Expression of the human alpha-galactosidase A in Escherichia coli K-12

We used the prokaryotic expression vector, ptrpL1, for the expression in Escherichia coli K-12 of a cDNA clone specific for the human lysosomal hydrolase, alpha-galactosidase A. The 5' terminus of the cDNA clone was engineered so that an ATG codon precedes the first codon of the mature form of the enzyme. A clone with elevated expression of this human enzyme was constructed by increasing the distance between the Shine-Dalgarno site and the ATG start codon from 6 to 8 bp. Clones with alpha-galactosidase A specific cDNA encoding the proenzyme produce a protein of 45 kDa, the size expected for the intact proenzyme. The 45-kDa protein is specifically precipitated by antibody to alpha-galactosidase A, and its expression is repressed by tryptophan and induced by 3-beta-indoleacrylic acid as expected for this expression vector. The human enzyme is produced in E. coli in a catalytically active form at levels sufficient to support the growth of cells using alpha-galactosides as sole sources of carbon and energy. In addition, bacterial colonies that produce the human enzyme turn blue in the presence of 5-bromo-4-chloro-3-indolyl-alpha-D-galactopyranoside.     

Biochemical and functional characterization of cation dependent (Mr 46,000) goat mannose 6-phosphate receptor

The Mannose 6-phosphate receptor (MPR’s) proteins are important for transporting lysosomal enzymes from trans-golgi to the pre-lysosomal compartment. These are conserved in the vertebrates from fish to mammals. We have cloned the full length cDNA for the goat MPR 46 protein and compared its sequences to the other known vertebrate MPR 46 proteins. In the present study the full-length cDNA for the goat MPR 46 protein was expressed in MPR deficient cells. The expressed protein was purified on the multivalent phosphomannan gel in the presence of divalent metal ions. The apparent molecular mass of the expressed protein was found to be ∼46 kDa and also exhibits oligomeric nature as observed in the other species, by using an MSC1 antibody (that recognizes the MPR 46 from molluscs to mammals) as well as with a peptide specific antibody corresponding to amino acid residues (218–237) of the cytoplasmic tail of human MPR 46 protein. Furthermore the distribution of the expressed protein was visualized by immunofluorescence using MSC1 and LAMP1 antibody. Additionally in the goat MPR 46 expressing cells, the sorting function of the expressed protein to sort cathepsin D to lysosomes was studied by confocal microscopy using cathepsin D antiserum and LAMP1 antibody. The binding of goat MPR 46 to cathepsin D was shown in far Western blotting and the mannose 6-phosphate dependent binding was shown by co-immunoprecipitation.     

Nucleotide sequence of the melA gene, coding for alpha-galactosidase in Escherichia coli K-12.

Melibiose uptake and hydrolysis in E.coli is performed by the MelB and MelA proteins, respectively. We report the cloning and sequencing of the melA gene. The nucleotide sequence data showed that melA codes for a 450 amino acid long protein with a molecular weight of 50.6 kd. The sequence data also supported the assumption that the mel locus forms an operon with melA in proximal position. A comparison of MelA with alpha-galactosidase proteins from yeast and human origin showed that these proteins have only limited homology, the yeast and human proteins being more related. However, regions common to all three proteins were found indicating sequences that might comprise the active site of alpha-galactosidase.     

Lipoproteins in pinnipeds: analysis of a high molecular weight form of apolipoprotein E

We have examined the apolipoprotein content of the lipoproteins of several marine mammals by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Their apolipoprotein (apo) B-100, apoB-48, and apoA-I migrated to virtually the same position as the comparable human apolipoproteins. In cetaceans (bottlenose dolphins and killer whales), the molecular mass of the apoE was identical to that of human apoE (35 kDa). In contrast, in the lipoproteins of pinnipeds (harbor seals, sea lions, and walrus) there was no protein comparable in size to human apoE; however, there were two proteins in the 40- to 44-kDa range. The protein with the higher apparent molecular weight (44 kDa) was apoA-IV, as determined by NH2-terminal amino acid sequencing. Sequencing of the NH2-terminal 15 amino acids of the lower molecular weight protein (40-42 kDa) revealed no obvious homology with human apoE. However, a human apoE-specific monoclonal antibody, 1D7, bound to the 40- to 42-kDa protein, allowing us to identify that protein as apoE. Sequencing of sea lion apoE cDNA clones demonstrated that sea lion apoE is 311 amino acids in length, 12 residues longer than human apoE. All 12 additional residues are located in the NH2-terminal 31 amino acids, a region that has extremely low homology with the NH2-terminal portion of human apoE. The remainder of the sea lion apoE sequence is 74% homologous to human apoE. The sea lion apoE cDNA was expressed in Chinese hamster ovary (CHO) cells as well as CHO ldlD cells, a cell line that is deficient in O-glycosylation of proteins. The size of the sea lion apoE secreted by these two cell lines, compared with the apoE in sea lion plasma, indicated that the predominant form of apoE in sea lion plasma must be posttranslationally modified. Thus, our studies have demonstrated that the higher apparent molecular weight of pinniped (sea lion) apoE is due to a longer polypeptide chain as well as posttranslational modification of the protein.     

