Methylation of γ-carboxylated Glu (Gla) allows detection by liquid chromatography-mass spectrometry and the identification of Gla residues in the γ-glutamyl carboxylase.

γ-Carboxylated Glu (Gla) is a post-translational modification required for the activity of vitamin K-dependent (VKD) proteins that has been difficult to study by mass spectrometry due to the properties of this negatively charged residue. Gla is generated by a single enzyme, the γ-glutamyl carboxylase, which has broad biological impact because VKD proteins have diverse functions that include hemostasis, apoptosis, and growth control. The carboxylase also contains Glas, of unknown function, and is an integral membrane protein with poor sequence coverage. To locate these Glas, we first established methods that resulted in high coverage (92%) of uncarboxylated carboxylase. Subsequent analysis of carboxylated carboxylase identified a Gla peptide (729-758) and a missing region (625-647) that was detected in uncarboxylated carboxylase. We therefore developed an approach to methylate Gla, which efficiently neutralized Gla and improved mass spectrometric analysis. Methylation eliminated CO2 loss from Gla, increased the ionization of Gla-containing peptide, and appeared to facilitate trypsin digestion. Methylation of a carboxylated carboxylase tryptic digest identified Glas in the 625-647 peptide. These studies provide valuable information for testing the function of carboxylase carboxylation. The methylation approach for studying Gla by mass spectrometry is an important advance that will be broadly applicable to analyzing other VKD proteins.

Expression of human pancreatic polypeptide precursors from a dicistronic mRNA in mammalian cells.

The ability of eukaryotic ribosomes to reinitiate translation at downstream AUG codons on polycistronic mRNAs was used to select transfected CHO clones that secreted a precursor to the human pancreatic polypeptide (PP). In the in vitro constructed transcription unit, a viral promoter directed the synthesis of a dicistronic mRNA. The PP cDNA was placed in the 5'-part of this transcript, while a DHFR cDNA was placed 3' to the PP. This dicistronic expression unit was transfected into CHO cells, and methotrexate-resistant colonies were selected. RNA-blots verified that the PP precursor and DHFR were expressed from the same dicistronic mRNA. The CHO cells synthesized the hormone precursor and secreted it through the constitutive secretory pathway.

The physiology of vitamin K nutriture and vitamin K-dependent protein function in atherosclerosis.

Recent advances in the discovery of new functions for vitamin K-dependent (VKD) proteins and in defining vitamin K nutriture have led to a substantial revision in our understanding of vitamin K physiology. The only unequivocal function for vitamin K is as a cofactor for the carboxylation of VKD proteins which renders them active. While vitamin K was originally associated only with hepatic VKD proteins that participate in hemostasis, VKD proteins are now known to be present in virtually every tissue and to be important to bone mineralization, arterial calcification, apoptosis, phagocytosis, growth control, chemotaxis, and signal transduction. The development of improved methods for analyzing vitamin K has shed considerable insight into the relative importance of different vitamin K forms in the diet and their contribution to hepatic vs. non-hepatic tissue. New assays that measure the extent of carboxylation in VKD proteins have revealed that while the current recommended daily allowance for vitamin K is sufficient for maintaining functional hemostasis, the undercarboxylation of at least one non-hemostatic protein is frequently observed in the general population. The advances in defining VKD protein function and vitamin K nutriture are described, as is the potential impact of VKD proteins on atherosclerosis. Many of the VKD proteins contribute to atherogenesis. Recent studies suggest involvement in arterial calcification, which may be influenced by dietary levels of vitamin K and by anticoagulant drugs such as warfarin that antagonize vitamin K action.

The vitamin K-dependent carboxylase.

The carboxylase is an integral membrane glycoprotein that uses vitamin K to modify clusters of glutamyl residues (glu's) to gamma-carboxylated glutamyl residues (gla's) post-translationally in vitamin K-dependent (VKD) proteins as they pass through the endoplasmic reticulum. Carboxylation is required for VKD protein functions in hemostasis, bone metabolism, growth control and signal transduction. Carboxylation of multiple glu residues is accomplished via a processive mechanism, which occurs with at least some order and involves carboxylation of the carboxylase. The carboxylase has a high affinity binding site for VKD proteins, which in most cases is a VKD propeptide sequence; it also appears to have a low affinity site for those glu's undergoing catalysis. The propeptide activates binding of the glu's; together, the two contact points between the carboxylase and VKD protein increase the affinity of the carboxylase for vitamin K. Biochemical mapping to identify where these events occur in the carboxylase remains a challenge, despite the availability of recombinant protein. The affinity of the carboxylase for the propeptide of several VKD proteins that are coexpressed in liver varies over a 100-fold range. Treatment with anticoagulants such as warfarin that indirectly block carboxylation likely decreases the rate of VKD protein catalysis and increases the accumulation of VKD precursors, leading to a competitive state among these proteins, which results in the premature dissociation of undercarboxylated, inactive protein.

