Biomedical subjects

G Szakonyi

Publications and source records attributed to G Szakonyi.

8 recordsLinked to original sources

PubMed2005

Active membrane transport and receptor proteins from bacteria.

A general strategy for the expression of bacterial membrane transport and receptor genes in Escherichia coli is described. Expression is amplified so that the encoded proteins comprise 5-35% of E. coli inner membrane protein. Depending upon their topology, proteins are produced with RGSH6 or a Strep tag at the C-terminus. These enable purification in mg quantities for crystallization and NMR studies. Examples of one nutrient uptake and one multidrug extrusion protein from Helicobacter pylori are described. This strategy is successful for membrane proteins from H. pylori, E. coli, Enterococcus faecalis, Bacillus subtilis, Staphylococcus aureus, Microbacterium liquefaciens, Brucella abortus, Brucella melitensis, Campylobacter jejuni, Neisseria meningitides, Streptomyces coelicolor and Rhodobacter sphaeroides.

Bacterial Proteins↗

PubMed2002

Structure of complement receptor (CR) 2 and CR2-C3d complexes.

Using X-ray crystallography, we have determined the structure of the first two short consensus repeats (SCRs) of human complement receptor (CR) 2 in complex with C3d. These studies revealed: (i) a primary site of interaction for C3d within SCR2 of CR2, (ii) a hydrophobic patch holding SCR1 to SCR2 in a rigid V-shape, (iii) a dimer formed by interactions between SCR1 of each molecule, (iv) several non-linear sequences on C3d that interact with CR2 and (v) mutations of C3d amino acids within the co-crystal interface that resulted in decreased binding. In addition, a polymorphism that results in decreased C3d binding and introduces a new glycosylation site predicted to disrupt the dimer interface was found in the New Zealand White autoimmune mouse strain. Although the co-crystal complex results are in agreement with a subset of prior studies, our additional findings, which demonstrate an extended SCR1-SCR2 structure in solution and differences in the kinetics of ligand-receptor interactions with longer forms of CR2, have suggested a more complex receptor-ligand interaction. To characterize this interaction further, several approaches directed at the determination of solution phase interactions as well as the analysis of the three-dimensional structure of CR2 alone and key CR2 mutants will be necessary.

Animals↗

PubMed2001

Epitope mapping using the X-ray crystallographic structure of complement receptor type 2 (CR2)/CD21: identification of a highly inhibitory monoclonal antibody that directly recognizes the CR2-C3d interface.

Complement receptor type 2 (CR2)/CD21 is a B lymphocyte cell membrane C3d/iC3b receptor that plays a central role in the immune response. Human CR2 is also the receptor for the EBV viral membrane glycoprotein gp350/220. Both C3d and gp350/220 bind CR2 within the first two of 15-16 repetitive domains that have been designated short consensus/complement repeats. Many mAbs react with human CR2; however, only one currently available mAb is known to block both C3d/iC3b and gp350/220 binding. We have used a recombinant form of human CR2 containing the short consensus/complement repeat 1-2 ligand-binding fragment to immunize Cr2(-/-) mice. Following fusion, we identified and further characterized four new anti-CR2 mAbs that recognize this fragment. Three of these inhibited binding of CR2 to C3d and gp350/220 in different forms. We have determined the relative inhibitory ability of the four mAbs to block ligand binding, and we have used overlapping peptide-based approaches to identify linear epitopes recognized by the inhibitory mAbs. Placement of these epitopes on the recently solved crystal structure of the CR2-C3d complex reveals that each inhibitory mAb recognizes a site either within or adjacent to the CR2-C3d contact site. One new mAb, designated 171, blocks CR2 receptor-ligand interactions with the greatest efficiency and recognizes a portion of the C3d contact site on CR2. Thus, we have created an anti-human CR2 mAb that blocks the C3d ligand by direct contact with its interaction site, and we have provided confirmatory evidence that the C3d binding site seen in its crystal structure exists in solution.

Animals↗

PubMed2001

Structure of complement receptor 2 in complex with its C3d ligand.

Complement receptor 2 (CR2/CD21) is an important receptor that amplifies B lymphocyte activation by bridging the innate and adaptive immune systems. CR2 ligands include complement C3d and Epstein-Barr virus glycoprotein 350/220. We describe the x-ray structure of this CR2 domain in complex with C3d at 2.0 angstroms. The structure reveals extensive main chain interactions between C3d and only one short consensus repeat (SCR) of CR2 and substantial SCR side-side packing. These results provide a detailed understanding of receptor-ligand interactions in this protein family and reveal potential target sites for molecular drug design.

Amino Acid Sequence↗

PubMed2001

Cr2, a candidate gene in the murine Sle1c lupus susceptibility locus, encodes a dysfunctional protein.

The major murine systemic lupus erythematosus (SLE) susceptibility locus, Sle1, corresponds to three loci independently affecting loss of tolerance to chromatin in the NZM2410 mouse. The congenic interval corresponding to Sle1c contains Cr2, which encodes complement receptors 1 and 2 (CR1/CR2, CD35/CD21). NZM2410/NZW Cr2 exhibits a single nucleotide polymorphism that introduces a novel glycosylation site, resulting in higher molecular weight proteins. This polymorphism, located in the C3d binding domain, reduces ligand binding and receptor-mediated cell signaling. Molecular modeling based on the recently solved CR2 structure in complex with C3d reveals that this glycosylation interferes with receptor dimerization. These data demonstrate a functionally significant phenotype for the NZM2410 Cr2 allele and strongly support its role as a lupus susceptibility gene.