Cloning, expression, and chromosomal localization of the 140-kilodalton subunit of replication factor C from mice and humans.

We have isolated a full-length mouse cDNA encoding a lysine-rich protein of 1,131 amino acids with a calculated molecular mass of 126 kDa. The protein binds in a sequence-unspecific manner to DNA, is localized exclusively in the nucleus, and contains a putative ATP binding site and a stretch of 80 amino acids with homology to the carboxy terminus of prokaryotic DNA ligases. On the basis of the following facts, we conclude that the isolated cDNA encodes the 140-kDa subunit of mouse replication factor C (mRFC140). (i) The sequence around the ATP binding site shows significant homology to three small subunits of human replication factor C. (ii) Polyclonal antibodies raised against the protein encoded by this cDNA cross-react with the 140-kDa subunit of purified human replication factor C (hRFC140) and recognize in mouse cell extracts an authentic protein with an apparent molecular mass of 130 kDa. (iii) Sequence comparison with a human cDNA isolated by using tryptic peptide sequence information from purified hRFC140 revealed 83% identity of the encoded proteins. The mRFC140 gene is ubiquitously expressed, and two mRNAs approximately 5.0 and 4.5 kb long have been detected. The gene was mapped by in situ hybridization to mouse chromosome 5, and its human homolog was mapped to chromosome 4 (p13-p14).     

Signal sequence and DNA-mediated expression of human lysosomal alpha-galactosidase A

Twelve complementary DNA clones for human lysosomal alpha-galactosidase A were isolated from an Okayama-Berg library constructed from SV40-transformed human fibroblasts. The identity of these clones was confirmed by complete colinearity of the nucleotide-deduced amino acid sequence with that determined by direct chemical sequencing of human placental alpha-galactosidase A. Hybridization of the alpha-galactosidase A cDNA to genomic DNA from individuals with varying numbers of X chromosomes as well as from interspecies somatic-cell hybrids showed only a single locus in the genome at Xq 13.1-Xq 22. One cDNA clone (pcD-AG210) contained the complete coding sequence for both the signal peptide and mature alpha-galactosidase A. The signal peptide of 31 amino acids contains the expected hydrophobic domains consisting of Leu-Gly-Cys-Ala-Leu-Ala-Leu and Phe-Leu-Ala-Leu-Val and has Ala at the signal peptidase cleavage site. Twelve out of fifteen G residues flanking the 5' end of the cDNA in pcD-AG210 were removed and the truncated fragment was ligated into the original vector. This construct, pcD-AG502, encoded enzymatically active human alpha-galactosidase A in monkey COS cells.     

Glycosidases of Ehrlich ascites tumor cells and ascitic fluid--purification and substrate specificity of alpha-N-acetylgalactosaminidase and alpha-galactosidase: comparison with coffee bean alpha-galactosidase

