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 GlcNAc1 3 3Gal1 3 4GlcNAc 3 R structure and poly-N-acetyllactosamine extension.
ABSTRACTcDNA clones encoding chum salmon (Oncorhynchus keta) growth hormone (sGH) have been isolated from a cDNA library prepared from chum salmon pituitary gland poly(A)+ RNA. Synthetic oligodeoxynucleotide mixtures based on amino acid residues 23-28 of sGH were used as hybridization probes to select recombinant plasmids carrying the sGH coding sequence. The complete nucleotide sequence of sGH cDNA has been determined. The cDNA sequence codes for a polypeptide of 210 amino acids, including a putative signal sequence of 22 amino acids. The 5' and 3' untranslated regions of the message were 64 and 426 bases long, respectively. Mature sGH was efficiently expressed in Escherichia coli carrying a plasmid in which the sGH cDNA was under control of the E. coli trp promoter; sGH comprised about 15% of the total cellular protein in such bacteria. The partially purified sGH from E. coli stimulated the growth of rainbow trout and the activity was indistinguishable from that of natural sGH.Human growth hormone now can be produced by genetically engineered organisms and can be used as a therapeutic agent. Salmon growth hormone (sGH) can be synthesized by use of similar techniques, and the massive supply of sGH may be extremely important to fish culture. Growth hormone (GH), together with prolactin and chorionic somatomammotropin (placental lactogen), forms a set of proteins that are structurally related and have partially overlapping biological activities (1). Primary structure analysis of the peptides and of the genes suggests that these hormone genes evolved from a common ancestral origin (1-5). Therefore, these genes provide an excellent model system for studying structurefunction relationships, evolution, and regulation of expression. GH genes have been isolated from several mammalian species and characterized in detail (3,(6)(7)(8). To obtain information about the evolution and the mechanisms of organization of this set of genes, it is essential to compare the structures of these hormone genes isolated from many organisms at various evolutionary stages. No information, however, has been available about lower vertebrates such as fish.
Abstract. Cancer cells induce proliferation and local accumulation of immunosuppressive cells, such as FOXP3-positive cells known as regulatory T cells (Tregs), leading to tumorinduced immune tolerance. Although cancer chemotherapy is usually considered immunosuppressive, some chemotherapeutic agents activate an anticancer immune response. Therefore, we postulated that the number of tumor-infiltrating FOXP3-positive cells during primary systemic chemotherapy (PSC) correlates with therapeutic outcomes in patients with breast cancer. Between September 2000 and January 2005, we examined 93 patients with breast cancer diagnosed by core-needle biopsy and treated with PSC. Core-needle biopsy (CNB) and surgical resected specimens were stained with a FOXP3 mouse monoclonal antibody to compare the numbers of FOXP3-positive cells in the tumors before and after PSC. A median cut-off value of >16.3/high power field (HPF) and >6.6/HPF defined high numbers of Tregs in CNB and in surgical specimens, respectively. We then assigned the patients into 4 groups (HH, high number of FOXP3-positive cells in both CNB and surgical specimen; LL, low number in both specimens; HL, high in CNB and low in the surgical specimen; LH, low in CNB and high in surgical specimen). Lymph vessel invasion-positive, clinically non-responder and ER-negative tumors contained significantly more FOXP3-positive cells after PSC (p=0.04, p=0.03 and p=0.04, respectively). Prognosis was better among patients with low numbers than high numbers of FOXP3-positive cells both in CNB and in surgically resected specimens. In multivariate analysis, LL group demonstrated significantly better recurrence-free survival with risk ratio of 5.81 (95%CI, 1.09-107.5; p=0.04) rather than that of non-LL group (LH, HL and HH). These findings suggest that the number of FOXP3-positive cells identified during PSC represents a promising predictive factor that might also be an important therapeutic target for breast cancer.
ABSTRACTcDNAs encoding a and 13 subunits of salmon gonadotropins, sGTHI and sGTHII, have been isolated from the cDNA library prepared from salmon pituitary mRNA. sGTHIa, sGTHIf8, and sGTHIIf3 cDNAs encode polypeptides of 114, 137, and 142 amino acids, including signal peptides of 22, 24, and 23 amino acids, respectively. The deduced amino acid sequence for sGTHIa revealed rather high homology (66-69%) to mammalian a chains, whereas sGTHII8 and sGTHII13 show lower homology (30%) to each other and to mammalian (3 subunits. The existence of two distinct fl-subunit cDNAs in the teleost suggests that divergence of the GTH gene took place earlier than divergence of teleosts from the main line of evolution leading to tetrapods.
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