Complement-binding activity of myeloma and normal immune complexes

The comparison of complement-fixing capacity of simulated immune complexes formed by normal IgG and IgG, isolated from serum of patients with multiple myeloma, has been performed. In both cases a non-linear dependence of complement-fixing capacity on the complex molecular mass was demonstrated, it being higher for myeloma proteins. Complement supplementation to high molecular complexes leads to their collapse, with normal immune complexes destroyed at lower molecular masses. Heat-aggregation of myeloma immunoglobulins leads to the formation of simulated immune complexes of lower molecular mass compared to normal proteins.     

Comparison of the complement-binding activity of model immune complexes of different molecular weights

The authors studied and compared the complement-fixing activity of model immune complexes with different molecular mass. The complement-fixing activity of the complexes was found to be linearly independent of the molecular mass, being mainly determined by the size of the complex, and to be slightly dependent on the concentration of aggregated immunoglobulins. As far as the aggregates with a molecular mass over 20 IgG are concerned, addition of complement leads to the dissociation of the complexes.     

Experimental analysis of the complement-binding activity of gamma-globulin]

It was experimentally demonstrated that the anticomplimentary characteristics of gamma-globulin perparations was associated with disturbances of the colloid condition of the serum system and the absence of any stabilizing action of albumin and other serum proteins. It is also expressed as a result of a high complement-binding activity of protein aggregates. The anticomplementary characteristics of the nonaggregated part of protein in commercial preparations of gamma-globulin could be depressed by the addition of albumin or fresh serum; as to the anticomplementary characteristics of protein aggregates -- it remains unchanged. An intermolecular electrostatic interaction exists between albumin and gamma-globulin; it prevents sorption of the complement on Fc-fragment of IgG, whose destruction leads to the manifestation of the gamma-globulin complement-binding activity.     

Circulating immune complexes in pre-eclampsia.

Sixteen patients with severe pre-eclampsia had more IgG-containing and C1q-binding circulating soluble immune complexes than did 16 matched women with normal pregnancies. The clinical features of preeclampsia may be explained by damage due to such complexes, although raised complex levels were also present in a few women with normal pregnancies. As immune complexes are so heterogenous in terms of the type of antigen, class and subclass of immunoglobulin, size, and complement-binding capacity, further investigations are needed to determine their role in normal and pre-eclamptic pregnancies.     

The role of the immunoglobin G "hinge" region in heat aggregation and activation of complex-formation

A high degree of correlation between the capability of subclasses of human immunoglobulins G to form aggregates due to thermal treatment, and their complement-binding activity was established. On the basis of the experimental data obtained by the methods of light scattering, circular dichroism, microcalorimetry, it was supposed that "hinge" region of immunoglobulins G participates in the initial stage of thermal aggregation and in the activation of the process of complement binding.     

The proteome of neurofilament-containing protein aggregates in blood

Protein aggregation in biofluids is a poorly understood phenomenon. Under normal physiological conditions, fluid-borne aggregates may contain plasma or cell proteins prone to aggregation. Recent observations suggest that neurofilaments (Nf), the building blocks of neurons and a biomarker of neurodegeneration, are included in high molecular weight complexes in circulation. The composition of these Nf-containing hetero-aggregates (NCH) may change in systemic or organ-specific pathologies, providing the basis to develop novel disease biomarkers. We have tested ultracentrifugation (UC) and a commercially available protein aggregate binder, Seprion PAD-Beads (SEP), for the enrichment of NCH from plasma of healthy individuals, and then characterised the Nf content of the aggregate fractions using gel electrophoresis and their proteome by mass spectrometry (MS). Western blot analysis of fractions obtained by UC showed that among Nf isoforms, neurofilament heavy chain (NfH) was found within SDS-stable high molecular weight aggregates. Shotgun proteomics of aggregates obtained with both extraction techniques identified mostly cell structural and to a lesser extent extra-cellular matrix proteins, while functional analysis revealed pathways involved in inflammatory response, phagosome and prion-like protein behaviour. UC aggregates were specifically enriched with proteins involved in endocrine, metabolic and cell-signalling regulation. We describe the proteome of neurofilament-containing aggregates isolated from healthy individuals biofluids using different extraction methods.     

