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PubMed1992

Purification, analysis, and enzymatic activity of recombinant human synovial fluid phospholipase A2 and N-terminal variants.

Recombinant human synovial fluid phospholipase A2 (rPLA2) and several variants with N-terminal sequences modified by addition or deletion of one or two amino acid residues (ala or Met; Des-Asn1, Leu2) have been expressed in mammalian cells and in Escherichia coli, respectively, purified to homogeneity, and characterized. The observed values for the molecular mass of rPLA2 and variants are in complete agreement with the predicted values for a correctly folded structure containing seven disulfide bridges. Moreover, the relative proportions of the various types of secondary structures of the variants of rPLA2, as measured by CD spectroscopy, are similar to that found for native porcine pancreatic PLA2, indicating that the recombinant proteins are correctly folded. Enzymatic activities of rPLA2 with modified N-termini decreased to 1.3-0.005% of the activity of the mature rPLA2, emphasizing a key role of the N-terminus for catalytic activity.

Amino Acid Sequence↗

PubMed1988

[Enzyme histochemical characteristics of the variants of kidney cancer].

Forty-seven specimens of renal cell carcinoma were studied using enzymatic histochemical (8 dehydrogenases and 3 hydrolases) and modern mathematical processing techniques. It was found that morphological variants of the tumor differed in enzymatic activity. This finding determines the possibility of employing specific enzymatic histochemical reactions in differential diagnosis. By the degree of differentiation morphological variants of hypernephroma can be entered into 3 groups: 1) clear cell carcinoma; 2) granular adenocarcinoma and sarcomatoid carcinoma; 3) mixed tumors. For clear cell carcinoma the activity of dehydrogenases and hydrolases enhanced with lesser differentiation of the tumor and vice versa for granular adenocarcinoma and carcinosarcoma. Poorly differentiated clear cell carcinoma is characterized by higher activity of dehydrogenases and hydrolases while granular adenocarcinoma and carcinosarcoma by lower activity of the enzymes compared to variants with well-differentiated tumor. These differences are due to specific cell structure of hypernephroma variants as well as their histochemical properties. There appeared a tendency to reduction of enzymatic histochemical polymorphism with a decrease in the differentiation of the tumor variant.

Adenocarcinoma↗

PubMed2006

Crystal structure of monomeric native antithrombin reveals a novel reactive center loop conformation.

The poor inhibitory activity of circulating antithrombin (AT) is critical to the formation of blood clots at sites of vascular damage. AT becomes an efficient inhibitor of the coagulation proteases only after binding to a specific heparin pentasaccharide, which alters the conformation of the reactive center loop (RCL). The molecular basis of this activation event lies at the heart of the regulation of hemostasis and accounts for the anticoagulant properties of the low molecular weight heparins. Although several structures of AT have been solved, the conformation of the RCL in native AT remains unknown because of the obligate crystal contact between the RCL of native AT and its latent counterpart. Here we report the crystallographic structure of a variant of AT in its monomeric native state. The RCL shifted approximately 20 A, and a salt bridge was observed between the P1 residue (Arg-393) and Glu-237. This contact explains the effect of mutations at the P1 position on the affinity of AT for heparin and also the properties of AT-Truro (E237K). The relevance of the observed conformation was verified through mutagenesis studies and by solving structures of the same variant in different crystal forms. We conclude that the poor inhibitory activity of the circulating form of AT is partially conferred by intramolecular contacts that restrain the RCL, orient the P1 residue away from attacking proteases, and additionally block the exosite utilized in protease recognition.

Amino Acid Sequence↗

PubMed2002

[Clinical and structural parallels in changes of the myocardial conduction system in diphtheria patients].

AIM: Comparison of the results of clinical device investigations of the heart with morphological autopsy evidence in diphtheria. MATERIAL AND METHODS: Hearts of 309 patients with diphtheria aged 25 to 80 years and 60 hearts of patients who died of diphtheria were investigated using ECG, echo-CG, doppler echo-CG, Holter ECG monitoring, biochemical tests of blood. Structural study of cardiac conduction included examination of the sinus-atrial node, atrioventricular node and bundle regarding the adjacent myocardium. RESULTS: Variants of structural-functional state of the myocardium and conduction system are explained by variability of the pathological processes which arose due to individual features of the conduction system structure and location, relationships with the myocardium. In acute diphtheria (day 1-10) dystrophic, necrobiotic and vascular disorders prevailed followed on days 11-30 by inflammation ending in myocardiosclerosis. Nodes and bundles of the conduction system are affected less frequently than the myocardium. CONCLUSION: A correlation exists between structural state of the cardiac conduction system and variants of clinical affection of the heart in diphtheria.

