Dentonin, a fragment of MEPE, enhanced dental pulp stem cell proliferation.

Matrix extracellular phosphoglycoprotein (MEPE) is a SIBLING protein, found in bone and dental tissues. The purpose of this study was to determine whether a 23-amino-acid peptide derived from MEPE (Dentonin or AC-100) could stimulate dental pulp stem cell (DPSC) proliferation and/or differentiation. DPSCs were isolated from erupted human molars, and the mitogenic potential of Dentonin in DPSCs was measured by BrdU immunoassay and cell-cycle gene SuperArray. Differentiation of DPSCs with Dentonin was characterized by Western blot and by osteogenesis gene SuperArray. Dentonin enhanced DPSC proliferation by down-regulating P16, accompanied by up-regulation of ubiquitin protein ligase E3A and human ubiquitin-related protein SUMO-1. Enhanced cell proliferation required intact RGD and SGDG motifs in the peptide. This study shows that Dentonin can promote DPSC proliferation, with a potential role in pulp repair. Further studies are required to determine the usefulness of this material in vivo.

Determination of the primary structures of human skin chymase and cathepsin G from cutaneous mast cells of urticaria pigmentosa lesions.

This study establishes the primary structure of human skin chymase and provides further evidence for the presence of a cathepsin G-like proteinase within human mast cells. The amino acid sequence of human skin chymase was established by protein methods and by analysis of PCR amplification products obtained with cDNA-derived from urticaria pigmentosa (UP) lesions. UP is a disease characterized by skin lesions containing high numbers of mast cells. Proteolytic digests of human chymase purified from normal skin yielded 10 resolvable peptides that were sequenced by automated Edman degradation. The amino acid sequences for these peptides combined with the sequence obtained for the protein's NH2-terminal region (35 residues) accounted for 137 residues of the human skin chymase sequence. This partial amino acid sequence corresponded to the sequence of human heart chymase, a proteinase isolated from heart tissue with immunologic and hydrolytic properties similar to skin chymase. PCR amplification of UP-derived cDNA with primers based on the cDNA structure of heart chymase demonstrated a single amplification product of expected size which was subcloned and sequenced. The amino acid sequence (135 residues) deduced from this product was identical to that of heart chymase in the region between the primers. This sequence, along with that established for the purified protein, constituted 99% of the heart chymase primary structure, strongly indicating that human skin and heart chymases have identical primary structures. Amplification of the same UP-cDNA with primers coding for the NH2- and COOH-terminal sequences of human neutrophil cathepsin G also produced a specific amplification product which was sequenced. The deduced amino acid sequence between the primers was identical to that reported for neutrophil cathepsin G, indicating that the protein of cutaneous mast cells previously shown to be immunologically cross-reactive with neutrophil cathepsin G has a comparable amino acid sequence. UP-cDNA demonstrating amplification products for cathepsin G did not demonstrate amplification products for human neutrophil elastase, suggesting that the cathepsin G PCR amplification product was not derived from neutrophils or monocytes possibly contaminating the lesion. These studies provide further evidence that human skin mast cells contain two different chymotrypsin-like proteinases.

Laminin-binding protein 120 from brain is closely related to the dystrophin-associated glycoprotein, dystroglycan, and binds with high affinity to the major heparin binding domain of laminin.

When brain proteins separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose are probe with 125I-labeled laminin, a single broad band of approximately 120 kDa binds laminin specifically. We show here by two-dimensional electrophoresis and protein microsequencing that this band consists of two distinct laminin-binding proteins. One of these is the amyloid precursor protein. The other, laminin-binding protein (LBP) 120, is closely related to the dystrophin-associated glycoprotein, dystroglycan (156 kDa); 5 peptides from purified bovine brain LBP120, ranging in size from 7 to 19 residues, are up to 100% identical to the predicted amino acid sequence of muscle dystroglycan (ibraghimov-Beskrovanaya, O., Ervasti, J. M., Leveille, C. J., Slaughter, C. A., Sernett, S. W., and Campbell, K. P. (1992) Nature 355, 696-702). These protein microsequence data support the data of Ibraghimov-Beskrovnaya et al., which suggest that the dystroglycan precursor is processed into 120/156- and 43-kDa proteins. Moreover, the data suggest a revision in the position of the proposed cleavage site of the precursor. The glycosylation and extracellular localization of LBP120/dystroglycan are consistent with it being a cell surface laminin receptor. LBP120/dystroglycan, either as a native protein, or following SDS-PAGE and transfer to nitrocellulose, binds with high affinity (Kd = 90 nM) to a proteolytic fragment of laminin (E3) containing the major heparin binding domain. This binding is Ca(2+)-dependent and inhibited by low concentrations of heparin. Thus, LBP120/dystroglycan is a major non-integrin laminin receptor whose high affinity interaction with laminin may reflect a structural role in brain and muscle.