Mixed IgA-IgG aggregates as a model of immune complexes in IgA nephropathy

Patients with IgA nephropathy have circulating immune complexes containing IgA, IgG, and C3. We have mixed human IgG and IgA1 and heated them to form mixed aggregate. On sucrose density gradients IgG aggregates were 11 to 19S whereas IgA aggregates were either 11S or greater than 19S. Mixed aggregates had both an 19 and 11 S peak. The isoelectric point of aggregates with only IgG was 7 to 9 and of only IgA 4.5 to 5.5. The isoelectric point of mixed aggregates decreased as the percent IgA increased. IgG aggregates mixed with normal human serum caused 30% C3 activation (20 min, 37 degrees C) whereas IgA aggregates causes no activation. There was a linear decrease in C3 activation as the percent IgA increased. Mixed aggregates that contained either radiolabeled IgG or IgA were mixed with normal human serum (1 h, 37 degrees C) and then solubilized, reduced, and separated by 10% SDS-PAGE. Heavy m.w. bands, consistent with covalent bonding of C3b and C3bi to Ig H chain were only seen in lanes with labeled IgG. This was confirmed by Western blot analysis. A human dimeric IgA1 myeloma protein with rheumatoid factor activity was also studied. It caused 15% alternative pathway C3 activation but did not fix C3 to its H chain. Binding of aggregates (+/- C3) to E was tested. Aggregates with IgG C3 bound but IgA (+/- C3) did not. Addition of greater than 10% IgA to an IgG-C3 aggregate inhibited E binding. We conclude that IgG in mixed aggregates is the site of C3 fixation. In contrast, IgA does not fix C3 but instead lowers the isoelectric point, increases the size and inhibits binding to E. These properties would inhibit clearance and promote mesangial deposition and local C activation.     

Determination of aberrant O-glycosylation in the IgA1 hinge region by electron capture dissociation fourier transform-ion cyclotron resonance mass spectrometry

In a number of human diseases of chronic inflammatory or autoimmune character, immunoglobulin molecules display aberrant glycosylation patterns of N- or O-linked glycans. In IgA nephropathy, IgA1 molecules with incompletely galactosylated O-linked glycans in the hinge region (HR) are present in mesangial immunodeposits and in circulating immune complexes. It is not known whether the Gal deficiency in IgA1 proteins occurs randomly or preferentially at specific sites. To develop experimental approaches to address this question, the synthetic IgA1 hinge region and hinge region from a naturally Gal-deficient IgA1 myeloma protein have been analyzed by 9.4 tesla Fourier transform-ion cyclotron resonance mass spectrometry. Fourier transform-ion cyclotron resonance mass spectrometry offers two complementary fragmentation techniques for analysis of protein glycosylation by tandem mass spectrometry. Infrared multiphoton dissociation of isolated myeloma IgA1 hinge region peptides confirms the amino acid sequence of the de-glycosylated peptide and positively identifies a series of fragments differing in O-glycosylation. To localize sites of O-glycan attachment, synthetic IgA1 HR glycopeptides and HR from a naturally Gal-deficient polymeric IgA1 myeloma protein were analyzed by electron capture dissociation and activated ion-electron capture dissociation. Multiple sites of O-glycan attachment (including sites of Gal deficiency) in myeloma IgA1 HR glycoforms were identified (in all but one case uniquely). These results represent the first direct identification of multiple sites of O-glycan attachment in IgA1 hinge region by mass spectrometry, thereby enabling future characterization at the molecular level of aberrant glycosylation of IgA1 in diseases such as IgA nephropathy.     

Analysis of Lupinus albus heat-shock granule proteins in response to high temperature stress

An important aspect of heat-shock response of lupin (Lupinus albus cv. Rio Maior) is the formation of cytoplasmic granular aggregates, called heat-shock granules (HSGs). In this study, two-dimensional electrophoresis (2-DE) was used to detect the component proteins of HSG complexes formed in vivo. Evaluation of 2-DE revealed differential expression of several proteins under heat shock conditions when compared with control. Among them, small heat-shock proteins (sHSPs) of 15 to 30 kDa were found to be the major representative proteins along with other proteins of relative molecular mass ranging from 36 to 45 kDa and above.     

