An anti-human interleukin 5 receptor (hIL-5R) humanized immunoglobulin G1 (IgG1) and an anti-CD20 chimeric IgG1 produced by rat hybridoma YB2/0 cell lines showed more than 50-fold higher antibody-dependent cellular cytotoxicity (ADCC) using purified human peripheral blood mononuclear cells as effector than those produced by Chinese hamster ovary (CHO) cell lines. Monosaccharide composition and oligosaccharide profiling analysis showed that low fucose (Fuc) content of complex-type oligosaccharides was characteristic in YB2/0-produced IgG1s compared with high Fuc content of CHO-produced IgG1s. YB2/0-produced anti-hIL-5R IgG1 was subjected to Lens culinaris aggulutin affinity column and fractionated based on the contents of Fuc. The lower Fuc IgG1 had higher ADCC than the IgG1 before separation. In contrast, the content of bisecting GlcNAc of the IgG1 affected ADCC much less than that of Fuc. In addition, the correlation between Gal and ADCC was not observed. When the combined effect of Fuc and bisecting GlcNAc was examined in anti-CD20 IgG1, only a severalfold increase of ADCC was observed by the addition of GlcNAc to highly fucosylated IgG1. Quantitative PCR analysis indicated that YB2/0 cells had lower expression level of FUT8 mRNA, which codes ␣1,6-fucosyltransferase, than CHO cells. Overexpression of FUT8 mRNA in YB2/0 cells led to an increase of fucosylated oligosaccharides and decrease of ADCC of the IgG1. These results indicate that the lack of fucosylation of IgG1 has the most critical role in enhancement of ADCC, although several reports have suggested the importance of Gal or bisecting GlcNAc and provide important information to produce the effective therapeutic antibody. Antibody-dependent cellular cytotoxicity (ADCC),1 a lytic attack on antibody-targeted cells, is triggered upon binding of lymphocyte receptors (Fc␥Rs) to the constant region (Fc) of the antibodies. ADCC is considered to be a major function of some of the therapeutic antibodies, although antibodies have multiple therapeutic functions (e.g. antigen binding, induction of apoptosis, and complement-dependent cellular cytotoxicity) (1, 2).One IgG molecule contains two N-linked oligosaccharide sites in its Fc region (3). The general structure of N-linked oligosaccharide on IgG is complex-type, characterized by a mannosyl-chitobiose core (Man3GlcNAc2-Asn) with or without bisecting GlcNAc/L-fucose (Fuc) and other chain variants including the presence or absence of Gal and sialic acid. In addition, oligosaccharides may contain zero (G0), one (G1), or two (G2) Gal.Recent studies have shown that engineering the oligosaccharides of IgGs may yield optimized ADCC. ADCC requires the presence of oligosaccharides covalently attached at the conserved Asn 297 in the Fc region and is sensitive to change in the oligosaccharide structure. In the oligosaccharide, sialic acid of IgG has no effect on ADCC (4). The relationship between the Gal residue and ADCC is controversial. Boyd et al. (4) have shown that obvious change was not found in ADCC after removal of ...

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We previously established a novel mouse model for human aging and identified the genetic foundation responsible for it. A defect in expression of a novel gene, termed klotho (kl), leads to a syndrome resembling human aging in mice. The kl gene encodes a single-pass membrane protein whose extracellular domain carries homology to L L-glucosidases. In this report, we present the entire mouse kl gene organization. The mouse kl gene spans about 50 kilobases and consists of five exons. The promoter region lacks a TATA-box and contains four potential binding sites for SP1. We further show that two kl gene transcripts encoding membrane or secreted protein are generated through alternative transcriptional termination. These findings provide fundamental information for further study of the kl gene which may regulate aging in vivo.z 1998 Federation of European Biochemical Societies.

The DNA f lanking the 5 sequence of the mouse 1␣-hydroxylase gene has been cloned and sequenced. A TATA box has been located at ؊30 bp and aCCAAT box has been located at ؊79 bp. The gene's promoter activity has been demonstrated by using a luciferase reporter gene construct transfected into a modified pig kidney cell line, AOK-B50. Parathyroid hormone stimulates this promoter-directed synthesis of luciferase by 17-fold, whereas forskolin stimulates it by 3-fold. The action of parathyroid hormone is concentration-dependent. 1,25-Dihydroxyvitamin D 3 does not suppress basal promoter activity and marginally suppresses parathyroid hormone-driven luciferase reporter activity. The promoter has three potential cAMP-responsive element sites, and two perfect and one imperfect AP-1 sites, while no DR-3 was detected. These results indicate that parathyroid hormone stimulates 25-hydroxyvitamin D 3 -1␣-hydroxylase by acting on the promoter of the 1␣-hydroxylase gene.Vitamin D is a major actor in calcium homeostasis of higher animals (1). To carry out these functions, vitamin D must be metabolized to its biologically active hormonal form, 1,25-dihydroxyvitamin D 3 (1,25-(OH) 2 D 3 ). This is a two-step process requiring 25-hydroxylation in the liver and 1␣-hydroxylation in the kidney. The resulting hormone, 1,25-(OH) 2 D 3 , then binds to a nuclear vitamin D receptor (VDR) in target tissues, and the liganded receptor acts as a transcription factor to modulate the expression of specific genes encoding proteins that bring about the actions of vitamin D (2).The importance of the 1␣-hydroxylase enzyme is emphasized by the occurrence of a genetic disorder of vitamin D metabolism, vitamin D dependency rickets type I (VDDRI) (3). This disorder is characterized by very low serum 1,25-(OH) 2 D 3 levels despite normal vitamin D intakes (4) and is thought to be the result of a defect in the 1␣-hydroxylase gene (5). This link has recently been reinforced with the identification of the gene for human 1␣-hydroxylase (6). The gene was mapped to chromosomal region 12q13.1-q13.3, which contains the VDDRI disease locus.The 1␣-hydroxylation step is the most tightly regulated step in vitamin D metabolism. Several physiological factors interact to regulate 1␣-hydroxylase activity that, in turn, determines serum and tissue levels of 1,25-(OH) 2 D 3 . These regulators include parathyroid hormone (PTH) and hypophosphatemia, which stimulate 1␣-hydroxylase activity, and 1,25-(OH) 2 D 3 , which suppresses it (7-9).