Streptokinase-plasmin(ogen) activator assays.
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Amino Acid Sequence↗
Biomedical subjects
D J Davidson
Publications and source records attributed to D J Davidson.
Expression of human plasminogen cDNA in lepidopteran insect cells and analysis of asparagine-linked glycosylation patterns of recombinant plasminogens.
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Animals↗
The influence of the nature of the asparagine 289-linked oligosaccharide on the activation by urokinase and lysine binding properties of natural and recombinant human plasminogens.
Several strategies have been used to obtain recombinant (r) human plasminogens (HPg) containing different oligosaccharide side chains on its sole N-linked glycosylation site, present at Asn289. The approaches included expression of the cDNA for HPg in insect cell lines under various conditions, addition of glycosidase inhibitors during expression, and purification of specific glycoforms of HPg using affinity chromatography on an insolubilized lectin column. The activation kinetics for urokinase (UK) of each of the purified HPgs, as well as their relative abilities to bind to the ligand, epsilon-aminocaproic acid (EACA), were then determined. Removal of both N- and O-linked oligosaccharide from HPg resulted in a slight increase in the Kcat/Km for its activation, while a glycoform containing tetrasialyl-tetra-antennary complex oligosaccharide on Asn289 was a slightly poorer substrate for UK than plasma HPg, which contains bisialyl-biantennary complex carbohydrate on Asn289. The most dramatic differences were observed for HPgs with high mannose-type glycans on Asn289. (Man9GlcNAc2)-HPg possessed only approximately 6% of the kcat/Km of plasma HPg, whereas (Glc3Man9GlcNAc2)-HPg did not undergo activation at a significant rate by UK. Differences were also found in the relative abilities of the HPg glycoforms to interact with EACA. The most effective interactions were observed with HPgs containing complex-type glycans, and the least effective binding was found for HPgs with high mannose-type oligosaccharides. The full range of the binding effects is represented by a fourfold difference between HPg containing tetrasialyl-tetra-antennary glycan and HPg with (Glc3Man9GlcNAc2) assembled on Asn289. These results clearly demonstrate that the nature of the N-linked glycan assembled on HPg dramatically influences its ability to be activated by UK and to bind to omega-amino acid effector molecules.
Animals↗
alpha-Mannosidase-catalyzed trimming of high-mannose glycans in noninfected and baculovirus-infected Spodoptera frugiperda cells (IPLB-SF-21AE). A possible contributing regulatory mechanism for assembly of complex-type oligosaccharides in infected cells.
Incubation of a Spodoptera frugiperda (IPLB-SF-21AE) cell extract with the oligosaccharide Man9GlcNAc2, the aglucosyl derivative of the glycan that is normally transferred from the dolichol carrier to the relevant Asn residue in the nascent protein, results in its trimming to Man6GlcNAc2, an intermediate that is relatively stable to further alpha-D-mannosidase action in these cells. On the other hand, incubation of a similar extract from cells that had been infected for various times with a wild-type baculovirus (Autographa californica nuclear polyhedrosis virus) or a recombinant baculovirus (r-BAC)/human plasminogen (HPg) construct employed for expression of HPg led to rapid trimming of Man6GlcNAc2 to Man5GlcNAc2 and Man3GlcNAc2. These latter reactions displayed temporal effects, in that an enhancement of this latter trimming process occurred as a function of the time of infection of the cells with the wild-type and recombinant viral constructs. We have previously demonstrated that the nature of the oligosaccharide assembled on Asn289 of HPg expressed in several lepidopteran insect cell lines was dependent on the time of infection of the cells with r-BAC/HPg and that the amount of complex glycan found on this recombinant protein increased with an increase in infection times [Davidson, D. J., & Castellino, F. J. (1991) Biochemistry 30, 6167-6174].(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Structures of the asparagine-289-linked oligosaccharides assembled on recombinant human plasminogen expressed in a Mamestra brassicae cell line (IZD-MBO503).
