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V N Schumaker

Publications and source records attributed to V N Schumaker.

At least 19 recordsLinked to original sources
PubMed1992

Lipoprotein assembly. Apolipoprotein B size determines lipoprotein core circumference.

Apolipoprotein B (apoB) is an essential structural protein for the two triglyceride-rich lipoproteins synthesized by humans: chylomicrons and very low density lipoproteins. Although much is known about the role of apoB in clearance of lipoproteins from the circulation, relatively little is known about its role in the assembly of nascent lipoproteins. Therefore, we have investigated the relationship between the length of various N-terminal apoB fragments and the characteristics of the lipoproteins with which these fragments were associated. After the addition of puromycin, HepG2 cells secreted a discrete series of C-terminally truncated apoB fragments on lipoprotein particles including apoB25, apoB29, apoB31, apoB33, apoB36, apoB38, apoB42, apoB45, apoB49, apoB51, apoB55, apoB70, and apoB80. Also, using plasmids encoding apoB26, apoB33, apoB37, apoB42, and apoB48, C-terminally truncated apoB fragments were expressed and secreted after transient transfection of HepG2 cells. Lipoproteins bearing the metabolically labeled apoB fragments were isolated from the cell culture media and characterized in terms of size, density, flotation coefficient, and composition. Lipoprotein radii, calculated from their flotation coefficients and buoyant densities, were used to derive the circumference of the non-polar core of each lipoprotein species. When plotted as a function of apoB size, core circumference defined a straight line of near-zero intercept. The slope of this line was approximately 1 A of core circumference/1 kDa of apoB molecular mass. A model for the mechanism of lipoprotein assembly in HepG2 cells, consistent with the concept that apoB size determines lipoprotein core circumference, is proposed.

Apolipoproteins B↗
PubMed1992

Recombinant human complement subcomponent C1s lacking beta-hydroxyasparagine, sialic acid, and one of its two carbohydrate chains still reassembles with C1q and C1r to form a functional C1 complex.

In contrast to the human serum protein which is approximately one-half erythro-beta-hydroxyasparagine at asparagine 134 [Theilens et al. (1990) Biochemistry 29, 3570-3578], recombinant C1s expressed by insect cells after infection with recombinant baculovirus entirely lacks posttranslational modification at asparagine 134. It is also incompletely glycosylated, lacking, at least, sialic acid. Site-directed mutagenesis of one of the two sites of carbohydrate attachment (Asn 159 to Gln 159) yields a faster migrating recombinant C1s still abundantly secreted. Furthermore, the mutated protein displays good hemolytic activity when reassembled with C1q and either human serum or recombinant C1r, demonstrating that these posttranslational modifications are not critical for any of the multiple interactions between C1s and C1q, C1r, C2, and C4 required for reassembly of the C1 complex, activation, and initiation of the classical complement pathway. The 4.0S recombinant C1s dimerizes to yield 5.6S C1s2 in the presence of Ca2+ and forms the 9.1S C1s-C1r-C1r-C1s tetramer upon the addition of human serum C1r and the 15.6S C1 complex upon the addition of C1q to the tetramer. The recombinant C1s and human serum C1s have identical N-terminal amino acid sequences, indicating proper recognition by the insect signal peptidase. The recombinant C1s is secreted and isolated as the unactivated zymogen, and it may be activated by human serum C1r which cleaves at Arg422-Ile423 to yield the characteristic heavy and light chains. A very tight complex is formed between C1-inhibitor and the light chain of recombinant C1s.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Identification of a neutral lipid core in a transiently expressed and secreted lipoprotein containing an apoB-48-like apolipoprotein.

