Apoptosis is a physiological form of cell death that plays a role in various biological processes, including normal development, tissue homeostasis, and defense against pathogens (Thompson et al., 1995, Science 267:1456-1462). Different forms of apoptosis can be distinguished according to whether transcription and translation, i.e., gene expression, are involved. For example, Fas ligand (FasL) and tumor necrosis factor (TNF) promote cell death by recruiting and activating caspases at the plasma membrane in the absence of transcription and translation (Rathmell et al., 1999, Annu. Rev. Immunol. 17:781-828). In contrast, other apoptotic programs require gene expression. The p53 tumor suppressor induces apoptosis in response to genotoxic agents, resulting, at least in part, from transcriptional activation of p53-dependent genes (Polyak et al., 1997, Nature 389:300-305). Other transcription-dependent apoptosis programs include glucocorticoid-induced killing of thymocytes (Cohen et al., 1984, J. Immunol. 132:38-42) and cell death induced by signaling through the T-cell receptor (TCR) (Lenardo et al., 1999, Annu. Rev. Immunol. 17:221-253).
In some cells, apoptosis can be induced by deprivation of trophic factors. For example, transcription-dependent cell death occurs following withdrawal of nerve growth factor (NGF) (Martin et al., 1988, J. Cell Biol. 106:829-844). IL-3-dependent cell lines undergo apoptosis upon cytokine withdrawal, and IL-3 promotes survival of several lymphoid progenitors (Palacios et al., 1987, J. Exp. Med. 166:12-32; Palacios et al., 1985, Cell 41:727-734).
Neutrophil gelatinase associated lipocalin (NGAL), a member of the lipocalin family of proteins, is a secreted 25 kDa glycoprotein found in granules of human neutrophils (Kjeldsen et al., 1993, J. Biol. Chem. 268:10425-10432). Lipocalins have been characterized by their ability to bind small lipophilic substances. Lipocalins share a common three-dimensional β-barrel structure which functions, in at least some lipocalins, in binding a lipophilic ligand, e.g., a steroid, bilin, retinoid, or other lipid. For a review of structure and function in the lipocalin family, see Flower, 1996, Biochem. J. 318:1-14. Murine forms of NGAL (homologs) from mice and rats are known. NGAL in mice is known by various designations, including NGAL, 24p3 protein, SIP24, P25, lipocalin 2, and uterocalin. NGAL in rats is known as NGAL or alpha 2-microglobulin. NGAL increases 7- to 10-fold in cultured mouse kidney cells in response to viral infection (Hraba-Renevey et al., 1989, Oncogene 4:601-608). NGAL is a major secretory product of lipopolysaccharide-stimulated, cultured mouse macrophages (Meheus et al., 1993, J. Immunol. 151:1535-1547).
NGAL is a positive acute phase protein. It has been suggested that NGAL is a scavenger of bacterial products at sites of inflammation (Nielsen et al., 1996, Gut 38:414-420). It has also been suggested that NGAL has an immunomodulatory function involving the binding of lipophilic inflammatory mediators (Bundgaard et al., 1994, Biochem. Biophys. Res. Commun. 202:1468-1475). NGAL is synthesized constitutively at a particular developmental point during the maturation of granulocyte precursors in the bone marrow (Borregaard et al., 1995, Blood 85:812-817). In addition, NGAL synthesis can be induced in epithelial cells under certain conditions such as inflammation and malignancy (Neilsen et al., supra; Bartsch et al., 1995, FEBS Lett. 357:255-259; Bundgaard et al., supra).
A full-length cDNA encoding human NGAL protein has been cloned and sequenced (Bundgaard et al., supra). In addition, the human NGAL gene, which includes seven exons and six introns, has been cloned and sequenced, and its expression in various tissues has been analyzed (Cowland et al., 1997, Genomics 45:17-23). The human NGAL gene encodes a polypeptide of 197 amino acids, with a 19- or 20-amino acid signal sequence, and a mature NGAL polypeptide containing 178 amino acids (Bundgaard, supra). The motifs Gly-X-Trp (amino acids 48-50 in mature human NGAL) and Thr-Asp/Asn-Tyr (amino acids 132-134 in mature human NGAL) are present in all known lipocalins (Bundgaard et al., supra). On the basis of X-ray crystallography, it has been suggested that these motifs are important in the tertiary structure common to lipocalins, i.e., an eight-stranded antiparallel β-barrel surrounding a hydrophobic core (Cowan et al., 1990, Proteins: Structure Function and Genetics 8:44-61). The cysteine residues 95 and 194 in the human NGAL sequence are conserved, and have been reported to form an intramolecular disulfide bridge (Bundgaard, supra; Cowan et al., 1990, supra). Human NGAL contains a single N-glycosylation site (an asparagine residue) at position 65 of the mature amino acid sequence (approximately position 84 or 85 of the pre-NGAL polypeptide).