In this report, we have fortified and extended a previous investigation [Davidson, D. J., Fraser, M. J., & Castellino, F. J. (1990) Biochemistry 29, 5584-5590] in which we demonstrated for the first time that lepidopteran insect (Spodoptera frugiperda) cells (IPLB-SF-21AE) were capable of assembling N-linked complex oligosaccharide on a human protein (plasminogen), the cDNA of which had been inserted into these cells via recombinant DNA technology with a baculovirus vector. In order to investigate whether a more general capability of lepidopteran insect cells to produce complex oligosaccharide existed, and to identify the chemical nature of the types of oligosaccharides that such insect cells were able to assemble, we have infected Mamestra brassicae (IZD-MBO503) cells for 48 h with a recombinant (r) baculovirus containing the [R561E]human plasminogen (HPg) cDNA and characterized the nature of the glycopeptidase F (GF) released N-linked oligosaccharides contained on Asn289 of the r-HPg expressed by these cells. We found that approximately 63% of the total N-linked oligosaccharides were of the complex type, with bisialo-biantennary (28%), asialo-biantennary (7%), fucosylated bisialo-biantennary (25%), and fucosylated asialo-biantennary (3%) oligosaccharides representing the major complex-type carbohydrate species. The remainder of the oligosaccharides were of the high-mannose type, with (mannose)9(N-acetylglucosamine)2 (22%), (mannose)5(N-acetylglucosamine)2 (13%), and (mannose)3(N-acetylglucosamine)2 (2%) representing the major oligosaccharides observed.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Asparagine-linked oligosaccharide processing in lepidopteran insect cells. Temporal dependence of the nature of the oligosaccharides assembled on asparagine-289 of recombinant human plasminogen produced in baculovirus vector infected Spodoptera frugiperda (IPLB-SF-21AE) cells.
Previous studies from this laboratory have established that lepidopteran insect cells possess the glycosylation machinery needed to assemble N-linked complex-type oligosaccharides on Asn289 of recombinant human plasminogen (r-HPg). In the present paper, we show that the nature of N289-linked glycosylation of [R561E]r-HPg expressed in Spodoptera frugiperda (IPLB-SF-21AE) cells is dependent upon the length of time of infection of the cells with the recombinant baculovirus/HPg-cDNA construct. At the earliest postinfection (p.i.) time period studied, i.e., 0-20 h, virtually all (96%) of the oligosaccharides released with glycopeptidase F from N289 of the expressed r-HPg were of the high-mannose type and comprised nearly the full range of such structures, containing 3-9 mannose units. At a time window of 60-96 h, p.i., essentially all of the oligosaccharides (92% of the total) assembled on N289 of rHPg were of the biantennary, triantennary, and tetraantennary complex classes, with varying extents of outer arm completion. At an intermediate time period window, of 20-60 h, p.i., a mixture of complex-type oligosaccharides, totaling approximately 77% of the glycans, with various levels of branching and outer arm completion, and high-mannose type of oligosaccharides, totaling approximately 23% of the glycans, was assembled on N289 of the r-HPg produced. These studies demonstrate that lepidopteran insect cells contain the glycosyltransferase genes required for assembly of N-linked complex oligosaccharide and that these transferases are utilized under proper conditions. The time dependency of the assembly of complex-type oligosaccharides on r-HPg indicates that an activation of the appropriate glycosyl transferases and/or transferase genes can take place. Thus, one consequence of the infective process with the recombinant baculovirus/HPg-cDNA construct is to alter the normal glycosylation characteristics of insect cells and to allow complex-type oligosaccharide processing to occur.
Amidohydrolases↗
Oligosaccharide structures present on asparagine-289 of recombinant human plasminogen expressed in a Chinese hamster ovary cell line.
