble c-kit receptor expressed in CHO cells. The three soluble c-kit receptors each have a molecular weight of 98 kD. Quantitative binding experiments with 125I-SCF indicate that the soluble c-kit receptors obtained from human serum or OCIM1 cells have binding affinities for SCF of approximately 200 to 300 pmol/L, in contrast to the recombinant form, which has a binding affinity of approximately 1.5 nmol/L. All three forms of the soluble c-kit receptor were able to compete with c-kit receptors on OCIM1 cells for 125I-SCF binding. Thus human hematopoietic cells can produce a soluble form of the c-kit receptor that retains high-affinity SCF binding activity. We speculate that the soluble c-kit receptor may bind SCF and function as a receptor antagonist in vivo.

Spontaneous dissociation-association of monomers of the human-stem-cell-factor dimer.

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.

Soluble kit receptor in human serum.

c-kit encodes the transmembrane receptor tyrosine kinase (Kit) for the recently described ligand stem cell factor (SCF). We have developed an enzyme-linked immunosorbent assay for measuring soluble human Kit and we have used the assay to show high levels of soluble Kit in human serum. The distribution of soluble Kit levels was investigated among 112 normal human serum donors. The mean serum level (+/- SD) was found to be 324 +/- 105 ng/mL with the values falling between 163 ng/mL and 788 ng/mL. No correlation between soluble Kit levels and the sexes or ages of the donors was found. Partial purification using immunoaffinity chromatography allowed us to characterize the soluble Kit from pooled human serum. Antibodies generated to a 497-amino acid recombinant human soluble Kit corresponding to the N-terminal extracellular domain of the receptor recognized the serum-derived soluble Kit by immunoblotting. We found that the serum-derived soluble Kit is glycosylated, with mostly N-linked but also O-linked carbohydrate, and with terminal sialic acid residues. When compared with the recombinant human soluble Kit, the serum-derived material was similar both in size and glycosylation pattern. CNBr cleavage of the isolated serum-derived material followed by amino terminal sequencing confirmed the presence of five peptides expected for the extracellular portion of the Kit molecule. The immunoaffinity purified serum-derived soluble Kit inhibited binding of [125I]SCF to membrane-bound receptor in an in vitro assay. These results indicate that soluble Kit could modulate the activity and functions of SCF in vivo.