Molecular analysis of three novel G6PD variants: G6PD Pedoplis-Ckaro, G6PD Piotrkow and G6PD Krakow

We present three novel mutations in the G6PD gene and discuss the changes they cause in the 3-dimensional structure of the enzyme: 573C-->G substitution that predicts Phe to Leu at position 191 in the C-terminus of helix alphae, 851T-->C mutation which results in the substitution 284Val--> -->Ala in the beta+alpha domain close to the C-terminal part of helix alphaj, and 1175T-->C substitution that predicts Ile to Thr change at position 392.     

G6PD Avenches and G6PD Moosburg: biochemical and erythrocyte membrane characterization

Two new G6PD variants with severe enzyme deficiency in Switzerland (G6PD Avenches, G6PD I) and in Germany (G6PD Moosburg, G6PD II) are described. One patient had suffered from severe postpartal hyperbilirubinemia, the other one presented with chronic hemolysis and remittent hyperbilirubinemia. Both variants showed diminished electrophoretic mobility, both variants were heat labile. The Michaelis-Menten constants KM for glucose-6-phosphate and for NADP+ were normal. 2-Desoxy-glucose-6-phosphate was utilized by G6PD I in a higher and by G6PD II at a lower rate than by the normal enzyme. Desamino-NADP+ and galactose-6-phosphate were utilized by both variants at a normal rate. The electrophoretic separation of membrane proteins of G6PD II showed both in the presence and in the absence of 6-mercaptoethanol no difference concerning the formation of membrane protein aggregates between patient and normal control.     

Six variants of HLA-1327 identified by isoelectric focusing

Six variant forms of HLA-1327 were identified among 68 unrelated 1327-positive donors by isoelectric focusing (IEF) gel analysis. Each of the six IEF variants was distinguished by charge heterogeneity of desialated B27 heavy chains immunoprecipitated with specific monoclonal antibody (MAb). Charge differences varied from single to several charge units, indicating that these variants may have substantially different amino acid compositions. Informative family study showed that three B27 variant molecules were genetically determined. The uniqueness of these variant molecules was also demonstrable using a panel of alloantisera and MAbs recognizing 1327-associated epitopes. Six distinct serological reactivity patterns were observed. Five of these serological patterns correlated with four of the IEF-defined variants, two of these patterns being associated with one IEF variant form. The sixth serological pattern was shared by the remaining two IEF variants. Combining the results of the electrophoretic and serological analyses, it is apparent that there are more than six structural variants within the B27 alloantigen family. Some B27 variant forms were found only in individuals of particular racial origin, indicating that unique genetic variations might occur in different racial groups. In a preliminary analysis of patients with ankylosing spondylitis, no apparent correlation was observed between any specific B27 variants and disease susceptibility.     

Analysis of human erythrocyte glucose 6-phosphate dehydrogenase isozymes by isoelectric focusing in polyacrylamide gel

The method of isoelectric focusing in polyacrylamide gel was used to separate G6PD isozymes in crude hemolysates of human, rabbit, and rat erythrocytes. G6PD (B) from erythrocytes of a normal human male donor revealed six bands of activity. Their mean isoelectric points, using pH 3–10 and 5–8 range empholytes, were pI 7.04 for band I, pI 6.60 for band II, pI 6.37 for band III, pI 6.11 for band IV, pI 5.94 for band V, pI 5.79 for band VI. G6PD from rabbit and rat erythrocytes revealed completely different multiple band patterns. The method of isoelectric focusing in polyacrylamide gel is presented as a new way of detecting G6PD isozyme patterns.     

Molecular dynamics of G6PD variants from sub-Saharan Africa

Precision medicine uses genomic guidance to improve drug treatment safety and efficacy. Prior knowledge of genetic variant impact can enable such strategies, but current knowledge of African variants remains scarce. G6PD variants are linked to haemolytic adverse effects for a number of drugs commonly used in African populations. We have investigated a set of G6PD variants with structural bioinformatics techniques to further characterise variants with known effect, and gain insights into variants with unknown impact. We observed wide variations in patterns of root-mean-square deviation between wild-type and variant structures. Variants with known, highly deleterious impact show structural effects which may likely result in the destabilisation of the G6PD homodimer. The V68M and N126D variants (which are both common across African populations, and together form the A- haplotype) induce large conformational shifts in the catalytic NADP+ binding domain. We observed a greater impact for the haplotype than for each of the individual variants in these cases. A novel African variant (M207T) shows the potential to disrupt interactions within the protein core, urging further investigation. We explore how characterising the molecular impact of African G6PD variants can enable advanced strategies for precision medicine, as well as impact the use of novel therapeutics aiming to treat G6PD deficiency. This knowledge can assist in bridging current knowledge gaps, and aid to facilitate precision medicine applications in African populations.     

