Identification and expression of two forms of the human transforming growth factor-β-binding protein endoglin with distinct cytoplasmic regions

Endoglin is an homodimeric membrane antigen with capacity to bind transforming growth factor-β (TGF-β) and whose expression is up-regulated on myeloid cells upon differentiation to macrophages. We have isolated full-length cDNA clones from a λgtl0 library, prepared from phorbol 12-myristate 13-acetate-differentiated HL60 cells by screening with an endoglin-specific cDNA probe from endothelial cells. Sequencing of the largest clone (3073 bp), revealed that the leader sequence contains 25 residues and that the 586 amino acids of the extracellular and transmembrane domains were identical to those described for endothelial endoglin. However, the cytoplasmic tail encoded by this cDNA clone contains only 14 amino acids as opposed to the 47 residues previously reported, suggesting the existence of two alternative endoglin variants. The expression of these isoforms was demonstrated by polymerase chain reaction analyses on endothelial cells, myelomonocytic cell lines HL-60 and U-937, and placenta. Independent cDN A constructs corresponding to both forms were transfected into mouse fibroblasts leading to the expression of two distinct endoglin molecules. Both forms were shown to bind TGF-β1 and, when overexpressed in transfected mouse fibroblasts, to form disulfide-linked homodimers, indicating that the cysteine residues present in the extracellular domain are responsible for the dimerization.     

Molecular basis of the neutrophil glycoprotein NB1 (CD177) involved in the pathogenesis of immune neutropenias and transfusion reactions

The human granulocyte alloantigen NB1, recently clustered as CD177, is heterogenously expressed on neutrophils of 88-97% of healthy individuals. Since its molecular nature has remained unknown, we isolated NB1 glycoprotein from granulocyte lysate by immunoaffinity chromatography. MALDI-TOF mass spectrometry identified a 50,556 Da glycoprotein which was reduced to 43,069 Da after removal of N-linked carbohydrates. Following N-terminal amino acid sequencing and NB1-specific primer construction, rapid amplification of cDNA ends PCR yielded a 1,614-bp cDNA for NB1. COS-7 cells transfected with the cDNA expressed immunoreactive NB1 glycoprotein. A 1,311-bp sequence was identified to be the entire coding region. The 5' and 3' untranslated regions consist of 27 bp and 276 bp, respectively. The open reading frame codes for 437 amino acids of which the first 21 form the signal peptide. The remaining 416 residues form a N-terminal extracellular protein with two cysteine-rich domains, three N-linked glycosylation sites and short transmembrane and cytoplasmic segments including a glycosyl-phosphatidylinositol attachment (omega) site. Database searches revealed homology to Ly-6 (uPAR) domain, suggesting that NB1 belongs to urokinase plasminogen activator receptor/CD59/Ly-6 snake toxin superfamily.     

Modulation of mitochondrial aconitase on the bioenergy of human prostate carcinoma cells

A bioenergetic theory of prostate malignancy proposed that normal citrate-producing prostate epithelial cell become citrate-oxidizing cells, in which mitochondrial aconitase (mACON) is not limiting, providing the energy required for the onset and progression of malignancy and metastasis. However, no direct evidence has been approved to support the hypothesis. A full-length cDNA encoding human skeletal muscle mACON cDNA was cloned and sequenced. mACON cDNA contains 19-bp 5' untranslated region, a 2343-bp coding segment, and 376-bp 3' untranslated region. This precursor enzyme contains mitochondrial targeting sequence of 27 amino acid residues and mature enzyme of 753 amino acids residues. A human anti-mACON overexpression vector containing the 1171-bp mACON cDNA fragment in the reverse orientation was stable transfected into human prostate carcinoma cells, PC-3 and DU145 cells. Results showed that mACON antisense blocked 40-60% mACON expression and enzymatic activity which induced decrease in the intracellular ATP biosynthesis but increase citrate secretion in the human prostate carcinoma cells. mACON antisense-transfected cells have lower cell proliferation ratio than the mock of DNA-transfected cells. Our study demonstrated the key role of the mACON in the cellular bioenergy and cell proliferation of human prostate carcinoma cells.     

