Redefined substrate specificity of ST6GalNAc II: a second candidate sialyl-Tn synthase

The acceptor substrate specificities of ST6GalNAc I and II, which act on the synthesis of O-linked oligosaccharides, were reexamined using ovine submaxillary mucin, [Ala-Thr(GalNAc)-Ala]n polymer (n = 7-11). It has been suggested that only ST6GalNAc I can synthesize carbohydrate structures of sialyl-Tn-antigen; i.e., NeuAc alpha2-6GalNAc-O-Thr/Ser [Kurosawa et al., J. Biol. Chem. 269, 19048-19053 (1994)] based on the result that ST6GalNAc I, not ST6GalNAc II, exhibited activity toward asialoagalacto-fetuin. In this study, we present evidence that both ST6GalNAc I and II exhibit activity toward asialo-OSM (ovine submaxillary mucin) and [Ala-Thr(GalNAc)-Ala]n polymer (n = 7-11) which have only the GalNAc-O-Thr/Ser-structures. These results strongly indicate that not only ST6GalNAc I but also II are candidates for sialyl-Tn synthases.     

Molecular cloning and functional expression of two members of mouse NeuAcalpha2,3Galbeta1,3GalNAc GalNAcalpha2,6-sialyltransferase family, ST6GalNAc III and IV

Two cDNA clones encoding NeuAcalpha2,3Galbeta1,3GalNAc GalNAcalpha2, 6-sialyltransferase have been isolated from mouse brain cDNA libraries. One of the cDNA clones is a homologue of previously reported rat ST6GalNAc III according to the amino acid sequence identity (94.4%) and the substrate specificity of the expressed recombinant enzyme, while the other cDNA clone includes an open reading frame coding for 302 amino acids. The deduced amino acid sequence is not identical to those of other cloned mouse sialyltransferases, although it shows the highest sequence similarity with mouse ST6GalNAc III (43.0%). The expressed soluble recombinant enzyme exhibited activity toward NeuAcalpha2, 3Galbeta1, 3GalNAc, fetuin, and GM1b, while no significant activity was detected toward Galbeta1,3GalNAc or asialofetuin, or the other glycoprotein substrates tested. The sialidase sensitivity of the 14C-sialylated residue of fetuin, which was sialylated by this enzyme with CMP-[14C]NeuAc, was the same as that of ST6GalNAc III. These results indicate that the expressed enzyme is a new type of GalNAcalpha2,6-sialyltransferase, which requires sialic acid residues linked to Galbeta1,3GalNAc residues for its activity; therefore, we designated it mouse ST6GalNAc IV. Although the substrate specificity of this enzyme is similar to that of ST6GalNAc III, ST6GalNAc IV prefers O-glycans to glycolipids. Glycolipids, however, are better substrates for ST6GalNAc III.     

Molecular cloning of chick UCH-6 which shares high similarity with human UCH-L3: its unusual substrate specificity and tissue distribution.

A full-length cDNA encoding ubiquitin C-terminal hydrolase-6 (UCH-6) was isolated from the chick skeletal muscle cDNA library. The sequence of two peptides generated from purified UCH-6 matched perfectly with the predicted amino acid sequence. Nucleotide sequence analysis of the cDNA containing an open reading frame of 690 base pairs revealed that the protease consists of 230 residues with a calculated molecular mass of 26,315 Da. UCH-6 belonged to members of the UCH family containing highly conserved Cys, His, and Asp domains and showed 86% amino acid identity to human UCH-L3. Interestingly, most tissues examined contained significant amounts of UCH-6 mRNA, while human UCH-L3 is expressed only in the brain, lungs, and red cells. Moreover, UCH-6, unlike other UCH family enzymes including UCH-L3, could release free ubiquitin from ubiquitin-beta-galactosidase fusion proteins both in vivo and in vitro. The ubiquitous expression pattern and unusual substrate specificity of UCH-6 suggest that the enzyme may represent a distinct subfamily of UCH-L3.     