Isolation of the human gamma-carboxylase and a gamma-carboxylase-associated protein from factor IX-expressing mammalian cells.

A model system for the analysis of intracellular events governing the modification of individual vitamin K-dependent (VKD) proteins by the carboxylase has been developed using recombinant VKD protein-transfected cell lines. When untransfected 293 cells were analyzed by in vitro carboxylation followed by SDS-PAGE, endogenous VKD proteins were not detected. With 293 cells stably-transfected with recombinant native factor IX, most (> 95%) of the carboxylase was in complex with the factor IX, as assayed by adsorption of carboxylase activity to immobilized anti-factor IX antibody. In contrast, with 293 cells stably-transfected with recombinant factor IX deleted in the propeptide sequence (amino acids -18 to -4, delta pro factor IX), no association of factor IX with the carboxylase was observed. This observation was used to specifically isolate and identify the human carboxylase, and carboxylase-associated protein. When the carboxylase was purified from solubilized microsomes from either native factor IX, or delta pro factor IX, stably-transfected 293 cells, a single 98 kDa band was specifically obtained from native factor IX microsomes, but not from delta pro factor IX microsomes. This band was subsequently shown by Western and microsequencing analysis to comprise both the carboxylase and carboxylase-associated protein. This isolation, which represents the first isolation to near homogeneity of both the human carboxylase and the carboxylase from cell lines, will be valuable in isolating enzymatically active recombinant carboxylase, which has been refractile to other purification attempts. This system was also used to show that the human carboxylase in 293 cells is capable of binding and modifying two different liver-derived proteins. Protein C-producing 293 cells were generated from the same 293 progenitor cell line used to created the factor IX-expressing cells. With both factor IX- and protein C-transfected 293 cells, the secreted proteins were almost completely carboxylated, and in microsomes from each cell line the carboxylase was found in near quantitative complex with the two different VKD proteins. Thus the carboxylase modifies both VKD proteins. The approach described here for the analysis of the carboxylase from recombinant VKD protein-transfected cell lines should provide an important new system for studying protein carboxylation and VKD protein-carboxylase interaction.

Generation of adenovirus by transfection of plasmids.

Biologically active fragments of Adenovirus 5 (Ad5) DNA that span the entire genome have been cloned into plasmids. The covalently attached terminal protein was removed and Eco RI linkers added in a fashion that preserves the Ad5 terminal sequences. When plasmids containing overlapping fragments that represent the entire genome are cotransfected onto 293 cells, infectious virus is obtained. Generation of virus depends upon the release of the 0 or 100 mu Ad5 terminus from pBR322 DNA by Eco RI cleavage. During virus production the modified termini of the transfected fragments are corrected exactly to that of wt viral DNA. The above method for preparing adenovirus recombinants has been used to construct a mutant, Ad5 delta (78.9-84.3), lacking most of the non-essential EIII transcriptional unit. This mutant is phenotypically wild type with respect to burst size and kinetics of growth. Surprisingly, it inhibits wt viral growth upon mixed infections of HeLa or 293 cells, apparently at the level of DNA replication.

Expression of dihydrofolate reductase, and of the adjacent EIb region, in an Ad5-dihydrofolate reductase recombinant virus.

A gene with the Ad2 MLP and first leader, and appropriate RNA processing signals (splicing, polyadenylation) positioned around a mouse DHFR cDNA clone was substituted for the EIa region of Ad5, and virus stocks of Ad5 (DHFR-I) were prepared on 293 cells. A DHFR RNA of the expected size and structure was expressed late after infection of 293 cells by Ad5 (DHFR-I), at levels comparable to that of other Ad5 late messages. Although this DHFR mRNA was translated as efficiently as other Ad late mRNAs in vitro, it was only poorly translated in vivo. The substitution of the DHFR gene for the Ad5 EIa region results in aberrant transcriptional activity in the adjacent EIb sequences. The transcriptional levels of the EIb 1kb message were down approximately 10-fold. In addition, a novel pIX-encoding mRNA was produced, generated by the splicing of the Ad first late leader onto sequences 14 bp upstream from the pIX initiation codon. This new mRNA was found to be potently efficient for translation both in vivo and in vitro.

The effects of substituted pyrimidines in DNAs on cleavage by sequence-specific endonucleases.

