Saturation Mutagenesis of Human Interleukin-3

Abstract

A deletion variant of human interleukin-3, hIL-3 15-125 , was produced in the periplasmic space of Escherichia coli and had full activity in an AML193.1.3 cell proliferation assay. Libraries of random single-amino acid substitutions were constructed at each of 105 positions in the gene for hIL-3 . Approximately eight single-site substitutions at each position were produced in osmotic shock fractions and screened for activity. 15 mutants were found with bioactivity of 5-26-fold greater than that of native hIL-3. The majority of amino acids in hIL-3 15-125 could be substituted without substantial loss of activity. Substitution of residues predicted to be in the hydrophobic core of the protein often resulted in reduced activity and/or low accumulation levels. Only five residues predicted to be on the surface of the protein were intolerant of substitution and hence are candidates for sites of interaction with the receptor. We therefore propose that the majority of residues in hIL-3 serve a structural role and permit the display of a few key residues in the correct configuration for recognition by the receptor.Human interleukin-3 (hIL-3) 1 is a multilineage hematopoietic cytokine acting in the bone marrow to promote the growth of most lineages of blood cell precursors (1). Recently, exogenously administered hIL-3 has shown promise for the clinical relief of neutropenia and thrombocytopenia induced by cancer chemotherapy (2, 3). Sequence homology comparisons of hIL-3 with other proteins indicate that it is a member of the hematopoietic cytokine family (4 -6) and that it adopts a four-␣-helix bundle topology (7-10). The protein binds to a receptor comprising at least two nonidentical subunits (11, 12). Although the precise nature of interaction between hIL-3 and its receptor is not known, studies using site-specific mutants have shed some light on which portions of the protein are important for function (8,(13)(14)(15)(16)(17). In particular, mutagenesis of the adjacent helices A and D indicate that these regions are important for interaction with the receptor. This is similar to the findings for human interleukin-5 and human granulocyte-macrophage colony stimulating factor, whose receptors share a common ␤ subunit with the hIL-3 receptor (11,18,19). Other members of the hematopoietic cytokine family also have important residues in helices A and D (19 -23) and in helix C (20,22,24,25).In this paper we have undertaken an extensive mutagenesis of hIL-3 in order to discover mutants with enhanced proliferative activity and to define residues necessary for activity. Although alanine scanning mutagenesis has been successfully used to derive structure-activity information (20, 26 -32), we chose to perform a more extensive mutagenesis, permitting the incorporation of any of the possible 19 substitutions (33). MATERIALS AND METHODS Production of hIL-3 and Variants in the Escherichia coli Cytoplasm-General techniques for manipulation of DNA are described elsewhere (34). The hIL-3 gene (35) was obtained from British Biotechnolo...

Phage display mutagenesis of the chimeric dual cytokine receptor agonist myelopoietin

“…Active hIL-3 variants can be presented on phage, 14,15 and hIL-3 variants with 10-fold increased proliferative activity isolated by conventional mutagenesis have 20-fold enhanced affinity for the receptor complex. 32,40 Thus, display of native hIL-3 on phage is feasible, affinity maturation for the receptor complex could have identified variants with better agonist activities. There may be other cytokines, some perhaps unknown, that could be productively affinity matured.…”

“…31 Protein variants expressed from each distinct myelopoietin variant gene were further characterized. Individual variant myelopoietin proteins were produced in E. coli as described 32 and quantitated using a sandwich ELISA for the hIL-3 receptor agonist domain. 1,14 Quantitation using the hIL-3 agonist domain was chosen to avoid artifacts due to variability in antibody recognition that could potentially occur due to mutagenic changes in the hG-CSF receptor agonist domain.…”

Characterization of the microheterogeneities of PIXY321, a genetically engineered granulocyte‐macrophage colony‐stimulating factor/interleukin‐3 fusion protein expressed in yeast

“…PIXY321 joins a no adverse effect is expected. Specific mutations in the C-terminal region of rhu-IL-3 have no effect on the activity [36,37]. A group of human proteins including insulin, factor VII and GM-CSF that have successfully used S. cerevisiae as a host for indusvariant molecule lacking the last eight residues has the same biological activity as the original IL-3 molecule [38].…”

The enhanced in vitro hematopoietic activity of leridistim, a chimeric dual G-CSF and IL-3 receptor agonist

“…14,24 Employment of a combinatorial mutagenesis strategy of the human IL-3 molecule resulted in several analogs exhibiting increased proliferative activity and hematopoietic potency relative to native IL-3. [25][26][27] Recently, it has been shown that the addition of daniplestim, one of these potent IL-3 receptor agonists, to ex vivo CD34 + cell cultures resulted in a greater increase in granulopoietic post-progenitor cells, suggesting that IL-3 receptor agonists can synergize with other lineage-specific cytokines in promoting expansion and differentiation of CD34 + cells. 28 Studies conducted in non-human primate models have demonstrated that daniplestim can potentiate the in vivo effects of rhG-CSF on mobilization of progenitor cells and neutrophil restoration, and can also prevent thrombocytopenia.…”