Ehrlich ascites tumor cells and ascitic fluid were assayed for glycosidase activity. alpha-Galactosidase and beta-galactosidase, alpha- and beta-mannosidase, alpha-N-acetylgalactosaminidase, and beta-N-acetylglucosaminidase activities were detected using p-nitrophenyl glycosides as substrates. alpha-Galactosidase and alpha-N-acetylgalactosaminidase were isolated from Ehrlich ascites tumor cells on epsilon-aminocaproylgalactosylamine-Sepharose. alpha-Galactosidase was purified 160,000-fold and was free of other glycosidase activities. alpha-N-Acetylgalactosaminidase was also purified 160,000-fold but exhibited a weak alpha-galactosidase activity which appears to be inherent in this enzyme. Substrate specificity of the alpha-galactosidase was investigated with 12 substrates and compared with that of the corresponding coffee bean enzyme. The pH optimum of the Ehrlich cell alpha-galactosidase centered near 4.5, irrespective of substrate, whereas the pH optimum of the coffee bean enzyme for PNP-alpha-Gal was 6.0, which is 1.5 pH units higher than that for other substrates of the coffee bean enzyme. The reverse was found for alpha-N-acetylgalactosaminidase: the pH optimum for the hydrolysis of PNP-alpha-GalNAc was 3.6, lower than the pH 4.5 required for the hydrolysis of GalNAc alpha 1,3Gal. Coffee bean alpha-galactosidase showed a relatively broad substrate specificity, suggesting that it is suited for cleaving many kinds of terminal alpha-galactosyl linkages. On the other hand, the substrate specificity of Ehrlich alpha-galactosidase appears to be quite narrow. This enzyme was highly active toward the terminal alpha-galactosyl linkages of Ehrlich glycoproteins and laminin, both of which possess Gal alpha 1, 3Gal beta 1,4GlcNAc beta-trisaccharide sequences. The alpha-N-acetylgalactosaminidase was found to be active toward the blood group type A disaccharide, and trisaccharide, and glycoproteins with type A-active carbohydrate chains.     

A novel 45 kDa secretory protein from rat olfactory epithelium: primary structure and localisation.

cDNA clones encoding the 45 kDa protein were isolated from a rat olfactory epithelium cDNA library and their inserts were sequenced. The reconstructed protein sequence comprises 400 amino acids with a calculated molecular mass of 46,026 Da. A homology was revealed between the amino acid sequence of the 45 kDa protein and the proteins involved in the transfer of hydrophobic ligands. Using in situ hybridisation, the 45 kDa protein mRNA expression was detected in the layer of supportive cells of olfactory epithelium, apical region of trachea, surface layer of the ciliated bronchial epithelium in lung and in skin epidermis.     

Purification, identification, and cDNA cloning of Cha o 2, the second major allergen of Japanese cypress pollen.

The second major allergen of Chamaecyparis obtusa (Japanese cypress) pollen, Cha o 2, has been purified and its cDNA cloned. Of patients with pollinosis caused by C. obtusa, 82.5% produce IgE antibodies which react with purified Cha o 2. The purified protein has a molecular mass of 46 kDa and its 12 N-terminal amino acid sequence displays a high homology with that of Cry j 2, the second major allergen of Cryptomeria japonica pollen. cDNA clones coding for Cha o 2 have been isolated using Cry j 2 cDNA as a probe. Cha o 2 cDNA clones were sequenced and found to code a putative 50-residue signal sequence and a 464-residue mature protein with a molecular weight of 50 kDa. Two possible N-linked glycosylation sites were found in the sequence. The deduced amino acid sequence of Cha o 2 shows 74.3% identity with that of Cry j 2. In its primary structure, Cha o 2 shows significant identity with those of the polygalacturonases of avocado, tomato, and maize as well as Cry j 2.     

Identification of human L-fucose kinase amino acid sequence

Fucose is a major component of complex carbohydrates. L-Fucose kinase (fucokinase) takes part in the salvage pathway for reutilization of fucose from the degradation of oligosaccharides. The amino acid sequence of human fucokinase was derived from a cDNA encoding a protein of hitherto unidentified function. Human fucokinase polypeptide chain consists of 990 amino acids with a predicted molecular mass of 107 kDa. The C-terminal part of its amino acid sequence showed sequence motifs typical for sugar kinases. Fucokinase full-length protein and a deletion mutant lacking the first 363 amino acids of the N-terminus were expressed in Escherichia coli BL21 cells. Both proteins displayed fucokinase activity. These results reveal that the discovered cDNA encodes the fucokinase protein and they confirm that a functional kinase domain is located in the C-terminal part of the enzyme.     

