琥珀酰辅酶A转移酶的表达纯化及抗体制备

目的:琥珀酰辅酶A转移酶(SCOT)是酮体代谢过程中的关键限速酶,此酶缺陷多由SCOT基因突变引起,患者多有酮症酸中毒表现。为了进一步研究SCOT的功能,采用原核表达系统表达并纯化重组SCOT,制备SCOT多克隆抗体。方法:选择蛋鸡、肉鸡模式生物为研究对象,通过生物信息学对其抗原性和属间同源性进行分析,通过RT-PCR从鸡的骨骼肌cDNA中扩增了SCOT基因N端半长片段,克隆到表达载体pET28b中,在大肠杆菌BL21(DE3)中诱导表达,并用镍离子螯合柱(Ni-NTA)纯化重组SCOT;用纯化的重组SCOT免疫小鼠后得到多克隆抗体。结果:Western印迹表明,制备的SCOT抗体具有较高的特异性,可特异性识别鸡的SCOT蛋白,同时可特异性识别小鼠和人的相应SCOT蛋白。结论:SCOT多克隆抗体的制备为后续在鸡、鼠和人中研究SCOT基因提供基础。     

Clinical and molecular characterization of five patients with succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency

Succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency is an inborn error of ketone body metabolism and causes episodic ketoacidosis. We report clinical and molecular analyses of 5 patients with SCOT deficiency. Patients GS07, GS13, and GS14 are homozygotes of S405P, L327P, and R468C, respectively. GS17 and GS18 are compound heterozygotes for S226N and A215V, and V404F and E273X, respectively. These mutations have not been reported previously. Missense mutations were further characterized by transient expression analysis of mutant cDNAs. Among 6 missense mutations, mutants L327P, R468C, and A215V retained some residual activities and their mutant proteins were detected in immunoblot analysis following expression at 37 °C. They were more stable at 30 °C than 37 °C, indicating their temperature sensitive character. The R468C mutant is a distinct temperature sensitive mutant which retained 12% and 51% of wild-type residual activities at 37 and 30 °C, respectively. The S226N mutant protein was detected but retained no residual activity. Effects of missense mutations were predicted from the tertiary structure of the SCOT molecule. Main effects of these mutations were destabilization of SCOT molecules, and some of them also affected catalytic activity. Among 5 patients, GS07 and GS18 had null mutations in both alleles and the other three patients retained some residual SCOT activities. All 5 developed a first severe ketoacidotic crisis with blood gas pH < 7.1, and experienced multiple ketoacidotic decompensations (two of them had seven such episodes). In general, the outcome was good even following multiple ketoacidotic events. Permanent ketosis or ketonuria is considered a pathognomonic feature of SCOT deficiency. However, this condition depends not only on residual activity but also on environmental factors.     

cDNA cloning, genomic structure, chromosomal mapping, and functional expression of a novel human alanine aminotransferase

Alanine aminotransferase (ALT) catalyzes the reversible transamination between alanine and 2-oxoglutarate to form pyruvate and glutamate, and thereby has a key role in the intermediary metabolism of glucose and amino acids. Two ALT isoenzymes are known to exist, but only one ALT gene has been cloned, GPT. In this study, we cloned a homolog of GPT and named it GPT2, and the corresponding protein ALT2. GPT2 shares 69% identity and 78% similarity at the protein level to the previously cloned GPT. The human gene GPT2 encodes a 3.9-kb mRNA, consists of 12 exons, spanning approximately 50 kb of the genome, and maps to chromosome 16q12.1. GPT2 and GPT differ in mRNA expression in that GPT2 is highly expressed in muscle, fat, and kidney, whereas GPT is mainly expressed in kidney, liver, and heart. In addition, GPT2 seems to be the predominant form of GPT at the mRNA level in these tissues. Expression of ALT2 protein in Escherichia coli produced a functional recombinant enzyme that catalyzes alanine transamination, confirming that the enzyme is an ALT. The more abundant expression of GPT2 than GPT, especially in muscle and fat, suggests a unique and previously unrecognized role of this gene product in glucose, amino acid, and fatty acid metabolism and homeostasis.     

