June Eisenman

Identification of a ligand for the c-kit proto-oncogene

We report the purification and N-terminal amino acid sequence of a novel mast cell growth factor, termed MGF, from the supernatants of a murine stromal cell line. A panel of interleukin 3-dependent cell lines were screened for responsiveness to partially purified MGF in [3H]thymidine incorporation assays; proliferative stimulation of these cells in response to MGF correlated with expression of mRNA for the c-kit protooncogene. MGF was shown to be a ligand for c-kit by cross-linking 125I-labeled MGF to c-kit-expressing cells with subsequent immunoprecipitation of the complex with antiserum specific for the C-terminus of c-kit. This establishes MGF as a ligand for the c-kit protein.

Molecular cloning of mast cell growth factor, a hematopoietin that is active in both membrane bound and soluble forms

We have previously reported the identification of a novel mast cell growth factor (MGF) that was shown to be a ligand for c-kit and is encoded by a gene that maps near the steel locus on mouse chromosome 10. We now report the cloning of cDNAs encoding the MGF protein. The MGF protein encoded by this cDNA can be expressed in a biologically active form as either a membrane bound protein or as a soluble factor. The soluble protein promotes the proliferation of MGF-responsive cell lines and, in the presence of erythropoietin, stimulates the formation of macroscopic [corrected] erythroid and multilineage hematopoietic colonies.

Structure-Function Studies of Interleukin 15 using Site-specific Mutagenesis, Polyethylene Glycol Conjugation, and Homology Modeling

Interleukin (IL)-15 is a multifunctional cytokine that shares many biological activities with IL-2. This functional overlap, as well as receptor binding subunits shared by IL-15 and IL-2, suggests tertiary structural similarities between these two cytokines. In this study, recombinant human IL-15 was PEGylated via lysine-specific conjugation chemistry in order to extend the circulation half-life of this cytokine. Although PEGylation did extend the β-elimination circulation half-life of IL-15 by greater than 50-fold, the biological activity of polyethylene glycol (PEG)-IL-15 was significantly altered. Specifically, PEG-IL-15 lost its ability to stimulate the proliferation of CTLL but took on the properties of a specific IL-15 antagonist in vitro. In comparing sequence alignments and molecular models for IL-2 and IL-15, it was noted that lysine residues resided in regions of IL-15 that may have selectively disrupted receptor subunit binding. We hypothesized that PEGylation of IL-15 interferes with β but not α receptor subunit binding, resulting in the IL-15 antagonist activity observed in vitro. The validity of this hypothesis was tested by engineering site-specific mutants of human IL-15 as suggested by the IL-15 model (IL-15D8S and IL-15Q108S block β and γ receptor subunit binding, respectively). As with PEG-IL-15, these mutants were unable to stimulate CTLL proliferation but were able to specifically inhibit the proliferation of CTLL in response to unmodified IL-15. These results supported our model of IL-15 and confirmed that interference of β receptor subunit binding by adjacent PEGylation could be responsible for the altered biological activity observed for PEG-IL-15.

A Proteomic Approach for the Identification of Cell-surface Proteins Shed by Metalloproteases

Proteolytic cleavage (shedding) of extracellular domains of many membrane proteins by metalloproteases is an important regulatory mechanism used by mammalian cells in response to environmental and physiological changes. Here we describe a proteomic system for analyzing cell surface shedding. The method utilized short-term culture supernatants from induced cells as starting material, followed by lectin-affinity purification, deglycosylation, and polyacrylamide gel electrophoresis separation. Relative quantitation of proteins was achieved via isotope dilution. In this study, a number of proteins already known to be shed were identified from activated monocytes and endothelial cells, thereby validating the method. In addition, a group of proteins were newly identified as being shed. The method provides an unbiased means to screen for shed proteins.

Isolation and Isotope Labeling of Cysteine- and Methionine-containing Tryptic Peptides Application to the Study of Cell Surface Proteolysis

Inexpensive methods were developed for isolating and isotopically labeling tryptic peptides that contain either cysteine or methionine. After covalently capturing cysteine-containing peptides with pyridyl disulfide reactive groups on agarose beads, extensive wash steps were applied, and the attached peptides were released using a reducing agent. This approach results in less nonspecifically bound peptides and eliminates the possibility of generating avidin peptide background ions that can arise when using methods based on biotin and avidin (e.g. isotope-coded affinity tag). The thiols were alkylated using either N-ethyl- or N-D5-ethyl-iodoacetamide, both of which can be synthesized in a single step using inexpensive reagents. This isotopic labeling does not greatly increase the peptide mass, nor does it affect the peptide ion charge state in electrospray ionization. In addition, methionine-containing peptides were captured using commercially available methionine-reactive beads, and relative quantitation of peptides was achieved by isotopic labeling of amino groups using activated esters of either nicotinic acid or D4-nicotinic acid. These methods were used to study the metalloprotease-mediated shedding of cell surface proteins from a mouse monocyte cell line that had been treated with a phorbol ester and lipopolysaccharide. In addition to the identification of proteins previously determined to be inducibly shed, three new shed proteins were identified: CD18, ICOS ligand, and tumor endothelial marker 7-related protein.