Identification of a gene selectively expressed in the brain, which encodes a putative transmembrane protein and a soluble cytoplasmic isoform.

Subtractive cloning procedures led to the identification of a variety of transcripts expressed in mammalian brain. However, little is known about the encoded proteins and the regulation of gene expression. Here, we describe the isolation and characterisation of a single-copy gene (83.5) of 21.7 kb which is specifically expressed in porcine brain. In situ hybridisation and immunohistochemistry experiments showed a distinct pattern of gene expression in neuronal cell types in different parts of the brain. The gene contains two mini exons, confirming neural-specific expression. cDNA cloning experiments revealed two species of mRNA differing in their 5'-regions. These transcripts are generated by two distinct transcription start sites that are under the control of different potential promoter regions as shown by primer-extension experiments. The amino acid sequences of the deduced proteins predict that one mRNA species encodes a novel type-I transmembrane protein, whereas the other transcript encodes only a part of its cytoplasmic domain. In Western-blot experiments, we detected two proteins of the predicted size and cellular localisation in porcine brain. The precise function of these proteins remains to be determined. However, our findings suggest that they may be generated by alternative promoter usage, leading to the expression of a membrane protein and its truncated cytoplasmic isoform.

Cloning and characterization of a porcine protein kinase gene and relationship to a class of heat shock proteins.

We have determined the genomic sequence of a porcine protein kinase (PPK) gene, including 1,844 bp upstream of the transcription initiation site. The gene spans over 19 kb and consists of 18 exons and 17 introns. The 5' regulatory region contains a characteristic heat shock element in the first intron, a weak heat shock element 1,464 bp upstream of the transcription initiation site, an atypical TATA box, and further consensus sequences typical for eukaryotic promoters such as an SP-1 binding site. Southern blot analysis indicates that PPK exists as a single-copy gene in the porcine haploid genome. The PPK gene is transcribed in all investigated tissues as shown by Northern blotting and reverse transcriptase polymerase chain reaction. Comparison of the protein and cDNA sequences of PPK to other sequences in DNA and protein databases indicates significant homology to a class of heat shock proteins, the glucose-regulated proteins (GRP94). In addition, nucleotide sequences at the 5' terminus of the PPK gene show strong homology to the GRP94 family. Domains highly conserved with human tumor rejection antigen (GP96) or glucose-regulated protein (GRP94) genes are identified within the 5' terminus and the first intron of the PPK gene. These findings suggest that these proteins are either identical or represent a family of closely related proteins.

Identification and characterization of a novel hsc70-like gene in the human lung tumor cell line HS24.

We determined the cDNA sequence and analyzed the genomic structure of a novel human gene designated HS24/p52, which shows significant similarity to the ATP-binding domain of stress-70 proteins in the human lung tumor cell line HS24. The 2,203-nucleotide-long cDNA sequence is divided into an incomplete 10-nucleotide 5' nontranslated region, a 1,425-nucleotide open reading frame which codes for 474 amino acids and a 768-nucleotide 3' nontranslated region. The first 404 of the deduced 474 amino acids resemble the amino-terminal regions of Hsp70 proteins from different species. Furthermore, single amino acid and short amino acid stretches, which are thought to be essential for the ATPase mechanism and ATP-binding activity in Hsp70 proteins, are conserved in this sequence, too. The carboxy-terminal 70 amino acids exhibit no significant similarity to hsp70 nor to any other known protein sequences. The HS24/p52 gene contains at least five introns, which differ significantly from hsc70 genes with regard to their size and location within the coding sequences. The total size of this gene is more than 15 kbp. Polymerase chain reaction (PCR) experiments showed that this gene is expressed in different human cell lines and tissues and it also seems to be highly conserved between human and mouse.

Organization of the 5'-End of the porcine gamma-glutamyl transpeptidase gene and identification of three different mRNAs in the kidney.

Three different mRNAs coding for the porcine gamma-glutamyl transpeptidase (GGT) in the kidney were identified by 5'-RACE-PCR. These differ in their 5'-noncoding region. Genomic Southern blot analysis has demonstrated the existence of a single GGT gene in the porcine genome. Thus, the existence of multiple mRNAs can only be explained by the use of different promoters or alternative splicing. Four GGT-specific genomic clones containing the complete 5'-end of the gene were isolated and characterized, revealing six exons common to all three mRNAs. Four of these exons were located in the coding region comprising the codons for amino acids 1 to 138. Two exons and an intervening sequence were identified upstream from these six common exons representing the unique 5'-ends of the three mRNAs. The coding exons show a significant sequence homology to mouse, rat, and human GGT cDNA, whereas exons 1 and 3 display no homology.