Adult↗

PubMed1999

The crystal structures of the complexes between bovine beta-trypsin and ten P1 variants of BPTI.

The high-resolution X-ray structures have been determined for ten complexes formed between bovine beta-trypsin and P1 variants (Gly, Asp, Glu, Gln, Thr, Met, Lys, His, Phe, Trp) of bovine pancreatic trypsin inhibitor (BPTI). All the complexes were crystallised from the same conditions. The structures of the P1 variants Asp, Glu, Gln and Thr, are reported here for the first time in complex with any serine proteinase. The resolution of the structures ranged from 1.75 to 2.05 A and the R-factors were about 19-20 %. The association constants of the mutants ranged from 1.5x10(4) to 1.7x10(13) M-1. All the structures could be fitted into well-defined electron density, and all had very similar global conformations. All the P1 mutant side-chains could be accomodated at the primary binding site, but relative to the P1 Lys, there were small local changes within the P1-S1 interaction site. These comprised: (1) changes in the number and dynamics of water molecules inside the pocket; (2) multiple conformations and non-optimal dihedral angles for some of the P1 side-chains, Ser190 and Gln192; and (3) changes in temperature factors of the pocket walls as well as the introduced P1 side-chain. Binding of the cognate P1 Lys is characterised by almost optimal dihedral angles, hydrogen bonding distances and angles, in addition to considerably lower temperature factors. Thus, the trypsin S1 pocket seems to be designed particularly for lysine binding.

Amino Acid Substitution↗

PubMed1989

MR imaging of the ankle: normal variants.

Thirty asymptomatic ankles were studied with high-resolution surface coil magnetic resonance (MR) imaging to identify normal structures. Several normal variants were demonstrated, including (a) Cortical irregularity of the posterior tibiotalar joint (should not be mistaken for osteonecrosis) in 27 of 30 cases; (b) normal posterior talofibular ligament with irregular and frayed superior edge simulating a tear in 13 of 30 cases; (c) normal navicular insertion of the posterior tibial tendon with heterogeneous signal intensity in 14 of 30 cases; (d) deltoid ligament inhomogeneity in seven of ten cases; and (e) fluid in the shared peroneal tendons sheath (may be confused with a longitudinal tendon tear) in three of 30 cases. Ankle imaging with the use of MR is still a relatively new procedure. Further investigation is needed to better define normal anatomy and normal variants.

Adolescent↗

PubMed2002

Structural analysis of the two horseradish peroxidase catalytic residue variants H42E and R38S/H42E: implications for the catalytic cycle.

The crystal structures of horseradish peroxidase C (HRPC) active-site mutants H42E and R38S/H42E co-crystallized with benzhydroxamic acid (BHA) and ferulic acid (FA), respectively, have been solved. The 2.5 A crystal structure of the H42E-BHA complex reveals that the side-chain O atoms of Glu42 occupy positions that are very similar to the positions of the two side-chain N atoms of the distal histidine in the wild-type HRPC-BHA structure. The mutation disturbs the hydrogen-bonding network extending from residue 42 to the distal calcium ion and results in the absence of the water molecule that is usually ligated to this ion in plant peroxidases. Consequently, the distal calcium ion is six- rather than seven-coordinated. In the 2.0 A R38S/H42E structure the position of Glu42 is different and no FA is observed in the distal haem pocket. This is a consequence of the absence of the Arg38 side chain, which limits the flexibility of the Glu42 side chain and modulates its acidity, making it unsuitable as a general acid-base catalyst in the reaction cycle. The water ligated to the distal calcium ion is present, showing that the wild-type distal hydrogen-bonding network is preserved. These results show why a glutamic acid residue can substitute for the conserved distal histidine in HRPC and that Arg38 plays a significant role in controlling the positioning and ionization state of the residue at position 42. Furthermore, these structures indicate that changes in the distal cavity are conveyed through the distal hydrogen-bonding network to the distal calcium site.

Amino Acid Sequence↗

PubMed1976

Genetic variants of human erythrocyte glucose-6-phosphate dehydrogenase. Kinetic and thermodynamic parameters of variants A, B, and A- in relation to quaternary structure.

