Human transforming growth factor type beta 2: production by a prostatic adenocarcinoma cell line, purification, and initial characterization

Human type beta 2 transforming growth factor (hTGF-beta 2) was purified from tamoxifen-supplemented, serum-free medium conditioned by the human prostatic adenocarcinoma cell line PC-3. The purification of hTGF-beta 2 was monitored in a growth inhibition assay and was achieved by batch purification on methylsilyl-controlled pore glass, followed by gel permeation chromatography and reversed-phase high-performance liquid chromatography. The overall recovery of hTGF-beta 2 was 75% of the initial activity and yielded 22 micrograms of hTGF-beta 2/L of conditioned medium. The concentration of hTGF-beta 2 required for half-maximal inhibition of Mv 1 Lu mink lung epithelial cells (CCl-64) was approximately 5 pM when assayed in the presence of 10% fetal bovine serum. The purified hTGF-beta 2 has a molecular weight of 24,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and consists of two disulfide-linked, apparently identical polypeptide chains, with a molecular weight of 13,000. The amino-terminal sequence of hTGF-beta 2 was determined. Alignment of the amino acid sequences of hTGF-beta 2 and hTGF-beta reveals statistically significant sequence homology. On the basis of the extensive amino acid sequence homology, we propose the term TGF-beta 2 for this newly isolated polypeptide. The reported results suggest that TGF-beta (TGF-beta 1) and TGF-beta 2 may have evolved from a common progenitor.     

The induction of endochondral bone formation by transforming growth factor-beta(3): experimental studies in the non-human primate Papio ursinus.

Transforming growth factor-beta(3) (TGF-beta(3)), a multi-functional growth modulator of embryonic development, tissue repair and morphogenesis, immunoregulation, fibrosis, angiogenesis and carcinogenesis, is the third mammalian isoform of the TGF-beta subfamily of proteins. The pleiotropism of the signalling proteins of the TGF-beta superfamily, including the TGF-beta proteins per se, are highlighted by the apparent redundancy of soluble molecular signals initiating de novo endochondral bone induction in the primate only. In the heterotopic bioassay for bone induction in the subcutaneous site of rodents, the TGF-beta(3) isoform does not initiate endochondral bone formation. Strikingly and in marked contrast to the rodent bioassay, recombinant human (h)TGF-beta(3), when implanted in the rectus abdominis muscle of adult non-human primates Papio ursinus at doses of 5, 25 and 125 mug per 100 mg of insoluble collagenous matrix as carrier, induces rapid endochondral bone formation resulting in large corticalized ossicles by day 30 and 90. In the same animals, the delivery of identical or higher doses of theTGF-beta(3) protein results in minimal repair of calvarial defects on day 30 with limited bone regeneration across the pericranial aspect of the defects on day 90. Partial restoration of the bone induction cascade by the hTGF-beta(3) protein is obtained by mixing the hTGF-beta(3) device with minced fragments of autogenous rectus abdominis muscle thus adding responding stem cells for further bone induction by the hTGF-beta(3) protein. The observed limited bone induction in hTGF-beta(3)/treated and untreated calvarial defects in Papio ursinus and therefore by extension to Homo sapiens, is due to the influence of Smad-6 and Smad-7 down-stream antagonists of the TGF-beta signalling pathway. RT-PCR, Western and Northern blot analyses of tissue specimens generated by the TGF-beta(3) isoform demonstrate robust expression of Smad-6 and Smad-7 in orthotopic calvarial sites with limited expression in heterotopic rectus abdominis sites. Smad-6 and -7 overexpression in hTGF-beta(3)/treated and untreated calvarial defects may be due to the vascular endothelial tissue of the arachnoids expressing signalling proteins modulating the expression of the inhibitory Smads in pre-osteoblastic and osteoblastic calvarial cell lines controlling the induction of bone in the primate calvarium.     

