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.     

A new hereditary single band variant of the Gc system

Summary A new single band variant (Gc Ar) or the Gc subtypes not identical with the known Gc variants has been detected in the plasma of a healthy blood donor by isoelectric focusing. Using this technique the variant is represented by a single band which has a similar isoelectric point to the Gc 1C2 anodal band. It is well known that the single band Gc phenotypes remain unaltered after neuraminidase treatment. Nevertheless, the new single band variant (Gc Ar) is altered after neuraminidase treatment as is Gc 2A3. After neuraminidase treatment, the Gc Ar band is affected and moved to the nearby position of the Gc 2 band. Investigation of the proband's family shows that the variant occurs combined with the common alleles Gc 1F, Gc 1S and that it has an autosomal dominant inheritance.     

Unusual sialilation of three different rare genetic variants of serum DBP: Gc 1A17, Gc 1A16, and Gc 1A11

Summary The proteins of three anodal Gc1 variants, Gc 1A16, 1A11, and 1A17, are characterized by the most acidic isoelectric points observed so far among the different Gc mutants. Stepwise removal of N-acetylneuraminic acid (NANA) by treatment with neuraminidase was performed to estimate the degree of sialilation of these Gc variants. The results indicate that both proteins, the anodal and the cathodal component of these Gc 1 mutants, carry sialic acid residues. This observation is remarkable in so far as usually only the anodal component of the Gc 1 protein contains NANA and only a single residue. From the experiments carried out it can be deduced that Gc 1A16 has two NANA residues in the anodal and one NANA residue in the cathodal component. Gc 1A16 was found in four members of three generations in a Danish family; the variant segregated as a Mendelian trait. More difficult to interprete are the results obtained with the variants Gc 1A11 and Gc 1A17. Gc 1A11 probably has three NANA residues in the anodal and two NANA residues in the cathodal component. Gc 1A11 has been observed in two mother-child pairs and is presumably also a simple genetic trait. Gc 1A17 has also several NANA residues in both Gc proteins; it is suggested that the anodal component has either three or four NANA residues and the cathodal component either two or three NANA residues. Family information on this variant is not yet available.     

Gc X and Gc Y revealed by immunofixation electrophoresis

Summary Immunofixation electrophoresis is used to define two variants in the Gc system: Gc X and Gc Y. Gc X has one band with a mobility between Gc 1-1 and Gc 2-2 while Gc Y has two bands migrating faster than the cathodal band of Gc 1.     

Detection of genetic variation with radioactive ligands. II. Genetic variants of vitamin D-labeled group-specific component (Gc) proteins.

A novel technique for detecting electrophoretic and quantitative variants of group-specific component (Gc) proteins is described. The technique, in vitro labeling with radioactive vitamin D followed by polyacrylamide gel electrophoresis and autoradiography (PAGE autoradiography), permits sensitive, high resolution detection of Gc variants by virtue of a physiologically significant property: the ability of Gc to bind vitamin D and 25-hydroxyvitamin D. Using this procedure, anodal Gc variants, with mobility similar to Gc Aborigine and Gc Eskimo, were observed in Chinese, Japanese, African Pygmies, and American Blacks. The gene frequency of these variants ranges from 2.6% to 15%; they were not previously known to be polymorphic in these populations. In addition to qualitative variants, individual variation in Gc band density ratios is documented and discussed. These studies not only illustrate the utility of PAGE autoradiography in screening Gc, but also confirm that a major functional role of Gc in man and other animals is the transport of vitamin D and vitamin D metabolites.     

Variants of the group-specific component system as demonstrated by immunofixation electrophoresis. Report of a new variant, Gc Boston (Ge B).

Immunofixation electrophoresis is a relatively simple and reliable method for the genetic phenotyping of the group-specific component (Gc) of serum. This method permits direct comparison of electrophoretic mobilities and band concentrations, with no interference by other proteins. The variants Gc Ab and Gc Y appear identical by this technique; the Eskimo variant appears to be similar to Gc D but not to Gc Ab as previously reported. Gc Norway, also designated Gc 1C, is electrophoretically cathodal to the slower band of Gc 1 and therefore appears to be a distinct variant. A new variant, Gc Boston, is single banded with mobility between the two bands of Gc 1.     

