Primary structure of rat secretory acid phosphatase and comparison to other acid phosphatases

Overlapping cDNA clones encoding rat prostatic acid phosphatase (rPAP) were isolated by using two human prostatic acid phosphatase (hPAP)-encoding cDNAs to screen rat prostatic cDNA libraries. The isolated cDNAs encompassed a total of 1626 nucleotides (nt), of which 1143 nt corresponded to the protein coding sequence encoding a mature polypeptide of 350 amino acids (aa) and a 31-aa long signal peptide-like sequence. The deduced Mr of the mature rPAP was 40,599. RNA blot analysis indicated the presence of three mRNA species (4.9, 2.3 and 1.5 kb in size) in the rat prostate. The deduced aa sequences of rPAP and hPAP show 75% identity, whereas the similarity between rPAP and human lysosomal acid phosphatase (hLAP) is only 45%. Furthermore, the sequence similarity between rPAP and rat lysosomal acid phosphatase (rLAP) is 46% at the aa level. Similar to hPAP, but unlike hLAP and rLAP, the rPAP sequence lacks a membrane-anchoring domain indicating the secretory character of this phosphatase. All six cysteines present in the overlapping areas of the mature rPAP, hPAP, rLAP and hLAP proteins are positionally conserved, suggesting that these residues are important for the tertiary structure of acid phosphatases (APs). The previously reported active site residues, two arginines and one histidine, are also conserved in these APs.     

Human prostatic acid phosphatase: cDNA cloning, gene mapping and protein sequence homology with lysosomal acid phosphatase

The cDNAs encoding human prostatic acid phosphatase were cloned and characterized. The mRNAs contain 3' noncoding regions of heterogeneous sizes 646, 1887 or 1913 nucleotides. A dimer and a monomer of the conserved Alu-repeats are present in the longer 3' noncoding sequences. The complete sequence of 354 amino acids for the mature enzyme was determined by sequencing both cDNA and protein. Human prostatic and lysosomal acid phosphatases exhibit 50% sequence homology, including five Cys residues and two putative N-linked glycosylation sites. The Acp-3 gene coding for human prostatic acid phosphatase was mapped onto chromosome 3 in this investigation. The Acp-2 gene coding for lysosomal acid phosphatase has previously been located on chromosome 11, while the Acp-1 gene coding for red blood cell acid phosphatase is on chromosome 2.     

Structure of human prostatic acid phosphatase gene.

Two cDNA clones containing the complete protein-coding sequence of 1,188 nucleotides as well as the 5' and 3' non-coding regions of human prostatic acid phosphatase (PAP) were isolated and sequenced. The size of PAP mRNAs from benign prostate hyperplasia and cancerous prostate was estimated to be 3.2Kb, indicating that the 3' downstream polyadenylation signal was used. Several genomic clones containing parts of the human PAP gene were isolated and the nucleotide sequence of ten exons and their flanking regions was determined. The protein-coding sequence of the human PAP gene was interrupted by nine introns. The positions of all nine introns present in the human PAP gene were homologous to those of the first nine introns in the human lysosomal acid phosphatase (LAP) gene. However, the last (11th) exon of the LAP gene encoding the COOH-terminal domain, which includes a transmembrane segment, was found to be absent in human PAP gene. Southern blot analysis of ten mammalian genomic DNAs gave multiple EcoRI fragments. The data of human genomic DNAs were consistent with the total length of the PAP gene of at least 50 kilobases.     

Human lysosomal acid phosphatase: cloning, expression and chromosomal assignment.