Molecular cloning and expression of mouse GD1alpha/GT1aalpha/GQ1balpha synthase (ST6GalNAc VI) gene

A novel member of the mouse CMP-NeuAc:beta-N-acetylgalactosaminide alpha2,6-sialyltransferase (ST6GalNAc) subfamily, designated ST6GalNAc VI, was identified by BLAST analysis of expressed sequence tags. The sequence of the cDNA clone of ST6GalNAc VI encoded a type II membrane protein with 43 amino acids composing the cytoplasmic domain, 21 amino acids composing the transmembrane region, and 269 amino acids composing the catalytic domain. The predicted amino acid sequence showed homology to the previously cloned ST6GalNAc III, IV, and V, with common amino acid sequences in sialyl motif L and S among these four enzymes. A fusion protein with protein A and extracts from L cells transfected with ST6GalNAc VI in an expression vector showed enzyme activity of alpha2,6-sialyltransferase for GM1b, GT1b, and GD1a but not toward glycoproteins. Thin layer chromatography-immunostaining revealed that the products were GD1alpha, GQ1balpha, and GT1aalpha. Northern blotting revealed that this gene was expressed in a wide range of mouse tissues such as colon, liver, heart, spleen, and brain. It is concluded that this enzyme is a novel sialyltransferase involved in the synthesis of alpha-series gangliosides in the nervous tissues and many other tissues.     

Molecular cloning and expression of Galbeta1,3GalNAc alpha2, 3-sialyltransferase from human fetal liver.

Based on the sequences of the highly conserved segments in the previously cloned sialyltransferases, a cDNA encoding Galbeta1, 3GalNAc alpha2,3-sialyltransferase (SIATFL) has been isolated from human fetal liver. Expression analysis of the gene has been performed with various carcinoma cell lines, fetal tissues, fetal and adult liver and both hepatoma and the surrounding tissue from the same liver. The SIATFL gene was expressed poorly in fetal liver and in adult liver, slightly in hepatoma and highly in the surrounding tissue of hepatoma. The cDNA encoding the putative active domain was expressed in COS-1, Escherichia coli, and Pichia pastoris. The recombinant protein expressed in COS-1 could catalyse the transfer of NeuAc from CMP-NeuAc to asialo-fetuin. No enzyme activity was detected with a 32-kDa protein in E. coli and both 32-kDa and 41-kDa proteins in P. pastoris. These results suggested that correct glycosylation of the enzyme might play a key role in its folding that may be directly related to the enzymatic activity.     

Molecular cloning and characterization of GalNAc 4-sulfotransferase expressed in human pituitary gland

We have previously cloned chondroitin-4-sulfotransferase (C4ST) cDNA from mouse brain. In this paper, we report cloning and characterization of GalNAc 4-sulfotransferase (GalNAc4ST), which transfers sulfate to position 4 of the nonreducing terminal GalNAc residue. The obtained cDNA contains a single open reading frame that predicts a type II transmembrane protein composed of 424 amino acid residues. Identity of the amino acid sequence between GalNAc4ST and human C4ST was 30%. When the cDNA was transfected in COS-7 cells, sulfotransferase activity toward carbonic anhydrase VI was overexpressed but no sulfotransferase activity toward chondroitin or desulfated dermatan sulfate was increased over the control. Sulfation of carbonic anhydrase VI by the recombinant GalNAc4ST occurred at position 4 of the GalNAc residue of N-linked oligosaccharides. The recombinant GalNAc4ST transferred sulfate to position 4 of GalNAc residue of p-nitrophenyl GalNAc, indicating that this sulfotransferase transfers sulfate to position 4 at the nonreducing terminal GalNAc residue. Dot blot analysis showed that the message of GalNAc4ST was expressed strongly in the human pituitary, suggesting that the cloned GalNAc4ST may be involved in the synthesis of the nonreducing terminal GalNAc 4-sulfate residues found in the N-linked oligosaccharides of pituitary glycoprotein hormones.     