Restoration of transforming growth factor-beta type II receptor reduces tumorigenicity in the human adrenocortical carcinoma SW-13 cell line.

Transforming growth factor-beta (TGF-beta) is a potent growth suppressor. Acquisition of TGF-beta resistance has been reported in many tumors, and has been associated with reduced TGF-beta receptor expression. In this study, we examined TGF-beta 1, TGF-beta type I receptor (TbetaRI) and TGF-beta type II receptor (TbetaRII) expression in SW-13 adrenocortical carcinoma cells by Northern and Western blot analysis. SW-13 cells did not express TbetaRII mRNA or protein. We have investigated the role of TbetaRII in modulating tumorigenic potential using stably transfected SW-13 cells with TbetaRII expression plasmid. TbetaRII-positive SW-13 cell growth was inhibited by exogenous human TGF-beta1 (hTGF-beta1) in a dose-dependent manner. In contrast, SW-13 cells and control clones transfected with empty vector remained hTGF-beta1-insensitive. Xenograft examination in athymic nude mice demonstrated that TbetaRII-positive SW-13 cells reduced tumor-forming activity. Reconstructing the TbetaRII can lead to reversion of the malignant phenotype of TbetaRII-negative human adrenocortical carcinoma, which contains SW-13 cells. Reduced TbetaRII expression may play a critical role in determining the malignant phenotype of human adrenocortical carcinoma.     

Human adenine phosphoribosyltransferase. Complete amino acid sequence of the erythrocyte enzyme

We defined the amino acid sequence of adenine phosphoribosyltransferase isolated from human erythrocytes. Peptide fragments formed by cleavage at arginine, lysine, glutamic acid, and methionine were purified by high pressure liquid chromatography and sequenced by manual Edman degradation. The complete primary structure of human adenine phosphoribosyltransferase was established by sequence analysis of 19 peptide fragments. Presumed homology between the human and rodent enzymes was used to order fragments that had inadequate overlapping sequences. The enzyme has 179 residues with a calculated subunit molecular weight of 19,481. Mass spectrometry indicated that the NH2-terminal residue is acetylated. Human adenine phosphoribosyltransferase has sequence homology with xanthine-guanine phosphoribosyltransferase from Escherichia coli in 110-amino acid region encompassing the NH2-terminal section of the enzyme.     

Complementary DNA for human glioblastoma-derived T cell suppressor factor, a novel member of the transforming growth factor-beta gene family.

Human glioblastoma cells secrete a peptide, termed glioblastoma-derived T cell suppressor factor (G-TsF), which has suppressive effects on interleukin-2-dependent T cell growth. As shown here, complementary DNA for G-TsF reveals that G-TsF shares 71% amino acid homology with transforming growth factor-beta (TGF-beta). In analogy to TGF-beta it is apparently synthesized as the carboxy-terminal end of a precursor polypeptide which undergoes proteolytic cleavage to yield the 112 amino-acid-long mature form of G-TsF. Comparison of the amino-terminal sequence of G-TsF with that of porcine TGF-beta 2 and bovine cartilage-inducing factor B shows complete homology, which indicates that we have cloned the human analogue of these factors. It is tempting to consider a role for G-TsF in tumor growth where it may enhance tumor cell proliferation in an autocrine way and/or reduce immunosurveillance of tumor development.     

Human uncoupling protein gene: structure, comparison with rat gene, and assignment to the long arm of chromosome 4