Mechanism of cell transfection with plasmid/chitosan complexes

Chitosan is useful as a non-viral vector for gene delivery. Although there are several reports supporting the use of chitosan for gene delivery, studies regarding effects on transfection and the chitosan-specific transfection mechanism remain insufficient. In this report, the level of expression with plasmid/chitosan was observed to be no less than that with plasmid/lipofectin complexes in SOJ cells. The transfection mechanism of plasmid/chitosan complexes as well as the relationship between transfection activity and cell uptake was analyzed by using fluorescein isothiocyanate-labeled plasmid and Texas Red-labeled chitosan. In regard to effects on transfection, there were several factors to affect transfection activity and cell uptake, for example: the molecular mass of chitosan, stoichiometry of complex, as well as serum concentration and pH of transfection medium. The level of transfection with plasmid/chitosan complexes was found to be highest when the molecular mass of chitosan was 40 or 84 kDa, ratio of chitosan nitrogen to DNA phosphate (N/P ratio) was 5, and transfection medium contained 10% serum at pH 7.0. We also investigated the transfection mechanism, and found that plasmid/chitosan complexes most likely condense to form large aggregates (5-8 microm), which absorb to the cell surface. After this, plasmid/chitosan complexes are endocytosed, and possibly released from endosomes due to swelling of lysosomal in addition to swelling of plasmid/chitosan complex, causing the endosome to rupture. Finally, complexes were also observed to accumulate in the nucleus using a confocal laser scanning microscope.     

Protein translocation in the endoplasmic reticulum. Ultraviolet light induces the noncovalent association of nascent peptides with translocon proteins.

We have photolyzed cell-free translation systems synthesizing beta-lactamase with 254-nm ultraviolet light. In the presence of canine rough microsomes (RM), incomplete chains of beta-lactamase became enriched relative to the full-length molecule in pellet fractions obtained following photolysis and alkaline carbonate extraction. In addition, high molecular weight aggregates were present on SDS-polyacrylamide gels and occurred only when translocation-competent microsomal membranes were used in translation mixtures. The incomplete chains and high molecular weight aggregates were not obtained when RM were inactivated by reaction with N-ethylmaleimide. The incomplete chains did not bind to concanavalin A-Sepharose, indicating that they had not sedimented as a result of being covalently cross-linked to membrane glycoproteins. Both photolysis and alkaline carbonate extraction were required to produce the results. Nascent peptides that were not exposed to alkaline carbonate following photolysis did not appear as high molecular weight bands on SDS-polyacrylamide gels. The high molecular weight aggregates therefore represent denatured protein complexes that contain nascent peptides and microsomal translocon proteins. The results suggest that the translocon is a large proteinaceous complex and that at least a portion of it, when denatured, migrates at a molecular mass of approximately 205 kDa.     

Investigation of aggregation and assembly of alkali lignin using iodine as a probe

Molecular iodine has been introduced into the alkali lignin (AL) solutions to adjust the π-π aggregation, and the effect of lignin-iodine complexes on the aggregation and assembly characteristics of AL have been investigated by using fluorescence, UV-vis spectroscopy, light scattering, and viscometric techniques. Results show that AL form π-π aggregates (i.e., J-aggregates) in THF driven by the π-π interaction of the aromatic groups in AL, and the π-π aggregates undergo disaggregation in THF-I(2) media because of the formation of lignin-iodine charge-transfer complexes. By using iodine as a probe to investigate the aggregation behaviors and assembly characteristics, it is estimated that about 18 mol % aromatic groups of AL form π-π aggregates in AL molecular aggregates. When molecular iodine is introduced into the AL solutions, lignin-iodine complexes occur with charge-transfer transition from HOMO of the aromatic groups of AL to the LUMO of iodine. The formation of lignin-iodine complexes reduces the affinity of the aromatic groups approaching each other due to the electrostatic repulsion and then eliminates the π-π interaction of the aromatic groups. The disaggregation of the π-π aggregates brings a dissociation behavior of AL chains and a pronounced molecular expansion. This dissociation behavior and molecular expansion of AL in the dipping solutions induce a decrease in the adsorbed amount and an increase in the adsorption rate, when AL is transferred from the dipping solution to the self-assembled adsorbed films. Consequently, the adsorption behavior of AL can be controlled by adjusting the π-π aggregation. Above observations give insight into the occurrence of J-aggregation of the aromatic groups in the AL molecular aggregates and the disaggregation mechanism of AL aggregates induced by the lignin-iodine complexes for the first time. The understanding can provide an academic instruction in the efficient utilization of the alkali lignin from the waste liquor and also leads to further development in expanding functionalities of the aromatic compounds through manipulation of the π-π aggregation.     