The rates of cleavage of DNAs containing substituents at position 5 of thymine or cytosine have been measured for a variety of sequence-specific endonucleases, so as to determine which features in the DNA sequence are being probed. Phage phi e DNA fully substituted with 5-hydroxymethyluracil is cleaved more slowly by enzymes whose recognition sequences contain A-T base pairs than are DNAs containing thymine, but both types of DNA are cleaved at similar rates by enzymes recognizing sequences composed only of G-C base pairs. Phage PBS2 DNA with uracil completely substituted for thymine is cleaved slowly by several enzymes which recognize sequences containing A-T base pairs (endonucleases Hpa I, HindII, and HindIII), while the rates of cleavage by other enzymes (endonucleases EcoRI and BamHI) are not affected. Phage lambda- and P22 DNAs containing 5-bromouracil are cleaved more slowly by several enzymes (endonucleases HindIII, Hpa I, BamHI) than are thymine-containing DNAs. Enzymes that recognize sequence isomers with the composition G:C:2A:2T (endonucleases EcoRI, Hpa I, HindIII) are not equally affected by substitution at position 5 of thymine, suggesting that they differ in their contacts with A-T base pairs. DNA containing glucosylated 5-hydroxymethylcytosine in place of cytosine is resistant to cleavage by all the endonucleases examined.

Quantitation of the various termini generated by type II restriction endonucleases using the polynucleotide kinase exchange reaction.

Parameters of the polynucleotide kinase-catalyzed exchange reaction between [gamma-32P]ATP and 5'-phosphoryl DNAs have been measured with the termini generated by the following endonucleases: EcoRI (Berkner, K. L., and Folk, W. R. (1977) J. Biol. Chem. 252, 3176-3184), Hpa II, BamHI, and HindIII (external termini); HindII and Hpa I (blunt terminal); Hae II and Hha I (internal termini). In every case, exchange is reproducible and proportional to the number of termini. However, in most cases, the exchange reaction does not proceed to the theoretical maximum. External termini and single-stranded DNAs are labeled more rapidly and to approximately 5-fold higher levels than blunt or internal termini. Concentrations of 100 to 200 microns ADP and 12 microns ATP are optimal for labeling all types of termini with the exchange reaction.

Overmethylation of DNAs by the EcoRI methylase.

EcoRI methylase is able to catalyze methy incorporation into DNA at sequences other than the canonical EcoRI site. At high enzyme concentrations and over a wide range of pH and ionic strengths, EcoRI methylase modifies polyoma DNA (which contains one EcoRI site) at a number of sites. This modification prevents EcoRI endonuclease activity, and thus is presumably at or near the EcoRI sequences (5') NAATTN.

EcoRI cleavage and methylation of DNAs containing modified pyrimidines in the recogintion sequence.

The effects of substituents at position 5 in the pyrimidine ring of a variety of phage DNAs upon EcoRI endonuclease and methylase activities have been examined. The replacement of cytidine in DNA with glucosylated hydroxymethylcytidine confers resistance to cleavage by the EcoRI endonuclease. Substitution of thymidine in DNA by hydroxy-methyluridine(a change in the methyl at position 5 of thymidine for a hydroxymethyl) lowers the maximal velocity of endonucleolytic cleavage 20-fold, but has no detectable effect upon the Km. Substitution of thymidine in DNA by uridine (a change in the methyl at position 5 of thymidine for a hydrogen atom) has no effect upon either the maximal velocity or the Km. The effect of these modifications upon EcoRI methylase activity was markedly different. DNA containing glucosylated hydroxymethylcytidine is methylated as well as normal DNA. DNA containing uridine or hydroxy-methyluridine, in place of thymidine, is much more poorly methylated than normal DNA. These different sensitivities of the EcoRI endonuclease and methylase to modifications in the pyrimidine rings of DNA suggest there are significant differences in the manner by which these enzymes recognize and bind to the canonical EcoRI sequence.

An assay for the rates of cleavage of specific sites in DNA by restriction endonucleases: its use to study the cleavage of phage lambda DNA by EcoRI and phage P22 DNA containing thymine or 5-bromouracil by HindIII.

A method to measure the rates of cleavage of specific sites in DNAs by restriction endonucleases is described. Partial digests are prepared by incubating DNAs with limiting amounts of endonuclease. The termini generated by cleavage are labeled with 32P by the polynucleotide kinase-exchange reaction. The labeled termini are then identified by completing the digestion with the same endonuclease and separating the products by gel electrophoresis. As the products of complete digestion of DNA are often easily separated and can be unequivocally identified, this procedure permits comparison of the rates of cleavage of specific sites in DNAs; furthermore, because detection of the products of cleavage utilizes radioautography and does not depend upon their size, or amount, only small amounts of DNA need to be utilized. This method has been used to examine the cleavage of phage lambda DNA by EcoRI endonuclease, and to demonstrate that 5-bromouracil substitution in phage P22 DNA reduces the rate of cleavage of most sites by HindIII endonuclease approximately threefold and the rate of cleavage of one site approximately tenfold.

Vitamin K-dependent carboxylation of the carboxylase.