The presence of core lipids in lipoproteins expressed and secreted by transfected HepG2 cells was demonstrated by measuring the densities of these lipoproteins before and after treatment with a bacterial lipase specific for neutral lipids. HepG2 cells were reproducibly transfected with pRSV/B48, containing a truncated human apolipoprotein B-100 (apoB-100) cDNA (nucleotides 1 to 6860, where nucleotide 129 is the start of translation). Northern blots of cellular message probed with apoB-48 showed abundant transcription of an apoB-48-sized message as well as endogenous apoB-100 message. When grown in the presence of [35S]methionine, pRSV/B48-transfected cells secreted lipoproteins containing an apoB-48-like apolipoprotein. This lipoprotein banded at a density of 1.11 g/ml in isopycnic NaBr gradients. Electron microscopy of the apoB-48-containing lipoproteins demonstrated spherical particles with an average diameter of 124A. A sedimentation rate of 8.4S was measured by sucrose gradient sedimentation. When the apoB-48-containing particles were treated with a bacterial lipase (from Chromobacterium viscosum), shown to hydrolyze triglycerides and cholesteryl esters but not phospholipids, their density increased to 1.18 g/ml, consistent with removal of core lipids. When the secreted lipoprotein was modeled as a spherical particle containing a single molecule of apoB-48, a triglyceride-filled core, and a surface monolayer of phospholipid and protein, the hydrodynamic properties were consistent with the observed sedimentation coefficient, buoyant densities before and after lipase treatment, and the diameter as seen with the electron microscope. These data indicate that transfected HepG2 cells assembled and secreted lipoproteins possessing the same physical structure as naturally occurring lipoproteins.

Apolipoprotein B-48↗
PubMed1991

Spontaneous activation of serum C1 in vitro. Role of C1 inhibitor.

The temperature and ionic strength dependence of the spontaneous activation of C1 were determined for normal human serum, and the free energy, enthalpy, and entropy of spontaneous activation were calculated. The half-life of C1 in human serum was approximately 15 h at 37 degrees C. This half-life was markedly extended by dilution with C1-depleted serum, and an extrapolated upper limit of 40 to 50 h was reached at infinite dilution. Thus, the spontaneous activation of C1 in serum appeared to involve a dilution-sensitive reaction as well as a dilution-insensitive, first order reaction. A reaction mechanism was developed combining: 1) first order spontaneous activation of C1; 2) second order, C1-catalyzed activation of C1; and 3) second order inactivation of C1 by C1-inhibitor. A steady state equation was derived from this reaction mechanism, which provided a reasonable fit to the experimental data. The equation predicts that when the C1-inhibitor concentration decreases so that the steady state condition is lost, the concentration of C1 builds up quickly, and activation of most of the C1 occurs rapidly.

Complement Activation↗
PubMed1991

Isolation of human complement subcomponents C1r and C1s in their unactivated, proenzyme forms.

We have modified a standard isolation procedure for C1r and C1s, which employs IgG-Sepharose affinity chromatography followed by DEAE chromatography. As usual, all steps were performed at low temperature and two proteolytic inhibitors, PMSF and NPGB, were added during affinity chromatography on IgG-Sepharose. The novel condition was to keep the pH at pH 6.1 during the entire procedure, where activation was markedly depressed. In addition, purification was improved by washing the IgG-Sepharose column with a buffer free of added divalent cations immediately prior to elution of the C1r and C1s with EDTA. The final yields of highly purified C1r and C1s were about 20%; little or no activated material was detected in these highly purified fractions.

Chromatography, Affinity↗
PubMed1991

Measurement of macromolecular interactions between complement subcomponents C1q, C1r, C1s, and immunoglobulin IgM by sedimentation analysis using the analytical ultracentrifuge.