How do surfactants and DTT affect the size, dynamics, activity and growth of soluble lysozyme aggregates?

The early intermediates in the protein aggregation pathway, the elusive soluble aggregates, play a pivotal role in growth and maturation of ordered aggregates such as amyloid fibrils. Blocking the growth of soluble oligomers is an effective strategy to inhibit aggregation. To decipher the molecular mechanisms and develop better strategies to arrest aggregation, it is imperative to understand how the size, molecular dynamics, activity and growth kinetics of soluble aggregates are affected when aggregation is inhibited. With this objective, in the present study we have investigated the influence of additives such as SDS, CTAB (cetyltrimethylammonium bromide) and DTT (dithiothreitol) on the slow aggregation of HEWL (hen eggwhite lysozyme) at pH 12.2. For this purpose, techniques such as steady-state and time-resolved fluorescence anisotropy of covalently labelled dansyl probe, gel-filtration chromatography, estimation of free thiol groups, thioflavin T and ANS (8-anilinonaphthalene-1-sulfonic acid) fluorescence, CD and atomic-force microscopy were employed to monitor the soluble oligomers over a period spanning 30 days. The results of the present study reveal that: (i) the spontaneous formation of soluble aggregates is irreversible and abolishes activity; (ii) the initial growth of aggregates (0-24 h) is promoted by a gradual increase in the exposure of hydrophobic surfaces; (iii) subsequently intermolecular disulfide bonds are critical for the assembly and stability of aggregates; (iv) the tight molecular packing inside large aggregates which contributed to slow (approximately 5 ns) and restricted segmental motion of dansyl probe was clearly loosened up in the presence of additives, enabling fast (1-2 ns) and free motion (unlike DTT, the size of lysozyme complexes with surfactants, was large, due to a conglomeration of proteins and surfactants); (v) the aggregates show reduced helical content compared with native lysozyme, except in the presence of SDS; and (vi) DTT was more potent than SDS/CTAB in arresting the growth of aggregates.     

Stimulant of antibody producers isolated from the supernatants of cell cultures of normal human bone marrow and in various diseases

The biological activity of a stimulant of antibody producers (SAP) isolated from normal human bone marrow was studied and compared with that of a stimulant of antibody producers from the bone marrow of patients with acute myeloblastic leukemia, acute lymphoblastic leukemia, lymphosarcoma, and multiple myeloma. The activity of the SAP from human bone marrow in health was similar to that of analogous transmitters from the bone marrow of other species of the mammals and birds. The activity of the SAP in patients with multiple myeloma was elevated, whereas in patients with acute myeloblastic leukemia, it was lowered.     

Mobility of sodium dodecyl sulphate - protein complexes.

Reduced and unreduced lysozyme aggregates formed by formaldehyde cross-linking comprise a set of model compounds for studying the effects of protein conformation on the electrophoretic mobilities of sodium dodecyl sulphate-protein complexes. The reduced aggregates were indistinguisable from normal proteins, but the unreduced aggregates migrated anomalously fast by about 14%. Contrary to expectations, plots of logarithm Rf versus Kr (retardation coefficient) failed to reveal an unusual conformation for the unreduced aggregates. Thus the anomalous mobility caused by several intramolecular disulphide bonds escaped detection by the above two diagnostic plots. Also included in this paper is a discussion of the implications of these results with regard to current models for sodium dodecyl sulphate-protein complexes.     

