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.

We report here the identification of mouse betaklotho (betakl), which encodes a type I membrane protein with high resemblance to Klotho (KL). Both betaKL and KL consist of two internal repeats with homology to family 1 glycosidases, while these essential glutamates for the enzymatic activities were not conserved. The identical pattern of substitution and variation in the substituted amino acids between these two proteins indicate that they likely to form a unique family within the glycosidase family 1 superfamily. During mouse embryonic development, strong betakl expression was detected in the yolk sac, gut, brown and white adipose tissues, liver and pancreas, and in the adult, predominantly in the liver and pancreas. Despite the high structural similarity between betaKL and KL, their expression profiles were considerably different and betakl expression was not induced in kl-deficient mouse mutants.

We have isolated three types of cDNAs encoding novel ␤1,3-N-acetylglucosaminyltransferases (designated ␤3Gn-T2, -T3, and -T4) from human gastric mucosa and the neuroblastoma cell line SK-N-MC. These enzymes are predicted to be type 2 transmembrane proteins of 397, 372, and 378 amino acids, respectively. They share motifs conserved among members of the ␤1,3-galactosyltransferase family and a ␤1,3-N-acetylglucosaminyltransferase (designated ␤3Gn-T1), but show no structural similarity to another type of ␤1,3-N-acetylglucosaminyltransferase (iGnT). Each of the enzymes expressed by insect cells as a secreted protein fused to the FLAG peptide showed ␤1,3-N-acetylglucosaminyltransferase activity for type 2 oligosaccharides but not ␤1,3-galactosyltransferase activity. These enzymes exhibited different substrate specificity. Transfection of Namalwa KJM-1 cells with ␤3Gn-T2, -T3, or -T4 cDNA led to an increase in poly-N-acetyllactosamines recognized by an anti-i-antigen antibody or specific lectins. The expression profiles of these ␤3Gn-Ts were different among 35 human tissues. ␤3Gn-T2 was ubiquitously expressed, whereas expression of ␤3Gn-T3 and -T4 was relatively restricted. ␤3Gn-T3 was expressed in colon, jejunum, stomach, esophagus, placenta, and trachea. ␤3Gn-T4 was mainly expressed in brain. These results have revealed that several ␤1,3-Nacetylglucosaminyltransferases form a family with structural similarity to the ␤1,3-galactosyltransferase family. Considering the differences in substrate specificity and distribution, each ␤1,3-N-acetylglucosaminyltransferase may play different roles.A family of human ␤1,3-galactosyltransferases (␤3Gal-Ts) 1 consisting of five members (␤3Gal-T1, -T2, -T3, -T4, and -T5) was recently identified (1-4). The first ␤1,3-galactosyltransferase (␤3Gal-T1), which catalyzes the formation of type 1 oligosaccharides, was isolated by us using an expression cloning approach (1). Expression patterns of ␤3Gal-T1 and type 1 oligosaccharides strongly suggested the existence of ␤3Gal-T1 homologs. For instance, type 1-derived oligosaccharides such as sialyl-Le a were known to be expressed in colon and pancreatic cancer cell lines, whereas expression of ␤3Gal-T1 was detected in brain, but not in cancer cells. Our early approach using Southern hybridization failed to detect the existence of ␤3Gal-T1 homologous genes. However, recent accumulation of nucleotide sequence information on human cDNAs and genes such as expressed sequence tags (ESTs) enabled us to search homologous genes that do not have high similarity as detected by hybridization, but show significant similarity. A homology search based on the nucleotide or amino acid sequence of ␤3Gal-T1 led to the isolation of ␤3Gal-T2, -T3, and -T4, indicating that ␤3Gal-Ts form a family (1-3).␤3Gal-T2 catalyzed a similar reaction, but showed different substrate specificity compared with ␤3Gal-T1. The activity of ␤3Gal-T3 has not been detected, whereas the corresponding mouse enzyme exhibits weak ␤3Gal-T activity for both GlcNAc and GalNAc (5). On the other...

The structure and biosynthesis of poly-Nacetyllactosamine display a dramatic change during development and oncogenesis. Poly-N-acetyllactosamines are also modified by various carbohydrate residues, forming functional oligosaccharides such as sialyl Le x . Herein we describe the isolation and functional expression of a cDNA encoding ␤-1,3-N-acetylglucosaminyltransferase (iGnT), an enzyme that is essential for the formation of poly-Nacetyllactosamine. For this expression cloning, Burkitt lymphoma Namalwa KJM-1 cells were transfected with cDNA libraries derived from human melanoma and colon carcinoma cells. Transfected Namalwa cells overexpressing the i antigen were continuously selected by f luorescenceactivated cell sorting because introduced plasmids containing Epstein-Barr virus replication origin can be continuously amplified as episomes. Sibling selection of plasmids recovered after the third consecutive sorting resulted in a cDNA clone that directs the increased expression of i antigen on the cell surface. The deduced amino acid sequence indicates that this protein has a type II membrane protein topology found in almost all mammalian glycosyltransferases cloned to date. iGnT, however, differs in having the longest transmembrane domain among glycosyltransferases cloned so far. The iGnT transcript is highly expressed in fetal brain and kidney and adult brain but expressed ubiquitously in various adult tissues. The expression of the presumed catalytic domain as a fusion protein with the IgG binding domain of protein A enabled us to demonstrate that the cDNA encodes iGnT, the enzyme responsible for the formation of GlcNAc␤1 3 3Gal␤1 3 4GlcNAc 3 R structure and poly-N-acetyllactosamine extension.