Affinity differences for vitamin D metabolites associated with the genetic isoforms of the human serum carrier protein (DBP)

Human vitamin D binding protein (DBP) displays considerable polymorphism with 120 described alleles. Among these, three alleles are frequently observed, Gc 1F (pI 4.94–4.84), Gc 1S (pI 4.95–4.85) and Gc 2 (pI 5.1). Differences between these genetic forms of the protein in affinity for vitamin D metabolites have been detected by electrophoretic methods. The constant affinity (Ka) values determined in this study confirm these differences. The affinities of six rare variants were also examine. Those of the DBP genetic forms to the vitamin D derivatives 25-OH-D₃ and 1,25-(OH)₂-D₃ seem to be related to the isoelectric point of the proteins: a high affinity corresponding to a low isoelectric point. The Gc 1A9 and 1A11 mutants were associated with higher affinity for the vitamin D derivatives and the Gc 1C1 and 1C21 mutants were deficient.     

The vitamin D binding of the common and rare variants of the group-specific component (Gc)

Summary The vitamin D₃ binding properties of the common and rare Gc variants were examined. Vitamin D₃ labeled with ¹⁴C was added to serum. Gc phenotypes were demonstrated autoradiographically following separation by immunofixation electrophoresis on agarose. This qualitative analysis did not reveal differences in vitamin D₃ binding by the group-specific components of the common types Gc1-1, Gc 2-1, and Gc2-2. The double-band variants Gc Darmstadt, Gc Y/Ab, Gc Toulouse, Gc Norway, and Gc Caucasian were examined; the phenotypes Gc Ab-Ab, Gc Ab-1, Gc Ab-2, Gc T-1, Gc T-2, Gc Norw-2, and Gc 1-Cau showed normal D₃ binding. The double bands of Gc Darmstadt in the phenotype D-2 appeared somewhat weak. The singleband mutants Gc Wien, Gc Chippewa, Gc Opava, and Gc Z were analyzed; the phenotypes Gc W-1, Gc W-2, Gc Chip-1, Gc Chip-2, Gc 1-Op, Gc Op-2, Gc 1-Z, and Gc 2-Z showed normal D₃ binding. A mutant in the Gc system with clearly defective vitamin D₃ binding properties remains to be delineated.     

The genetic variants of group-specific component (vitamin D-binding protein) possess different binding characteristics for immobilized Cibacron Blue 3-GA.

The elution profiles of several variants of the Gc protein have been studied after chromatography on immobilized Cibacron Blue 3-GA. The allele products belonging to the Gcl type were retarded and eluted with a Ve/Vo at 1.5, as previously reported for the Gcl-1 phenotype [Chapuis-Cellier, Gianazza & Arnaud (1982) Biochim. Biophys. Acta 709, 353-357]. The allele products belonging to the Gc2 type were further retarded (Ve/Vo at 2.6), and both Gcl and Gc2 allele products were clearly separated in heterozygous individuals. This observation allows the isolation and purification of Gc variants in heterozygous individuals which carry the combination Gcl variant-Gc2, Gcl-Gc2 variant, or Gcl variant-Gc2 variant. In contrast, the corresponding holoproteins did not bind to the gel and were eluted in the void volume. This suggests that the interaction of Gc with immobilized Cibacron Blue 3-GA involves the binding site of the protein for 25-hydroxycholecalciferol and that the dye behaves as a 'pseudoligand' for the protein. In addition, our data suggest that the different elution profiles of the variants could reflect a different affinity of the gene products for the dye.     