Cloning and characterisation of a zona pellucida 3 cDNA from a marsupial, the brushtail possum Trichosurus vulpecula

The mammalian zona pellucida (ZP) is an extracellular glycoprotein coat that plays vital roles throughout fertilisation and preimplantation development. Like that of eutherian mammals the brushtail possum ZP is composed of three glycosylated proteins of 137 kDa, 92 kDa and 62 kDa. The 62 kDa protein is a ZP3 orthologue based on its nucleotide and deduced amino acid sequence. The brushtail possum ZP3 cDNA isolated in this study is 1305 nucleotides with an open reading frame encoding a 422 amino acid peptide of 45.7 kDa. Possum ZP3 has a 46% amino acid identity with eutherian ZP3 and shares similar structural characteristics including 12 conserved cysteine residues, N-linked glycosylation sites and hydrophobic regions. Like human and rabbit ZP1 an altered furin cleavage site upstream of the C-terminal hydrophobic domain also occurs in possum ZP3 (S-R-K-R), suggestive of processing by a furin-related endoprotease. Expression of brushtail possum ZP3 is limited to the ovary. Characterisation of brushtail possum ZP3 will enable examination of its functional role in marsupial fertilisation and its effectiveness as an immunocontraceptive agent.     

Isolation and characterization of the cDNA for pulmonary surfactant-associated protein-B (SP-B) in the rabbit

Pulmonary surfactant contains phospholipids including dipalmitoyl-phosphatidylcholine and three surfactant-associated proteins designated SP-A, SP-B and SP-C. A cDNA for rabbit SP-B has been isolated from a fetal (30 days gestation) rabbit lung cDNA library constructed in lambda gt11. The cDNA and deduced amino acid sequences show strong homology with the cDNAs and predicted 40 kDa proproteins for human and canine SP-B. Strong homology is also observed with the amino acid sequences directly determined for the mature 8 kDa bovine and porcine SP-B isolated from lung lavage. SP-B is remarkable for its high cysteine and proline content and for the hydrophobic nature of the organic solvent-soluble, mature protein. In vitro translation of sense but not antisense RNA transcribed from the cDNA led to the production of 40 kDa and 32 kDa proteins. These proteins were immunoprecipitated by an antibody raised against bovine SP-B. Northern blot analysis revealed the mRNA for rabbit SP-B appears in fetal rabbit lung late in gestation and falls slightly in the neonate.     

The sequence of human retinal S-antigen reveals similarities with alpha-transducin

The complete amino acid sequence of human retinal S-antigen (48 kDa protein), a retinal protein involved in the visual process has been determined by cDNA sequencing. The largest cDNA was 1590 base pairs (bp) and it contained an entire coding sequence. The similarity of nucleotide sequence between the human and bovine is approximately 80%. The predicted amino acid sequence indicates that human S-antigen has 405 residues and its molecular mass is 45050 Da. The amino acid sequence homology between human and bovine is 81%. There is no overall sequence similarity between S-antigen and other proteins listed in the National Biomedical Research Foundation (NBRF) protein data base. However, local regions of sequence homology with alpha-transducin (T alpha) are apparent including the putative rhodopsin binding and phosphoryl binding sites. In addition, human S-antigen has sequences identical to bovine uveitopathogenic sites, indicating that some types of human uveitis may in part be related to the animal model of experimental autoimmune uveitis (EAU).     

Characterization of the cell-cycle-regulated protein calcyclin from Ehrlich ascites tumor cells. Identification of two binding proteins obtained by Ca2(+)-dependent affinity chromatography

The nearly complete amino acid sequence obtained for murine calcyclin from Ehrlich ascites tumor cells reveals a very strong similarity with the rat and human sequences previously deduced from corresponding cDNA clones. While mouse and rat calcyclins are identical, the human protein shows at three positions a conservative amino acid replacement. Using a mouse calcyclin affinity matrix, two proteins with molecular masses of about 36 kDa have been purified from Ehrlich ascites tumor cells. The interaction between these two proteins and the immobilized calcyclin is strictly Ca2(+)-dependent. Immunological criteria and partial sequence data identify the two calcyclin-binding proteins as the phospholipid-binding protein annexin II (p36) and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase. These observations suggest that calcyclin may exert its physiological function by a Ca2(+)-dependent interaction with cellular targets, e.g. annexin II or glyceraldehyde-3-phosphate dehydrogenase.     