The uncoupling protein (UCP) gene encodes a unique mammalian mitochondrial proton carrier that induces heat production in brown adipocytes. Human UCP gene was isolated and its organization analyzed. A comparison was made with rat UCP gene. Human UCP gene spans 13 Kb and contains a transcribed region that covers 9 Kb of the human genome. All of the exons were also sequenced except the extreme end of the 3' untranslated region. Two Kb DNA upstream the TATA box were also sequenced. This region contains several fragments that are highly homologous to the gene of rat UCP. Neither CCAAT sequence nor Sp 1 binding motif were detected. Human UCP gene is split into six exons. The complete amino acid sequence of the protein was determined. Human UCP has 305 amino acids and a molecular weight of 32,786. It has no N-terminal targeting sequence. It is 79% homologous to rat UCP both at nucleotidic and amino acid levels. The primary structure of UCP is significantly homologous to the primary structure of the human T1 ADP/ATP carrier, particularly in the C-terminal extremity, which is supposed to contain a nucleotide-binding site in both proteins. Human UCP gene is single type, as it is in rodents. Two genomic fragments were used to detect a 1.9 Kb mRNA in human perirenal brown adipose tissue. Using in situ hybridization, UCP gene was assigned in humans to chromosome 4 in q31. Interestingly, the T1 gene encoding the heart-skeletal muscle ADP/ATP carrier has recently been shown to be on the same chromosome (Li et al. Biol Chem 264:13998, 1989).     

Amino acid sequences of the human kidney cathepsins H and L

The complete amino acid sequences of human kidney cathepsin H (EC 3.4.22.16) and human kidney cathepsin L (EC 3.4.22.15) were determined. Cathepsin H contains 230 residues and has an Mr of 25116. The sequence was obtained by sequencing the light, heavy and mini chain and the peptides produced by cyanogen bromide cleavage of the single-chain form of the enzyme. The glycosylated mini chain is a proteolytic fragment of the propeptide of cathepsin H. Human cathepsin L has 217 amino acid residues and an Mr of 23720. Its amino acid sequence was deduced from N-terminal sequences of the heavy and light chains and from the sequences of cyanogen bromide fragments of the heavy chain. The fragments were aligned by comparison with known sequences of cathepsins H and L from other species. Cathepsins H and L exhibit a high degree of sequence homology to cathepsin B (EC 3.4.22.1) and other cysteine proteinases of the papain superfamily.     

Molecular cloning and characterization of the human and porcine transforming growth factor-beta type III receptors.

Full-length cDNAs for the transforming growth factor-beta (TGF-beta) type III receptors were isolated from porcine uterus and human placenta cDNA libraries. The human TGF-beta type III receptor coding region encodes a protein of 849 amino acids with a single transmembrane domain and a short stretch of the intracellular domain. Potential glycosaminoglycan attachment sites were found in the extracellular domain. The overall amino acid sequence identities with those of the porcine and rat TGF-beta type III receptors were 83% and 81%, respectively. A high degree of sequence conservation was observed in the transmembrane and intracellular domains, which also have sequence similarity with human endoglin. In addition, two portions with 29 and 52 amino acids in the extracellular domain were found to be substantially similar with human endoglin.     

Characterization of Human Brain S100 Protein Fraction: Amino Acid Sequence of S100β

Two major components of human brain S100 fraction were purified by HPLC and an amino acid sequence was elucidated for the S100 beta component. Human S100 proteins showed absorption spectra and amino acid compositions similar to S100 alpha and S100 beta from bovine brain. However, the relative amounts of the human proteins were 4% S100 alpha and 96% S100 beta by weight, while the bovine protein distribution was 47% S100 alpha and 53% S100 beta by weight. An amino acid sequence of human S100 beta was established by analysis of overlapping fragments generated by cyanogen bromide and trypsin cleavage. Three amino acid sequence differences between the human and bovine S100 beta were found at residues 7, 62, and 80. These differences were chemically conservative and compatible with minimum single base changes in the codon structures. These results document that S100 beta is a conserved protein among mammals and provide the necessary foundation for current clinical studies.     

Characterization of cDNA clones encoding rabbit and human serum paraoxonase: the mature protein retains its signal sequence.

