Jolanda Schreurs

Use of the silkworm, Bombyx mori, and an insect baculovirus vector for high-level expression and secretion of biologically active mouse interleukin-3

Using the virus vector derived from a baculovirus of Bombyx mori (Bm), we constructed an infectious recombinant virus carrying the mouse interleukin-3 (IL-3) cDNA placed downstream from the polyhedrin promoter. Silkworms infected in vivo with recombinant virus or the silkworm-derived BmN cell line infected in vitro secreted large amounts of IL-3 into hemolymph or culture medium, respectively. On a per volume basis, about 20-fold more activity was found in the culture supernatants of the infected BmN cells and 10000-fold more activity was detected in the hemolymph as compared to supernatants obtained from COS7 monkey cells transfected with plasmid pcD-IL3 using the SV40 early promoter [Yokota et al., Proc. Natl. Acad. Sci. USA 81 (1984) 1070-1074]. Three distinct species of Il-3 of molecular masses, 18, 20 and 22 kDa were produced and all were converted to a 15-kDa protein by N-glycanase digestion, indicating that silkworm cells glycosylated IL-3. The N-terminal amino acid sequences of the IL-3 purified from tissue culture medium and hemolymph were identical to that of mammalian-derived IL-3, showing that silkworm cells recognized the mammalian signal sequence and cleaved it at the correct position. The purified silkworm-produced IL-3 had biological activities indistinguishable from IL-3 produced by mammalian cells as assessed by mast-cell proliferation assays, colony-formation assays using mouse bone marrow cells, and by receptor-binding assays using [125I]IL-3.

Mass spectrometric determination of glycosylation sites and oligosaccharide composition of insect-expressed mouse interleukin-3

The primary structure of Baculovirus-expressed mouse interleukin-3 produced in infected Bombyx mori larvae was characterized by liquid secondary ion mass spectrometry and 252Cf-plasma desorption mass spectrometry in combination with selected protein microchemical reactions. Interleukin-3 was found to consist of at least two glycoprotein species of ca. 17,000 dalton. Characterization of tryptic and S. aureus V8 protease peptides by Edman degradation combined with plasma desorption mass spectrometry showed that two N-glycosylation sites. Asn-16 and Asn-86, were present. N-Glycan residues were shown by liquid secondary ion mass spectrometry and high-performance liquid chromatography to consist of mannose, fucose, and glucosamine. The presence of galactosamine indicated that O-glycosylated residues were present, in addition to the N-glycosylated residues. Glucose was also present, which indicated incomplete processing of the insect-expressed N-linked oligosaccharides.

Cytokines in infectious disease: The battle between host and pathogen

The immune system is poised like a fulcrum to respond quickly to challenge by infectious agents, but can produce excess inflammatory signals or excess suppressive signals when out of balance. During the past year, significant progress has been made in our understanding of how certain pathogens promote immune suppression and shift the balance from the host in their favor. Understanding the mechanisms that underlie excessive inflammatory responses or the suppressive effects of the micro-organism will aid in the development of new therapies.

Cytokine receptors: a new superfamily of receptors.

Publisher Summary This chapter discusses the characteristics that define the structure and composition of the receptors and their ability to bind ligand and transduce signals, known as “signal transduction pathways,” and provides the evidence for oncogenic processes dependent on aberrant cytokine–receptor interactions. Hematopoiesis is a dynamic process that produces all the blood cells from multipotential stem cells. As the development of various lineages of cells is regulated by a number of cytokines, two types of hematopoiesis may be distinguished based on cytokine production: constitutive versus inducible. Normal constitutive hematopoiesis in the bone marrow is regulated by soluble cytokines produced by stromal cells as well as by direct contact with membrane or extracellular matrix-associated proteins on the stromal cells. Cytokines are produced from multiple types of cells and, in turn, target a variety of cells that manifest cell-type specific as well as pleiotropic activities. A complicated cellular network among various kinds of blood cells is formed through the various cytokines.