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1.
Valentina A. Spiteri James Doutch Robert P. Rambo Jayesh Gor Paul A. Dalby Stephen J. Perkins 《Biophysical journal》2021,120(9):1814-1834
The human immunoglobulin G (IgG) class is the most prevalent antibody in serum, with the IgG1 subclass being the most abundant. IgG1 is composed of two Fab regions connected to a Fc region through a 15-residue hinge peptide. Two glycan chains are conserved in the Fc region in IgG; however, their importance for the structure of intact IgG1 has remained unclear. Here, we subjected glycosylated and deglycosylated monoclonal human IgG1 (designated as A33) to a comparative multidisciplinary structural study of both forms. After deglycosylation using peptide:N-glycosidase F, analytical ultracentrifugation showed that IgG1 remained monomeric and the sedimentation coefficients s020,w of IgG1 decreased from 6.45 S by 0.16–0.27 S. This change was attributed to the reduction in mass after glycan removal. X-ray and neutron scattering revealed changes in the Guinier structural parameters after deglycosylation. Although the radius of gyration (RG) was unchanged, the cross-sectional radius of gyration (RXS-1) increased by 0.1 nm, and the commonly occurring distance peak M2 of the distance distribution curve P(r) increased by 0.4 nm. These changes revealed that the Fab-Fc separation in IgG1 was perturbed after deglycosylation. To explain these changes, atomistic scattering modeling based on Monte Carlo simulations resulted in 123,284 and 119,191 trial structures for glycosylated and deglycosylated IgG1 respectively. From these, 100 x-ray and neutron best-fit models were determined. For these, principal component analyses identified five groups of structural conformations that were different for glycosylated and deglycosylated IgG1. The Fc region in glycosylated IgG1 showed a restricted range of conformations relative to the Fab regions, whereas the Fc region in deglycosylated IgG1 showed a broader conformational spectrum. These more variable Fc conformations account for the loss of binding to the Fcγ receptor in deglycosylated IgG1. 相似文献
2.
Ruodan Nan Stuart Tetchner Elizabeth Rodriguez Po-Jung Pao Jayesh Gor Imre Lengyel Stephen J. Perkins 《The Journal of biological chemistry》2013,288(26):19197-19210
The sub-retinal pigment epithelial deposits that are a hallmark of age-related macular
degeneration contain both C3b and millimolar levels of zinc. C3 is the central protein of
complement, whereas C3u is formed by the spontaneous hydrolysis of the thioester bridge in C3.
During activation, C3 is cleaved to form active C3b, then C3b is inactivated by Factor I and Factor
H to form the C3c and C3d fragments. The interaction of zinc with C3 was quantified using analytical
ultracentrifugation and x-ray scattering. C3, C3u, and C3b associated strongly in >100
μm zinc, whereas C3c and C3d showed weak association. With zinc, C3 forms soluble
oligomers, whereas C3u and C3b precipitate. We conclude that the C3, C3u, and C3b association with
zinc depended on the relative positions of C3d and C3c in each protein. Computational predictions
showed that putative weak zinc binding sites with different capacities exist in all five proteins,
in agreement with experiments. Factor H forms large oligomers in >10 μm zinc. In
contrast to C3b or Factor H alone, the solubility of the central C3b-Factor H complex was much
reduced at 60 μm zinc and even more so at >100 μm zinc. The
removal of the C3b-Factor H complex by zinc explains the reduced C3u/C3b inactivation rates by zinc.
Zinc-induced precipitation may contribute to the initial development of sub-retinal pigment
epithelial deposits in the retina as well as reducing the progression to advanced age-related
macular degeneration in higher risk patients. 相似文献
3.
Lee YC Flora R McCafferty JA Gor J Tsaneva IR Perkins SJ 《Journal of molecular biology》2003,333(4):677-682
In the context of the bacterial RuvABC system, RuvA protein binds to and is involved in the subsequent processing of a four-way DNA structure called Holliday junction that is formed during homologous recombination. Four crystal structures of RuvA from Escherichia coli (EcoRuvA) showed that it was tetrameric, while neutron scattering and two other crystal structures for RuvA from Mycobacterium leprae (MleRuvA) and EcoRuvA showed that it was an octamer. To clarify this discrepancy, sedimentation equilibrium experiments by analytical ultracentrifugation were carried out and the results showed that MleRuvA existed as a tetramer-octamer equilibrium between 0.2-0.5 mg/ml in 0.1 M NaCl with a dissociation constant of 4 muM, and is octameric at higher concentrations. The same experiments in 0.3 M NaCl showed that MleRuvA is a tetramer up to 3.5 mg/ml, indicating that salt bridges are involved in octamer formation. Sedimentation equilibrium experiments with EcoRuvA showed that it was tetrameric at low concentration in both salt buffers but the protein was insoluble at high-protein concentrations in 0.1 M NaCl. It is concluded that free RuvA exists in an equilibrium between tetrameric and octameric forms in the typical concentration range and buffer found in bacterial cells. 相似文献
4.
