Elizabeth A. Mendiaz

Identification, purification, and biological characterization of hematopoietic stem cell factor from buffalo rat liver-conditioned medium

We have identified a novel growth factor, stem cell factor (SCF), for primitive hematopoietic progenitors based on its activity on bone marrow cells derived from mice treated with 5-fluorouracil. The protein was isolated from the medium conditioned by Buffalo rat liver cells. It is heavily glycosylated, with both N-linked and O-linked carbohydrate. Amino acid sequence following removal of N-terminal pyroglutamate is presented. The protein has potent synergistic activities in semisolid bone marrow cultures in conjunction with colony-stimulating factors. It is also a growth factor for mast cells. In two companion papers, we present the sequences of partial SCF cDNAs, identify SCF as a c-kit ligand, and map the SCF gene to the Sl locus of the mouse.

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 Å 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.

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 × 108 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.

Purification and characterization of soluble forms of human and rat stem cell factor recombinantly expressed by Escherichia coli and by Chinese hamster ovary cells

Stem cell factor (SCF) is a novel, early-acting hematopoietic factor. It was isolated from the medium of a rat cell line in a soluble, processed form (Zsebo et al., 1990, Cell 63, 195). The cloned human and rat genes encode the soluble form plus additional C-terminal amino acids including a hydrophobic transmembrane domain (Martin et al., 1990, Cell 63, 203). We have recombinantly expressed forms of human and rat SCF corresponding to the soluble, processed form in Escherichia coli and in Chinese hamster ovary (CHO) cells. After expression in E. coli, folding and oxidation of the SCF polypeptides are required. The SCFs expressed in CHO cells are secreted into the medium in active state and, like the natural SCF, are glycosylated. Purification of the recombinant SCFs is described. Biological and biochemical characterization includes activity toward responsive human and mouse cell lines, N-terminal amino acid sequences, disulfide bond linkages, and sites of glycosylation.

Disulfide structures of human interleukin-6 are similar to those of human granulocyte colony stimulating factor

The amino acid sequences of human interleukin-6 and granulocyte colony stimulating factor are approximately 30% homologous in the N-terminal region. The relative positions of four half-cystines in human interleukin-6 (IL-6) match four of the five in human granulocyte colony stimulating factor. Labeling experiments of recombinant interleukin-6 with tritiated iodoacetate confirmed that the molecule forms two intramolecular disulfide bonds and contains no detectable level of free sulfhydryls. By isolation and characterization of tryptic and subtilytic peptides obtained from different proteolytic digestions, the disulfide bonds of the IL-6 molecule were assigned to Cys44-Cys50 and Cys73-Cys83. The two disulfide bridges form two small loops which are separated by 22 amino acids. These structures are similar to those of recombinant granulocyte colony stimulating factor.

Cloning and nucleotide sequence of the N-acetylmuramidase M1-encoding gene from Streptomyces globisporus

The gene (acm) encoding N-acetylmuramidase M1 (ACM) was cloned of Streptomyces globisporus ATCC No. 21553. The nucleotide sequence of the acm gene was determined and found to code for an ORF of 294 amino acids (aa). Comparison of aa sequence deduced from the acm gene with the N-terminal sequence of the extracellular enzyme suggests that ACM is synthesized with a 77-aa leader peptide. A comparison of the ACM aa sequence with the aa sequences of other proteins in the NBRF data base reveals that ACM has strong similarity to the N-O-diacetylmuramidase secreted by the fungus Chalaropsis.