Serum paraoxonase hydrolyzes the toxic metabolites of a variety of organophosphorus insecticides. High serum paraoxonase levels appear to protect against the neurotoxic effects of organophosphorus substrates of this enzyme [Costa et al. (1990) Toxicol. Appl. Pharmacol. 103, 66-76]. The amino acid sequence accounting for 42% of rabbit paraoxonase was determined by (1) gas-phase sequencing of the intact protein and (2) peptide fragments from lysine and arginine digests. From these data, two oligonucleotide probes were synthesized and used to screen a rabbit liver cDNA library. A clone was isolated and sequenced, and contained a 1294-bp insert encoding an open reading frame of 359 amino acids. Northern blot hybridization with RNA isolated from various rabbit tissues indicated that paraoxonase mRNA is synthesized predominately, if not exclusively, in the liver. Southern blot experiments suggested that rabbit paraoxonase is coded by a single gene and is not a family member of closely related genes. Human paraoxonase clones were isolated from a liver cDNA library by using the rabbit cDNA as a hybridization probe. Inserts from three of the longest clones were sequenced, and one full-length clone contained an open reading frame encoding 355 amino acids, four less than the rabbit paraoxonase protein. Each of the human clones appeared to be polyadenylated at a different site, consistent with the absence of the canonical polyadenylation signal sequence. Of potential significance with respect to the paraoxonase polymorphism, the derived amino acid sequence from one of the partial human cDNA clones differed at two positions from the full-length clone. Amino-terminal sequences derived from purified rabbit and human paraoxonase proteins suggested that the signal sequence is retained, with the exception of the initiator methionine residue [Furlong et al. (1991) Biochemistry (preceding paper in this issue)]. Characterization of the rabbit and human paraoxonase cDNA clones confirms that the signal sequences are not processed, except for the N-terminal methionine residue. The rabbit and human cDNA clones demonstrate striking nucleotide and deduced amino acid similarities (greater than 85%), suggesting an important metabolic role and constraints on the evolution of this protein.     

Amino acid sequence of human liver cathepsin B

The complete amino acid sequence of cathepsin B (EC 3.4.22.1) from human liver was determined. The 252-residue sequence was obtained by automated solid-phase Edman degradation of the light and heavy chain resulting from limited proteolysis of the single-chain enzyme and of fragments produced by cyanogen bromide and enzymatic cleavage of the heavy chain. Human liver cathepsin B has 83.7% identical residues with the corresponding enzyme from rat liver. Comparison of both mammalian cathepsin B sequences with the sequence of papain provides further evidence that lysosomal and plant cysteine proteinases have evolved from a common ancestor and share a similar catalytic mechanism.     

Human skeletal-muscle aldolase: N-terminal sequence analysis of CNBr- and o-iodosobenzoic acid-cleavage fragments

Fructose-1,6-bisphosphate aldolase was purified from human skeletal-muscle by affinity elution chromatography. Four CNBr-cleavage fragments were purified by gel filtration, and their N-terminal amino acid sequences were determined. Cleavage with o-iodosobenzoic acid at the three tryptophan residues also yielded fragments suitable for N-terminal sequence analysis. Thus, the sequence of 272 of the 363 residues was established. These sequence results allow many of the discrepancies between the two published rabbit skeletal-muscle aldolase sequences to be resolved. The human aldolase sequence reported here is 96% identical to a "consensus" rabbit aldolase sequence. A comparison with a partial sequence of Drosophila aldolase (103 residues) shows 80% identity. The determination of the amino acid sequence of human aldolase is important for the interpretation of the crystal structure of this enzyme.     

Molecular structure of human protamine P4 (HP4), a minor basic protein of human sperm nuclei

Protamine HP4 is a minor protein which was purified from human sperm nuclei. It was characterized by its amino acid composition, peptide mapping after digestion with highly specific endoproteinases and finally by its amino acid sequence. Protamine HP4 contains high amounts of arginine, cysteine and histidine. The primary structure of the protein was established by sequence analysis of intact protamine and of its fragments. HP4 is a P2-type protamine of 58 residues (Mr 7783) structurally related to human protamines HP2 and HP3 from which it only differs by an amino-terminal extension of one and four residues, respectively. These three protamines exhibit a close structural relationship with mouse protamine mP2. The heterogeneity of protamines in human sperm nuclei is discussed.     