The nuclear-encoded human NADH:ubiquinone oxidoreductase NDUFA8 subunit: cDNA cloning, chromosomal localization, tissue distribution, and mutation detection in complex-I-deficient patients

We report the cloning of the cDNA sequence of the nuclear-encoded NDUFA8 subunit of NADH: ubiquinone oxidoreductase, the first mitochondrial respiratory chain complex. The NDUFA8 open reading frame (ORF) includes 519 bp and encodes 172 amino acids (Mr=20.1 kDa). The human cDNA sequence shows 86.2% identity with the bovine sequence, whereas the human NDUFA8 amino acid sequence is 87.8% similar to its bovine PGIV protein counterpart. Both human and bovine NDUFA8 contain a conserved cysteine motif. Polymerase chain reaction analysis of rodent/human somatic cell hybrids maps the human NDUFA8 gene to chromosome 9. A multiple tissue blot has revealed the highest NDUFA8 mRNA expression in human heart, skeletal muscle, and fetal heart. Mutation analysis of the NDUFA8 fibroblast cDNA in 20 patients with an isolated enzymatic complex I deficiency in cultured skin fibroblasts has revealed two polymorphisms, one within the ORF and the other in the 3’ untranslated region of the NDUFA8 cDNA sequence. The allelic frequency of both polymorphisms was similar in controls and complex-I-deficient patients. Received: 17 April 1998 / Accepted: 22 July 1998     

Molecular cloning and chromosomal mapping of a cDNA encoding human 80K-L protein: major substrate for protein kinase C.

We have isolated and sequenced complementary DNA (cDNA) for the human 80K-L protein, a major substrate for protein kinase C and the human homologue of an 80- to 87-kDa bovine protein named MARCKS (myristoylated alanine-rich C kinase substrate). The human 80K-L cDNA encodes a protein of 332 amino acids with a calculated molecular weight of 31,534. Homology comparisons of the nucleotide sequences of the cDNAs indicated that their 3'-untranslated regions are more homologous than the coding regions. Spot blot hybridization using flow-sorted human chromosomes indicated that the gene encoding the 80K-L protein, designated MACS, maps to the q15----qter region of human chromosome 6, and it also suggested that a genomic region with a sequence homologous to the 3'-untranslated region of the 80K-L mRNA exists on chromosome 21.     

cDNA cloning and chromosomal localization (4q11-13) of a gene for statherin, a regulator of calcium in saliva.

On the basis of the known amino acid sequence of statherin, a human salivary protein, mixed synthetic oligonucleotides were synthesized and used to screen a cDNA library constructed from human parotid-gland mRNA. A cDNA clone coding for statherin was isolated from this library and has been completely sequenced. The cDNA represents a full-length (or nearly full-length) copy of an approximately 640-bp statherin mRNA. Statherin appears to be coded by a single-copy gene that maps to chromosome 4q11-4q13 when somatic-cell hybrids are used.     

Identification and characterization of a succinyl-coenzyme A (CoA):benzoate CoA transferase in Geobacter metallireducens

Geobacter metallireducens is a Fe(III)-respiring deltaproteobacterium and serves as a model organism for aromatic compound-degrading, obligately anaerobic bacteria. In this study, a genetic system was established for G. metallireducens using nitrate as an alternative electron acceptor. Surprisingly, disruption of the benzoate-induced bamY gene, encoding a benzoate coenzyme A (CoA) ligase, reproducibly showed an increased biomass yield in comparison to the wild type during growth with benzoate but not during growth with acetate. Complementation of bamY in trans converted the biomass yield back to the wild-type level. Growth of the bamY mutant with benzoate can be rationalized by the identification of a previously unknown succinyl-CoA:benzoate CoA transferase activity; it represents an additional, energetically less demanding mode of benzoate activation. The activity was highly enriched from extracts of cells grown on benzoate, yielding a 50-kDa protein band; mass spectrometric analysis identified the corresponding benzoate-induced gene annotated as a CoA transferase. It was heterologously expressed in Escherichia coli and characterized as a specific succinyl-CoA:benzoate CoA transferase. The newly identified enzyme in conjunction with a benzoate-induced succinyl-CoA synthetase links the tricarboxylic acid cycle to the upper benzoyl-CoA degradation pathway during growth on aromatic growth substrates.     

