Molecular Basis of Salivary Proline-rich Protein and Peptide Synthesis: Cell-free Translations and Processing of Human and Macaque Statherin mRNAs and Partial Amino Acid Sequence of their Signal Peptides

1987 ◽  
Vol 66 (2) ◽  
pp. 462-466 ◽  
Author(s):  
F.G. Oppenheim ◽  
D.I. Hay ◽  
D.J. Smith ◽  
G.D. Offner ◽  
R.F. Troxler
Keyword(s):  
Calcium Phosphate ◽  
Macaca Fascicularis ◽  
Electrophoretic Analysis ◽  
Proteolytic Processing ◽  
Oral Environment ◽  
Reticulocyte Lysate ◽  
Polyprotein Precursor ◽  
Phosphate Salts ◽  
Calcium Phosphate Precipitation

Acidic proline-rich phosphoproteins and phosphopeptides are abundant components of parotid and submandibular salivary secretions in man and in the subhuman primate, Macaca fascicularis. The major acidic proline-rich proteins and the proline-rich phosphopeptide, statherin, of man and macaques have been shown to be potent inhibitors of calcium phosphate precipitation and are thought to function in the oral environment by maintaining saliva supersaturated with respect to calcium phosphate salts. Little is known about the biosynthesis of these proline-rich phosphoproteins and peptides, and the aim of the present work was to determine the structural relationship between statherin precursors and native human and macaque statherin. RNA was isolated from human submandibular gland, and poly(A+) mRNA was selected by affinity chromatography on oligo(dT) cellulose and translated in a reticulocyte lysate. Electrophoretic analysis of the translation products revealed that this mRNA directed the synthesis of a large number of polypeptides with M,s ranging from 5000 to 70,000. Immunoprecipitates, prepared with an antiserum directed against human statherin, contained a single component with a Mr of 7800, approximately 2000 daltons larger than native statherin. Radiosequencing of the in vitro precursor of statherin in immunoprecipitates demonstrated the presence of a 19-residue signal peptide. These results suggest that statherin is derived from a unique structural gene, and does not result from proteolytic processing of a large polyprotein precursor.

scholarly journals Proteolytic processing of Semliki Forest virus-specific non-structural polyprotein by nsP2 protease

2001 ◽  
Vol 82 (4) ◽  
pp. 765-773 ◽  
Author(s):  
Andres Merits ◽  
Lidia Vasiljeva ◽  
Tero Ahola ◽  
Leevi Kääriäinen ◽  
Petri Auvinen
Keyword(s):  
Mammalian Cells ◽  
Active Sites ◽  
Insect Cells ◽  
Semliki Forest Virus ◽  
Point Mutations ◽  
Proteolytic Processing ◽  
Wild Type ◽  
In Trans ◽  
Polyprotein Precursor

The RNA replicase proteins of Semliki Forest virus (SFV) are translated as a P1234 polyprotein precursor that contains two putative autoproteases. Point mutations introduced into the predicted active sites of both proteases nsP2 (P2) and nsP4 (P4), separately or in combination, completely abolished virus replication in mammalian cells. The effects of these mutations on polyprotein processing were studied by in vitro translation and by expression of wild-type polyproteins P1234, P123, P23, P34 and their mutated counterparts in insect cells using recombinant baculoviruses. A mutation in the catalytic site of the P2 protease, C478A, (P2CA) completely abolished the processing of P12CA34, P12CA3 and P2CA3. Co-expression of P23 and P12CA34 in insect cells resulted in in trans cleavages at the P2/3 and P3/4 sites. Co-expression of P23 and P34 resulted in cleavage at the P3/4 site. In contrast, a construct with a mutation in the active site of the putative P4 protease, D6A, (P1234DA) was processed like the wild-type protein. P34 or its truncated forms were not processed when expressed alone. In insect cells, P4 was rapidly destroyed unless an inhibitor of proteosomal degradation was used. It is concluded that P2 is the only protease needed for the processing of SFV polyprotein P1234. Analysis of the cleavage products revealed that P23 or P2 could not cleave the P1/2 site in trans.