Regions of an Eimeria tenella antigen contain sequences which are conserved in circumsporozoite proteins from Plasmodium spp. and which are related to the thrombospondin gene family

Coccidiosis, caused by Eimeria spp., is a major disease of economic importance to the poultry industry. The cloning and characterisation of genes coding for antigens of those species infecting chickens is an initial step in the identification of protective antigens suitable for the development of a genetically engineered vaccine. This report describes the molecular characterisation of an antigen of E. tenella produced by the recombinant lambda amp3 bacteriophage EtHL6. Three native polypeptides corresponding to the EtHL6 antigen, with sizes between 110 and 94 kDa, have been identified on both sporozoites and second generation merozoites of E. tenella by mouse antisera raised against the EtHL6 fusion protein. The DNA insert is a 722-bp EcoRI fragment encoding a polypeptide comprising three tandem blocks of amino acids which are highly homologous to each other. Each region, A, B and C, contains a strongly hydrophilic domain and two pairs of cysteine residues. Computer analysis has identified similarities with a group of proteins which include the circumsporozoite antigen and thrombospondin-related anonymous protein (TRAP) of malaria parasites, human thrombospondin, mouse properdin and the terminal components of the complement pathway.     

Cloning of cDNA for the bovine IL-2 receptor (bovine Tac antigen).

We have cloned the Tac analog of the bovine IL-2 receptor (IL-2R) cDNA. Using mouse and human cDNA probes, we isolated five bovine IL-2R clones from a lambda gt11 bovine long-term lymphocyte cDNA library. Three of the clones had inserts of 2600 base pairs (bp), the same size as the bovine IL-2R mRNA visualized on Northern blots. The full-length cDNA contain a 190-bp 5' untranslated region, followed by a 825-bp coding region, and a 3' untranslated region that contain 1600 bp. Comparison of the bovine and human IL-2R-coding sequences revealed 71% homology at the nucleotide level. The 3' and 5' non-coding regions were not as homologous, apart from a specific site in the 5'-untranslated region that contained a 5'-upstream start codon. In this region, 24 of 26 nucleotides were identical for the human and bovine cDNAs. Further analysis of the bovine IL-2R sequence also revealed the following: (i) the hydrophobic domains of the IL-2R protein were more conserved between species than the hydrophilic domains, (ii) the predominant site of intracellular IL-2R phosphorylation in mouse and human was a conserved Ser which was not conserved in the bovine sequence, and (iii) there exists a statistically significant amino acid homology with the AIDS gag protein.     

用标记磁微粒技术克隆牛CD14基因

为获得牛ＣＤ１４ 的全长编码基因，以探讨牛ＣＤ１４ 基因结构与功能的关系，进一步阐明其作用机理，我们用抗牛ＣＤ１４ 的单克隆抗体（ｍＡｂ）ＣＣＧ３３ 标记的磁微粒作为探针，从转染有牛肺泡巨噬细胞ｃＤＮＡ文库的ＣＯＳ７细胞中进行筛选。并对获得的牛ＣＤ１４ ｃＤＮＡ进行了克隆和序列分析。结果表明，牛ＣＤ１４ 基因全长为１１２２ ｂｐ，共编码３７３ 个氨基酸，其中含有信号肽序列和３ 个可识别的Ｎ连接糖基化位点，但缺少穿膜结构域及细胞内区。证实牛ＣＤ１４ 以糖基磷脂酰肌醇（ＧＰＩ） 锚定在靶细胞膜上；其氨基酸序列与人和小鼠ＣＤ１４ 的同源性分别为７３－５ ％ 和６１－４ ％ ，三者共有的保守序列约为５５％ 。     

The isolation, characterization and cloning of a globin-like, host-protective antigen from the excretory-secretory products of Trichostrongylus colubriformis.