RpoS and oxidative stress conditions regulate succinyl‐CoA: 3‐ketoacid‐coenzyme A transferase (SCOT) expression in Burkholderia pseudomallei

Burkholderia pseudomallei, a pathogenic gram‐negative bacterium, causes the severe human disease melioidosis. This organism can survive in eukaryotic host cells by escaping reactive oxygen species via the regulation of stress responsive sigma factors, including RpoS. In B. pseudomallei, RpoS has been reported to play a role in the oxidative stress response through enhanced activity of OxyR and catalase. In this study, the RpoS dependent oxidative stress responsive system was further characterized using comparative proteomic analysis. The proteomic profiles of wild‐type B. pseudomallei following exposure to H₂O₂ and between wild‐type and the rpoS mutant strains were analyzed. Using stringent criteria, 13 oxidative responsive proteins, eight of which are regulated by RpoS, were identified with high confidence. It was observed that ScoA, a subunit of the SCOT enzyme not previously shown to be involved directly in the oxidative stress response, is significantly down‐regulated after hydrogen peroxide treatment. ScoA and ScoB have been predicted to be organized in a single operon using computational methods: in this study it was confirmed by RT‐PCR that these genes are indeed co‐transcribed as a single mRNA. The present study is the first to report a role for RpoS in the down‐regulation of SCOT expression in response to oxidative stress in B. pseudomallei.     

cDNA cloning, mRNA distribution and heterogeneity, chromosomal location, and RFLP analysis of human osteopontin (OPN)

A human osteopontin (OP) cDNA was isolated from a library made from primary cultures of human bone cells. The distribution of osteopontin mRNA in human tissues was investigated by Northern analysis and showed that the human message was predominant in cultures of bone cells and in decidua cells isolated at 6-12 weeks of gestation. Immunohistochemical analysis confirmed that OP expression is high in decidua cells as well as in the endometrial glands of a non-pregnant secretory-phase human uterus. Two variants of the OP message were evident on the basis of DNA sequencing and polymerase chain reaction amplification of bone and decidua cell mRNA. The peptides potentially translated by the variant messages differ by the presence (OP1b) or absence (OP1a) of 14 amino acids at residue 58 of the molecule. The deduced human protein sequence shows a conservation between species in the position of the Arg-Gly-Asp (RGD) cell attachment site. Chromosomal mapping of the osteopontin gene (OPN) using human-rodent cell hybrids demonstrated a location on chromosome 4 in the human genome. In situ hybridization of metaphase chromosomes using radiolabeled OP1a as a probe indicated that the gene is located on a region of 4q that is near the centromere. A high-frequency restriction fragment length polymorphism was evident in the DNA from 29 unrelated individuals using the enzyme BglII. Analysis of total genomic DNA by digestion with several restriction enzymes, Southern blotting, and hybridization with the human osteopontin cDNA indicated that the gene is a single copy with an approximate length of 5.4-8.2 kb.     

cDNA cloning, characterization and chromosome mapping of the gene encoding human cartilage associated protein (CRTAP)

We have recently isolated and characterized cDNA clones coding for a novel developmentally regulated avian and mouse embryo protein, CASP for Cartilage Associated Protein. Here we describe the isolation and characterization of the gene coding for the human CASP. The comparison of the putative human and mouse protein sequences with the chick sequence revealed an overall high identity (89% and 51%, respectively). Homology search with known DNA and protein sequences showed that CASPs are related to two mammalian nuclear proteins. Here we demonstrate definitively that CASPs are distinct from these nuclear proteins. However, sequence comparison analyses suggest that all of these proteins belong to a new family. In all human tissues examined two CASP mRNA species were detected, whereas a single mRNA and three mRNAs were found in chick and mouse, respectively. The human CASP gene (CRTAP) was assigned to chromosome 3p22 by fluorescence in situ hybridization.     

