Electrophoretic properties of human IgG and its subclasses on sodium dodecyl-sulfate-polyacrylamide gel electrophoresis and immunoblots

Fasler, Stephan; Skvaril, F; Lutz, Hans

doi:10.1016/0003-2697(88)90061-9

Cited by 33 publications

(14 citation statements)

References 20 publications

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“…The occurrence of a band with an apparent molecular mass of 26 kDa on SDS gels (instead of 13 kDa as calculated from the sequenced N terminus) is obviously the consequence of dimer formation. Such aggregates, which remain stable under the conditions of denaturing and reducing electrophoresis, have been described also for other proteins (24). Dimer formation is also supported by the crystallographic analysis; the packing parameters reflect the presence of two molecules in the asymmetric unit.…”

Section: Discussionsupporting

confidence: 57%

Crystallization and Preliminary Diffraction Data of a Major Pollen Allergen

Bufe

Betzel

Schramm

et al. 1996

Journal of Biological Chemistry

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Group V major allergen Phl p 5b of timothy grass pollen induces allergic rhinitis and bronchial asthma in 90% of grass pollen-allergic patients. In addition to its allergenicity ribonuclease activity has recently been attributed to this 29-kDa protein.The allergen was expressed in Escherichia coli and subsequently purified. Spontaneous conversion of these preparations to a mixture of various forms with molecular sizes between 10 and 29 kDa was consistently observed. Surprisingly, crystals could be grown from this heterogenous preparation. Single crystals, redissolved and analyzed by SDS-polyacrylamide gel electrophoresis and immunoblot, yielded one distinct low molecular weight protein, which was identified by amino acid sequencing as the C-terminal 13-kDa portion of the allergen. Histamine release assays with single crystal solutions using basophils of an allergic patient demonstrated allergenicity comparable with that of the holoallergen. By contrast, RNase activity of the crystallized C-terminal form was 23 times higher than that of the full-length parent allergen. Crystals were used to collect preliminary diffraction data; the space group was evaluated to I4 1 22 with cell dimensions of a ‫؍‬ 87.7 Å, b ‫؍‬ 87.7 Å, and c ‫؍‬ 59.6 Å.We conclude that preferential crystal growth of the 13-kDa form is indicative of a compact conformation of this particular C-terminal portion of the allergen. Thus, we show here that protein crystallization is not only a prerequisite for structural analyses, but it also can provide a unique separation technique to localize the functional domain of a major allergen.

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Section: Discussionsupporting

confidence: 57%

Crystallization and Preliminary Diffraction Data of a Major Pollen Allergen

Bufe

Betzel

Schramm

et al. 1996

Journal of Biological Chemistry

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“…3A, lane 5). Heterogeneity of the heavy chain has also been described for human IgG myeloma proteins (Fasler et al, 1988). Three of these polypeptides (70, 66, and 60 kD) appeared to combine a positive staining for glycoproteins with PSL ligand activity (lanes 3 and 4).…”

Section: Rat Ige Has a Glycan Moiety That Binds Pslmentioning

confidence: 67%

Sugar-Binding Activity of Pea Lectin Expressed in White Clover Hairy Roots

et al. 1995

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Dénarié et al., 1992). Nodulation is regulated by a two-way exchange of plant and bacterial signal molecules (Fisher and Long, 1992;Spaink et al., 1993). Signal exchange begins in the rhizosphere. Rhizobia sense flavonoids secreted by seeds and by the host plant root, which activate the constitutively expressed nodD gene product (reviewed by Schlaman et al., 1992). This step is a determinant of host plant specificity in some Rhizobium-host plant combinations (Horvath et al., 1987; Spaink et al., 1987). Transcription of structural nod genes is under the control of NodD and results in production and secretion of specific lipochitin oligosaccharides (Nod factors), which act as host plant-specific inducers of nodule morphogenesis (reviewed by Dénarié and Cullimore, 1993;Spaink et al., 1993). Most probably, Nod factors constitute the primary determinants of host-plant specificity of nodulation.Root lectins are plant factors involved in nodulation. Lectins are nonenzymatic sugar-binding (g1yco)proteins characterized by sugar-binding specificity. For example, root PSL is Glc/Man specific (Díaz et al., 1990) and white clover root lectin is 2-deoxyglucose specific (Sherwood et al., 1984). In general, the interaction between RI uiciae and white clover does not proceed beyond root hair curling, and these bacteria are not able to penetrate host plant cells by infection thread formation (Yao and Vincent, 1969;Djordjevic et al., 1986;Huang et al., 1993). However, RI viciae is able to induce infection thread formation and nodulation in a significant percentage of white clover plants with roots transformed with the psl gene, as reported by Díaz et al. (1989Díaz et al. ( , 1995. This heterologous nodulation does not take place when a psl mutant, which encodes a non-sugar-binding protein, is used for transformation of white clover roots (Van Eijsden et al., 1992. These results strongly suggest that (a) PSL is functionally expressed in transgenic white clover roots, (b) conservation of the sugar-binding activity of PSL is necessary for infection of white clover by RI viciae, and (c) root lectin helps to break a host plant-specific barrier for nodulation. The relationship between Nod factor recognition by host plants and Rhizobium recognition by host lectin is not yet clear.PSL is an ellipsoidal, nonglycosylated dimeric lectin with two sugar-binding sites, each located near the top of two identical monomers (Einspahr et al., 1988). PSL is encoded by a single gene (Gatehouse et al., 1987;Kaminski et al., 1987) and is synthesized as a single translation product, a prolectin with a molecular mass of 28 kD. Posttranslational modifications result in a protein that dissociates into two p subunits (18 kD each) and two 01 subunits (6 kD each, Higgins et al., 1983)