Goraczko AJ 《Journal of molecular modeling》2005,11(4-5):271-277
The location of the most abundant peak of the molecular-ion pattern often differs from the molecular mass published in scientific databases. The location is also distinct from the value expected from average atomic masses. The cause of this phenomenon is a large number of atoms of carbon, sulfur, chlorine, bromine, silicon and boron. This due to the natural isotope abundances of some elements forming organic compounds. A parameter called location of the most abundant peak of an isotopometric cluster (LAPIC) denotes the location of the most abundant (the main) peak of an isotopomeric cluster, which is determined, e.g., by mass spectrometry and can be important for medium- and high-molecular mass compounds. The equations for LAPIC calculation are presented for elements usually observed in organic compounds. The LAPIC with elemental formula helps effectively, e.g., in mass spectra interpretation since the prediction of LAPIC allows the correct connection of the main peak of the investigated ion with the expected ion formula and the mass of the ion considered. This solution can be a substitute for the much more complex method of isotopometric analysis applied in mass spectra interpretation. [Figure: see text]. Differences of the most abundant peak location (Delta LAPIC(C)=f(n)) for carbon aggregates C(n). 相似文献
5.
Lee Y Haas KM Gor DO Ding X Karp DR Greenspan NS Poe JC Tedder TF 《Journal of immunology (Baltimore, Md. : 1950)》2005,175(12):8011-8023
C3d can function as a molecular adjuvant by binding CD21 and thereby enhancing B cell activation and humoral immune responses. However, recent studies suggest both positive and negative roles for C3d and the CD19/CD21 signaling complex in regulating humoral immunity. To address whether signaling through the CD19/CD21 complex can negatively regulate B cell function when engaged by physiological ligands, diphtheria toxin (DT)-C3d fusion protein and C3dg-streptavidin (SA) complexes were used to assess the role of CD21 during BCR-induced activation and in vivo immune responses. Immunization of mice with DT-C3d3 significantly reduced DT-specific Ab responses independently of CD21 expression or signaling. By contrast, SA-C3dg tetramers dramatically enhanced anti-SA responses when used at low doses, whereas 10-fold higher doses did not augment immune responses, except in CD21/35-deficient mice. Likewise, SA-C3dg (1 microg/ml) dramatically enhanced BCR-induced intracellular calcium concentration ([Ca2+]i) responses in vitro, but had no effect or inhibited [Ca2+]i responses when used at 10- to 50-fold higher concentrations. SA-C3dg enhancement of BCR-induced [Ca2+]i responses required CD21 and CD19 expression and resulted in significantly enhanced CD19 and Lyn phosphorylation, with enhanced Lyn/CD19 associations. BCR-induced CD22 phosphorylation and Src homology 2 domain-containing protein tyrosine phosphatase-1/CD22 associations were also reduced, suggesting abrogation of negative regulatory signaling. By contrast, CD19/CD21 ligation using higher concentrations of SA-C3dg significantly inhibited BCR-induced [Ca2+]i responses and inhibited CD19, Lyn, CD22, and Syk phosphorylation. Therefore, C3d may enhance or inhibit Ag-specific humoral immune responses through both CD21-dependent and -independent mechanisms depending on the concentration and nature of the Ag-C3d complexes. 相似文献
6.
Azubuike I. Okemefuna Lasse Stach Sudeep Rana Akim J. Ziai Buetas Jayesh Gor Stephen J. Perkins 《The Journal of biological chemistry》2010,285(2):1041-1052
C-reactive protein (CRP) is an acute phase protein of the pentraxin family that binds ligands in a Ca2+-dependent manner, and activates complement. Knowledge of its oligomeric state in solution and at surfaces is essential for functional studies. Analytical ultracentrifugation showed that CRP in 2 mm Ca2+ exhibits a rapid pentamer-decamer equilibrium. The proportion of decamer decreased with an increase in NaCl concentration. The sedimentation coefficients s20,w0 of pentameric and decameric CRP were 6.4 S and in excess of 7.6 S, respectively. In the absence of Ca2+, CRP partially dissociates into its protomers and the NaCl concentration dependence of the pentamer-decamer equilibrium is much reduced. By x-ray scattering, the radius of gyration RG values ranged from 3.7 nm for the pentamer to above 4.0 nm for the decamer. An averaged KD value of 21 μm in solution (140 mm NaCl, 2 mm Ca2+) was determined by x-ray scattering and modeling based on crystal structures for the pentamer and decamer. Surface plasmon resonance showed that CRP self-associates on a surface with immobilized CRP with a similar KD value of 23 μm (140 mm NaCl, 2 mm Ca2+), whereas CRP aggregates in low salt. It is concluded that CRP is reproducibly observed in a pentamer-decamer equilibrium in physiologically relevant concentrations both in solution and on surfaces. Both 2 mm Ca2+ and 140 mm NaCl are essential for the integrity of CRP in functional studies and understanding the role of CRP in the acute phase response. 相似文献
7.