Partial purification and characterization of the components of the neutral ribonuclease II-inhibitor system of normal and distrophic mouse skeletal muscle

The complexes between a proteinaceous inhibitor and neutral ribonuclease II (EC 3.127.5) purified from low ionic strength extracts of normal and dystrophic mouse muscle are essentially indistinguishable in (a) purification behavior, (b) apparent molecular weights of approximately 50 000, (c) thermal denaturation (50% loss of activity in 5 min at 73.5 degrees C), (d) isoelectric points (pH 4.8), and (e) procedures for reversible resolution into free inhibitor and free RNase II. The free RNase II species are also similar whether obtained by resolution of the purified complexes or by direct isolation of free enzyme from dystrophic muscle. All have apparent molecular weights of 11 500 compared with 13 700 for bovine pancreatic RNase A; all retain 80% of activity after 5 min at 95 degrees C. The active RNase II prepared directly from muscle, by resolution of inhibitor complexes or by organic mercurial treatment of the inhibitor complexes, all have identical pH-activity profiles in 200 mM KC1 with an optimum near pH 7.0. In comparison RNase A has an optimum pH near 7.5 and its activity decreases more rapidly as KC1 concentration is increased above 50 mM KC1. RNase II inhibitor obtained by resolution of the purified complexes or by direct isolation in the free form from normal muscle extracts has an apparent molecular weight of 42 000 and is very sensitive to heat; it loses all activity at 40 degrees C in 5 min. These studies (a) provide methods for obtaining useful amounts of the components of the neutral RNase II - inhibitor system from muscle, (b) provide the first method reported for the reversible resolution of RNase II - inhibitor complexes, (c) fail to show any distinct difference between corresponding components of the system from normal and dystrophic mice, (d) establish interesting differences between the apparently homologous enzymes, murine muscle neutral RNase II, and bovine pancreatic RNase A, and (e) provide a substantially lower molecular weight estimate for RNase II inhibitor from muscle than has been reported for the inhibitor from liver, kidney, and placenta.     

Some characteristics of aggregates of IgG and plasma proteins in heat-treated factor VIII concentrates

Eight batches of commercial heat-treated and one untreated factor VIII concentrate from 6 producers were analyzed for their content of IgG, IgG subclasses, IgG aggregates and the presence of other plasma proteins combined with the IgG as well as for anticomplement activity. Methods used were thin-layer gel filtration, immuno-gel filtration, spot immuno-precipitate assay in a double antibody version and an agarose plate haemolysis inhibition assay of complement fixation. The IgG content varied from 0.1-6.90 g/l. In all preparations IgG existed as monomers and aggregates. Associated with the IgG were also found, at significantly increased amounts compared to normal serum and intravenous immunoglobulin, one to four of the following plasma proteins; fibronectin, fibrinogen, von Willebrand factor antigen, Clq, albumin and IgA. Three batches from two producers had high anticomplementary activity, presumably caused by the IgG aggregates. Two of these deviated strikingly from normal human serum pools in percent distribution of IgG subclasses. It is hypothesized that these aggregates can induce side effects or cause immunological aberrations.     

Trifluoperazine inhibits formation of adhesion plaque complexes and cell sorting in aggregates of fibroblasts

Trifluoperazine (TFP) at 5μM completely blocks the formation of adhesion plaque complexes (adhaerens junctions) between aggregating fibroblasts; the drug at this same concentration did not prevent the cells from producing aggregates of normal size and appearance. Implicit in this finding is that aggregation does not rely on adhesion plaque complex formation. When thymidine-³ H labelled 16C and unlabelled BHK fibroblast cells were experimentally combined to form aggregates in which the cells were initially uniformly distributed, the 16C cells, which produced adhesion plaque complexes within minutes and in greater numbers than did BHK cells, congregated in an internal position after the aggregates had been cultured for 12h. This redistribution of the cells, indicated by the positioning of the labelled 16C nuclei, did not occur when the aggregates were exposed to TFP. Thus cell sorting, unlike aggregation, seems to be reliant on the formation of adhesion plaque complexes.