A 2112-bp cDNA clone (lambda CT29) encoding the entire sequence of the human lysosomal acid phosphatase (EC 3.1.3.2) was isolated from a lambda gt11 human placenta cDNA library. The cDNA hybridized with a 2.3-kb mRNA from human liver and HL-60 promyelocytes. The gene for lysosomal acid phosphatase was localized to human chromosome 11. The cDNA includes a 12-bp 5' non-coding region, an open reading frame of 1269 bp and an 831-bp 3' non-coding region with a putative polyadenylation signal 25 bp upstream of a 3' poly(A) tract. The deduced amino acid sequence reveals a putative signal sequence of 30 amino acids followed by a sequence of 393 amino acids that contains eight potential glycosylation sites and a hydrophobic region, which could function as a transmembrane domain. A 60% homology between the known 23 N-terminal amino acid residues of human prostatic acid phosphatase and the N-terminal sequence of lysosomal acid phosphatase suggests an evolutionary link between these two phosphatases. Insertion of the cDNA into the expression vector pSVL yielded a construct that encoded enzymatically active acid phosphatase in transfected monkey COS cells.     

Covalent structure, disulfide bonding, and identification of reactive surface and active site residues of human prostatic acid phosphatase

The pairing of the half-cysteine residues of human prostatic acid phosphatase was established by proteolytic digestion and analysis of the resulting peptide mixtures by fast atom bombardment mass spectrometry (FAB-MS). An independently derived, full length cDNA clone was used as the basis for the interpretation of the FAB-MS data. The sequence of the native protein is that predicted from the present cDNA sequence, except for the carboxyl-terminal end and some possible post-translational deamidations. Isolated human prostatic acid phosphatase was found to have multiple carboxyl-terminal ends, terminating in Thr, Glu, and Asp, corresponding to residues 349-351 of the 354-residue protein that is predicted from the cDNA sequence after removal of a leader peptide. The protein contains no free sulfhydryl groups. The identical monomer chains of the dimeric native enzyme are found to contain three disulfide bonds, specifically Cys-129 to Cys-340, Cys-183 to Cys-281, and Cys-315 to Cys-319. In view of the conserved positions of cysteines in the homologous human and rat liver lysosomal acid phosphatases, an identical disulfide bonding pattern may be predicted for those proteins. The location of a potential antigenic site was established by selective labeling of proximate tyrosine residues predicted to be on the surface. A conserved RHGXRXP sequence is present in the prostatic, lysosomal, Escherichia coli, and yeast acid phosphatases and is predicted to be of mechanistic significance. In addition, residue Arg-54 is shown to be an active site residue by reaction of the enzyme with phenylglyoxal. Interestingly, this residue is present in a sequence RXRY (R,H) that is also present in lysosomal phosphatase and in recently described protein tyrosine phosphatases.     

Molecular cloning of cDNAs encoding two isoforms of the catalytic subunit of protein phosphatase 2A

Clones coding for the catalytic subunit of one of the major protein phosphatases (type 2A) were isolated from a porcine cDNA library. Sequence analysis indicated that two different mRNA species coded for this enzyme. The deduced amino acid sequences of the two forms (alpha and beta) of the enzyme were 98% identical and showed 95% identity with the partial sequence of the rabbit enzyme determined by amino acid sequencing. The use of specific oligonucleotide probes indicated that the mRNAs coding for the alpha and beta forms were about 2 kilobases in length, present in equal amounts in a porcine cell line (LLC-PK1), and were the products of two distinct genes. Southern analysis using the coding region of the alpha phosphatase cDNA as a probe suggested the existence of additional related phosphatase genes.     

Isolation and sequence analysis of a cDNA clone encoding a type-1 protein phosphatase catalytic subunit: homology with protein phosphatase 2A

A 1.5 kb clone containing the full-length coding sequence of a type-1 protein phosphatase catalytic subunit has been isolated from a rabbit skeletal muscle cDNA library constructed in lambda gt10. The protein sequence deduced from the cDNA contains 311 residues and has a molecular mass of 35.4 kDa. A single mRNA species at 1.6 kb was visualized by Northern blotting. The type-1 protein phosphatase was strikingly homologous to protein phosphatase 2A, 49% of the amino acids between residues 11 and 280 being identical. The first 10 and last 31 residues were dissimilar. Residues 1-101 of the type-1 protein phosphatase also showed 21% sequence identity with a region of mammalian alkaline phosphatases.     