The protease inhibitory properties of the Alzheimer's beta-amyloid precursor protein.

We have expressed the 57-amino acid Kunitz domain of the Alzheimer's beta-amyloid precursor protein (APP751) as a bacterial fusion protein. The protease inhibitory properties of the purified fusion protein, BX9, were virtually identical in all respects tested to those of purified secreted APP751. Both proteins strongly inhibited pancreatic trypsin (Kis = 0.2 and 0.3 nM) and less well epidermal growth factor-binding protein (Kis = 1 and 3.5 nM), alpha-chymotrypsin (Kis = 3 and 6 nM), and the gamma-subunit of nerve growth factor (Kis = 8 and 9 M). Neither protein appreciably inhibited plasma and pancreatic kallikreins, thrombin, lung tryptase, neutrophil elastase, or cathepsin G. The remarkable similarity of the protease inhibitory profile of BX9 to that of secreted APP751 suggests that proper intramolecular disulfide bond formation has occurred in the bacterial fusion protein and leads to the conclusion that the amyloid precursor protein Kunitz domain is a relatively specific inhibitor of only a few trypsin-like arginine esterases.

Cleavage of amyloid beta peptide during constitutive processing of its precursor.

The amyloid beta peptide (A beta P) is a small fragment of the much larger, broadly distributed amyloid precursor protein (APP). Abundant A beta P deposition in the brains of patients with Alzheimer's disease suggests that altered APP processing may represent a key pathogenic event. Direct protein structural analyses showed that constitutive processing in human embryonic kidney 293 cells cleaves APP in the interior of the A beta P, thus preventing A beta P deposition. A deficiency of this processing event may ultimately prove to be the etiological event in Alzheimer's disease that gives rise to senile plaque formation.

Purification and characterization of a trophic factor for embryonic peripheral neurons: comparison with fibroblast growth factors.

The validation of NGF as a physiologically important neurotrophic factor has led to intense efforts to identify novel polypeptide growth factors for neurons. We report here the details of a greater than 80,000-fold purification of a neurotrophic molecule, referred to as growth-promoting activity (GPA), from chicken sciatic nerves. The final product of the purification migrated as a protein band of 21.5 kd, its apparent pI was approximately 4.8, and the ED50 of the most active preparation was approximately 10 pg/ml. Amino acid sequence of a proteolytic digestion fragment of GPA revealed homology with the recently published sequences for rabbit and rat sciatic nerve CNTF. Thus this molecule may be the chicken form of CNTF. Analysis of the specificity of action of GPA showed that, in addition to E8 ciliary ganglion neurons, the factor was able to support short-term survival of E8 dorsal root ganglion and E12 sympathetic neurons. This range of specificities of biological action was also seen with both acidic and basic FGF in the presence of heparin. The biological activity of GPA differed from that of FGF in that it was not potentiated by heparin and did not stimulate mitogenesis in chick fibroblasts.

Detection of methylated asparagine and glutamine residues in polypeptides.

A residue of gamma-N-methylasparagine (gamma-NMA) is found at position beta-72 of many phycobiliproteins. delta-N-Methylglutamine is present in some bacterial ribosomal proteins. gamma-NMA was synthesized by reacting the omega-methyl ester of aspartate with methylamine and delta-N-methylglutamine by reaction of pyroglutamate with methylamine. These derivatives and the omega-methyl esters of aspartate and glutamate were characterized by melting point, by thin-layer chromatography, by amino acid analysis, by NMR spectroscopy, and after conversion to the phenylthiohydantoin (PTH) derivative. The gamma-NMA residues in peptides from allophycocyanin, C-phycocyanin, and B-phycoerythrin were stable under the conditions of automated sequential gas-liquid phase Edman degradation. On HPLC, PTH-gamma-NMA co-eluted with PTH-serine and was accompanied by a minor component eluting just prior to dimethylphenylthiourea. Similar results were obtained on manual derivatization of synthetic gamma-NMA to prepare the PTH derivative. The PTH-delta-N-methylglutamine standard eluted near the position of dimethylphenylthiourea under the usual conditions employed for the identification of PTH-amino acid derivatives in automated protein sequencing.