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“…SDS-polyacrylamide gel electrophoresis was performed as outlined previously (11). Samples were reduced for 3 min in a boiling water bath, alkylated, and run at 8% total acrylamide.…”

Section: Methodsmentioning

confidence: 99%

Complement Factor D, Albumin, and Immunoglobulin G Anti-Band 3 Protein Antibodies Mimic Serum in Promoting Rosetting of Malaria-Infected Red Blood Cells

et al. 2007

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Rosetting of Plasmodium falciparum-infected red blood cells (parasitized RBC [pRBC]) with uninfected RBC has been associated in many studies with malaria morbidity and is one form of cytoadherence observed with malarial parasites. Rosetting is serum dependent for many isolates of P. falciparum, including the strains FCR3S1.2 and Malayan Camp studied here. We identified the three naturally occurring components of sera which confer rosetting. Complement factor D alone induced 30 to 40% of de novo rosetting. Its effect was additive to that of 0.5 mg/ml albumin and to that of 15 ng/ml of naturally occurring antibodies to the anion transport protein, band 3. The three components together mediated rosetting as effectively as 10% serum. De novo rosetting experiments showed that naturally occurring anti-band 3 antibodies as well as factor D were effective only when added to pRBC. Factor D appeared to cleave a small fraction of a protein expressed on the surface of pRBC.Cerebral malaria occurs from infections with Plasmodium falciparum, the parasite species that shows sequestration in the periphery. Sequestration is a means by which parasitized red blood cells (pRBC) escape from their clearance from the reticuloendothelial system. It is mediated by cytoadherence and rosetting of pRBC with uninfected RBC. Clinical studies and autopsies have indeed revealed rosettes in blood vessels from malaria victims (19,20,40,43), and parasites isolated from patients with severe disease formed more and larger rosettes than those from uncomplicated cases (4, 47). For many parasite isolates, in vitro rosetting is dependent on serum factors that are also present in nonimmune, healthy persons. Thus, it is more than a coincidence that the mortality from cerebral malaria is highest in very young children, who have not developed immunity against the parasite but have partially established their innate immune systems. The serum factors that have been shown to mediate rosetting are immunoglobulins (15,43,45), albumin (48), and a third so far unidentified small component which does not bind to concanavalin A (45). We embarked on a study of these serum factors in more detail because neither the specificities of the involved innate immunoglobulins (naturally occurring antibodies [NAbs]) nor the role of complement has been investigated. . When the parasitemia reached 5 to 10%, the culture was diluted to achieve 0.2 to 0.5% parasitemia and culture was continued. Twice a month, cultured parasites were enriched for the rosetting phenotype (49). MATERIALS AND METHODSRosette disruption. A rosetting parasite culture containing P. falciparum late stages at a parasitemia of 5 to 10% was centrifuged for 5 min at 500 ϫ g. Cells were washed twice with phosphate-buffered saline (PBS)-glucose (pH 7.4) and resuspended to a 20% hematocrit in PBS-glucose containing 5 mM sodium citrate, and rosettes were disrupted by passing the resuspended cells 20 times through a 23-gauge 0.6-mm-diameter needle. Cells were then washed once with PBS-glucose containing 5 mM sodium ...

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Electrophoretic properties of human IgG and its subclasses on sodium dodecyl-sulfate-polyacrylamide gel electrophoresis and immunoblots

Cited by 33 publications

References 20 publications

Crystallization and Preliminary Diffraction Data of a Major Pollen Allergen

Crystallization and Preliminary Diffraction Data of a Major Pollen Allergen

Sugar-Binding Activity of Pea Lectin Expressed in White Clover Hairy Roots

Complement Factor D, Albumin, and Immunoglobulin G Anti-Band 3 Protein Antibodies Mimic Serum in Promoting Rosetting of Malaria-Infected Red Blood Cells

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