cDNA sequence of a Drosophila melanogaster gene, Dfur1, encoding a protein structurally related to the subtilisin-like proprotein processing enzyme furin

Screening a genomic library of Drosophila melanogaster DNA with a human fur cDNA probe resulted in the isolation of DNA clones that apparently belonged to two different DNA regions of the Drosophila genome. Subsequently, corresponding Drosophila cDNA clones were isolated. Nucleotide sequence analysis indicated that these cDNA clones originated from two different genes, which were called Dfur1 and Dfur2. From overlapping Dfur1 cDNA clones, a composite cDNA could be constructed and analysis of its nucleotide sequence revealed the coding sequence for a protein of 899 amino acid residues. This protein, designated Dfurin1, exhibited striking sequence homology to human furin and contained the same protein domains except for the cysteine-rich region. Furthermore, unlike human furin, Dfurin1 possessed an extended amino-terminal region in which a potential transmembrane anchor was present.     

Gene expression and prostate specificity of human prostatic acid phosphatase (PAP): evaluation by RNA blot analyses.

A fragment of a complementary DNA (cDNA) clone for human prostatic acid phosphatase (PAP) (EC 3.1.3.2.) was used to study the expression of corresponding mRNA in human tissues. The specificity of its expression in benign prostatic hyperplasia (BPH) and prostatic carcinoma tissues were indicated in RNA blot analyses. The PAPcDNA probe did not recognize any specific mRNAs in RNAs extracted from human liver cancer, lung cancer, pancreatic cancer, placenta, breast cancer cells (MCF-7), mononuclear blood cells or acute promyelocytic leukemia cells (HL-60), according to Northern blot analysis. mRNA for PAP was detected in the androgen-dependent human prostatic cancer cell line LNCaP, but not in the androgen-insensitive human prostatic cancer cell line PC-3. In contrast, lysosomal acid phosphatase (LAP) mRNA was detected in both of these human prostatic cancer cell lines. Our findings indicate a high specificity for the PAP gene in prostatic tissue. The mean abundance for the PAPmRNA expression was 0.26 for prostatic carcinoma samples (n = 11) and 0.46 for BPH samples (n = 8) according to slot-blot analysis. The differences observed between the different categories of prostatic tissue in PAPmRNA abundances call for additional studies on regulation of its expression.     

Sequencing, cloning, and expression of human red cell-type acid phosphatase, a cytoplasmic phosphotyrosyl protein phosphatase.

Low molecular weight phosphotyrosyl protein phosphatases of human placenta and human red cell were purified and sequenced by a combination of Edman degradation and tandem mass spectrometry. Screening of a human placental lambda gt11 cDNA library yielded overlapping cDNA clones coding for two distinct human cytoplasmic low molecular weight phosphotyrosyl protein phosphatases (HCPTPs). The two longest clones, designated HCPTP1-1 and HCPTP2-1, were found to have identical nucleotide sequences, with the exception of a 108-base pair segment in the middle of the open reading frame. Polymerase chain reaction studies with human genomic DNA suggest that the difference between HCPTP1-1 and HCPTP2-1 does not result from alternative RNA splicing. Studies with a human chromosome 2-specific library confirmed that these sequences are located on chromosome 2, which is known to be the location of red cell acid phosphatase locus ACP1. The coding sequences of HCPTP1-1 and HCPTP2-1 were placed downstream from a bacteriophage T7 promoter and the proteins were expressed in Escherichia coli. The resulting recombinant enzymes (designated HCPTP-A and HCPTP-B, respectively) showed molecular weights of 18,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and both of them exhibited immunoreactivity with antisera raised against authentic human placental and bovine heart enzymes. The expressed proteins were highly active towards the phosphatase substrates p-nitrophenyl phosphate, beta-naphthyl phosphate, and O-phospho-L-tyrosine, but not alpha-naphthyl phosphate, threonine phosphate, or O-phospho-L-serine. HCPTP-A and -B possessed effectively identical amino acid compositions, immunoreactivities, inhibition by formaldehyde, and kinetic properties when compared with two human red cell acid phosphatase isoenzymes. It is concluded that HCPTP-A and -B are the fast and slow forms of red cell acid phosphatase, respectively, and that this enzyme is not unique to the red cell but is instead expressed in all human tissues.     