Two new sheep transferrin variants and the effect of neuraminidase

Transferrin phenotypes were determined in six breeds of sheep by starch gel electrophoresis. Two new variants, Tf H_Czech and Tf K_Czech, were found and some evidence of their genetic control was obtained. Tf H_Czeeh was detected only in Sumava sheep; it has an intermediate mobility between Tf A and Tf B. Tf K_Czech was found only in Tsigais; it was localized between Tf B and Tf C. The frequencies of corresponding alleles were very low.
Individual transferrin variants (I, A, H_Czech, B, K_czech, C, D, E, and P) were treated with neuraminidase. Electrophoretic mobility of the strong band was decreased by two steps in each case. It suggests that in the strong Tf band two sialic acid residues are accessible to the enzyme.     

Group-specific component (Gc): subtypes in the Finnish population. Description of a new allele and an apparent mother-child incompatibility

The group-specific component (Gc) subtypes were determined in 575 adult Finns by immunoblotting after isoelectric focusing in agarose gel. The gene frequencies were Gc1S = 0.661, Gc1F = 0.139 and Gc2 = 0.200. This material included one rare allele, a more acidically focusing Gc 2 (named Gc 2A18). The phenotypes of 200 mother-child pairs studied were in accordance with the three-allelic mode of inheritance. An apparent mother-child incompatibility observed during routine paternity testing is reported.     

Quantitative studies of the interaction of cholecalciferol ((vitamin D3) and its metabolites with different genetic variants of the serum binding protein for these sterols.

Cholecalciferol (vitamin D3) and its 25-hydroxy metabolite are transported in plasma bound to a specific protein, the binding protein for cholecalciferol and its metabolites (DBP). DBP is identical with the group-specific component (Gc) proteins, which are known to display genetic polymorphism. Studies were conducted to explore whether or not major differences in the transport of cholecalciferol and its biological metabolites might exist among persons with different Gc phenotypes. Detailed quantitative studies were first carried out on the interaction of 25(OH)D3 with DBP in 21 different samples of serum, representing eight different Gc phenotypes. The studies used a filter disc assay method that provided highly reproducible quantitative results with cholecalciferol-related sterols. The Gc phenotypes studied included the three common types (Gc 1-1, 2-1, and 2-2) and several uncommon genetic variants (Gc Ab-Ab, Ab-1, Ab-2, Chip-1, and Chip-2). The binding affinities for 25(OH)D3 observed with these different sera were all fairly similar to each other. More extensive studies were then conducted to compare the binding of four cholecalciferol-related sterols to each of three genetic variants of DBP, by using sera from homozygous persons with the Gc 1-1, Gc 2-2 and Gc Ab-Ab phenotypes. The ligands tested included cholecalciferol, 25(OH)D3, 1,25(OH)2D3, and 24(R) 25(OH)2D3. The affinities of the three genetic types of DBP/Gc protein were found to be similar for each of the four cholecalciferol-related sterols. The apparent association constants for 25(OH)D3 and 24,25(OH)2D3 were similar (approx. 1--2 x 10(8) M-1); lesser affinities were observed for 1,25(OH)2D3 (kA approx. 1 x 10(7) M-1) and for cholecalciferol (kA approx. 3--4 x 10(5) M-1). Thus the common genetic variants of DBP/Gc protein, and the uncommon genetic variants studied here, all appear to have similar binding properties for cholecalciferol and its several metabolites.     

Post-translational heterogeneity of the human vitamin D-binding protein (group-specific component)

The vitamin D-binding protein in human serum (the group-specific component) is an alpha 2-globulin which is genetically polymorphic in all populations studied. Previous work (J. Svasti and B. H. Bowman (1978) J. Biol. Chem. 253, 5188-5194, and J. Svasti, A. Kurosky, A. Bennett, and B. H. Bowman (1979) Biochemistry 18, 1611-1617) has shown that the electrophoretic variations of the proteins controlled by two allelic genes, Gc1 and Gc2, are due to at least three amino acid substitutions between Gc1 and Gc2 (Svasti et al. (1979] and to heterogeneity in the Gc1 phenotype arising from carbohydrate dissimilarities. Gc1 migrates electrophoretically as two protein bands, while Gc2 migrates cathodally as a single band. This study demonstrates a post-translational glycosylation difference occurring in a single area of the Gc1 sequence which accounts for the heterogeneity observed previously. The glycosylation site, a threonine residue, appears to be in a sequence which differs between Gc1 and Gc2. The O-glycosidic bond, which is typical of mucins, is rare in plasma proteins. The cyanogen bromide fragment containing the galactosamine-containing carbohydrate in Gc1 was partially sequenced through 20 residues from the amino terminus. No detectable galactosamine could be found in the homologous cyanogen bromide fragment in Gc2. A new purification procedure for the vitamin D-binding protein in human plasma has been developed. Three chromatographic steps provide purified protein.     