An 18-kDa component from the excretory-secretory (ES) products of adults of Trichostrongylus colubriformis was isolated and characterized, and was shown to induce 60-84% protection of guinea pigs from challenge infection following a single intraperitoneal injection. Amino-terminal sequence analysis of gel-purified protein enabled oligonucleotides to be synthesized and used to screen a lambda gt10 cDNA library made from young adult worm mRNA, and to synthesize full-length clones from cDNA using the polymerase chain reaction (PCR). The full-length clones coded for a 20-kDa precursor protein of 173 amino acids which had a strongly hydrophobic leader sequence of 15 residues. The mature protein sequence of 158 amino acid residues was rich in charged amino acids (32%), including 8 oppositely charged pairs of amino acids. The protein sequence contained no half-cystine residues and no potential N-glycosylation sites. Unlike 2 other fully characterized ES components which are expressed only in the parasitic stages, mRNA coding for the 20-kDa component was present in both the parasitic and free-living stages of T. colubriformis. The parasite protein had approximately 20% identity with globins from human and from the larvae of the insect Chironomus thummi thummi. The homology included the invariant distal histidine and phenylalanine, and a number of other residues highly conserved in globins.     

Cloning and expression of a gamma-interferon-inducible gene in monocytes: a new member of a cytokine gene family

To define activation-specific sequences in human peripheral blood lymphocytes (PBL), a cDNA library was constructed by subtractive hybridization using resting and stimulated PBL pairs. Stimulation of PBL was achieved by triggering with mitogenic anti-CD2 (T11) monoclonal antibodies. Differential library screening with cDNA probes derived from stimulated versus resting PBL led to identification of two novel sequences, termed HC11 and HC14. The predicted primary and secondary structure of HC11 deduced from the translated nucleotide sequence suggests that the gene encodes a secreted protein of 99 amino acids (aa), including a 23 aa residue leader sequence. Surprisingly, Northern blot analysis demonstrated that HC11 mRNA is induced predominantly in peripheral blood non-T cells. Subsequently, we observed that the HC11 mRNA is induced in macrophages and the monocytic line U-937 by gamma-IFN, raising the possibility that T cell-derived gamma-IFN induced upon anti-CD2 stimulation activated monocytes to express HC11 RNA. In support of this notion, neutralizing anti-gamma-IFN monoclonal antibody inhibits the induction of HC11 mRNA in PBL activated through anti-CD2 antibodies. These findings suggest that there is a molecular cascade involving T cell-produced lymphokines and monokines which serve as a means for intercellular communication. Transient expression of HC11 cDNA results in a readily detectable specific set of protein bands in SDS-PAGE analysis of supernatants from radio-labeled COS cells, consistent with HC11 encoding a secreted product(s). Protein sequence comparison reveals homology with other members of a recently described inducible cytokine family whose functions are yet to be defined.     

Molecular cloning, reconstruction and expression of the gene encoding the alpha-chain of the bovine CD8--definition of three peptide regions conserved across species.

We report the cloning of a cDNA encoding the alpha-chain of the bovine CD8 (BoCD8 alpha). A bovine thymus cDNA library was hybridized at low stringency with a human CD8 alpha cDNA clone. The first round of screening of 5 x 10(4) independent colonies yielded 12 clones containing incomplete BoCD8 alpha genes. Two further rounds of colony hybridization were conducted, each using as a probe the 5' fragment from the longest BoCD8 alpha clone previously isolated. The final screening yielded a clone containing a 2 kilobase (kb) insert. We mapped and sequenced the 2 kb BoCD8 alpha clone and compared it with the published sequences of the genes encoding the human, mouse and rat CD8 alpha. Sequence analysis confirmed that the clone under study encoded the BoCD8 alpha. The overall similarity of the BoCD8 alpha coding region with the human CD8 alpha coding sequence is 74.7% at the nucleotide level and 62.1% at the protein level. Lower levels of similarity are found with the mouse and rat CD8 alpha. Interestingly, three separate highly homologous regions are clearly defined at the peptide level in bovine versus human and mouse versus rat comparisons. Two of the regions are highly conserved among all species analysed, while the most 5' region is not. We speculate that the latter region may contain the binding site of CD8 alpha to the alpha 3 domain of major histocompatibility complex (MHC) class I molecules. Sequence analysis showed that the 2 kb BoCD8 alpha clone contains an incomplete coding region, i.e. lacks six bases corresponding to the first two amino acids of the leader region. To allow efficient translation and processing of the BoCD8 alpha gene, we constructed a chimeric gene containing the coding sequence of the BoCD8 alpha clone and synthetic sequences corresponding to the first two amino acids of the human CD8 alpha leader sequence. The chimeric gene was subcloned in the pKSV10 expression vector. The pKSV10-BoCD8 alpha construct is efficiently expressed both transiently in COS cells and stably in L cells, as determined by Northern blot and by FACS analysis, using the ILA-51 monoclonal antibody to BoCD8 alpha. The latter result formally proves that the ILA-51 antibody does indeed recognize the product of the BoCD8 alpha gene, as previously suggested on serological grounds.     