Complementary DNA sequence of rabbit CAP18--a unique lipopolysaccharide binding protein

CAP18 is a novel 18 kDa cationic protein [pI approximately 10] originally purified from rabbit granulocytes using as an assay the agglutination of lipopolysaccharide (LPS) coated erythrocytes. cDNA clones encoding CAP18 were isolated from a rabbit bone marrow cDNA library using a PCR generated oligonucleotide probe derived from the N-terminal amino acid sequence. The deduced amino acid sequence reveals a putative signal sequence of 29 amino acids and a mature protein of 142 amino acid residues. The predicted size of the encoded protein is 16.6 kDa with a pI of 10. There are no N-linked glycosylation sites. The CAP18 sequence bears no homology with other known LPS-binding proteins including human bacterial permeability increasing protein (BPI)(1) and rabbit LPS binding protein (LBP)(2).     

Characterization of cDNA encoding mouse DNA repair protein O6-methylguanine-DNA methyltransferase and high-level expression of the wild-type and mutant proteins in Escherichia coli.

A mouse cDNA clone encoding O6-methylguanine-DNA methyltransferase (MGMT), responsible for repair of mutagenic O6-alkylguanine in DNA, was cloned from a lambda gt11 library. On the basis of an open reading frame in cDNA, the mouse protein contains 211 amino acids with a molecular mass of 22 kDa. The size and the predicted N-terminal sequence of the mouse protein were confirmed experimentally. The deduced amino acid sequence of the mouse MGMT is 70% homologous to that of the human MGMT. Cysteine-149 was shown to be the only alkyl acceptor residue in the mouse protein, in confirmation of the prediction based on conserved sequences of different MGMTs. Mouse MGMT protein is recognized by some monoclonal antibodies specific for human MGMT. Site-directed mutagenesis was utilized to reclone the mouse cDNA in a T7 promoter-based vector for overexpression of the native repair protein in Escherichia coli. The mouse protein has a tetrapeptide sequence, Pro-Glu-Gly-Val at positions 56-59, absent in the human protein. Neither deletion of this tetrapeptide nor substitution of valine-169 with alanine affected the activity of the mutant proteins.     

Purification and characterization of alpha-N-acetylgalactosaminidase from skipjack liver

By means of a simple procedure involving two gel filtrations and an ion-exchange chromatography, alpha-N-acetylgalactosaminidase was purified to an electrophoretically homogeneous form from skipjack liver, in which the enzyme is the dominant glycosidase. The final alpha-N-acetylgalactosaminidase preparation contained practically no other glycosidase activities except alpha-galactosidase activity, which amounted to 0.8% of the alpha-N-acetylgalactosaminidase activity and may be an intrinsic activity of the enzyme. The molecular weight of the enzyme was estimated to be 80,000 at pH 4.2 and 40,000 at pH 7.2 by molecular sieve chromatography, and to be 35,000 by SDS-polyacrylamide gel electrophoresis. The enzyme was most active at pH 4 and was inactive above pH 7. These results suggest that skipjack alpha-N-acetylgalactosaminidase exists as an active dimer at acidic pH and as inactive monomer at neutral or alkaline pH. The enzyme efficiently liberated the N-acetylgalactosamine unit from ovine submaxillary glycoprotein which had been desialylated by neuraminidase. The Km value and maximum velocity were 4.28 mM and 409 mumol/min X mg for p-nitrophenyl alpha-N-acetylgalactosaminide, and 0.0543 mM and 1.19 mumol/min X mg for ovine submaxillary asialoglycoprotein.     

Expression and characterization of the human mitochondrial leucyl-tRNA synthetase

A cDNA clone encoding the human mitochondrial leucyl-tRNA synthetase (mtLeuRS) has been identified from the EST databases. Analysis of the protein encoded by this cDNA indicates that the protein is 903 amino acids in length and contains a mitochondrial signal sequence that is predicted to encompass the first 21 amino acids. Sequence analysis shows that this protein contains the characteristic motifs of class I aminoacyl-tRNA synthetases and regions of high homology to other mitochondrial and bacterial LeuRS proteins. The mature form of this protein has been cloned and expressed in Escherichia coli. Gel filtration indicates that human mtLeuRS is active in a monomeric state, with an apparent molecular mass of 101 kDa. The human mtLeuRS is capable of aminoacylating E. coli tRNA(Leu). Its activity is inhibited at high levels of either monovalent or divalent cations. K(M) and k(cat) values for ATP:PP(i) exchange and for the aminoacylation reaction have been determined.