The interactions between the complement components and with immunoglobulins are greatly enhanced by lowering the ionic strength and become readily measurable by physical techniques. Thus, the binding between C1q and IgM was previously shown to be appreciable (k = 1 x 10(6) M-1) at 0.084 M ionic strength (Poon, P.H., Phillips, M.L., and Schumaker, V.N. (1985) J. Biol. Chem. 260, 9357-9365). We have now found that, at 0.128 M ionic strength, the binding between human C1- (the activated first component of complement) and IgM was strong at physiological concentrations (k = 1 x 10(7) M-1), while under the same conditions binding between C1q and IgM was not observed. To explore the nature of the interactions responsible for this enhanced binding by C1- over C1q, mixtures of the various subcomponents of C1- were studied alone and with IgM. C1r2 did not bind to C1q, even when the ionic strength was reduced to 0.098 M, nor did the presence of C1r2 enhance the binding of C1q to IgM. In contrast, two C1s2 independently bound to C1q (k = 1 x 10(6) M-1), and caused a marked increase in its association with IgM (k = 5 x 10(6) M-1) at 0.098 M ionic strength. No detectable interaction was found between C1s2 and/or C1r2 and IgM in the absence of C1q. Moreover, there was no detectable interaction between the C1(-)-like complex formed between C1r2C1s2 and the collagenous C1q stalks (pepsin-digested C1q) and IgM. These data suggest that the binding of C1s2 to C1q, either alone or together with C1r2, induces a conformational change in C1q which results in additional C1q heads binding to complementary sites on IgM.

Complement C1q↗
PubMed1991

Mapping apolipoprotein B on the low density lipoprotein surface by immunoelectron microscopy.

Apolipoprotein B (apoB) was mapped using electron microscopy to visualize pairs of monoclonal antibodies binding to the low density lipoprotein (LDL) surface. The sites at which these monoclonals bind the apoB polypeptide sequence had already been established. The angular distances between all possible pairs of binding sites except one allowed the relative placement of six epitopes on the LDL sphere. We conclude that apoB extends over at least a hemisphere of the LDL surface since four epitopes are located in the Northern Hemisphere at sites arbitrarily designated as the North Pole, the Aleutian Islands, Bogotá, and in the Atlantic Ocean, while two are found in the Southern Hemisphere at Buenos Aires and at Madagascar. ApoB appears to possess a restricted flexibility, since these relative epitope locations show a substantial standard deviation in latitude and longitude. Mapping of additional epitopes may provide an answer to the question of whether apoB circumnavigates the LDL sphere.

Antibodies, Monoclonal↗
PubMed1991

Identification of the ancestral haplotype for apolipoprotein B suggests an African origin of Homo sapiens sapiens and traces their subsequent migration to Europe and the Pacific.

The probable ancestral haplotype for human apolipoprotein B (apoB) has been identified through immunological analysis of chimpanzee and gorilla serum and sequence analysis of their DNA. Moreover, the frequency of this ancestral apoB haplotype among different human populations provides strong support for the African origin of Homo sapiens sapiens and their subsequent migration from Africa to Europe and to the Pacific. The approach used here for the identification of the ancestral human apoB haplotype is likely to be applicable to many other genes.

Africa↗
PubMed1990

Ecdysteroids increase the yield of recombinant protein produced in baculovirus insect cell expression system.

Spodoptera frugiperda cells are the hosts of wild type and recombinant virus in the baculovirus insect cell expression system. The expression of the foreign gene could be enhanced by the addition of ecdysteroids and increased amount of recombinant protein was secreted into the medium. The time and concentration dependence of this effect was followed in the case of 20-hydroxyecdysone-, makisterone and ecdysone. 20-hydroxyecdysone proved to be the most efficient, producing a three fold increase in the level of recombinant protein secreted into the medium, as it was measured by ELISA. This effect was also confirmed by tracing the L-(35S)methionine incorporation into the gene product. Makisterone was also effective in stimulation, while ecdysone proved to be ineffective.

Animals↗
PubMed1990

Rapid typing of apolipoprotein B DNA polymorphisms by DNA amplification. Association between Ag epitopes of human apolipoprotein B-100, a signal peptide insertion/deletion polymorphism, and a 3'flanking DNA variable number of tandem repeats polymorphism of the apolipoprotein B gene.