Partial characterization of horse transferrin heterogeneity with respect to the atypical type, Tf C

In starch gel electrophoresis of horse sera each transferrin variant is formed by a strong anodal band and a weaker cathodal band. An 'atypical' variant, Tf C, has two zones of about equal intensity. Family data show that Tf C is genetically controlled by an allele Tf C at the Tf locus. Frequencies of transferrin alleles in various horse breeds are also presented.
After isolation and fractionation of individual transferrin variants (Tf O, Tf D, Tf C) on DEAE-Sephad Summary ex, additional weak bands were detected. The two main zones of each variant were isolated in a pure state and treated with neuraminidase. In all three variants studied the electrophoretic mobility of the slower band (2a) was decreased in two steps, and the faster band (4b) in four steps. The mobilities of bands derived from the fast zone (4b) were slower than mobilities of corresponding bands derived from the slow zone (2a). These results suggest the presence of two sialic acid residues in the slow zone, and of four residues in the fast zone. Residual heterogeneity was independent of sialic acid.     

A monoclonal antibody against human vitamin-D-binding protein for the analysis of genetic variation in the group-specific component system (Gc)

Summary We have developed a murine hybridoma cell line that is stable in secreting a monoclonal antibody (hDBP-1) directed against the group-specific component (Gc) molecule. The hDBP-1 is monospecific for Gc and does not crossreact with human albumin, which has 23% of its amino acid residues identical with vitamin-D-binding protein (DBP). The subclass of the antibody is IgG1 for the heavy chain, the light chain being of the kappa type. Isoelectric focusing discloses four major bands for the hDBP-1 with isoelectric points between pH 6.5 and 7.8. Binding to the antigen at different pH values was determined: there is high affinity in the physiological range and no binding at pH 3.5 and lower. In the presence of high salt concentrations, binding was reduced to about 50% at 1.5 M NaCl. The hDBP-1 recognizes the common human Gc types and the Gc of all apes and old world monkeys. No reaction was observed with the Gc of other mammals such as horses, cattle, rats, rabbits, sheep, goats and pigs. By testing hDBP-1 against 77 of the more than 120 known rare human Gc variants, it could be shown that this monoclonal antibody cannot recognize seven of these rare variants and can only poorly recognize nine. The binding site of hDBP-1 to Gc is not related to the binding site of Gc with G-actin: it recognizes Gc, the binary complex between Gc and G-actin, as well as the ternary complex between Gc, G-actin and DNase I. Competition assays with vitamin D₃ and Gc in enzyme-linked immunosorbent assay indicate that the epitope of hDBP-1 on the Gc molecule may be related to the vitamin-D₃-binding site.     