The oligosaccharide structures linked to Asn289 of a recombinant (r) variant (R561S) human plasminogen (HPg) expressed in Chinese hamster ovary (CHO) cells, after transfection of these cells with a plasmid containing the cDNA coding for the variant HPg, have been determined. Employing high-performance anion-exchange liquid chromatography mapping of the oligosaccharide units cleaved from the protein by glycopeptidase F, compared with elution positions of standard oligosaccharides, coupled with monosaccharide compositional determinations and analyses of sequential exoglycosidase digestions and specific lectin binding, we find that considerable microheterogeneity in oligosaccharide structure exists at this sole potential N-linked glycosylation site on HPg. A variety of high-mannose structures, as well as bi-, tri-, and tetraantennary complex-type carbohydrate, has been found, in relative amounts of 1-25% of the total oligosaccharides. The complex-type structures contain variable amounts of sialic acid (Sia), ranging from 0 to 5 mol/mol of oligosaccharide in the different glycan structures. Neither hybrid-type molecules, N-acetylglucosamine bisecting oligosaccharides, nor N-acetyllactosaminyl-repeat structures were found to be present in the complex-type carbohydrate pool in observable amounts. Of interest, a significant portion of the Sia exists an outer arm structures in an (alpha 2,6) linkage to the penultimate galactose, a novel finding in CHO cell-directed glycosylation.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Oligosaccharide processing in the expression of human plasminogen cDNA by lepidopteran insect (Spodoptera frugiperda) cells.
A comparison has been made between the Asn289-linked oligosaccharide structures of human plasma plasminogen and a recombinant human plasminogen, expressed in lepidopteran insect (Spodoptera frugiperda) cells, after infection of these cells with a recombinant baculovirus containing the entire human plasminogen cDNA. Using anion-exchange liquid chromatography mapping of the oligosaccharide units cleaved from the proteins by glycopeptidase F, compared with elution positions of standard oligosaccharide structures, coupled with monosaccharide compositional analysis, we find that the human plasma protein contained only bisialo-biantennary complex-type carbohydrate and asialo-biantennary complex carbohydrate, confirming earlier work published by this laboratory. The glycosylation pattern of the insect cell expressed recombinant human plasminogen showed considerable microheterogeneity, with identifiable high-mannose carbohydrate (Man9GlcNAc2) and truncated high-mannose oligosaccharide (Man5GlcNAc2, Man4GlcNAc2, and Man3GlcNAc2). Of major importance, approximately 40% of the oligosaccharide population consisted of complex carbohydrate (bisialo-biantennary), identical in structure with that of the human plasma protein. This is the first direct identification of complex carbohydrate in proteins produced in insect cells and demonstrates that trimming and processing of high-mannose carbohydrate into complex-type oligosaccharide can occur. Our data indicate that both normal and alternate pathways exist in these cells for incorporation and trimming of high-mannose oligosaccharides and that mannosidases, as well as galactosyl-, hexosaminidasyl-, and sialyltransferases are present, and/or can be induced, in these cells. From these observations, we conclude that amino acid sequences and/or protein conformational properties can control oligosaccharide processing events.
Animals↗
Plasminogen activator activities of equimolar complexes of streptokinase with variant recombinant plasminogens.
The steady-state kinetic characteristics of the amidolytic and plasminogen activator activities of equimolar streptokinase (SK)-human plasminogen (HPg) and SK-human plasmin (HPm) complexes have been determined, exploiting the generation and use of cleavage site resistant mutants of HPg to stabilize plasminogen within the complex. Whereas amidolytic kinetic constants for equimolar complexes of SK with the following proteins, viz., plasma HPm, insect (i) cell-expressed wild-type (wt) recombinant (r) HPm, R561E-irHPg, and Chinese hamster ovary cell (c)-expressed R561S-crHPg, are similar, it has been found that the various SK-HPg complexes are far better enzymes than SK-HPm complexes for activation of bovine plasminogen, a species of plasminogen that is resistant to activation by SK, alone. In addition, it is emphasized that as a result of mutating the cleavage site in plasminogen, it is possible to express this protein in mammalian cells, and thus provide it for use in complex with SK as a more efficient plasminogen activator than plasma plasminogen, which is rapidly converted to HPm within the SK complex. This finding has important implications in the assessment of thrombolytic therapeutic reagent employing SK-plasminogen and SK-plasmin complexes.
Amidohydrolases↗
Solid-phase assay for the detection of low-abundance enzymes, and antibodies to enzymes in immune reactions, using acid sphingomyelinase as a model.