Five new Gc variants detected by isoelectric focusing in agarose gel

Summary Five new genetically determined Gc variants were observed by isoelectric focusing. Seven rare variants 1A4, 1C1, 1C3, 1C9, 1C11, 2A2, and 2A5 were also found in the material comprising Danish ans Swedish paternity cases. All the variants were further analysed by electrophoresis in agarose gel. Two of the new variants had double bands of which the anodal one was susceptible to neuraminidase treatment (Gc 1C13 and 1C14). The three other new variants appeared as a single band, which was unaffected by neuraminidase treatment (Gc 2A9, 2C5, and 2C6). The Gc Ar variant originally detected by electrophoresis was reexamined by isoelectric focusing and named 2C4.     

Characterization of normal and abnormal variants of galactose-1-phosphate uridylyltransferase (EC 2.7.7.12) by isoelectric focusing

Isoelectric focusing (IEF) in polyacrylamide gels has been used to study the isozymes of human galactose-1-phosphate uridylyltransferase (GALT) in erythrocytes and fibroblasts. In addition to the usefulness of IEF in differentiating normal, Duarte variant, and galactosemic homozygotes and heterozygotes, the ability of IEF to distinguish the residual GALT activity in two different galactosemic fibroblast lines and in revertants from them is demonstrated.     

Characterization by isoelectric focusing of chorion protein variants inDrosophila melanogaster and their use in developmental and linkage analysis

Summary Drosophila melanogaster chorion proteins are characterized on one-dimensional isoelectric focusing (IF) gels. The six major chorion components previously identified on SDS gels are shown to resolve into at least 11 components in our IF system. IF screening of 102 geographic strains ofDrosophila melanogaster revealed seven cases of variation in major chorion components. Two strains, Crimea and Falsterbo, which were monomorphic for a variant B1 protein and two strains, Skafto and Lausanne, which were monomorphic for a variant C1 protein, were chosen for further study. After IF developmental analysis of F1 hybrids had indicated that the sources of the variation resided in the structural genes for these proteins, each variant was crossed to a multiply marked and inverted strain (BLT) to determine the linkage group of the variant gene. To localize genes to more specific sites multiply marked 3rd (SKERO) or X-chromosomal (CB1) (X-PLE) mapping strains were used. In both Crimea and Falsterbo the gene for the B1 protein is located near map location 26 on the 3rd chromosome. In both Lausanne and Skafto the C1 gene is located on the X chromosome. Hence, for the first time, we have demonstrated genetically the non-linkage of two chorion genes, B1 and C1.     

Characterization of isoforms of human mitochondrial creatine kinase by isoelectric focusing

High enzyme activity of mitochondrial creatine kinase (creatine-N-phosphotransferase, mCK, EC 2.7.3.2) was detected in serum from a patient with advanced carcinoma of the rectum and its isoforms were characterized by means of isoelectric focusing (IEF). Three forms of mCK, membrane-bound (pI 6.9–7.0), octameric (pI 7.0–7.9) and dimeric (pI 6.7, 6.8, 6.9 and 7.0), were detected in the fresh serum. These three forms of mCK were converted to five dimeric isoforms, and these were characterized as one reduced form (pI 7.0) and four oxidized (pI 6.6, 6.7, 6.8 and 6.9) forms upon treatment with urea, hydrogen peroxide or 2-mercaptoethanol (2-ME). The C-terminal of the mCKs was concluded to be a lysine residue because the mCKs treated with carboxypeptidase B migrated to positions closer to the anode than did those not treated with carboxypeptidase B. Therefore, four bands were concluded to represent one reduced-delysined isoform (pI 6.4) and three oxidized-delysined isoforms (pI 6.1, 6.2 and 6.3). The broad octameric mCK band disappeared and a narrow band focused at pI 6.8–6.9 appeared upon probable delysination of the mCKs. Thus, the number of lysine residues at the C-terminal of the octamer was concluded to be variable due to variable catalysis by carboxypeptidase N in the plasma. mCKs seemed to be inactivated during conversion from a membrane-bound form to dimeric oxidized-delysined forms via the octameric, dimeric reduced and oxidized forms.     

Procedure for simultaneous phenotyping of genetic variants in cow's milk by isoelectric focusing

A method is described which allows the simultaneous separation of all polymorphic protein fractions in cow's milk in one single run. The separation could be achieved by isoelectric focusing in ultrathin-layer polyacrylamide gels. The method is especially suitable for screening purposes because it combines low costs, high resolution and short separation time.     

G6PD Toronto

Members of a Toronto family of Northern European ancestry were found to have a deficiency of red cell and white cell glucose 6-phosphate dehydrogenase (G6PD) activity. The hemizygous propositus had neonatal hyperbilirubinemia and subsequent episodes of hemolytic jaundice associated with respiratory infections and exposure to paint fumes. Characterization of the enzyme revealed that it was a variant which had not been previously described, and it was named 6GPD Toronto.This study was supported by grants MT 696 from the Medical Research Council of Canada and GM 15253 from the National Institutes of Health.     