Buffalo (Bubalus bubalis) interleukin-2: sequence analysis reveals high nucleotide and amino acid identity with interleukin-2 of cattle and other ruminants.

A 4400-bp genomic sequence and a 332-bp truncated cDNA sequence of the interleukin-2 (IL-2) gene of Indian water buffalo (Bubalus bubalis) were amplified by polymerase chain reaction and cloned. The coding sequence of the buffalo IL-2 gene was assembled from the 5' end of the genomic clone and the truncated cDNA clone. This sequence had 98.5% nucleotide identity and 98% amino acid identity with cattle IL-2. Three amino acid substitutions were observed at positions 63, 124 and 135. Comparison of the predicted protein structure of buffalo IL-2 with that of human and cattle IL-2 did not reveal significant differences. The putative amino acids responsible for IL-2 receptor binding were conserved in buffalo, cattle and human IL-2. The amino acid sequence of buffalo IL-2 also showed very high identity with that of other ruminants, indicating functional cross-reactivity.     

A 135-kilodalton surface antigen of Mycoplasma hominis PG21 contains multiple directly repeated sequences.

A monoclonal antibody was used to characterize a 135-kDa surface-located membrane protein (Lmp1) generally present in Mycoplasma hominis strains. The monoclonal antibody, 552, was applied to identify the corresponding gene in an expression library of M. hominis PG21 DNA. The M. hominis PG21 lmp1 gene was sequenced, and its gene product was characterized with the goal of elucidating the structure and function of Lmp1. A total of 7,196 bp in the lmp1 region was sequenced. An open reading frame of 4,032 bp, encoding a protein of 1,344 amino acids with a calculated molecular weight of 147,000, was identified. Analysis of the deduced amino acid sequence predicted a hydrophilic protein with a basic pI (10.0). The N-terminal 24 amino acids were a typical leader sequence. Downstream from the first 726 nucleotides, six similar direct repeats of 471 nucleotides were found. In repeat 7, a single-base substitution, C-->A, gave rise to the stop codon of lmp1. Thus, the C-terminal 945 amino acids were encoded by the 471-bp direct repeats. As evidenced by Southern blot analysis, the gene encoding the 135-kDa antigen is part of a multigene family. One of the genes, lmp2, was situated directly downstream from lmp1 where the direct repeats continued.     

Human cystathionine {beta}-synthase cDNA: sequence, alternative splicing and expression in cultured cells

Cystathionineß-synthase (CBS) deficiency is the majorcause of homocystinuria in humans. The most frequent symptomsof homocystinuria include: dislocated optic lenses, vasculardisorders, skeletal abnormalities and mental retardation. Patientswith this deficiency have elevated levels of homocyst(e)ine,methionine and low cysteine in their body fluids. These abnormallevels often partially or fully normalize upon treatment withpharmacological doses of vitamin B₆ To Investigate the molecularand biochemical basis for these conditions, It was necessaryto determine the nucleotide and polypeptide sequence of CBS.We report here the human CBS cDNA sequence of 2,554 nucleotidesencoding the CBS subunit of 551 amino acids. An intron of 214bp appears to be retained in the 3'-untranslated region of mostof the fibroblast and liver mRNA. We also report a frequentMspl polymorphism in the 3'-untranslated sequence and two synonymousmutations in the coding region: 699C/T (Y233Y) and 1080C/T (A360A).The amino acid sequence similarity of human and rat CBS is greaterthan 90% the enzyme also exhibits 52% similarity to O-acetylserine(thiol)-lyasefrom bacteria and plants. Lastly, we demonstrate that expressionof the human enzyme in CHO cells yields enzymatically activeprotein of the expected size with a half-life of {small tilde}14hrs.