Molecular cloning and chromosomal mapping of the human homologue of MYB binding protein (P160) 1A (MYBBP1A) to 17p13.3

We have previously isolated and characterized murine MYB binding protein (p160) 1a, a protein that specifically interacts with the leucine zipper motif within the negative regulatory domain of the c-Myb proto-oncoprotein. We now describe the molecular cloning of the human MYBBP1A cDNA and chromosomal localization to 17p13.3 by fluorescence in situ hybridization analysis. Given the likely presence of a tumor suppressor gene (or genes) within this region of chromosome 17, the position of MYBBP1A was further mapped by radiation hybrid analysis and was found to lie between markers D17S1828 and D17S938. A P1 artificial chromosome clone containing the 5' region of MYBBP1A was isolated and indicates a physical linkage between MYBBP1A and the 15-lipoxygenase gene (ALOX15). A novel, polymorphic (CA)(25) dinucleotide repeat was also isolated from this PAC and may serve as a useful marker for MYBBP1A and this region of chromosome 17.     

Mouse cytosolic and mitochondrial deoxyribonucleotidases: cDNA cloning of the mitochondrial enzyme,gene structures,chromosomal mapping and comparison with the human orthologs

Two of the five known mammalian 5'-nucleotidases show a preference for the dephosphorylation of deoxynucleoside-5'-phosphates. One is a cytoplasmic enzyme (dNT-1), the other occurs in mitochondria (dNT-2). The human mitochondrial enzyme, recently discovered and cloned by us, is encoded by a nuclear gene located on chromosome 17 p11.2 in the critical region deleted in the Smith-Magenis syndrome (SMS), a genetic disease of unknown etiology. Looking for a model system to study the possible involvement of dNT-2 in the disease, we have cloned the cDNA of the mouse ortholog. The deduced protein sequence is 84% identical to the human ortholog, has a very basic NH(2)-terminus, a very high calculated probability of being imported into mitochondria and contains the DXDXT/V motif conserved among nucleotidases. Expression in Escherichia coli of the predicted processed form of the protein produced an active deoxyribonucleotidase. We also identified in genomic sequences present in the data base the structures of the murine genes for the cytosolic and mitochondrial deoxyribonucleotidases (Nt5c and Nt5m). PAC clones for the two loci were isolated from a library and used for chromosomal localization by fluorescent in situ hybridization. Both genes map on chromosome 11: Nt5c at 11E and Nt5m at 11B, demonstrating the presence of the dNT-2 locus in the mouse shaker-2 critical region, the murine counterpart of the human SMS region. We performed pair-wise dot-plot and PIP (percent identity plot) analyses of mouse and human deoxyribonucleotidase genes, and found a strong conservation that extends also to some intronic sequences of possible regulatory significance.     

A gene coding for 3-deoxy-D-manno-octulosonic-acid transferase in Escherichia coli. Identification, mapping, cloning, and sequencing

An autoradiographic assay applicable to colonies immobilized on filter paper was developed for obtaining temperature-sensitive mutants of Escherichia coli defective in the transfer of 3-deoxy-D-manno-octulosonic acid (KDO) from CMP-KDO to a tetraacyldisaccharide 1,4'-bisphosphate precursor of lipid A, designated lipid IVA. Cell-free extracts from two mutants found in a population of 30,000 mutagen-treated cells showed normal KDO transferase activity when assayed at 30 degrees C, but almost no activity at 42 degrees C. The mutation was mapped by mating one of the mutants with different Hfr strains and analyzing genetic linkage of KDO transferase activity to selectable markers. The lesion was located to a position between 80 and 84 min on the E. coli chromosome. A plasmid from the Clarke and Carbon collection (Clarke, L., and Carbon, J. (1976) Cell 9, 91-99), pLC17-24, known to contain genes from the rfa region (81 min), was shown to overexpress KDO transferase activity 4-5 times and to correct the mutation when the plasmid was conjugated into the mutant strains. The KDO transferase gene, designated kdtA, was subcloned from pLC17-24 into a multicopy vector. The resulting plasmid, pCL3, overproduced transferase activity approximately 100-fold. The kdtA gene was shown to code for a 43-kDa polypeptide, as judged by radiolabeling of minicells. Its DNA sequence was determined. The results demonstrate that overexpression of this single gene product greatly stimulates the incorporation of two stereochemically distinct KDO residues during lipopolysaccharide biosynthesis in extracts of E. coli.