The development of a solid-phase immunosorbent assay, suitable for use with enzyme antigens, is described. Acid sphingomyelinase and a mouse monoclonal anti-sphingomyelinase antibody have been used to determine optimal conditions for the assay. The assay involves immobilization of a second antibody (anti-mouse IgG) in the wells of a polyvinyl microtiter plate. Soluble immune complexes of first antibody (monoclonal anti-sphingomyelinase) and antigen (sphingomyelinase), incubated in separate vials, are then reacted in the anti-mouse IgG-coated assay wells, and the extent of the cross-reaction between antibody and antigen is measured by direct assay of enzyme retained in the well. A necessary condition of the assay is that antibody must not inhibit enzyme activity, which makes it especially suitable for monoclonal antibodies. The assay finds useful application in hybridoma fluid screening, equivalence point determination, and demonstration of cross-reacting enzyme from various tissue sources.
Animals↗
Studies on the structure of sphingomyelinase. Amino acid composition and heterogeneity on isoelectric focusing.
Sphingomyelinase, purified to apparent homogeneity from human placenta, is an acidic protein, as judged from its amino acid composition and by isoelectric focusing of the carboxymethylated protein. The amino acid composition is characterized by an approximately equal content of hydrophobic and polar amino acid residues. The reduced-alkylated polypeptides were separated into two groups. Most of the polypeptides were heterogeneous with pI values of 4.4-5.0, but an additional more minor component was observed at pI 5.4. Liquid isoelectric focusing resolved the purified enzyme into a single major component (pI 4.7-4.8), a minor component (pI 5.0-5.4) and a plateau region of activity (pI 6-7). On thin-layer isoelectric focusing, the protein profile obtained from each of these regions was the same. In addition, the substrate specificity, Km values and effect of inhibitory substances were identical. We conclude that sphingomyelinase is an acidic, microheterogeneous protein that likely exists as a holopolymer of a single major polypeptide chain. the heterogeneity of the intact protein on isoelectric focusing appears to reflect this microheterogeneity, which is influenced by a tendency to associate with itself and with detergents such as Triton X-100.
Amino Acids↗
The active site of lysosomal sphingomyelinase: evidence for the involvement of hydrophobic and ionic groups.
The natural substrate for sphingomyelinase contains hydrophobic and polar moieties. In this study, we have employed pH rate studies and examined hydrophobic compounds and phosphorylated esters for their effect on sphingomyelinase activity in an attempt to determine some of the kinetic properties of this enzyme. Sphingomyelinase, purified from human placentae, undergoes noncompetitive inhibition by octylglucoside and Nonidet P-40, two nonionic detergents containing terminal octyl groups. The effect of these detergents at the hydrophobic binding site is somewhat different from that of Triton X-100, which contains an isooctyl terminal group, and this may serve to identify a structural basis for the effects. Sphingomyelinase activity is also modulated by several nucleotides. Inhibition by 5'-adenosine monophosphate (5'-AMP) is also noncompetitive. Other nucleotide monophosphates (such as 5'-uridine monophosphate (5'-UMP), 5'-cytidine monophosphate (5'-CMP), 2'-adenosine monophosphate (2'-AMP), and 3'-adenosine monophosphate (3'-AMP) and phosphorylated intermediates (such as phosphorylcholine, phosphorylethanolamine and hexose phosphates) have a lower inhibitory effect. The data suggest that the inhibition by 5'-AMP involves the combined effect of the phosphate group and the purine ring, structural requirements which may also be satisfied by bis(4-methylumbelliferyl)phosphate, a synthetic enzyme substrate. Studies of pH rate indicate that the maximal velocity for the hydrolysis of sphingomyelin is independent of pH over the range 3.5-6.2 while the Km value shows a pH dependence. The Km value is lowest from pH 4.0-5.2 and rises at pH values outside this range. The log Vmax/Km and pKm relationships, when plotted as a function of pH, have been used to identify the dissociation constants for the binding of sphingomyelin by the enzyme. These occur at pK values of 4.1 and 5.5. The activity of sphingomyelinase is also reduced when the enzyme is photooxidized in the presence of methylene blue or rose bengal and carbamylated by diethylpyrocarbonate (DEPC). These results are interpreted to show that 1). the enzyme contains a hydrophobic binding site which involves linear aliphatic moieties containing at least eight carbon atoms; 2) two ionic groups are involved in formation of the enzyme substrate complex, one of which is presumed to be the carboxylate group of aspartate or glutamate (represented by pK 4.1) and the second may be the protonated imidazolium group of histidine (represented by pK 5.5); and 3) since the maximal velocity shows no pH dependence, the interactions involving the hydrophobic and ionic groups affect only the binding of the substrate to the enzyme and formation of the enzyme-substrate complex.