Molecular cloning and sequencing of human palmitoyl-CoA ligase and its tissue specific expression

A complimentary DNA clone encoding the entire human palmitoyl-CoA ligase has been isolated from a liver cDNA library and sequenced in it's entirety. The predicted product is a 699 amino acid protein. Southern analysis utilizing the human palmitoyl-CoA ligase gene as a probe revealed varying degrees of similarity amongst various mammalian species. The palmitoyl-CoA ligase gene is highly expressed in liver, heart, skeletal muscle and kidney, and to a lesser extent in brain, lung, placenta and pancreas. The expression of palmitoyl-CoA ligase in various tissue parallels the function of this enzyme in the metabolism of fatty acids in these tissues.     

德国小蠊两个海藻糖合成酶基因的克隆、组织分布及温度诱导表达分析

【目的】海藻糖合成酶（trehalose-6-phosphate synthase, TPS）是参与昆虫血糖-海藻糖合成的关键酶。本研究旨在克隆德国小蠊 Blattella germanica TPS基因,研究TPS基因在德国小蠊不同组织中的表达模式及在不同温度处理下的表达情况。【方法】通过RACE技术克隆德国小蠊TPS基因全长序列,利用荧光定量PCR的方法检测TPS基因在德国小蠊5龄幼虫不同组织中的表达模式及在高温(40℃和46℃处理30 min)及低温(0℃和10℃处理1 h)逆境下的表达量变化。【结果】从德国小蠊中克隆获得2个TPS基因,分别命名为 BgTPS1 (GenBank登录号：KR050213) 和 BgTPS2 (GenBank登录号：KR050214)。其中,BgTPS1基因cDNA序列全长2 987 bp,开放阅读框 (ORF) 2 502 bp,编码833个氨基酸;BgTPS2基因cDNA序列全长3 212 bp,开放阅读框2 469 bp,编码822个氨基酸。BgTPS1和BgTPS2基因都主要在5龄幼虫脂肪体中表达,且BgTPS2基因的表达量为BgTPS1基因表达量的3.9倍。在两种不同极端温度诱导下,BgTPS1和BgTPS2基因mRNA均上调表达。其中,BgTPS2 的表达量始终显著高于 BgTPS1。在0℃时,BgTPS1和BgTPS2的表达量最高。【结论】德国小蠊5龄幼虫中存在2个TPS基因。两个TPS基因均在脂肪体中高表达,且BgTPS2基因的表达量显著高于BgTPS1基因;低温和高温诱导下均能促进两个基因的表达量上升。该结果为进一步明确昆虫海藻糖的合成途径及其在昆虫对温度逆境的反应中的作用研究奠定了基础。     

家蚕丝氨酸蛋白酶抑制剂基因serpin-6的克隆、序列分析和组织表达

本文利用RT-PCR和RACE方法,获得了家蚕Bombyx mori丝氨酸蛋白酶抑制剂基因（serpin）的开放读码框(ORF) 和5′UTR序列。根据该基因与其他昆虫serpin的同源性关系将其命名为家蚕serpin-6 基因,GenBank登录号EU159447。为了研究serpin-6与家蚕免疫反应的相关机理,对serpin-6基因的组织表达和免疫刺激反应进行了研究。结果表明该基因在家蚕的头部和生殖腺中mRNA表达量非常高,在中肠,脂肪体,丝腺和血淋巴表达量较低。用脂多糖(LPS) 刺激5龄第3 d家蚕后,serpin-6基因在脂肪体和血淋巴中都被显著诱导表达,且都在刺激6 h后表达量最高。推测该基因在家蚕细胞免疫反应中起着一定的作用。     

