Excessive centrifugal fields damage high density lipoprotein.

HDL is typically isolated ultracentrifugally at 40,000 rpm or greater, however, such high centrifugal forces are responsible for altering the recovered HDL particle. We demonstrate that this damage to HDL begins at approximately 30,000 rpm and the magnitude of loss increases in a rotor speed-dependent manner. The HDL is affected by elevated ultracentrifugal fields resulting in a lower particle density due to the shedding of associated proteins.

Mass spectral analysis of the apolipoproteins on mouse high density lipoproteins. Detection of post-translational modifications.

Using mass spectrometry, we have recently reported on molecular masses of the apolipoproteins associated with porcine and equine HDL. In addition to obtaining accurate masses for the various apolipoproteins, we also were able to detect mass variations due to post-translational modifications. In the present study, we have used these same approaches to characterize the apolipoproteins in two inbred mouse strains, C57BL/6 and BALB/c.

Mass spectral analysis of domestic and wild equine apoA-I and A-II: detection of unique dimeric forms of apoA-II.

In pigs, humans, chimpanzees and probably other great apes, a cysteine at residue 6 enables apolipoprotein A-II to form a homodimer. However, the apoA-IIs of other primates, lacking a cysteine residue, are monomeric. We have already reported that horse apoA-IIs form homodimers due also to a cysteine at residue 6.

Mass spectral analysis of pig (Sus scrofa) apo HDL: Identification of pig apoA-II, a dimeric apolipoprotein.

Comparative studies of mammalian high density lipoproteins have clearly indicated that the major apolipoprotein is apoA-I and in some mammals apoA-II is the second major apolipoprotein. However, in pigs, apoA-II has been considered to be either present in trace amounts or absent. Recently, cDNA sequences for pigs A-II have been entered into the database.

Virtual two-dimensional gel electrophoresis of high-density lipoproteins.

High-density lipoproteins (HDLs) isolated by immunoaffinity chromatography and separated by immobilized pH gradient-isoelectric focusing (IPG-IEF) were examined by mass spectrometry directly, applying a new proteomics technology, virtual two-dimensional (2-D) gel electrophoresis. A preliminary examination of HDL particles has revealed at least 42 unique masses for protein species with isoelectric points between pH 5.47-5.

Sequence of horse (Equus caballus) apoA-II. Another example of a dimer forming apolipoprotein.

Apolipoprotein A-II, the second major apolipoprotein of human HDL, also has been observed in a variety of mammals; however, it is either present in trace amounts or absent in other mammals. In humans and chimpanzee, and probably in other great apes, apoA-II with a cysteine at residue 6 is able to form a homodimer. In other primates as well as other mammals, apoA-II, lacking a cysteine residue, is monomeric.

Rat McA-RH7777 cells efficiently assemble rat apolipoprotein B-48 or larger fragments into VLDL but not human apolipoprotein B of any size.

Studies of truncated apoB peptides in human subjects with familial hypobetalipoproteinemia, as well as of puromycin-generated spectra of nascent apoB peptides in rat and hamster liver, suggest that a minimum size is required for N-terminal fragments of apoB to be efficiently assembled into full-sized VLDL. We report here results of experiments undertaken to examine this phenomenon in greater detail by expressing individual carboxyl-truncated human apoB constructs in McArdle cells. Thus, apoB-29, -32, -37, -42, -47, -53, -70 and full length apoB-100 were transiently expressed in rat McA-RH7777 hepatoma cells, or human apoB-31 and apoB-53 were stably expressed in the same cells, and the secreted VLDL particles were characterized by kinetic gradient ultracentrifugal flotation.

Studying low-density lipoprotein-monoclonal antibody complexes using dynamic laser light scattering and analytical ultracentrifugation.

Monoclonal antibody complexes have proven very useful in the study of low-density lipoproteins (LDLs). Thus, complexes composed of two different monoclonal antibodies, selected from a panel of 11 different antibodies, and LDL have been employed to map apolipoprotein B (apoB) on the surface of the LDL. In this way, apoB was found to surround the LDL as a ribbon with a bow [Chatterton, J.

The apolipoprotein B R3531C mutation. Characteristics of 24 subjects from 9 kindreds.