Isolation and sequence analysis of a cDNA clone encoding the entire catalytic subunit of a type-2A protein phosphatase

A 2.5 kb clone containing the full-length coding sequence of a type-2A protein phosphatase catalytic subunit has been isolated from a rabbit skeletal muscle cDNA library constructed in lambda gt10. The sequence of the protein deduced from the cDNA contains 309 residues (35.58 kDa). A major mRNA species at 2.0 kb and a minor component at 2.8 kb were visualized by Northern blotting in both skeletal muscle and liver. The type-2A enzyme showed weak homology with mammalian alkaline phosphatases between residues 55 and 95. The protein sequence of the type-2A phosphatase from rabbit skeletal muscle differs from that reported for the bovine adrenal enzyme in three regions.     

Isolation and characterization of human cDNA clones encoding the alpha and the alpha' subunits of casein kinase II

Casein kinase II is a widely distributed protein serine/threonine kinase. The holoenzyme appears to be a tetramer, containing two alpha or alpha' subunits (or one of each) and two beta subunits. Complementary DNA clones encoding the subunits of casein kinase II were isolated from a human T-cell lambda gt10 library using cDNA clones isolated from Drosophila melanogaster [Saxena et al. (1987) Mol. Cell. Biol. 7, 3409-3417]. One of the human cDNA clones (hT4.1) was 2.2 kb long, including a coding region of 1176 bp preceded by 156 bp (5' untranslated region) and followed by 871 bp (3' untranslated region). The hT4.1 clone was nearly identical in size and sequence with a cDNA clone from HepG2 human hepatoma cultured cells [Meisner et al. (1989) Biochemistry 28, 4072-4076]. Another of the human T-cell cDNA clones (hT9.1) was 1.8 kb long, containing a coding region of 1053 bp preceded by 171 bp (5' untranslated region) and followed by 550 bp (3' untranslated region). Amino acid sequences deduced from these two cDNA clones were about 85% identical. Most of the difference between the two encoded polypeptides was in the carboxy-terminal region, but heterogeneity was distributed throughout the molecules. Partial amino acid sequence was determined in a mixture of alpha and alpha' subunits from bovine lung casein kinase II. The bovine sequences aligned with the 2 human cDNA-encoded polypeptides with only 2 discrepancies out of 535 amino acid positions. This confirmed that the two human T-cell cDNA clones encoded the alpha and alpha' subunits of casein kinase II. Microsequence data determined from separated preparations of bovine casein kinase II alpha subunit and alpha' subunit [Litchfield et al. (1990) J. Biol. Chem. 265, 7638-7644] confirmed that hT4.1 encoded the alpha subunit and hT9.1 encoded the alpha' subunit. These studies show that there are two distinct catalytic subunits for casein kinase II (alpha and alpha') and that the sequence of these subunits is largely conserved between the bovine and the human.     

Cloning, sequence, and developmental expression of a type 5, tartrate-resistant, acid phosphatase of rat bone.