Secretion of active human mucus proteinase inhibitor by Aspergillus niger after KEX2-like processing of a glucoamylase-inhibitor fusion protein.

We report the production of human mucus proteinase inhibitor (MPI) by the filamentous fungus Aspergillus niger to test the ability of this host organism to secrete low molecular weight, highly disulfide-bonded proteins in biologically active conformation. Fungal transformants have been obtained with an expression cassette consisting of a chimeric gene founded on a mpi cDNA, encoding mature MPI, fused in frame to sequences encoding A. niger glucoamylase (glaA), separated by a KEX2-like processing sequence. Expression of the glucoamylase fusion gene in these transformants resulted in secretion of MPI into the growth medium with yields up to 3 mg 1-1. N-terminal sequence analysis of the purified inhibitor confirmed that the glucoamylase-MPI fusion protein was correctly processed to mature MPI by a KEX2-type endopeptidase present in A. niger. Furthermore, recombinant MPI retains full inhibitory activity against chymotrypsin and leukocyte elastase indicating that the protein was folded properly.

L-aspartate oxidase from Escherichia coli. I. Characterization of coenzyme binding and product inhibition.

This paper reports the biochemical characterization of the flavoprotein L-aspartate oxidase from Escherichia coli. Modification of a previously published procedure allowed overexpression of the holoenzyme in an unproteolysed form. L-Aspartate oxidase is a monomer of 60 kDa containing 1 mol of noncovalently bound FAD/mol protein. A polarographic and two spectrophotometric coupled assays have been set up to monitor the enzymatic activity continuously. L-Aspartate oxidase was subjected to product inhibition since iminoaspartate, which results from the oxidation of L-aspartate, binds to the enzyme with a dissociation constant (Kd) equal to 1.4 microM. The enzyme binds FAD by a simple second-order process with Kd 0.67 microM. Site-directed mutagenesis of the residues E43, G44, S45, F47 and Y48 located in the putative binding site of the isoallossazinic portion of FAD reduces the affinity for the coenzyme.

gamma-Glutamyl transpeptidase-cellular expression in populations of normal human mononuclear cells and patients suffering from leukemias.

The expression of the ectoenzyme gamma-glutamyl transpeptidase (EC2.3.2.2., gamma GT) was investigated by flow cytometry on populations of peripheral blood mononuclear cells (PBMC) from healthy subjects and patients suffering from several types of leukemia before and under chemotherapy. In unstimulated PBMC, 28% of these cells were found to be gamma GT positive. The highest expression was measured on monocytes (CD14/gamma GT+ cells: 60%). Within the subsets of T lymphocytes (CD3/gamma GT+ cells: 18%) we saw no clear differences between CD4+ and CD8+ cells. B lymphocytes, NK cells, and activated cells showed low expressions (up to 10%). Treatment of PBMC with mitogens, alpha-IFN, IL-2, and GM-CSF did not affect the enzyme expression on normal mononuclear cells (MNC). However, a rapid increase of gamma GT+ cells was found in the presence of glutathione (GSH) and n-acetyl cysteine (nAC), particularly on monocytes, B cells, and NK cells. Comparing 40 healthy subjects and untreated patients suffering from leukemias, a significantly higher expression of gamma GT+ cells in the total MNC populations (B-CLL: 57%, CML: 62% gamma GT+ cells) was observed in B-chronic lymphocytic leukemia (B-CLL) and chronic myelogenous leukemia (CML), whereas other leukemias did not show clear differences. Most interestingly, the gamma GT expression was diminished in all populations of CML cells after 5 h of incubation in the presence of 10 units/ml IFN-alpha. These data suggest a possible protective role of gamma GT in MNC and a regulatory function of this enzyme in the development of CML.

A protein kinase isolated from porcine brain microvessels is similar to a class of heat-shock proteins.