The values of Vmax and Km for the three genetic variants A, B, and A- of erythrocyte glucose-6-phosphate dehydrogenase have been determined at 10 different pH values in the range from 5.5 to 9.5, and at four different temperatures in the range from 18.5-40.0 degrees. The log Vmax versus pH curve for each of the enzymes shows a monotonic increase between pH 5.5 and 7, and a plateau from pH 7.5 upwards. These curves, and their temperature dependence, are compatible with the presence of a single ionizable group which, in its conjugate acid form, renders the enzyme-substrate complex inactive. The pK of this group is 6.94 at 18.5 degrees, and its enthalpy of ionization is 7.0 kcal mol-1. The log Km versus pH curves show a broad plateau between pH 6.2 and 8.2, interrupted by a sharp minimum at pH 7.2 for variant B, while variants A and A- show sharp maxima at pH 7.2 and 7.45, respectively. It is proposed that this unusual behavior depends on the dissociation of the tetrameric enzyme to dimers in this pH region. Specifically, it is shown that a sharp maximum or minimum of Km can arise if cooperative uptake or release of protons is linked to dimer formation, and if the degree of cooperativity is different for the free enzyme compared to the enzyme-substrate complex. The pH dependence of the equilibrium between the tetrameric and the dimeric form of the enzyme has been determined by gel filtration for the same three genetic variants B, A, and A-. In agreement with previous ultracentrifugal data, the enzyme is a tetramer in acid solution and a dimer in alkaline solution. The pH at which half of the enzyme is in dimeric form, under our experimental conditions, is 7.15 +/- 0.05 for variants A and B, and 7.35 +/- 0.05 for variant A-. These pH values correspond closely, for all three variants, to the sharp extrema in the pH dependence of their Km values for glucose 6-phosphate. From the measured dissociation equilibria, it can be inferred that the tetramer-dimer transition entails cooperative release of protons. The degree of cooperativity estimated from these data agrees closely with the independent estimate based on the pH dependence of Km.

Erythrocytes↗

PubMed2001

Identification of base substitutions in ten types of rare variants of phosphoglucomutase-1 (PGM1) encountered in Japanese.

In a previous starch-gel electrophoresis study of erythrocyte phosphoglucomutase-1 (PGM1) in 23,095 Japanese from Hiroshima and Nagasaki, we detected 14 types of rare variant alleles. To determine sequence differences in these rare alleles, cell lines were established from peripheral B-lymphocytes from 24 unrelated individuals in whom nine types of rare variants are presumed to exist on the basis of earlier electrophoresis studies. cDNAs reverse transcribed from mRNAs extracted from these cell lines were amplified by polymerase chain reaction and sequences determined. Amino acid substitution types were deduced from each cDNA sequence. Although two individuals were reported to have an identical electromorph (PGM1 4HR3), sequence analysis revealed that alleles encoding these electromorphs possessed different base substitutions, and one was renamed PGM1 4HR4. As the amino acid substitution of ten different variants could be deduced by cDNA sequence in this study, the effect of each amino acid substitution on enzyme activity could be precisely simulated. The secondary structure of each variant predicted by computer simulations revealed that very decreased activity observed on PGM1 4HR2 protein was caused by significant secondary structure change introduced by the amino acid substitution. On the basis of the crystal structure, the amino acid substitutions of the ten types of rare variants seem to be outside the active center of this enzyme.

Alleles↗

PubMed2005

New approaches to understanding the genetics of asthma.

Several key conditions that are necessary to identify disease susceptibility genes in common diseases such as asthma are now available, including (1) increasingly comprehensive genomic information on gene location, genomic structure, and sequence variants, from the Human Genome Project (and from other species); (2) better understanding of the biologic functions of relevant genes and inflammatory and immunity pathways important in asthma; (3) newer high throughput and accurate technologies for DNA sequencing and SNP genotyping; (4) improved statistical methods for analyzing genetic data from families and populations; and (5) availability of methods to characterize function of sequence variants and study biologic responses. Collectively, these conditions will allow the prioritization of candidate genes based on available knowledge of map position and biologic relevance; obtain genomic structure of these genes; and study sequence variants in these genes in populations to facilitate the identification of genes that are important in the development and expression (severity) of asthma and associated phenotypes. Although, it is still a labor-intensive and expensive project to identify susceptibility genes in common diseases such as asthma, the new techniques that are now being used will greatly facilitate gene mapping. The techniques discussed in this article include genome-by-genome analysis in family data, such as those listed in Box 2. This analysis has already been shown to be a powerful too in mapping genes for another common disease (prostate cancer) with interesting preliminary results for asthma. Second, the use of man-mouse homology mapping that has proven very useful in cardiovascular studies is beginning to be applied to asthma and related phenotypes. finally with new available technology, genome-wide screens using very dense SNP maps are now a reality and a significant new development in family linkage and case-control association studies. In summary, these new approaches should be considered in designing studies to detect genes that are important in asthma and allergy.