Tartrate-resistant acid phosphatase (TRAP) is a characteristic constituent of osteoclasts and some mononuclear preosteoclasts and, therefore, used as a histochemical and biochemical marker for osteoclasts and bone resorption. We now report the isolation of a 1397-base pair (bp) full-length TRAP/tartrate-resistant acid ATPase (TrATPase) cDNA clone from a neonatal rat calvaria lambda gt11 cDNA library. The cDNA clone consists of a 92-bp untranslated 5'-flank, an open reading frame of 981 bp and a 324-bp untranslated 3'-poly(A)-containing region. The deduced protein sequence of 327 amino acids contains a putative cleavable signal sequence of 21 amino acids. The mature polypeptide of 306 amino acids has a calculated Mr of 34,350 Da and a pI of 9.18, and it contains two potential N-glycosylation sites and the lysosomal targeting sequence DKRFQ. At the protein level, the sequence displays 89-94% homology to TRAP enzymes from human placenta, beef spleen, and uteroferrin and identity to the N terminus of purified rat bone TRAP/TrATPase. An N-terminal amino acid segment is strikingly homologous to the corresponding region in lysosomal and prostatic acid phosphatases. The cDNA recognized a 1.5-kilobase mRNA in long bones and calvaria, and in vitro translation using, as template, mRNA transcribed from the full-length insert yielded an immunoprecipitated product of 34 kDa. In neonatal rats, TRAP/TrATPase mRNA was highly expressed in skeletal tissues, with much lower (less than 10%) levels detected in spleen, thymus, liver, skin, brain, kidney, brain, lung, and heart. In situ hybridization demonstrated specific labeling of osteoclasts at endostal surfaces and bone trabeculae of long bones. Thus, despite the apparent similarity of this osteoclastic TRAP/TrATPase with type 5, tartrate-resistant and purple, acid phosphatases expressed in other mammalian tissues, this gene appears to be preferentially expressed at skeletal sites.     

Cloning, expression, and catalytic mechanism of the low molecular weight phosphotyrosyl protein phosphatase from bovine heart.

The first representative of a group of mammalian, low molecular weight phosphotyrosyl protein phosphatases was cloned, sequenced and expressed in Escherichia coli. Using a 61-mer oligonucleotide probe based on the amino acid sequence of the purified enzyme, several overlapping cDNA clones were isolated from a bovine heart cDNA library. A full-length clone was obtained consisting of a 27-bp 5' noncoding region, an open reading frame encoding the expected 157 amino acid protein, and an extensive 3' nontranslated sequence. The identification of the clone as full length was consistent with results obtained in mRNA blotting experiments using poly(A)+ mRNA from bovine heart. The coding sequence was placed downstream of a bacteriophage T7 promoter, and protein was expressed in E. coli. The expressed enzyme was soluble, and catalytically active and was readily isolated and purified. The recombinant protein had the expected Mr of 18,000 (estimated by SDS-PAGE), and it showed cross-reactivity with antisera that had been raised against both the bovine heart and the human placenta enzymes. The amino acid sequence of the N-terminal region of the expressed protein showed that methionine had been removed, resulting in a sequence identical to that of the enzyme isolated from the bovine tissue, with the exception that the N-terminal alanine of the protein from tissue is acetylated. A kinetically competent phosphoenzyme intermediate was trapped from a phosphatase-catalyzed reaction. Using 31P NMR, the covalent intermediate was identified as a cysteinyl phosphate. By analogy with the nomenclature used for serine esterases, these enzymes may be called cysteine phosphatases.     

Isolation and characterization of a cDNA clone encoding the anti-viral protein from Phytolacca americana

Phytolacca anti-viral protein (PAP) was purified from Phytolacca leaves and the N-terminal was sequenced. A cDNA library was made from mRNAs isolated from Phytolacca leaves and cDNA clones for PAP were identified using oligonucleotide probes derived from the N-terminal amino acid sequence. The PAP-cDNA clone was sequenced from both directions. The predicted amino acid sequence of PAP was compared with the amino acid sequences of other ribosome-inactivating proteins. The identities of these proteins to PAP ranged from 29 to 38%, and a region was found in each with a sequence similar to the PAP sequence (AIQMVSEAARFKYI). Southern blot analysis indicates that PAP is encoded by a multi-gene family.Abbreviations MAP Mirabilis jalapa anti-viral protein - PAP Phytolacca anti-viral protein - SO6 30 kDa ribosome-inactivating protein from the seeds of Saponaria officinalis     