Efficient association of in vitro translation products with purified stable Candida tropicalis peroxisomes

1987 ◽  
Vol 7 (5) ◽  
pp. 1848-1855
Author(s):  
G M Small ◽  
T Imanaka ◽  
H Shio ◽  
P B Lazarow
Keyword(s):  
Candida Tropicalis ◽  
Proteolytic Processing ◽  
In Vitro Translation ◽  
Moderate Number ◽  
Brij 35 ◽  
Reticulocyte Lysate ◽  
Molecular Information ◽  
Acyl Coenzyme A

Newly synthesized peroxisomal proteins enter preexisting peroxisomes posttranslationally in vivo, generally without proteolytic processing. An efficient reconstitution of this process in vitro together with cloned DNAs for peroxisomal proteins would make possible investigation of the molecular information that targets proteins to peroxisomes. We have previously reported the isolation of clones for Candida tropicalis peroxisomal proteins; here we describe the association (and possible import) of peroxisomal proteins with peroxisomes in vitro. C. tropicalis was grown in a medium containing Brij 35, resulting in the induction of a moderate number of medium-sized peroxisomes. These peroxisomes, isolated in a sucrose gradient, had a catalase latency of 54% and were sufficiently stable to be concentrated and used in an import assay. The reticulocyte lysate translation products of total RNA from oleate-grown cells were incubated with the peroxisomes at 26 degrees C in the presence of 50 mM KCl, protease inhibitors, 0.5 M sucrose, 2.5 mM MOPS (morpholinepropanesulfonic acid) (pH 7.2), and 0.5 mM EDTA. Ten major translation products (which could be immunoprecipitated with antiserum against peroxisomal protein) became progressively associated with the peroxisomes during the first 30 min of incubation (some up to approximately 70%). These include acyl coenzyme A oxidase and the trifunctional protein hydratase-dehydrogenase-epimerase. This association did not occur at 4 degrees C nor did it occur if the peroxisomes were replaced with mitochondria.

scholarly journals Cleavage targets and the D-arginine-based inhibitors of the West Nile virus NS3 processing proteinase

2005 ◽  
Vol 393 (2) ◽  
pp. 503-511 ◽  
Author(s):  
Sergey A. Shiryaev ◽  
Boris I. Ratnikov ◽  
Alexei V. Chekanov ◽  
Sergey Sikora ◽  
Dmitri V. Rozanov ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Protein A ◽  
Three Dimensional ◽  
Proteolytic Processing ◽  
Three Dimensional Model ◽  
West Nile ◽  
Highly Active ◽  
Polyprotein Precursor ◽  
Global Threat

Mosquito-borne WNV (West Nile virus) is an emerging global threat. The NS3 proteinase, which is essential for the proteolytic processing of the viral polyprotein precursor, is a promising drug target. We have isolated and biochemically characterized the recombinant, highly active NS3 proteinase. We have determined that the NS3 proteinase functions in a manner that is distantly similar to furin in cleaving the peptide and protein substrates. We determined that aprotinin and D-arginine-based 9–12-mer peptides are potent inhibitors of WNV NS3 with Ki values of 26 nM and 1 nM respectively. Consistent with the essential role of NS3 activity in the life cycle of WNV and with the sensitivity of NS3 activity to the D-arginine-based peptides, we showed that nona-D-Arg-NH2 reduced WNV infection in primary neurons. We have also shown that myelin basic protein, a deficiency of which is linked to neurological abnormalities of the brain, is sensitive to NS3 proteolysis in vitro and therefore this protein represents a convenient test substrate for the studies of NS3. A three-dimensional model of WNV NS3 that we created may provide a structural guidance and a rationale for the subsequent design of fine-tuned inhibitors. Overall, our findings represent a foundation for in-depth mechanistic and structural studies as well as for the design of novel and efficient inhibitors of WNV NS3.