Isolation, by partial pepsin digestion, of the three collagen-like regions present in subcomponent Clq of the first component of human complement.

1. Digestion of human subcomponent C1q with pepsin at pH4.45 for 20h at 37 degrees C fragmented most of the non-collagen-like amino acid sequences in the molecule to small peptides, whereas the entire regions of collagen-like sequence that comprised 38% by weight of the subcomponent C1q were left intact. 2. The collagen-like fraction of the digest was eluted in the void volume of a Sephadex G-200 column, was was showm to be composed of two major fragments when examined by electrophoresis on polyacrylamide gels run in buffers containing sodium dodecyl sulphate. These fragments were separated on CM-cellulose at pH4.9 in buffers containing 7.5M-urea. 3. Human subcomponent C1q on reduction and alkylation yields equimolar amounnts of three chains, which have been designated A, B and C [Reid et al. (1972) Biochem. J. 130, 749-763]. One of the pepsin fragments was shown to be composed of the N-terminal 95 residues of the A chain linked, via residue A4, by a single disulphide bond to a residue in the sequence B2-B6 in the N-terminal 91 residues of the B chain. The second pepsin fragment was shown to be composed of a disulphide-linked dimer of the N-terminal 94 residues of the C chain, the only disulphide bond being located at residue C4.4. The mol. wts. of the unoxidized and oxidized pepsin fragments were estimated from their amino acid compositions to be 20 000 and 18 200 for the A-B and C-C dimers and 11 400, 8800 and 9600 for the collagen-like fragments of the A, B and C chains respectively. Estimation of the molecular weights of the peptic fragments by polyacrylamide-gel electrophoresis run in the presence of sodium dodecyl sulphate gave values that were approx. 50% higher than expected from the amino acid sequence data. This is probably due to the high collagen-like sequence content of these fragments.     

Molecular cloning of a human immunoglobulin lambda chain variable sequence

We have cloned a human V lambda cDNA sequence from an Ig lambda-producing human Burkitt lymphoma cell line (BL2) by taking advantage of a cloned constant region gene as a primer for cDNA synthesis instead of an oligo(dT) primer. The amino acid sequence deduced from the nucleotide sequence of V lambda clones is highly related to that of the NEW V lambda protein of subgroup I. Southern blot hybridization of human DNAs with the V lambda I probe showed at least 12 hybridizing V lambda fragments. These fragments are amplified in K562 cells which derive from a case of chronic myelogenous leukemia and contain an amplified c-abl oncogene and amplified C lambda sequences.     

Identification of a new P450 expressed in human lung: complete cDNA sequence, cDNA-directed expression, and chromosome mapping

A cDNA coding for a P450 expressed in human lung was isolated from a lambda gt11 library constructed from human lung mRNA using a cDNA probe to rat P450 IVA1. The cDNA-deduced amino acid sequence of this P450, designated IVB1, consisted of 511 amino acids and had a calculated molecular weight of 59,558. The IVB1 amino acid sequence bore 51%, 53%, and 52% similarities to rat IVA1, IVA2, and rabbit P450p-2, respectively. Comparison of the primary amino acid sequence of human IVB1 with rat IVA and rabbit p-2 P450 sequences revealed a region of absolute sequence identity of 17 amino acids between residues 304 and 320. However, the functional significance of this conserved sequence is unknown. Human IVB1 also appears to be related to P450 isozyme 5 that has been extensively characterized in rabbits. The IVB1 cDNA was inserted into a vaccinia virus expression vector and the enzyme expressed in human cell lines. The expressed enzyme had an absorption spectrum with a lambda max at 450 nm when reduced and complexed with carbon monoxide, typical of other cytochrome P450s. Unlike rabbit P450 isozyme 5, however, human IVB1 was unable to activate the promutagen 2-aminofluorene. Human lung microsomal P450s were also unable to metabolize this compound despite the presence of IVB1 mRNA in three out of four human lungs analyzed. In contrast to its expression in lung, IVB1 mRNA was undetectable in livers from 14 individuals, including those from which the lungs were derived. IVB1-related mRNA was also expressed in rat lung and was undetectable in untreated rat liver.(ABSTRACT TRUNCATED AT 250 WORDS)     