Adenosine Monophosphate↗
Monoclonal antibodies against human placental sphingomyelinase.
Monoclonal antibodies against human placental acid sphingomyelinase have been raised. The antibodies are all of the IgG1 type, and are quite specific for the enzyme. One of the antibodies has been used to demonstrate a common antigenic identity of three polypeptides (mol.wts. 90,000, 72,000, and 48,000) of a purified sphingomyelinase preparation.
Antibodies, Monoclonal↗
Complex kinetics of bis(4-methylumbelliferyl)phosphate and hexadecanoyl(nitrophenyl)phosphorylcholine hydrolysis by purified sphingomyelinase in the presence of Triton X-100.
We have examined the hydrolysis of the synthetic phosphodiesters, bis(4-methylumbelliferyl)phosphate and hexadecanoyl(nitrophenyl)phosphorylcholine, by purified placental sphingomyelinase (sphingomyelin cholinephosphohydrolase, EC 3.1.4.12) in the presence of Triton X-100. Triton X-100 enhanced activity with bis(4MU)phosphate at all concentrations tested. At very low concentrations of detergent, bis(4MU)phosphate hydrolysis approached zero. Our results indicate that bis(4MU)phosphate does not form a micelle with Triton X-100. The observed enhancement of bis(4MU)phosphate activity with Triton X-100 is likely due to a direct effect of detergent on the enzyme itself. HDNP-phosphorylcholine formed its own micelle (or liposome) in the absence of Triton X-100 and, at substrate concentrations below 4 mM, hydrolysis was inhibited by Triton X-100. The extent of this inhibition varied with detergent concentrations but could be totally eliminated at substrate values above 4 mM. For theoretical reasons kinetic constants which could be obtained with the HDNP-phosphorylcholine substrate at concentrations above 4 mM are not considered to be truly representative of the real values. We conclude that neither substrate is recommended to describe the true kinetic parameters pertaining to purified sphingomyelinase. In addition, bis(4MU)phosphate may not be suitable as an aid for diagnosis of sphingomyelinase deficiency states.U
Animals↗
Where is that cancer? Part two: detection by lymphography, CAT scans, ultrasound, and other technics.
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Angiography↗
A convenient ferricyanide estimation of reducing sugars in the nanomole range.
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Blood Glucose↗
Expression of three recombinant proteins using baculovirus vectors in 23 insect cell lines.
Recombinant Autographa california baculoviruses expressing genes for pseudorabies virus glycoprotein (gp50T), human plasminogen (HPg), and beta-galactosidase (beta-gal) were used to infect 23 cell lines or strains. The objectives were to compare amounts of recombinant proteins expressed in the cell lines, compare yields from clones and parent lines, investigate the effects of long-term culture in serum-free medium on production, and determine if some lines yield gp50T with different glycosylation patterns. For HPg, IZD-MB0503 had the highest yield and four other lines (IPLB-TN-R2, IPLB-SF-1254, IPLB-LdEIta, and CM-1) had levels above that of SF-9 cells. For gp50T, four lines (IPLB-HvT1, IPLB-SF21AE, IPLB-SF21AE-15, and IPLB-SF-1254) had higher amounts than SF-9 cells. Some lines yielded gp50T with molecular mass about 1000 daltons larger than that from SF-9 cells, which suggests increased oligosaccharide processing. Equally high levels of beta-gal were expressed in three lines (SF-9, IZD-MB0503, and BCIRL-PX2-HNV3). The major conclusion is that no single cell line produced highest yields for all three recombinant proteins. Four lines were cultured in serum-free medium for 31-34 passages and then infected with the three recombinant viruses. For most cell line-recombinant combinations, the yields in serum-free medium were equal to or better than those in serum-supplemented medium. Medium composition had a much stronger effect on foreign gene expression than on susceptibility of cells to wild-type virus.
Animals↗