Purification, microheterogeneity, and stability of human lipid transfer protein

A method for the purification of lipid transfer protein (LTP) from human plasma was developed with the aid of succinylated low density lipoprotein-Sepharose affinity column chromatography. The purified LTP exhibited a single main band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, upon isoelectric focusing on polyacrylamide gel, the preparations consistently showed nine bands with isoelectric points ranging from 4.6 to 5.4. The treatment of LTP with Clostridium perfringens neuraminidase shifted these multiple bands toward higher pH regions due to the release of sialic acid. Extensive treatment with neuraminidase resulted in the appearance of a major band with the isoelectric point of 5.6. The purified LTP was rapidly inactivated upon incubation at 37 degrees C due to the denaturation at the "air"-water interface. Various factors promoting or preventing this interfacial denaturation were elucidated. When purified LTP was stored at 4 degrees C, plasma neuraminidase co-purified with LTP became activated, resulting in the gradual desialylation of LTP. It seemed that the LTP preparations of apparent homogeneity are associated with a trace amount of an inactive form of plasma neuraminidase. The inclusion of 4 mM 2-mercaptoethanol or 0.2% EDTA in the storage media completely prevented the activation of plasma neuraminidase. These agents, however, did not significantly inhibit the already activated neuraminidase. When LTP was stored at -20 degrees C in very low ionic strength media, such as 0.001% EDTA (pH 7.4) and at high protein concentrations, the loss of the activity was minimal even after prolonged storage.     

Distribution of group-specific component/vitamin-D-binding protein subtypes in Saudi Arabia

The distribution of group-specific component (Gc) phenotypes and the Gc allele frequencies were investigated in 1,082 individuals from five different provinces of Saudi Arabia by the combined use of isoelectric focusing and immunofixation. Between provinces variations in the Gc allele frequencies were found. Gc1S decreased and Gc1F increased from the northwest to the southeast in Saudi Arabia. The overall frequencies in Saudi Arabia were 0.236 for Gc1F, 0.610 for Gc1S, 0.150 for Gc2 and 0.004 for rare alleles. The observed frequencies did not differ significantly from those found in other population samples from the Middle East. In nine samples rare Gc variants were found.     

Three new orosomucoid (ORM) variants revealed by isoelectric focusing and print immunofixation

Summary Phenotypes of orosomucoid (ORM) in human sera have been analysed by isoelectric focusing and print immunofixation. After neuraminidase treatment the band patterns indicated that the polymorphism of the structural locus ORM1 is controlled by three autosomal codominant alleles. According to the previous nomenclature they were called ORM1^*F1, ORM1^*F2, and ORM1^*S. In a study of 272 unrelated individuals from southern Germany, five of the six expected common ORM1 subtypes were observed. Furthermore, we found three ORM variant phenotypes which have not been reported previously. These variants were characterized by additional bands in a cathodal position. One variant had additional double bands and presumably represents a rare ORM1 variant named ORM1S1. Two variants had additional single bands. They were assigned tentatively to the ORM2 gene locus. While the common gene product of ORM2 may be called ORM2A, the two variants are named ORM2B1 and ORM2B2, respectively. ORM2B1 has, thus far, been found only in a single individual; the variants ORM1S1 and ORM2B2 were found in a father-child pair and a mother-child pair, respectively. The frequency for variants tentatively assigned to the ORM2 locus is very low and was calculated to be 0.0037.     

Protein subtype polymorphisms (Gc and Tf) in a Catalan population from Gerona (northeastern Spain)

The Tf and Gc polymorphic subtype variants have been examined by means of isoelectric focusing in a population sample from two subpyrenean regions in the province of Gerona (Northeast Spain). The estimated allele frequencies were Tf*C1 = 0.774, Tf*C2 = 0.167, TF*C3 = 0.055, TF*B = 0.004; Gc*1F = 0.129, Gc*1S = 0.555 and Gc*2 = 0.316. These values are in general similar to those so far reported in other Spanish populations. The comparisons between our data and those published in Spain, indicate that the present sample is closer to Barcelona than to the other groups compared.     

Gc subtypes identified by isoelectric focusing in Baboons (Papio hamadryas)

Summary Phenotyping for Gc variants by conventional electrophoresis in 835 Papio hamadryas baboons demonstrated a monomorphic population. Gc subtyping by polyacrylamide IEF gels, pH 4–6, on 394 of these baboons revealed the existence of two common alleles which we named Gc _Papio ¹ and Gc _Papio ² . Pedigree data confirmed the inheritance of a single locus, two allele system and the observed gene frequencies were 0.593 for Gc _Papio ¹ and 0.407 for Gc _Papio ² .