The Effect of Silicic Acid on Calcium Phosphate Precipitation

1989 ◽  
Vol 68 (9) ◽  
pp. 1355-1359 ◽  
Author(s):  
J.J.M. Damen ◽  
J.M. Ten Cate
Keyword(s):  
Crystal Growth ◽  
Calcium Phosphate ◽  
Silicic Acid ◽  
Inhibitory Effect ◽  
Polysilicic Acid ◽  
Lag Period ◽  
Additional Support ◽  
Silica Suspensions ◽  
Calcium Phosphate Precipitation

So that a possible involvement in the mineralization of dental plaque could be investigated, the effects of silicic acid on calcium phosphate precipitation were assessed in vitro. By measuring the decrease in Ca2+ concentration (by means of ion-selective electrodes), we determined both spontaneous precipitation and seeded crystal growth from solutions that contained 1 mmol/L calcium, 7.5 mmol/L phosphate, 50 mmol/L Hepes pH 7.2, and various amounts of silicic acid. Polymerized silicic acid, but not its monomer, was found both to cause a 60% reduction in the lag period that precedes spontaneous precipitation and to enhance the growth rate of seeded hydroxyapatite crystals. Silica suspensions showed effects similar to those of polysilicic acid. In all cases, the precipitated material was found to be hydroxyapatite. Whereas seeded brushite crystals grew slowly without silicic acid, hydroxyapatite was the only mineral detected after crystal growth in the presence of silicic acid. Apparently, polysilicic acid acted as a substrate for hydroxyapatite nucleation, inducing secondary nuclei on both hydroxyapatite and brushite crystals. The finding that polysilicic acid could overcome part of the inhibitory effect of a phosphoprotein on calcium phosphate precipitation gave additional support for the idea that polysilicic acid and silica may promote the formation of dental calculus.

scholarly journals In vitro synthesis and membrane insertion of bovine MP26, an integral protein from lens fiber plasma membrane.

1983 ◽  
Vol 96 (3) ◽  
pp. 633-638 ◽  
Author(s):  
D L Paul ◽  
D A Goodenough
Keyword(s):  
Plasma Membranes ◽  
Proteolytic Processing ◽  
Membrane Insertion ◽  
Membrane Association ◽  
Lens Fiber ◽  
In Vitro Synthesis ◽  
Reticulocyte Lysate ◽  
Immune Precipitation ◽  
Dog Pancreas

Synthesis of MP26, the principal protein of lens fiber plasma membranes, was directed in the reticulocyte lysate system by poly A mRNA enriched from whole bovine lens RNA using oligo (dt)-cellulose chromatography. Synthesized MP26 was enriched by immune precipitation. The in vitro-synthesized MP26 had an electrophoretic mobility indistinguishable from that of the native molecule. MP26 showed a cotranslational requirement for dog pancreas microsomes in order for membrane association to occur. Microsome-associated in vitro-synthesized MP26 showed a sensitivity to digestion with chymotrypsin which was similar to the sensitivity of native MP26 in isolated lens fiber plasma membranes, indicating correct insertion of the MP26 into the microsome. Synthesis and membrane insertion of MP26 using N-formyl-[35S]methionyl tRNA as label demonstrated that no proteolytic processing or significant glycosylation accompanied membrane insertion. Chymotryptic cleavage of membrane-inserted, N-formyl-[35S]methionine-labeled MP26 resulted in loss of label, suggesting that the N-terminal of the in vitro-synthesized MP26 faces the cytoplasm.

scholarly journals Efficient association of in vitro translation products with purified stable Candida tropicalis peroxisomes.