Vitamin K-dependent (VKD) proteins require modification by the VKD-gamma-glutamyl carboxylase, an enzyme that converts clusters of glus to glas in a reaction that requires vitamin K hydroquinone, for their activity. We have discovered that the carboxylase also carboxylates itself in a reaction dependent on vitamin K. When pure human recombinant carboxylase was incubated in vitro with 14CO2 and then analyzed after SDS/PAGE, a radiolabeled band corresponding to the size of the carboxylase was observed. Subsequent gla analysis of in vitro-modified carboxylase by base hydrolysis and HPLC showed that all of the radioactivity could be attributed to gla residues. Quantitation of gla, asp, and glu residues indicated 3 mol gla/mol carboxylase. Radiolabeled gla was acid-labile, confirming its identity, and was not observed if vitamin K was not included in the in vitro reaction. Carboxylase carboxylation also was detected in baculovirus-(carboxylase)-infected insect cells but not in mock-infected insect cells, which do not express endogenous VKD proteins or carboxylase. Finally, we showed that the carboxylase was carboxylated in vivo. Carboxylase was purified from recombinant carboxylase BHK cells cultured in the presence or absence of vitamin K and analyzed for gla residues. Carboxylation of the carboxylase only was observed with carboxylase isolated from BHK cells cultured in vitamin K, and 3 mol gla/mol carboxylase were detected. Analyses of carboxylase and factor IX carboxylation in vitro suggest a possible role for carboxylase carboxylation in factor IX turnover, and in vivo studies suggest a potential role in carboxylase stability. The discovery of carboxylase carboxylation has broad implications for the mechanism of VKD protein carboxylation and Warfarin-based anti-coagulant therapies that need to be considered both retrospectively and in the future.

Effect of the tripartite leader on synthesis of a non-viral protein in an adenovirus 5 recombinant.

The EIa region of an Adenovirus 5 recombinant has been substituted by a modular gene encoding dihydrofolate reductase (DHFR). In this recombinant, the mouse DHFR cDNA was positioned behind sequences of the major late promoter and the complete tripartite leader. The leader sequences end in the normal 5' splice site (SS) of the third leader, so that RNA splicing joins the tripartite leader to a 3' splice site immediately upstream of the DHFR cDNA. At late stages of infection, high levels of DHFR mRNAs were synthesized. At early times in the late stage, this mRNA was efficiently translated; however, at later times translation of DHFR decreased probably due to poor competition with other late mRNAs. Synthesis of DHFR protein from an analogous Adenovirus 5 recombinant containing only the first late leader was studied in parallel. Equivalent levels of DHFR mRNA were expressed after infection with this recombinant virus; however, the efficiency of DHFR translation was at least 20 fold lower than that of the DHFR mRNA containing the tripartite leader. This suggests that the tripartite leader sequence is important for translation in the late stage of infection. As reported previously, the Ad5 recombinant containing only the first leader vastly overexpresses polypeptide IX from a novel mRNA, formed by the splicing of the first leader in the modular DHFR gene to the 3' splice site in the EIb region. Cells infected with this recombinant synthesize very little normal mRNA from the EIb region. Here, we demonstrated that coinfection of 293 cells with this recombinant and wild type Adenovirus 5 also results in decreased EIb mRNA synthesis. We propose that the overproduction of polypeptide IX suppresses mRNA expression from the EIb and IX promoter sites, probably by an autoregulation loop active during lytic growth.

Synthesis, purification, and characterization of an Arg152----Glu site-directed mutant of recombinant human blood clotting factor VII.

Coagulation factor VII circulates in blood as a single-chain zymogen of a serine protease and is converted to its activated two-chain form, factor VIIa, by cleavage of an internal peptide bond located at Arg152-Ile153. Previous studies using serine protease active-site inhibitors suggest that zymogen factor VII may possess sufficient proteolytic activity to initiate the extrinsic pathway of blood coagulation. In order to assess the putative intrinsic proteolytic activity of single-chain factor VII, we have constructed a site-specific mutant of recombinant human factor VII in which arginine-152 has been replaced with a glutamic acid residue. Mutant factor VII was purified in a single step from culture supernatants of baby hamster kidney cells transfected with a plasmid containing the sequence for Arg152----Glu factor VII using a calcium-dependent, murine anti-factor VII monoclonal antibody column. Purified mutant factor VII was indistinguishable from plasma-derived or recombinant wild-type factor VII by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and migrated as a single band with an apparent molecular weight of 50,000. The average specific activity of several mutant factor VII preparations was 0.00025 unit/micrograms, or 0.01% of that observed for recombinant wild-type factor VII preparations. The clotting activity of mutant factor VII was, however, completely inhibited following incubation with dansyl-Glu-Gly-Arg chloromethyl ketone, suggesting that the apparent clotting activity of mutant factor VII was due to a contaminating serine protease.(ABSTRACT TRUNCATED AT 250 WORDS)