Molecular cloning and expression of human L-pipecolate oxidase

In higher eukaryotes L-lysine can be degraded via two distinct routes including the saccharopine pathway and the L-pipecolate pathway. The saccharopine pathway is the primary route of degradation of lysine in most tissues except the brain in which the L-pipecolate pathway is most active. L-pipecolate is formed from L-lysine via two enzymatic reactions and then undergoes dehydrogenation to Delta(1)-piperideine-6-carboxylate. At least in humans and monkeys, this is brought about by the enzyme L-pipecolate oxidase (PIPOX) localized in peroxisomes. In literature, several patients have been described with hyperpipecolic acidaemia. The underlying mechanism responsible for the impaired degradation of pipecolate has remained unclear through the years. In order to resolve this question, we have now cloned the human L-pipecolate oxidase cDNA which codes for a protein of 390 amino acids and contains an ADP-betaalphabeta-binding fold compatible with its identity as a flavoprotein. Furthermore, the deduced protein ends in -KAHL at its carboxy terminus which constitutes a typical Type I peroxisomal-targeting signal (PTS I).     

Molecular cloning, expression and tissue distribution of hamster diacetyl reductase. Identity with L-xylulose reductase

Using rapid amplification of cDNA ends PCR, a cDNA species for diacetyl reductase (EC 1.1.1.5) was isolated from hamster liver. The encoded protein consisted of 244 amino acids, and showed high sequence identity to mouse lung carbonyl reductase and hamster sperm P26h protein, which belong to the short-chain dehydrogenase/reductase family. The enzyme efficiently reduced L-xylulose as well as diacetyl, and slowly oxidized xylitol. The K(m) values for L-xylulose and xylitol were similar to those reported for L-xylulose reductase (EC 1.1.1.10) of guinea pig liver. The identity of diacetyl reductase with L-xylulose reductase was demonstrated by co-purification of the two enzyme activities from hamster liver and their proportional distribution in other tissues.     

Molecular cloning,expression and tissue distribution of hamster diacetyl reductase. Identity with l-xylulose reductase

Using rapid amplification of cDNA ends PCR, a cDNA species for diacetyl reductase (EC 1.1.1.5) was isolated from hamster liver. The encoded protein consisted of 244 amino acids, and showed high sequence identity to mouse lung carbonyl reductase and hamster sperm P26h protein, which belong to the short-chain dehydrogenase/reductase family. The enzyme efficiently reduced l-xylulose as well as diacetyl, and slowly oxidized xylitol. The K_m values for l-xylulose and xylitol were similar to those reported for l-xylulose reductase (EC 1.1.1.10) of guinea pig liver. The identity of diacetyl reductase with l-xylulose reductase was demonstrated by co-purification of the two enzyme activities from hamster liver and their proportional distribution in other tissues.     

鲈鱼钙蛋白酶基因cDNA克隆及表达分析

钙蛋白酶超家族（Calpain,CAPN,EC3.4.22.17）是由一类钙依赖性半胱氨酸蛋白酶组成.其在细胞周期、细胞迁移、信号转导以及细胞凋亡中都有着广泛的作用.研究了鲈鱼催化亚基CAPN2的基因结构.鲈鱼（Lateolabrax japonicus） CAPN2 cDNA全长2373 bp,5′非翻译区74 bp、3′非翻译区205 bp、开放阅读框2094 bp,编码一个由697个氨基酸组成的蛋白质,分子量为77.94kDa,等电点为5.02.氨基酸序列分析表明,鲈鱼CAPN2具有较高的保守性,与大西洋鲑（Hippoglossus hippoglossus）、虹鳟（Oncorhynchus mykiss）、斑马鱼（Danio rerio）、斑点叉尾鮰（Ictalurus punctatus）、猪（Sus scrofa）、人（Homo sapiens）、大鼠（Mus musculus）等 7个物种的同源性分别为89%、81%、77%、75%、63%、62%、62%.用RT-PCR分析CAPN2基因在10个组织中均有表达,肌、心、肾表达较高,脑、鳃、肠次之,肝、眼、脂肪、脾表达最低.     

Molecular cloning of cDNA encoding human prointerleukin-6

The data are presented on the cloning and sequencing of cDNA coding for human interleukin-6. The variability of cDNA proIL-6 cloned from different cellular sources was studied. The variability of cDNA proIL-6 may be expressed as heterogeneity of 5'- and 3'-end sequences of cDNA as well as single base-pair changes.