We present here rapid and efficient methods for the analysis of multiple variable apolipoprotein (apo) B loci using polymerase chain reaction based techniques. For illustrative purposes, we have applied these methods to establish an association between these polymorphisms and the apo B Ag immunological epitopes. The 5 DNA polymorphisms include 3 restriction endonuclease sites (for XbaI, EcoRI and MspI), a variable number of tandem repeat (VNTR) locus at the 3' end of the apo B gene, and an insertion/deletion polymorphism involving the signal peptide region of apo B. The latter two newly described polymorphisms are directly detectable following amplification and may have physiological effects on apo B expression because of their critical locations. All of these sites were typed using flanking oligonucleotides and the newly developed polymerase chain reaction. Amplification products were typed either directly (3' VNTR and signal peptide insertion/deletion alleles), or following specific enzyme digestion (for the restriction sites), or by allele specific oligonucleotides. The detailed methods presented will prove generally useful for rapidly typing DNA variation in the apo B gene. Using these techniques, we found a significant linkage disequilibrium between the Ag(t/z) locus and the 3' VNTR, and the Ag(c/g) locus and the signal peptide length polymorphism. Future association studies using these DNA polymorphisms should take into consideration that observed effects may be related to its linkage disequilibrium with the Ag loci and vice versa.

Alleles↗
PubMed1990

Electron microscopic study of ring-shaped, bivalent hapten, bivalent antidansyl monoclonal antibody complexes with identical variable domains but IgG1, IgG2a and IgG2b constant domains.

We have studied by electron microscopy a fascinating series of antidansyl monoclonal antibodies developed by Dangl et al. (Cytometry 2, 395-401, 1982) which have the same variable domain but different constant domains. Three of the four subclasses of mouse IgG were represented, IgG1, IgG2a and IgG2b. Previously, Oi et al. (Nature 307, 136-140, 1984) had examined the flexibilities of these antibodies by time-resolved fluorescence depolarization and found that IgG1 was least flexible, IgG2a was intermediate and IgG2b was the most flexible. In this communication we examine the conformations of circular complexes formed between these antibodies and a bivalent hapten, bis-dansyl cadaverine. The circular complexes were predominantly composed of two antibodies linked into a ring by two bivalent haptens, and are referred to as dimers, since only the antibody molecules are seen with the electron microscope. A few trimers and an occasional tetramer were also present in these preparations. For the least flexible IgG1, almost all (greater than 99%) of the circular dimers were "open-hinge" complexes with a hinge angle between the Fab arms of 100-120 degrees. For the intermediate IgG2a, most of the dimers were "open-hinge" complexes, but a larger percentage, 4 to 5%, had closed hinges with a hinge angle approaching 0 degrees. For the most flexible IgG2b, over 40% of the dimers were "closed-hinge" complexes. A model is proposed to explain these differences based upon orientation of the hapten in the combining site and differences in hinge structure.

Animals↗
PubMed1989

Expression of hemolytically active human complement component C1r proenzyme in insect cells using a baculovirus vector.

The gene of human C1r has been expressed in a baculovirus-insect-cell system via the pAc373 transplacement vector. The full-length cDNA copy was inserted into the pAc373 vector downstream from the strong polyhedrin promoter of the baculovirus, Autographa californica nuclear polyhedrosis virus (AcNPV). Spodoptera frugiperda cells were cotransfected with the resultant plasmid, pAcC1r, and the wild-type AcNPV DNA. Recombinant viruses, which drove the expression of C1r protein, were selected by plaque morphology and ELISA. Insect cells infected with the recombinant virus produced and secreted human C1r protein, at a level of 1-2 mg/l of medium. The expressed C1r was isolated from the medium by chromatofocusing. On reducing gels only a single Coomassie-staining band was observed, and this band migrated at 80-83 kD characteristic of the unactivated C1r proenzyme. Its identification as C1r was immunologically confirmed on Western blots. C1 reconstituted from purified C1r expressed in insect cells together with human C1q and C1s proved biologically active in a hemolytic assay. Thus, the baculovirus-insect-cell system is capable of expressing and secreting a sophisticated, multifunctional human complement subcomponent in its biologically activatable form.

Animals↗
PubMed1989

Conformation of apolipoprotein B after lipid extraction of low density lipoproteins attached to an electron microscope grid.