Asthma↗

PubMed2024

Loss-of-function in RBBP5 results in a syndromic neurodevelopmental disorder associated with microcephaly.

PURPOSE: Epigenetic dysregulation has been associated with many inherited disorders. RBBP5 (HGNC:9888) encodes a core member of the protein complex that methylates histone 3 lysine-4 and has not been implicated in human disease. METHODS: We identify 5 unrelated individuals with de novo heterozygous variants in RBBP5. Three nonsense/frameshift and 2 missense variants were identified in probands with neurodevelopmental symptoms, including global developmental delay, intellectual disability, microcephaly, and short stature. Here, we investigate the pathogenicity of the variants through protein structural analysis and transgenic Drosophila models. RESULTS: Both missense p.(T232I) and p.(E296D) variants affect evolutionarily conserved amino acids located at the interface between RBBP5 and the nucleosome. In Drosophila, overexpression analysis identifies partial loss-of-function mechanisms when the variants are expressed using the fly Rbbp5 or human RBBP5 cDNA. Loss of Rbbp5 leads to a reduction in brain size. The human reference or variant transgenes fail to rescue this loss and expression of either missense variant in an Rbbp5 null background results in a less severe microcephaly phenotype than the human reference, indicating both missense variants are partial loss-of-function alleles. CONCLUSION: Haploinsufficiency of RBBP5 observed through de novo null and hypomorphic loss-of-function variants is associated with a syndromic neurodevelopmental disorder.

Humans↗

PubMed2000

Propensities for the formation of individual disulfide bonds in hen lysozyme and in the size and stability of disulfide-associated submolecular structures.

Hen lysozyme single-disulfide variants were constructed to characterize the structures associated with the formation of individual native disulfide bonds. Circular dichroism spectra and the effective concentration of protein thiol groups showed that the propensity for structure formation was relatively high for Cys-6-Cys-127 and Cys-30-Cys-115 disulfides. The urea concentration dependence of individual effective concentrations showed that the apparent sizes of the structures were 14-50% of the whole molecule. The intrinsic stability of each submolecular structure in a reduced form of protein, obtained by subtracting the entropic contribution of cross-linking, was highest for Cys-64-Cys-80 and lowest for Cys-76-Cys-94 disulfide bonds.

Animals↗

PubMed1987

Misdiagnosis in normal radiographic anatomy: eight structural configurations simulating disease entities in dogs and cats.

Radiographic diagnosis is a combination of accurately identifying normal and abnormal variants, projecting anatomic structures, and describing abnormal findings. Disease conditions may easily be confused with normal structures. Eight conditions and normal counterparts that are presented illustrate the similarities and differences between normal and abnormal.

Animals↗

PubMed1990

In-vitro translation of cucumoviral satellites. III. Translational efficiencies of cucumber mosaic virus-associated RNA 5 sequence variants can be related to the predicted secondary structures of their first 55 nucleotides.

The cucumber mosaic virus (CMV) satellites D- and S-CARNA 5 (CARNA 5 = Cucumber mosaic virus-Associated RNA 5), their full-length cDNA clone transcripts, and DNA clone transcripts of their open reading frames (ORFs) were used as mRNAs in the wheat-germ in-vitro translation system. Natural D-CARNA 5 yielded an anomalously large polypeptide, while transcripts made from cDNA clones of D-CARNA 5 or its first ORF had no mRNA activity. Transcripts made from the second major ORF in D-CARNA 5 yielded a smaller product, consistent with its size. Natural S-CARNA 5 and its cDNA clone transcripts both yielded the two polypeptides previously reported, while transcripts of its only major ORF yielded exclusively the smaller of the two products. The potential for an alternate initiation codon, 36 nucleotides upstream, being the source of the larger of the two polypeptides was tested. The differences in the translational properties of D- and S-CARNA 5 were related to the predicted secondary structures of the first 55 nucleotides in these CARNA 5 sequence variants. The calculated free energies of the predicted hairpins correlated inversely with their in-vitro translational activities.

Base Sequence↗

PubMed1999

Hydrogen-exchange stabilities of RNase T1 and variants with buried and solvent-exposed Ala --> Gly mutations in the helix.