Isolation and sequence determination of cDNA clones for porcine and human lipoamide dehydrogenase. Homology to other disulfide oxidoreductases

A 2.3-kilobase cDNA clone encoding lipoamide dehydrogenase was isolated from a porcine adrenal medulla library in the vector pCD by screening with four synthetic oligonucleotide probes corresponding to amino acid sequence from tryptic peptides of porcine lipoamide dehydrogenase. A 450-bp fragment of the porcine cDNA was used to screen a human small cell lambda gt10 library at reduced stringency. Overlapping human cDNA clones of various lengths were isolated, the largest of which was again 2.3 kilobases in length. Sequencing of both porcine and human cDNAs revealed a short 5'-untranslated region followed by 1530-bp of coding region and 700 bp of 3'-untranslated region preceding a poly(A) tail. The porcine cDNA displayed coding regions corresponding to the known tryptic peptides and a 35-amino acid leader sequence involved in targeting of the protein to the mitochondria. The human lipoamide dehydrogenase cDNA is 96% identical to the porcine at the amino acid level. Alignment of the deduced amino acid sequence of human lipoamide dehydrogenase with human erythrocyte glutathione reductase and mercuric reductase from Tn501 revealed extensive homologies throughout the primary sequence, suggesting that secondary and tertiary structure is also similar among these three enzymes.     

Bacillus licheniformis APase I gene promoter: a strong well-regulated promoter in B. subtilis

The 5' regulatory region and the portion of the structural gene coding for the amino-terminal sequence of alkaline phosphatase I (APase I) were isolated from Bacillus licheniformis MC14 using a synthetic oligodeoxynucleotide deduced from the amino acid sequence of the enzyme. The DNA sequence analysis of this region revealed an open reading frame of 129 amino acids containing the amino-terminal sequence of the mature APase protein. The protein sequence was preceded by a putative signal sequence of 32 amino acid residues. The predicted amino acid sequence of the partial APase clone as well as the experimentally determined amino acid sequence of the enzyme indicated that B. licheniformis APase retains the important features conserved among other APases of Bacillus subtilis, Escherichia coli, Saccharomyces cerevisiae, and various human tissues. Heterologous expression studies of the promoter using a fusion with the lacZ gene indicated that it functions as a very strong inducible promoter in B. subtilis that is tightly regulated by phosphate concentration.     

Amino acid sequence of human histidine-rich glycoprotein derived from the nucleotide sequence of its cDNA

A lambda gt 11 library containing cDNA inserts prepared from human liver mRNA has been screened with an affinity-purified antibody to human histidine-rich glycoprotein (HRG) and then with a restriction fragment isolated from the 5' end of the largest cDNA insert obtained by antibody screening. A number of positive clones were identified and shown to code for HRG by DNA sequence analysis. A total of 2067 nucleotides were determined by sequencing 3 overlapping cDNA clones, which included 121 nucleotides of 5'-noncoding sequence, 54 nucleotides coding for a leader sequence of 18 amino acids, 1521 nucleotides coding for the mature protein of 507 amino acids, a stop codon of TAA, and 352 nucleotides of 3'-noncoding sequence followed by a poly(A) tail of 16 nucleotides. The length of the noncoding sequence of the 3' end differed in several clones, but each contained a polyadenylylation or processing sequence of AATAAA followed by a poly(A) tail. More than half of the amino acid sequence of HRG consisted of five different types of internal repeats. Within the last 3 internal repeats (type V), there were 12 tandem repetitions of a 5 amino acid segment with a consensus sequence of Gly-His-His-Pro-His. This repeated portion, referred to as a "histidine-rich region", contained 53% histidine and showed a high degree of similarity to a histidine-rich region of high molecular weight kininogen.