To further characterize a protein kinase present in porcine brain microvessels, a cDNA library using porcine microvessel poly(A) RNA was screened with polyclonal antibodies raised against the native protein kinase. Since no full-length cDNA clone could be obtained, the missing sequence information was completed using two subsequent polymerase chain reactions. The amplified transcripts were cloned and the sequence determined. Additionally, a genomic DNA library from porcine kidney was screened to substantiate the results obtained from the polymerase chain reaction. Earlier hints of a relation to a subclass of the family of heat-shock proteins (HSPs) based upon a close sequence similarity at its amino-terminus could be confirmed by comparison of the full-length cDNA sequences. Common protein kinase consensus sequences, a targeting sequence for proteins of the endoplasmic reticulum at the carboxy-terminus as well as a hydrophobic leader sequence in the amino-terminal region of the protein could also be identified. Furthermore, a set of membrane-associated substrate proteins of this enzyme could be detected in brain capillaries. The results indicate that at least some members of the HSP 90 subfamily undergo autophosphorylation and show protein kinase activity by phosphorylating substrate proteins in vitro.

Expression of apolipoprotein A-I in porcine brain endothelium in vitro.

Apolipoprotein (apo) A-I is the major protein component of high-density lipoproteins (HDLs), which are responsible for reverse cholesterol transport from peripheral tissues to the liver. A low level of plasma HDL is correlated with susceptibility to atherosclerosis and coronary heart disease. Mammalian apo A-I synthesis has been attributed mainly to liver and intestine. Recently, apo A-I expression has been shown in porcine brain capillaries, suggesting an independent lipid metabolism within the brain. In this study, protein synthesis and secretion were investigated in primary cultures of porcine brain microvascular endothelial cells and compared with those in large vessel endothelium. Active protein synthesis in vitro was demonstrated by metabolic labeling. Cerebral endothelial cells were shown to secrete apo A-I into the culture supernatant, whereas aortic endothelial cells were negative for apo A-I expression. Further studies of transcriptional regulation showed that cerebral endothelium was responsive to apo A-I-inducing agents, such as cholesterol, insulin, and retinoic acid, as previously shown in human hepatoma HepG2 cells. Thus, cultures of porcine cerebral endothelial cells may represent a suitable model for physiological studies of apo A-I-regulation with regard to brain lipid metabolism and blood-brain barrier function. To investigate the interspecies conservation of regulatory elements, 178 bp of the 5' flanking region of the porcine apo A-I gene was cloned using PCR techniques. Alignments of the cDNA, of the deduced apo A-I protein sequence, and of the 5' promoter region with the corresponding genomic sequences of different species show a high degree of similarity between the porcine and the primate apo A-I genes, thus indicating a similar function and possibly common regulatory mechanisms in those species. In contrast, the rodent and avian apolipoprotein A-I promoter sequences differed significantly.

Blood-brain barrier: a molecular approach to its structural and functional characterization.

Our approach to analyze molecular components of the blood-brain barrier led to the identification of additional transcripts which can be regarded as "BBB markers". Other candidates are presently analyzed in order to find hitherto unknown cell type-specific transcripts. We investigated the expression of these marker-genes in cell culture and found all genes still being transcribed after 10 days in primary cultures, although at a lower level. This is surprising, since other authors report the disappearance of BBB characteristics under such conditions. Moreover, the BBB marker gamma-GT is found to be not only expressed in BMEC, but also in the closely associated pericytes. The hitherto unknown physiological function of the enzyme, especially the abundance in pericytes is still under investigation. Since the method of subtractive cloning has been proven as a fruitful approach, we consider to establish further subtractive cDNA libraries, using different subtraction parameters. The PCR method is applicable for amplification of subtracted cDNA (Timblin et al., 1990) and we expect to find additional clones, mainly of lower abundance which are of functional importance for the BBB phenomenon. The described characterization of cultured BMEC now allows to proceed to study BBB-specific gene expression with special regard to regulatory elements. We will perform these experiments by use of enhancer trap vectors transfected into BMEC. The isolation of the corresponding genomic DNA fragments of the BBB markers is in progress.

Pericytes of the brain microvasculature express gamma-glutamyl transpeptidase.