Hydrogen-exchange rates were measured for RNase T1 and three variants with Ala --> Gly substitutions at a solvent-exposed (residue 21) and a buried (residue 23) position in the helix: A21G, G23A, and A21G + G23A. These results were used to measure the stabilities of the proteins. The hydrogen-exchange stabilities (DeltaG(HX)) for the most stable residues in each variant agree with the equilibrium conformational stability measured by urea denaturation (DeltaG(U)), if the effects of D(2)O and proline isomerization are included [Huyghues-Despointes, B. M. P., Scholtz, J. M., and Pace, C. N. (1999) Nat. Struct. Biol. 6, 210-212]. These residues also show similar changes in DeltaG(HX) upon Ala --> Gly mutations (DeltaDeltaG(HX)) as compared to equilibrium measurements (DeltaDeltaG(U)), indicating that the most stable residues are exchanging from the globally unfolded ensemble. Alanine is stabilizing compared to glycine by 1 kcal/mol at a solvent-exposed site 21 as seen by other methods for the RNase T1 protein and peptide helix [Myers, J. K., Pace, C. N., and Scholtz, J. M. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 3833-2837], while it is destabilizing at the buried site 23 by the same amount. For the A21G variant, only local NMR chemical shift perturbations are observed compared to RNase T1. For the G23A variant, large chemical shift changes are seen throughout the sequence, although X-ray crystal structures of the variant and RNase T1 are nearly superimposable. Ala --> Gly mutations in the helix of RNase T1 at both helical positions alter the native-state hydrogen-exchange stabilities of residues throughout the sequence.

Alanine↗

PubMed2002

Tat HIV-1 primary and tertiary structures critical to immune response against non-homologous variants.

Clinical studies show that in the absence of anti-retroviral therapy an immune response against the human immunodeficiency virus type 1 (HIV-1), transacting transcriptional activator (Tat) protein correlates with long term non-progression. The purpose of this study is to try to understand what can trigger an effective immune response against Tat. We used five Tat variants from HIV strains identified in different parts of the world and showed that mutations of as much as 38% exist without any change in activity. Rabbit sera were raised against Tat variants identified in rapid-progressor patients (Tat HXB2, a European variant and Tat Eli, an African variant) and a long term non-progressor patient (Tat Oyi, an inactive African variant). Enzyme-linked immunosorbent assay (ELISA) results showed that anti-Tat Oyi serum had the highest antibody titer and was the only one to have a broad antibody response against heterologous Tat variants. Surprisingly, Tat HXB2 was better recognized by anti-Tat Oyi serum compared with anti-Tat HXB2 serum. Western blots showed that non-homologous Tat variants were recognized by antibodies directed against conformational epitopes. This study suggests that the primary and tertiary structures of the Tat variant from the long term non-progressor patient are critical to the induction of a broad and effective antibody response against Tat.

AIDS Vaccines↗

PubMed1996

Structure-function relationships among wild-type variants of Staphylococcus aureus beta-lactamase: importance of amino acids 128 and 216.

beta-Lactamases inactivate penicillin and cephalosporin antibiotics by hydrolysis of the beta-lactam ring and are an important mechanism of resistance for many bacterial pathogens. Four wild-type variants of Staphylococcus aureus beta-lactamase, designated A, B, C, and D, have been identified. Although distinguishable kinetically, they differ in primary structure by only a few amino acids. Using the reported sequences of the A, C, and D enzymes along with crystallographic data about the structure of the type A enzyme to identify amino acid differences located close to the active site, we hypothesized that these differences might explain the kinetic heterogeneity of the wild-type beta-lactamases. To test this hypothesis, genes encoding the type A, C, and D beta-lactamases were modified by site-directed mutagenesis, yielding mutant enzymes with single amino acid substitutions. The substitution of asparagine for serine at residue 216 of type A beta-lactamase resulted in a kinetic profile indistinguishable from that of type C beta-lactamase, whereas the substitution of serine for asparagine at the same site in the type C enzyme produced a kinetic type A mutant. Similar bidirectional substitutions identified the threonine-to-alanine difference at residue 128 as being responsible for the kinetic differences between the type A and D enzymes. Neither residue 216 nor 128 has previously been shown to be kinetically important among serine-active-site beta-lactamases.

Alanine↗

PubMed1982

[Variants of histological and electron-microscopic structure of lobular carcinoma of the breast].

Histological and electron microscopic features of the structure of lobular carcinoma have been demonstrated on the material of 15 neoplasms of the mammary gland. It is suggested that alongside with the known classical and solid variants a microtubular variant of infiltrative lobular carcinoma be distinguished. In the opinion of the author, features of the cytological (small sizes, monomorphic type of cells and nuclei, clear cytoplasm) and electron microscopic (myofibrils, few organelles) structure of these tumors should constitute their main characteristic signs.

Adult↗