The secreted form of the Alzheimer's amyloid precursor protein with the Kunitz domain is protease nexin-II.

The A4 protein (or beta-protein) is a 42- or 43-amino-acid peptide present in the extracellular neuritic plaques in Alzheimer's disease and is derived from a membrane-bound amyloid protein precursor (APP). Three forms of APP have been described and are referred to as APP695, APP751 and APP770, reflecting the number of amino acids encoded for by their respective complementary DNAs. The two larger APPs contain a 57-amino-acid insert with striking homology to the Kunitz family of protease inhibitors. Here we report that the deduced amino-terminal sequence of APP is identical to the sequence of a cell-secreted protease inhibitor, protease nexin-II (PN-II). To confirm this finding, APP751 and APP695 cDNAs were over-expressed in the human 293 cell line, and the secreted N-terminal extracellular domains of these APPs were purified to near homogeneity from the tissue-culture medium. The relative molecular mass and high-affinity binding to dextran sulphate of secreted APP751 were consistent with that of PN-II. Functionally, secreted APP751 formed stable, non-covalent, inhibitory complexes with trypsin. Secreted APP695 did not form complexes with trypsin. We conclude that the secreted form of APP with the Kunitz protease inhibitor domain is PN-II.

Identification of a neuropeptide hormone that regulates sex pheromone production in female moths.

A pheromone biosynthesis activating neuropeptide (PBAN) hormone that controls sex pheromone production in female moths was identified from the brain-subesophageal ganglion complexes of the adult corn earworm, Heliothis zea. PBAN has 33 amino acid residues and a molecular weight of 3900. Its amino acid sequence has no significant homology with any of the fully characterized peptide hormones. The synthetic peptide, at a dose of between 2 and 4 picomoles, induced production of a normal quantity of sex pheromone in ligated H. zea females. The peptide also induced pheromone production in six other species of moths, thus indicating that this or similar peptides may be responsible for the regulation of pheromone production in moths.

Amino acid sequence of the serine active-site region of the medium-chain S-acyl fatty acid synthetase thioester hydrolase from rat mammary gland.

Medium-chain S-acyl fatty acid synthetase thioester hydrolase (thioesterase II), a discrete monomeric enzyme of 29 kDa, regulates the product specificity of the de novo lipogenic systems in certain specialized mammalian and avian tissues, such as mammary and uropygial glands. The amino acid sequence of a 57-residue region containing the active site of the rat mammary gland enzyme has been established by a combination of amino acid and cDNA sequencing. Thioesterase II was radiolabeled with the serine esterase inhibitor [1,3-14C]diisopropyl-fluorophosphate and digested sequentially with cyanogen bromide, Staphylococcus aureus V8 protease and trypsin. A radiolabeled tryptic peptide was isolated and sequenced by automated Edman degradation and the location of the active-site residue established. The amino acid sequence was confirmed by sequencing an overlapping, unlabeled peptide, obtained by V8 digestion of the whole enzyme, and by dideoxynucleotide sequencing of a thioesterase II cDNA clone isolated from a lambda gt11 expression library. The active center contains the motif Gly-Xaa-Ser-Xaa-Gly, characteristic of the serine esterase family of enzymes. A seven-residue region around the essential serine of the rat mammary thioesterase II, Phe-Gly-Met-Ser-Phe-Gly-Ser, is completely homologous with a region of the mallard uropygial thioesterase, recently analyzed by cDNA sequencing, indicating that this is likely to be the active site of the avian enzyme. Overall homology between the mammalian and avian enzymes for the 57-amino-acid residue region is 47% and suggests that the two enzymes may share a common evolutionary origin.

Chemical synthesis and expression in yeast of a gene encoding connective tissue activating peptide-III. A novel approach for the facile assembly of a gene encoding a human platelet-derived mitogen.