Alleles↗

PubMed1998

Expression of the sarco/endoplasmic reticulum Ca(2+)-transport ATPase protein isoforms during regeneration from notexin-induced necrosis of rat soleus muscle.

Expression levels of fast-twitch (SERCA1), slow-twitch (SERCA2a) and "housekeeping" (SERCA2b) isoforms of the sarcoplasmic reticulum Ca(2+)-transport ATPase were monitored during regeneration of rat soleus muscles following necrosis induced by the toxin notexin at the tissue level by Western blot analysis and at the cellular level by immunocytochemical analysis. Due to necrosis, levels of muscle-specific SERCA1 and SERCA2a isoforms dropped to low levels on the third day after injection of the toxin. Subsequently, during regeneration both isoforms recovered but with a different time course. Expression of the fast type SERCA1 increased first. This type showed its most pronounced increase between day 3 and 10. Expression of the slow type SERCA2a was biphasic. After an increase to approximately one third of the control value on days 5-10, it showed its main increase up to the control level between day 10 and 21. Expression levels of the house-keeping SERCA2b isoform remained relatively constant throughout the 4 weeks of regeneration. Between day 10 and 28, when new innervation is established, SERCA2a expression spread gradually over almost all fibers whereas the number of SERCA1-expressing fibers decreased and only a limited number of fibers co-expressed SERCA1 and SERCA2a. At 4 weeks of regeneration, expression of the fast isoform was found only in 12% of the fibers, whereas the slow form was found in 98% of the fibers. In the contralateral untreated soleus muscles, 26% SERCA1-positive and 81% SERCA2a-positive fibers were observed. Immunocytochemical analysis showed that SERCA1 and SERCA2a were co-expressed with fast and slow myosin isoforms in fibers of normal muscles but in regenerated muscle only slow myosin and slow SERCA isoforms correlated. The results show that during regeneration levels of fast and slow SERCA proteins change in a similar way as their mRNAs do. However, in regenerated soleus, unlike in normal muscle, expression of slow SERCA is coregulated only with the slow myosin isoform. This finding is in agreement with the fact that the number of slow type fibers is increased in regenerated soleus.

Animals↗

PubMed1998

Expression of sarcoplasmic/endoplasmic reticulum Ca2+ ATPases in the rat extensor digitorum longus (EDL) muscle regenerating from notexin-induced necrosis.

The level of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) mRNAs and proteins have been assessed by RT-PCR, immunoblotting and immunocytochemistry in the rat extensor digitorum longus (EDL) muscles during regeneration from notexin-induced necrosis. As a result of the necrosis, SERCA1 and SERCA2 declined on days 1 and 3 after administration of the toxin. Thereupon the mRNA of the fast isoform SERCA1 rapidly increased between days 5 and 10 to the normal level. The mRNA level of the "housekeeping" SERCA2b isoform increased markedly during the actual necrosis at days 1 and 5, probably due to invading cells. Then the mRNA level of the neonatal SERCA1b splice variant increased first, and exceeded the level of the adult SERCA1a transcript on day 5. At later stages of regeneration the neonatal form was gradually replaced by the adult SERCA1a form, thus recapitulating similar changes known to occur during normal ontogenesis. Along with SERCA1, the levels of the slow isoform (SERCA2a) mRNA and protein increased on day 5, but the SERCA2a mRNA levels never rose above 10% of SERCA1 and after 10 days gradually declined again. In the normal and regenerated muscles, SERCA1 was expressed in 97% of the fibres which accounted for the population of fast-twitch fibres (type IIa, type IIb and probably type IIx/d). SERCA2a was present in 6% of the fibres of normal muscle (mostly in the slow-twitch type I fibres). At the end of regeneration the number of fibres expressing SERCA2a was twice as high and were found in small groups, unlike in normal EDL, but about 50% of these clustered fibres also expressed SERCA1.

Animals↗

PubMed1997

Sonochemical enantioselective hydrogenation of ethyl pyruvate over platinum catalysts.

Chiral sonochemical hydrogenation of an aliphatic alpha-ketoester, ethyl pyruvate to ethyl lactate was carried out over various platinum catalysts in different solvents under atmospheric hydrogen pressure. The reaction rates and the enantiomeric excesses were determined over Pt/C, Pt/SiO2 and Pt/K-10 catalysts both under conventional and sonochemical conditions. The effect of ultrasounds on the catalytic activity and enantioselectivity was tested applying sonochemical pretreatment before the reaction. The ultrasonic irradiation was found to be highly advantageous in these hydrogenations. After insonation of the catalysts, the enantioselectivity was highly improved over Pt/SiO2 and Pt/K-10 catalysts. In addition, the reactions took place in quantitative yield and with complete chemoselectivity and the hydrogenation rates increased with one order of magnitude despite the very mild (atmospheric hydrogen pressure, room temperature) experimental conditions.

Journal Article↗