Two commonly occurring nucleotide base substitutions in Chinese G6PD variants

Using a direct PCR sequencing technique, we have identified two DNA base substitutions in 8 different biochemical G6PD variants of Chinese origin. Neither one of these abnormalities has been reported in other ethnic groups. An abnormality (C1) of G to T substitution at cDNA 1376 causing an amino acid change from Arg to Leu has been found in 3 variants. Another abnormality (C2) of G to A substitution at cDNA 1388 causing an amino acid change from Arg to His has been found in 5 variants. Both C1 and C2 are located in exon 12 of the G6PD gene and are only 12 base pairs apart. However, C1 is associated with a significant increase in the deamino-NADP utilization rate, whereas C2 is not. Taken together, our data suggest that C1 and C2 are very common among Chinese with a G6PD deficiency and exon 12 may define an important functional domain of the human G6PD.     

Changes in relative mobility of pancreatic amylase variants in isoelectric focusing

Using isoelectric focusing with one ampholytic solution, double- and single-banded amylase phenotypes were found in a sample of rhesus monkeys,Macaca mulatta. When applying different ampholytic solutions, these variants were shown to change their position relative to each other. Single-banded phenotypes showed either a position corresponding to one of the bands of the double-banded phenotype or to an intermediate one. Family studies, however, suggested that the differences between the observed patterns were not caused by genetic differences. This discloses a problem with respect to the interpretation of electrophoretic data, i.e. bands with different positions produced by isoelectric focusing may not necessarily represent genetic differences.     

Procedure for simultaneous phenotyping of genetic variants in cow's milk by isoelectric focusing*

A method is described which allows the simultaneous separation of all polymorphic protein fractions in cow's milk in one single run. The separation could be achieved by isoelectric focusing in ultrathin-layer polyacrylamide gels. The method is especially suitable for screening purposes because it combines low costs, high resolution and short separation time.     

Characterization of phospholipid-protein interactions by capillary isoelectric focusing with whole-column imaging detection

The integration of functional proteins in the phospholipid bilayer is one of the most crucial features of biological membrane architecture. Phospholipid-protein interactions play an important role in the functions of bounded proteins in the phospholipid membrane. When the phospholipid-protein interactions occur, the protein structure tends to alter, which can result in a change in the isoelectric points (pI) of protein. Capillary isoelectric focusing (cIEF) with whole-column imaging detection (WCID) is an attractive technique that has the features of simple operation, high resolution, and fast separation without focused band mobility for detection of amphoteric biomolecules. In this study, a cIEF-WCID method was developed to characterize the phospholipids-protein interactions by monitoring the protein cIEF profiles. Seven proteins with different pI and molecular mass , and a zwitterionic phosphatidylcholine (PC) with zwitterionic properties, were used to evaluate the feasibility of the cIEF-WCID approach in the study of phospholipid-protein interactions. The cIEF profiles changed in response to the changes in protein conformation, clearly exhibiting interactions between the PC vesicles and the targeted proteins. The formation of PC-protein complex was observed in the cIEF electropherograms. It was demonstrated that seven proteins displayed distinct interactions with the PC vesicles due to their different chemical and physical properties. The influences of the PC concentration, incubation time, and incubation temperature on the phospholipids-protein interactions were investigated. This study validated a novel analytical approach for the characterization of phospholipid-protein interactions.     

In vitro correction of erythrocyte glucose 6-phosphate dehydrogenase (G6PD) deficiency.

The permeability characteristics of egg phosphatidylcholine (EPC) vesicles to Eu³⁺ has been examined by ³¹P-nmr, in various mixed lipid systems. It has been found that incorporation of small amounts of sodium taurocholate (NaTC) in the EPC vesicle greatly increased the vesicular permeability to Eu³⁺. Incorporation of cholesterol in the EPC vesicle significantly inhibits the ability of NaTC to induce permeability alterations in this mixed system. It has been reported that at low EPC:NaTC ratios (2:1-0.65:1), mixed micelles of the components are formed [N. A. Mazer, R. F. Kwasnick, M. C. Carey, and G. B. Benedek, 1977, in Micellization, Solubization, and Microemulsions (Mittal, K. L., ed.) Vol. 1, pp. 483-402, Plenum Press, New York]. By examination of the ³¹P-nmr linewidths of EPC, at various ratios of EPC:NaTC, it is possible to follow the decrease in size of the EPC vesicle, as it becomes incorporated into the smaller NaTC micelles. The ³¹P{¹H} nuclear Overhauser enhancement of the simple EPC vesicle is not significantly different from this same parameter measured for EPC, when it exists in mixed micellar systems with NaTC. This indicates that there must be considerable internuclear head group interactions of EPC molecules in EPC-NaTC mixed micelles. This argues for sequestering of EPC in such micelles.