Latent high molecular weight complex of transforming growth factor beta 1. Purification from human platelets and structural characterization

Human transforming growth factor beta 1 (TGF-beta 1) was purified as a latent high Mr complex from human platelets by a six-step procedure. Analysis by sodium dodecyl sulfate (SDS)-gel electrophoresis under reducing conditions revealed that the complex was composed of at least three components with apparent Mr values of 13,000, 40,000, and 125,000-160,000. The 13-kDa subunit was part of a disulfide-bonded dimer and was identified by amino acid sequencing as TGF-beta 1. The 40-kDa subunit was identified as the amino-terminal part of the TGF-beta 1 precursor lacking the hydrophobic signal sequence. Partial sequencing of the 125-160-kDa protein revealed that it is distinct from known proteins. The 40-kDa and the 125-160-kDa subunits are linked by disulfide bonds, forming a complex with an apparent Mr of 210,000 on SDS gels under nonreducing conditions. Experiments with partial reduction revealed that each complex contains two 40-kDa components linked by disulfide bonds; in addition, the dimer is disulfide-linked to one 125-160-kDa binding protein. TGF-beta 1 binds noncovalently to the 210-kDa complex, and in bound form, TGF-beta 1 is inactive. Incubations of the latent form of TGF-beta 1 at extreme pH values, in 0.02% SDS or in 8 M urea, lead to activation of TGF-beta 1, whereas the complex was resistant to treatment with 5 M NaCl or heat (3 min at 95 degrees C).     

Sequence and localization of human NASP: conservation of a Xenopus histone-binding protein.

In this study the sequence and localization of human testicular NASP (nuclear autoantigenic sperm protein) are reported. NASP cDNA contains 2561 nt encoding a protein of 787 amino acids. The open reading frame contains 2446 nt followed by an ochre stop codon (TAA) and 104 nucleotides of untranslated sequence containing a poly(A) addition signal 10 bases upstream of the poly(A) tail. Northern blot analysis of human testis poly(A) mRNA indicates a message of approximately 3.2 kb. Multiple sequence alignment (MSA) analysis of the encoded human NASP amino acid sequence with the sequence for the Xenopus histone-binding protein N1/N2 and the rabbit NASP amino acid sequence demonstrates that the human sequence and the Xenopus sequence have extensive amino acid homology upstream of the rabbit initiation codon. Significantly, there is an 85% identity between the human and the rabbit NASP sequences when the alignment starts at the N-terminal of the rabbit sequence and at amino acid 101 of the human sequence. The nuclear translocation signal found in N1/N2 and rabbit NASP is completely conserved in human NASP. The first histone-binding domain of Xenopus is 70% identical and 90% similar to the human NASP domain. The second histone-binding domain of Xenopus is 48% identical and 71% similar to the human NASP domain. MSA analysis of the three sequences generated an unrooted ancestral tree with two branches, indicating that fewer amino acid changes have occurred between the Xenopus and the human sequences than between the Xenopus and the rabbit sequences. In the human testis, NASP is localized predominantly in primary spermatocytes and round spermatids. Spermatogonia, Sertoli cells, Leydig cells, peritubular cells, and other somatic cells do not stain. Human spermatozoa contain NASP in the acrosomal region. Following the acrosome reaction, some NASP remains in the equatorial and postacrosomal regions. We propose that mammalian testes and sperm contain a histone-binding protein which may play a role in regulating the early events of spermatogenesis.