Polymorphisms in factor H (FH), a major regulator of complement activation, and the accumulation of high zinc concentrations in the outer retina are both associated with age-related macular degeneration. FH is inhibited by zinc, which causes FH to aggregate. To investigate this, we quantitatively studied zinc-induced FH self-association by X-ray scattering and analytical ultracentrifugation to demonstrate uncontrolled FH oligomerisation in conditions corresponding to physiological levels of FH and pathological levels of zinc in the outer retina. By scattering, FH at 2.8-7.0 μM was unaffected until [Zn] increased to 20 μM, whereupon the radius of gyration, RG, values increased from 9 to 15 nm at [Zn] = 200 μM. The maximum dimension of FH increased from 32 to 50 nm, indicating that compact oligomers had formed. By ultracentrifugation, size-distribution analyses showed that monomeric FH at 5.57 S was the major species at [Zn] up to 60 μM. At [Zn] above 60 μM, a series of large oligomers were formed, ranging up to 100 S in size. Oligomerisation was reversed by ethylenediaminetetraacetic acid. Structurally distinct large oligomers were observed for Cu, while Ni, Cd and Fe showed low amounts of oligomers and Mg and Ca showed no change. Fluid-phase assays showed reduced FH activities that correlated with increased oligomer formation. The results were attributed to different degrees of stabilisation of weak self-dimerisation sites in FH by transition metals. The relevance of metal-induced FH oligomer formation to complement regulation and age-related macular degeneration is discussed. 相似文献
8.
Vibet S Goupille C Bougnoux P Steghens JP Goré J Mahéo K 《Free radical biology & medicine》2008,44(7):1483-1491
Docosahexaenoic acid (DHA, a lipid of marine origin) has been found to enhance the activity of several anticancer drugs through an oxidative mechanism. To examine the relation between chemosensitization by DHA and tumor cells antioxidant status, we used two breast cancer cell lines: MDA-MB-231, in which DHA increases sensitivity to doxorubicin, and MCF-7, which does not respond to DHA. Under these conditions, reactive oxygen species (ROS) level increased on anthracycline treatment only in MDA-MB-231. This was concomitant with a decreased cytosolic glutathione peroxidase (GPx1) activity, a crucial enzyme for protection against hydrogen and lipid peroxides, while major antioxidant enzyme activities increased in both cell lines in response to ROS. GPx-decreased activity was accompanied by an accumulation of glutathione, the GPx cosubstrate, and resulted from a decreased amount of GPx protein. In rat mammary tumors, when a DHA dietary supplementation led to an increased tumor sensitivity to anthracyclines, GPx1 activity was similarly decreased. Furthermore, vitamin E abolished both DHA effects on chemotherapy efficacy enhancement and on GPx1 inhibition. Thus, loss of GPx response to an oxidative stress in transformed cells may account for the ability of peroxidizable targets such as DHA to enhance tumor sensitivity to ROS-generating anticancer drugs. 相似文献
9.
Andrzej J. Gorączko 《Journal of molecular modeling》2009,15(6):759-764
The presence of doubly charged ions in mass spectra is detected only occasionally because their clusters are observed more
rarely than singly charged ones. The patterns connected with doubly charged ions are located in the spectrum below M/2. The
narrow shapes of such patterns as well as overlapping with other bands generate significant problems in their interpretation.
The method described here is based on modelling of the isotopomeric form of single- and double-charged mass ion clusters.
The present work attempts to explain the generation of the double charge disotopomeric patterns of high- as well as low-resolution
spectra. Predicting the high-resolution mass cluster is simpler than calculations of the low-resolution cluster. The high-resolution
cluster may represent the initial form of low-resolution pattern formation.
相似文献
Andrzej J. GorączkoEmail: |
10.
Nan R Farabella I Schumacher FF Miller A Gor J Martin AC Jones DT Lengyel I Perkins SJ 《Journal of molecular biology》2011,408(4):714-735
The Tyr402His polymorphism of complement factor H (FH) with 20 short complement regulator (SCR) domains is associated with age-related macular degeneration (AMD). How FH contributes to disease pathology is not clear. Both FH and high concentrations of zinc are found in drusen deposits, the key feature of AMD. Heterozygous FH is inhibited by zinc, which causes FH to aggregate. Here, zinc binding to homozygous FH was studied. By analytical ultracentrifugation, large amounts of oligomers were observed with both the native Tyr402 and the AMD-risk His402 homozygous allotypes of FH and both the recombinant SCR-6/8 allotypes with Tyr/His402. X-ray scattering also showed that both FH and SCR-6/8 allotypes strongly aggregated at > 10 μM zinc. The SCR-1/5 and SCR-16/20 fragments were less likely to bind zinc. These observations were supported by bioinformatics predictions. Starting from known zinc binding sites in crystal structures, we predicted 202 putative partial surface zinc binding sites in FH, most of which were in SCR-6. Metal site prediction web servers also suggested that SCR-6 and other domains bind zinc. Predicted SCR-6/8 dimer structures showed that zinc binding sites could be formed at the protein-protein interface that would lead to daisy-chained oligomers. It was concluded that zinc binds weakly to FH at multiple surface locations, most probably within the functionally important SCR-6/8 domains, and this explains why zinc inhibits FH activity. Given the high pathophysiological levels of bioavailable zinc present in subretinal deposits, we discuss how zinc binding to FH may contribute to deposit formation and inflammation associated with AMD. 相似文献