The expression of gamma-glutamyl transpeptidase (GGT) is a specific property of the brain capillary endothelium that constitutes the blood-brain barrier. We report here the detection of GGT, not only in endothelial cells, but also in pericytes, demonstrating that a brain capillary-specific pericyte population exists. We raised antibodies to GGT using a porcine brain microvessel GGT-protein-A (staphylococcal protein A) fusion protein as antigen which was expressed in Escherichia coli. The immunohistochemical analysis of the subcapillary distribution of GGT in porcine brain cortex and cerebellum sections by both light and electron microscopy revealed the expression of GGT in the capillary-adjacent pericytes in addition to the GGT-positive endothelial layer. We confirmed these data for cultured porcine brain microvascular endothelial cells and pericytes. GGT immunofluorescence could be detected in both cell types in culture. Endothelial cells exhibited a weak staining, whereas pericytes were strongly positive for GGT. Due to the high phagocytotic activity of pericytes and their location on the abluminal surface of the microvessels, we propose a possible protective or detoxifying function of GGT in cerebrovascular pericytes.

The calcium-binding protein MRP-8 is produced by human pulmonary tumor cells.

A calcium-binding protein was isolated from serum-free culture medium of human squamous carcinoma cells (HS 24). N-Terminal sequencing of the protein yielded 30 amino acids which were identical to the N-terminus of cystic fibrosis antigen. Northern blot analysis with an oligonucleotide derived from the N-terminus resulted in the detection of a transcript of approximately 600 bases. Screening of a HS 24-cDNA library with the same oligonucleotide led to the isolation of a 381-bp-cDNA encoding a protein of 93 amino acids. The corresponding protein has been identified as the calcium-binding protein MRP-8 usually found in Macrophages.

Expression and characterization of recombinant second domain variants of human mucus proteinase inhibitor (MPI).

The second, carboxyterminal domain of human mucus proteinase inhibitor (MPI) represents a strong antagonist of trypsin, chymotrypsin and leucocyte elastase. To modulate the inhibitory specificity and chemical stability of this domain, mutants have been prepared by site-directed mutagenesis of a cDNA fragment encoding for the carboxyterminal half of the inhibitor, followed by expression in E. coli. Inhibition assays with the purified recombinant domains revealed the possibility to create variants for potential pharmaceutical use.

Expression of the E. coli nadB gene and characterization of the gene product L-aspartate oxidase.

Quinolinic acid is synthesized in E. coli by the enzymes L-aspartate oxidase and quinolinate synthase A, the genes of which are named nadB and nadA. In our previous work we cloned and characterized the two genes (Flachmann, R., Kunz, N., Seifert, J., Gütlich, M., Wientjes, F.J., Läufer, A. & Gassen, H.G. (1988) Eur. J. Biochem. 175, 221-228). Here we report on the expression of the nadB gene under control of the inducible left promoter of the bacteriophage lambda. The yield of the active gene product L-aspartate oxidase was enhanced up to 20% of the soluble cell protein. The enzyme was purified to homogeneity in a three-step procedure and the reading frame of the L-aspartate oxidase gene was confirmed by Edman degradation of five cyanogen bromide peptides. L-Aspartate oxidase shows no classical Michaelis-Menten behaviour but is subject to a substrate inactivation. The apparent Km values were different for substrate concentrations below and above 1mM and were determined to 0.5 mM and 4.1mM, respectively. The active form of the enzyme is a monomer of 60,284 Da and contains one molecule of FAD and nine cysteine residues, four of which built up two disulfide bonds. The isoelectric point of the protein was determined to be at pH 5.6. Chemical modifications of the enzyme showed that at least one tyrosine and one histidine residue are essential for enzyme activity. The coenzyme-binding domain is located in the amino-terminal part of the polypeptide chain as revealed by a sequence comparison to other dinucleotide binding enzymes. Furthermore, there is evidence for a relationship to fumarate reductase and succinate dehydrogenase of E. coli.

Synthesis of apolipoprotein A-1 in pig brain microvascular endothelial cells.

In an approach toward the identification of hitherto unknown proteins involved in the function of the blood-brain barrier, we constructed a pig brain microvessel-derived cDNA library that is enriched in blood-brain barrier specific sequences by means of subtractive cloning. Sequence analysis of selected clones revealed that one of the cDNAs encoded porcine apolipoprotein (apo) A-1. The identity of apo A-1 mRNA was further confirmed by in vitro translation of RNA from brain microvascular endothelial cells and subsequent immunoprecipitation with an antibody against human apo A-1. We further investigated the expression of apo A-1 mRNA in several tissues and in endothelial cells of the pig. It is shown that cultured brain microvascular endothelial cells provide an in vitro model to study the expression and function of apo A-1 in the microvasculature of the brain.