A synthetic gene encoding the platelet-derived factor, connective tissue activating peptide-III (CTAP-III) (Castor, C.W., Miller, J.W., and Walz, D.A. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 765-769), has been expressed and secreted from Saccharomyces cerevisiae by using a yeast expression vector and an alpha-factor leader segment. Mitogenic activity reported for naturally derived CTAP-III has been demonstrated here for recombinant CTAP-III. Active CTAP-III has been purified to apparent homogeneity. Structural studies have confirmed its identity. A general approach for the facile synthesis of genes is presented which has permitted the assembly of the entire structural gene and flanking regions (280 base pairs) from 20 oligomers in a single annealing and ligation reaction pool.

Sequence of protein disulphide isomerase and implications of its relationship to thioredoxin.

The formation of disulphide bonds is essential to the structure and function of proteins. These bonds rapidly form either cotranslationally or immediately post-translationally in the lumen of the endoplasmic reticulum. Native disulphide pairing for such proteins has been achieved in vitro; however, the rates of reassembly are slow and the conditions non-physiological. To account for these observations, Anfinsen et al. proposed that a 'disulphide interchange protein' was the in vivo catalyst of disulphide bond rearrangement. Other groups discovered an activity with similar characteristics that catalysed the reductive cleavage of insulin and may be associated with insulin degradation, although this result has been disputed. The enzyme involved, protein disulphide isomerase (PDI; EC 5.3.4.1), may be the in vivo catalyst of disulphide bond formation. Here we describe the sequence of cloned rat liver PDI complementary DNA which predicts a protein with two distinct regions homologous with Escherichia coli thioredoxin, a known cofactor in oxidation-reduction reactions. Each of these regions contains the presumed active site sequence Trp-Cys-Gly-His-Cys-Lys, suggesting that PDI, similar in action to thioredoxin, catalyses disulphide bond interchange via an internal disulphide-sulphydryl interchange. The cDNA predicts a signal peptide consistent with the view that PDI is a luminal endoplasmic reticulum protein. PDI messenger RNA, although ubiquitous, is more highly concentrated in secretory cells.

Cloning and sequencing of the beta-lactamase I gene of Bacillus cereus 5/B and its expression in Bacillus subtilis.

The beta-lactamases of Bacillus cereus have attracted interest because they are secreted efficiently, because multiple enzymes are frequently present, and because their regulation has unusual features. beta-Lactamase I of strain 5/B is produced constitutively at a high level, and the exoenzyme appears to be several thousand daltons larger than the corresponding product of strain 569/H. We have cloned the gene for 5/B beta-lactamase I in Escherichia coli and B. subtilis and have sequenced the structural portion and the regulatory regions. The 5/B enzyme is produced at a low level in E. coli RR1(pRWY200) and remains cellbound. In B. subtilis it is formed in large amounts, and over 90% of it is released into the medium. There is a large degree of homology between the promoter and leader peptide regions of the 5/B and 569/H genes; both utilize UUG as the translation initiation codon (P. S. F. Mézes, R. W. Blacher, and J. O. Lampen, (J. Biol. Chem. 260:1218-1223, 1985). Although there are significant differences in the peptide segment where processing would be expected to occur, the NH2 terminus of the major 5/B product from B. subtilis BD170(pRWY215) is His-44, which is the same as the NH2 terminus of the major 569/H product from B. subtilis BD170(pRWM5).

Processing of Bacillus cereus 569/H beta-lactamase I in Escherichia coli and Bacillus subtilis.

The gene for Bacillus cereus 569/H beta-lactamase I, penPC, has recently been cloned and sequenced (Mézes, P. S. F., Yang, Y. Q., Hussain, M., and Lampen, J. O. (1983) FEBS Lett. 161, 195-200). A typical prokaryotic signal peptide but with no lipoprotein modification site, as present in the Bacillus licheniformis 749/C beta-lactamase, was indicated by the DNA sequence for this secretory protein. We have here purified the beta-lactamase I products found in Escherichia coli and Bacillus subtilis carrying penPC and have determined the first 20 NH2-terminal amino acids of each of the forms. Processing of the beta-lactamase I in E. coli occurs at a single site which is characteristic for cleavage by a signal peptidase. B. subtilis secreted two distinct products to the culture medium which were both smaller than the single product formed in E. coli. Sequencing of [35S]Met-labeled pre-beta-lactamase I from phenylethyl alcohol-treated cells of B. cereus 569/H indicated that UUG is being utilized as the initiation codon for penPC. The same result was obtained for the pre-beta-lactamase I from similarly treated cells of the closely related B. cereus 5/B strain.