1987 ◽  
Vol 7 (5) ◽  
pp. 1848-1855 ◽  
Author(s):  
G M Small ◽  
T Imanaka ◽  
H Shio ◽  
P B Lazarow
Keyword(s):  
Candida Tropicalis ◽  
Proteolytic Processing ◽  
In Vitro Translation ◽  
Moderate Number ◽  
Brij 35 ◽  
Reticulocyte Lysate ◽  
Molecular Information ◽  
Acyl Coenzyme A

Newly synthesized peroxisomal proteins enter preexisting peroxisomes posttranslationally in vivo, generally without proteolytic processing. An efficient reconstitution of this process in vitro together with cloned DNAs for peroxisomal proteins would make possible investigation of the molecular information that targets proteins to peroxisomes. We have previously reported the isolation of clones for Candida tropicalis peroxisomal proteins; here we describe the association (and possible import) of peroxisomal proteins with peroxisomes in vitro. C. tropicalis was grown in a medium containing Brij 35, resulting in the induction of a moderate number of medium-sized peroxisomes. These peroxisomes, isolated in a sucrose gradient, had a catalase latency of 54% and were sufficiently stable to be concentrated and used in an import assay. The reticulocyte lysate translation products of total RNA from oleate-grown cells were incubated with the peroxisomes at 26 degrees C in the presence of 50 mM KCl, protease inhibitors, 0.5 M sucrose, 2.5 mM MOPS (morpholinepropanesulfonic acid) (pH 7.2), and 0.5 mM EDTA. Ten major translation products (which could be immunoprecipitated with antiserum against peroxisomal protein) became progressively associated with the peroxisomes during the first 30 min of incubation (some up to approximately 70%). These include acyl coenzyme A oxidase and the trifunctional protein hydratase-dehydrogenase-epimerase. This association did not occur at 4 degrees C nor did it occur if the peroxisomes were replaced with mitochondria.

Calcium Phosphate Precipitation in Liposomal Suspensions

1989 ◽  
Vol 174 ◽  
Author(s):  
Edward D. Eanes
Keyword(s):  
Calcium Phosphate ◽  
Lipid Vesicles ◽  
Matrix Vesicles ◽  
Liposomal Membrane ◽  
Liposomal Membranes ◽  
Precipitation Reactions ◽  
Basic Features ◽  
Calcium Phosphate Precipitation

AbstractArtificial lipid vesicles (liposomes) provide an in vitro approach for microencapsulating calcium phosphate precipitation reactions in a manner similar to that which occurs in matrix vesicles, the initial loci for extracellular mineralization in many skeletal tissues. Apatitic precipitates readily form within the aqueous interiors of liposomes prepared from phosphatidylcholine, dicetylphosphate, and cholesterol when the liposomal membranes enclosing pH 7.4 buffered PO4 solutions are made permeable to external Ca2+ with ionophores. If the external Ca solution is rendered metastable with PO4, the apatitic precipitates rapidly expand to outside the liposomes as well. The present paper describes the basic features of liposomal mineralization and presents some specific examples on how compositional alterations in the liposomal membrane and external Ca solution can affect the progress of this mineralization.

Isolation from urine of pyrophosphate, a calcification inhibitor

1962 ◽  
Vol 203 (4) ◽  
pp. 671-675 ◽  
Author(s):  
Herbert Fleisch ◽  
Sylvia Bisaz
Keyword(s):  
Calcium Phosphate ◽  
Inorganic Pyrophosphate ◽  
Minimum Value ◽  
Phosphate Compound ◽  
Apatite Crystals ◽  
Urinary Inhibitors ◽  
The Mean ◽  
Calcium Phosphate Precipitation

The minimum value of the product, Ca X P, necessary for calcium phosphate precipitation to occur, was determined in vitro. The addition of 2.2% urine to the solutions raised the mean product from 53 to 105 (mg/100 ml)2, which shows that the urine contains inhibitors to hydroxyapatite precipitation. One of these was isolated and purified. Its characteristics are those of inorganic pyrophosphate. The mean level of urinary pyrophosphate was 2.24 mg P/liter, sufficient to inhibit precipitation of hydroxyapatite. Pyrophosphate is one of the substances permitting urine to be supersaturated with calcium and phosphate; other urinary inhibitors are still to be identified. It is suggested that the phosphate compound present in the plasma which inhibits hydroxyapatite precipitation is pyrophosphate. In bone, pyrophosphatase would act by locally destroying this calcification inhibitor, thus allowing the apatite crystals to form.

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