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The enzyme releasing active IL-1b from its 31 kDa precursor has been identified and studied in detail. Termed IL-1b converting enzyme (ICE), it is a serine protease whose only known physiological substrate is the inactive IL-1b precursor. ICE cleaves this precursor between Asp 116 and Ala 117, releasing the active IL-1b.
ICE is an oligomeric enzyme (its active form may be a tetramer). It contains two distinct polypeptide subunits, p20 (20 kDa) and p10 (10 kDa). These two subunit types associate very closely, and the protease’s active site spans residues from both. p10 and p20 are proteolytically-derived from a single 45 kDa precursor protein.
INTERLEUKIN-3: BIOCHEMISTRY AND BIOTECHNOLOGY
IL-3 is yet another cytokine whose biotechnological applications have attracted interest. This cytokine is produced primarily by T lymphocytes as well as mast cells and eosinophils. The mature molecule is a 133 amino acid glycoprotein of molecular mass 15-30 kDa. It induces its biological effects by binding a specific receptor — the IL-3 receptor (IL-3R) on sensitive cells. The IL-3R is composed of two subunits, a ligand-binding a-subunit and a b-subunit that appears to mediate signal transduction (Figure 5.5). Binding of IL-3 to its receptor induces phosphorylation of several (mostly unidentified) cellular proteins. These substrates are phosphorylated either on tyrosine residues or threonine and serine residues. The amino acid sequence of the intracellular part of the b-receptor subunit exhibits no homology to any known kinase, suggesting that phosphorylation is mediated by a distinct cytoplasmic kinase that is activated upon ligand binding. The IL-3R b-subunit also forms part of the IL-5 and GM-CSF receptors. Not surprisingly, all of these cytokines share at least some biological activities.
The IL-3 receptor is found on a wide range of haematopoietic progenitor cells (see Chapter 6). They are also present on monocytes and B lymphocytes. Its major biological activity relates to stimulation of growth of various cell types derived from bone marrow cells and which represent the immature precursors to all blood cells (Chapter 6). IL-3 thus appears to play a central role in stimulating the eventual formation of various blood cell types, in particular monocytes, mast cells, neutrophils, basophils and eosinophils, from immature precursor cells in the bone marrow. Several other cytokines (including IL-2, -4, -5, -6, -7, -11, -15 and CSFs) also play important costimulatory roles in the maturation of the range of blood cells.
Its growth and differentiation-inducing effects on early haematopoietic progenitor cells forms the basis of clinical interest in IL-3. Its administration to healthy patients results in increased blood leukocyte counts, although the concentration of all white blood cell types is not equally increased.
Figure 5.5. The IL-3 receptor. A high-affinity receptor is formed by association of an IL-3-binding a-subunit and a second b-subunit. Although both receptor constituents are transmembrane glycoproteins, the 70 kDa a-subunit exhibits only a minor intracellular domain, and is itself incapable of initiating signal transduction. The b-subunit (also 70 kDa) contains a significant intracellular domain, responsible for signal transduction. Ligand binding likely promotes association and activation of an intracellular kinase(s) which phosphorylates various substrates, including the b-subunit itself
Clinical trials continue to investigate IL-3’s ability to stimulate production of blood cells in patients suffering from bone marrow failure induced by, for example, chemotherapy. In some such studies, leukocyte, neutrophil and platelet recovery was significantly improved by i.v. or s.c. IL-3 administration. Additionally, in studies with cancer patients, fewer cycles of chemotherapy had to be postponed because of complications resulting from depressed bone marrow function when IL-3 was concurrently administered to the patient. As with many drugs, however, other trials using IL-3 have shown less encouraging results regarding its efficacy in stimulating blood cell production.
Clinical interest in IL-3 is also sustained due to its low toxicity. Few side effects other than minor bone pain and nausea are witnessed at typical therapeutic doses (usually in the region of 5 mg/kg body weight). Further trials will probably investigate the effects of IL-3 treatment in combination with additional cytokines, such as CSFs. Such approaches may prove to be more effective in stimulating overall blood cell production.
IL-4, also known as B cell stimulating factor, is produced by a variety of cell types, most notably mast cells and T cells. It is a 129 amino acid glycoprotein exhibiting a molecular mass of 15-19 kDa, depending upon the level of glycosylation. The protein forms a compact globular shape and exhibits four a-helical stretches as well as a short stretch of a (two-stranded) b-sheet. The overall conformation is stabilized by three disulphide bonds and the protein exhibits a high pI value, in the region of 10.5 (Figure 5.6).