We have examined the shape of apolipoprotein B (apoB) from low density lipoproteins (LDL) using a new method to prepare the electron microscope grids. After adsorption of the lipoproteins to a carbon-coated copper grid, lipids were extracted with ethanol-ether 4:1; an aqueous negative stain was then applied. When the LDL residue was examined after this treatment, apoB, together with residual lipid, appeared as an elongated flexible structure about 600-700 A in length consisting of multiple domains of variable width from 20-70 A. Occasionally, the elongated apoB formed an irregular ring-shaped structure, but most of the rings were open. When LDL were pretreated with glutaraldehyde, then adsorbed, extracted, and stained, most of the images were closed rings with an average contour length of 700 A, again consisting of multiple domains of variable sizes. These results are consistent with apoB being composed of multiple domains arranged in an elongated structure on the surface of the LDL, and with distant domains possessing a mutual affinity that favors their cross-linking.

Antibodies, Monoclonal↗
PubMed1989

Low density lipoprotein heterogeneity in the cebus monkey.

In studies of cebus monkey plasma lipoproteins, we have used an ultracentrifugally generated density gradient to isolate two distinct species of low density lipoproteins (LDL). Compositional analyses revealed that each of the ultracentrifugally isolated fractions was enriched in cholesteryl esters and contained a single apolipoprotein which in terms of its mobility on SDS gels corresponded to apolipoprotein B-100, the major apolipoprotein of human LDL. Hydrodynamic measurements carried out in the analytical ultracentrifuge showed that F1.20 values were 30.0 for LDL1 and 23.5 for LDL2. In a solution of density 1.0069 g/ml, the sedimentation rates were 5.9 and 7.2 S for LDL1 and LDL2, respectively. In addition to sedimentation velocity data, we describe a new approach for using these same data to obtain calculated values for molecular weight. The hydrated densities calculated for the two fractions were 1.033 and 1.045 g/ml and calculated molecular weights were 3.08 million for LDL1 and 2.42 million for LDL2. Hydrated density values were in excellent agreement with those calculated from compositional data. Electron microscopy data showed that LDL1 had a larger mean diameter of 26.7 nm than LDL2 which had a diameter of 19.3 nm. Native gel electrophoretic analyses of the two LDL fractions in 3.5% acrylamide showed that, consistent with its size, LDL1 had slower mobility than LDL2.

Animals↗
PubMed1989

Spontaneous activation of reconstituted and serum C1 and the role of C1-inhibitor.

Evidence will be presented that first order, spontaneous activation of solution C1 at 37 degrees C under physiological conditions is a very slow process with a half-life of the order of one day and perhaps considerably longer. In addition, negative evidence will be presented showing that the formation of functionally significant levels of a complex between C1-Inhibitor and unactivated C1 does not occur. Such a complex had been previously postulated to explain the strong inhibition of the spontaneous activation of C1 which was observed upon the addition of C1-Inhibitor. Rather, we shall demonstrate that C1 catalytically activates C1, and that a critical role for C1-inhibitor is to complex with C1 to eliminate this autocatalytic reaction.

Catalysis↗
PubMed1986

Human apolipoprotein B: chromosomal mapping and DNA polymorphisms of hepatic and intestinal species.

Apolipoprotein B (apoB) is the major protein component of low-density and very-low-density lipoproteins. We have recently isolated nonoverlapping cDNA clones for apoB and confirmed their identity by sequence comparisons. We now report the mapping of the human apoB gene (APOB) to the p23-p24 region of chromosome 2 by examination of human-mouse somatic cell hybrids and by in situ hybridization to human chromosomes. Thus, APOB is unlinked to members of the dispersed gene family encoding other apolipoprotein species or to the gene encoding the low-density lipoprotein receptor. Hybridization analysis with genomic DNA and liver and intestinal mRNA suggests that APOB encodes both the high-molecular-weight form of apoB (apoB100) incorporated into very-low-density lipoproteins in liver and the lower-molecular-weight form (apoB48) incorporated into chylomicrons in intestine. Restriction fragment length polymorphisms of APOB have been identified and should prove useful in examining the possibility that genetic variations of APOB are involved in dyslipoproteinemias and atherosclerosis.

Apolipoproteins B↗