Familial ligand-defective apolipoprotein B (apoB) is a group of disorders caused by mutations in the apoB gene. In this report the R3531C mutation is characterized further using a monoclonal antibody MB19/dynamic laser light scattering technique to measure ratios of Cys(3531) to normal low density lipoprotein (LDL) particles. All six subjects studied showed a preferential accumulation of particles carrying the defective apoB allotype.

One active C1r subunit is sufficient for the activity of the complement C1 complex: stabilization of C1r in the zymogen form by point mutations.

The binding of C1 (the first component of complement) to immune complexes leads to the autoactivation of C1r through the cleavage of the Arg463-Ile464 bond in the catalytic domain. Spontaneous activation of C1r (and C1) also occurs in the fluid phase, preventing the characterization of the zymogen form of C1r. To overcome this difficulty, the zymogen form of human C1r was stabilized by mutating the Arg in the Arg463-Ile464 bond to Gln.

Expression and characterization of a 159 amino acid, N-terminal fragment of human complement component C1s.

A 159 residue, N-terminal fragment of the human C1s complement component, C1s alpha(159), was expressed in the baculovirus, insect cell system. The protein was abundantly produced 3 days after infection, reaching levels as high as 40 microg/ml in cell culture media. It had a molecular weight of 18,100 (+/-4.

Secretion from cell culture of HDL and VLDL bearing apoB-33 with a large internal deletion.

Rat hepatoma McA-RH7777 cells synthesize and secrete two populations of apoB-containing lipoproteins: a larger, VLDL-sized population floating in the Sf 40-150 range and a smaller, LDL and HDL-sized population. Three permanently transfected cell lines of McA-RH7777 cells secreted (in addition to the endogenous lipoproteins) lipoproteins containing 1) a carboxyl-terminally truncated human apoB-53 (2377 amino acids in length); 2) a carboxyl-terminally truncated human apoB-31 (1420 amino acids in length); or 3) an internally deleted human apoB protein, apoB-18/95, containing a total of 1490 amino acid residues, equivalent in length to an apoB33. The apoB-18/95 protein contained amino acid residues 1-782 joined to 708 residues near the C-terminus of apoB (residues 36364343).

Oligomerization of a 45 kilodalton fragment of diphtheria toxin at pH 5.0 to a molecule of 20-24 subunits.

Diphtheria toxin (DT) is a 58 kDa protein, secreted by lysogenic strains of Corynebacterium diphtheriae, that causes the disease diphtheria in humans. The catalytic (C) domain of DT kills host cells by gaining entry into the cytoplasm and inhibiting protein synthesis. The translocation of the C domain across the endosomal membrane and into the cytoplasm of a host cell is mediated by the translocation (T) domain of DT.

Probing the structure of C1 with an anti-C1s monoclonal antibody: the possible existence of two forms of C1 in solution.

Anti-human C1s monoclonal antibody H1532, a mouse gamma-1-immunoglobulin elicited by a C1r2C1s2 immunogen, appeared to bind to the beta-domain of C1s by electron microscopy. In agreement with this observation, Western blotting demonstrated good binding to unreduced C1s, but no binding to the alpha or gamma-B domains. When added to solutions of the C1r2C1s2 tetramer, HI532 converted the 8.

A single copy of apolipoprotein B-48 is present on the human chylomicron remnant.

Individuals homozygous for the e2 allele encoding apolipoprotein E exhibit a remnant removal defect and accumulate substantial levels of intestinally derived particles containing apolipoprotein B-48 (apoB-48). Such lipoproteins were isolated from the plasma of E2/E2 individuals, and further purified by affinity chromatography using a polyclonal antibody specific for selective binding and removal of apoB-100-containing lipoproteins. The unbound lipoproteins, termed chylomicron remnants, were particles with average hydrated diameters of 31.

The human complement C1 complex has a picomolar dissociation constant at room temperature.

Periodic sampling of serum or reconstituted C1 initially diluted 1/2000 and 1/4000 (that is, to 0.1 and 0.05 nM) into a recombinant C1s-containing solution showed a gradual decline of hemolytic activity until equilibrium was approached, consistent with a simple dissociation, reassociation equilibrium, presumably C1 <--> C1q + C1r2C1s2.

Functional effects of domain deletions in a multidomain serine protease, C1r.

The C1r subcomponent of the first component of complement is a complex, multidomain glycoprotein containing five regulatory or binding modules in addition to the serine protease domain. To reveal the functional role of the N-terminal regulatory domains, two deletion mutants of C1r were constructed. One mutant comprises the N-terminal half of domain I joined to the second half of the highly homologous domain III, resulting in one chimeric domain in the N-terminal region, instead of domains I-III.

Immunoelectron microscopy of low density lipoproteins yields a ribbon and bow model for the conformation of apolipoprotein B on the lipoprotein surface.

In the present study, the relative positions of 11 anti-apolipoprotein B monoclonal antibodies have been mapped onto the surface of human low density lipoproteins by electron microscopy. As the epitopes recognized by these antibodies have been previously located on the primary sequence of apoB, these data provide a map of the configuration of the protein on the surface of the LDL. The first 89% of apoB-100 may be modeled as a thick ribbon that wraps once around the LDL, completing the encirclement by about amino acid residue 4050.

Identification of apolipoprotein B100 polymorphisms that affect low-density lipoprotein metabolism: description of a new approach involving monoclonal antibodies and dynamic light scattering.

Rare mutations in apolipoprotein B (apoB) can cause defective binding of low-density lipoproteins (LDLs) to the LDL receptor, leading to elevated plasma cholesterol levels and premature atherosclerosis. This communication describes a novel approach to study the effects of apoB mutations on LDL metabolism. Monoclonal antibody MB19 identifies a common polymorphism in apoB, an Ile/Thr substitution at residue 71, by binding with a 60-fold higher affinity to apoB(Ile71)-containing LDL.

Structure and function of several anti-dansyl chimeric antibodies formed by domain interchanges between human IgM and mouse IgG2b.

Two pairs of chimeric, domain-switched immunoglobulins with identical murine, anti-dansyl (5-dimethylaminonaphthalene-1-sulfonyl) variable domains have been generated, employing as parent antibodies a human IgM and a mouse IgG2b. The first pair of chimeric antibodies mu mu gamma mu and gamma gamma mu gamma was generated by switching the C mu 3 and C gamma 2 domains between IgM and IgG2b. The second pair of chimeras mu mu gamma gamma and gamma gamma mu mu were formed by switching both C mu 3 and C mu 4 with C gamma 2 and C gamma 3.

Familial ligand-defective apolipoprotein B. Identification of a new mutation that decreases LDL receptor binding affinity.

Detection of new ligand-defective mutations of apolipoprotein B (apoB) will enable identification of sequences involved in binding to the LDL receptor. Genomic DNA from patients attending a lipid clinic was screened by single-strand conformation polymorphism analysis for novel mutations in the putative LDL receptor-binding domain of apoB-100. A 46-yr-old woman of Celtic and Native American ancestry with primary hypercholesterolemia (total cholesterol [TC] 343 mg/dl; LDL cholesterol [LDL-C] 241 mg/dl) and pronounced peripheral vascular disease was found to be heterozygous for a novel Arg3531-->Cys mutation, caused by a C-->T transition at nucleotide 10800.

Analysis of the N-linked oligosaccharides of human C1s using electrospray ionisation mass spectrometry.

Information on the structures of the oligosaccharides linked to Asn residues 159 and 391 of the human complement protease C1s was obtained using mass spectrometric and monosaccharide analyses. Asn159 is linked to a complex-type biantennary, bisialylated oligosaccharide NeuAc2 Gal2 GlcNAc4 Man3 (molecular mass = 2206 +/- 1). Asn391 is occupied by either a biantennary, bisialylated oligosaccharide, or a triantennary, trisialylated species NeuAc3 Gal3 GlcNAc5 Man3 (molecular mass = 2861 +/- 1), or a fucosylated triatennary, trisialylated species NeuAc3 Gal3 GlcNAc5 Man3 Fuc1 (molecular mass = 3007 +/- 1), in relative proportions of approximately 1:1:1.

Human/mouse chimeric monoclonal antibodies with human IgG1, IgG2, IgG3 and IgG4 constant domains: electron microscopic and hydrodynamic characterization.

The unique structure of the human IgG3 constant region with its greatly extended hinge can clearly be seen in electron micrographs, which compare a series of recombinant proteins with the same murine anti-dansyl variable domain but constant domains from human IgG1, IgG2, IgG3 and IgG4. The hinge region of IgG3 was found to be very long, with some measurements extending to 100 A. It exhibited considerable flexibility allowing the Fc to be displaced far toward either side.