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.     

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.     

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.     

Comparative immunological and electrophoretic analysis of Gc protein in sera from various animals

1. On immunodiffusion, using an anti-human Gc antibody, serum Gc in all mammals tested revealed a partial identity with human Gc. 2. The relative cross-reactivities of serum Gc in monkeys, dogs, cats and rats with human Gc antiserum were found to be more than 70% while the serum Gc in other mammals (pigs, cattle, goats and a guinea pig) was less than 50%. 3. Testing, using the isoelectrofocusing method, showed specific patterns of Gc in the mammals. In the sera of cats and cattle, Gc polymorphisms were detected. 4. Neuraminidase treatment affected the isoelectrofocusing Gc patterns of pigs, goats and cattle, whereas those in other mammals remained unchanged.     

Group-specific component (Gc) 'subtypes' of Gc1 by isoelectric focusing in US blacks and whites.

Isoelectric focusing was applied to the Gc polymorphism. In agreement with Constans et al., we found two common 'subtypes' of Gc1 that could not be identified by conventional electrophoretic procedures. They are labeled Gc1F and Gc1S. Gc1F has a slightly lower isoelectric point than Gc1S. In groups of US blacks the allele frequencies were for Gc1F; 0.732 and for Gc1S; 0.147. In whites these figures were 0.149 and 0.572. We also found GcAb in blacks with a frequency of 0.015. The concentrations in serum of Gc protein as measured by radial immunodiffusion did not differ according to phenotype.     

Hereditary recombined three-allele variant of the Gc system

A three-allele variant with Gc 2, Gc 1F and Gc 1A2 alleles was detected in both a baby and his mother during paternity testing by isoelectric focusing. His father had a normal Gc phenotype, Gc 2-1F. Further examination of his mother's relatives revealed that his grandfather also had the same three-allele variant, while his grandmother and his aunt had normal Gc 2-1F and Gc 2-2. From these results, it was considered that the Gc 1F and Gc 1A2 alleles were on the same single chromosome. It was suggested that recombination had occurred between two chromosomes that had the Gc 1F and Gc 1A2 allele, respectively, forming the variant allele Gc 1F1A2 on a single chromosome.     

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.     

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.     

A faster migrating Gc-variant: Gc Darmstadt

Summary In three members of a family from Darmstadt (Germany) a faster migrating Gc variant has been observed. The variant phenotypes have been examined by routine immunoelectrophoresis (Fig. 1), by immunoelectrophoresis with prolonged separation times and with Gc-monospecific antisera (Fig. 2), by polyacrylamide gel electrophoresis (Fig. 3), and by antigen-antibody crossed electrophoresis (Fig. 4). By antigen-antibody crossed electrophoresis the new Gc variant was clearly distinguishable from the Gc Aborigine and from the Gc Chippewa variant. The variant was named Gc Darmstadt (Gc D). Gc Darmstadt has an electrophoretic migration rate intermediate between Gc Ab and Gc 1. In two sibs the type Gc D-2 was observed, the daughter of one of these sibs had the type Gc D-1. The analysis of several members of this family provided only limited information on the mode of inheritance of Gc Darmstadt (Fig. 5). Gc Darmstadt appears to be determined by a gene Gc^D which may be allelic to Gc¹ and Gc².

---

Zusammenfassung Bei drei Angehörigen einer Familie aus Darmstadt (Deutschland) wurde eine schneller wandernde Gc-Variante beobachtet. Die neue Variante, die eindeutig von Gc Aborigine und Gc Chippewa unterschieden werden kann, wurde Gc Darmstadt (Gc D) genannt. Bei elektrophoretischer Auftrennung liegt Gc Darmstadt zwischen Gc Ab und Gc 1. Gc Darmstadt ist sehr wahrscheinluch durch ein Gen Gc^D bedingt, das ein Allel zu Gc¹ und Gc² ist.

---

---

Supported by U.S.-PHS Grant AM 11796 and aided by a grant from the Deutsche Forschungsgemeinschaft, Bad Godesberg.     

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.     

Characterization of a knock-out mutation at the <Emphasis Type="Italic">Gc2</Emphasis> locus in wheat

Gametocidal (Gc) genes, introduced into common wheat from related Aegilops species, are selfish genetic elements that ensure their preferential transmission by inducing chromosomal breaks. Here we report the production and characterization of a knock-out mutation of the Gc2 gene transferred to wheat as a wheat-Aegilops sharonensis T4B-4S(sh)#1 translocation chromosome. In hemizygous Gc2/- condition, gametophytes lacking Gc2 suffer chromosomal fragmentation and produce non-functional gametes, which leads to sporophytic semisterility and exclusive transmission of the Gc2-carrier chromosome. We have identified one putative ethyl methylsulfonate (EMS)-induced Gc2 mutant that restores spike fertility and shows Mendelian segregation. Progeny screening mapped the mutation to the Gc2-carrier chromosome T4B-4S(sh)#1. C-banding and fluorescence in situ hybridization analyses showed that the loss of Gc2 function in the mutant is not due to a terminal deficiency. Analysis of first and second pollen mitoses in Gc2(mut) /- plants and C-banding analysis of testcross progenies showed that no chromosomal breakage occurs in the mutant. No gametophytic chromosomal breakage was observed in heterozygous Gc2(mut) /Gc2 plants, which had fully fertile spikes. These results suggest that Gc2 encodes two agents, one causing chromosomal breaks in gametophytes lacking Gc2 and another that protects the Gc2 carrier from breakage. The EMS-induced Gc2 mutant appears to be a knock-out of the gene encoding the "breaking" agent. These data are a first crucial step toward the molecular understanding of Gc2 action.     

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.     

Phenotype frequencies of vitamin D binding protein (Gc) and of posttransferrin-2 (Ptf-2) in Belgian cattle breeds*

The vitamin D binding protein (Gc) and posttransferrin-2 (Ptf-2) phenotypes have been determined in a number of Belgian cattle breeds. A very slow migrating variant of the Gc protein — Gc C — has been found in White and Red East Flemish breed. This variant was absent from the other breeds studied. This slow variant was identified as a vitamin D binding protein by autoradiography. The Gc C protein was shown to be controlled by a codominant autosomal allele Gc C at the Gclocus. The Gc C protein is probably identical with a fraction previously described in buffalo and an Italian cattle breed. The allele frequencies for the Gc and Pft-2 systems are reported for several Belgian breeds of cattle.     

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.     

Resistance of Neisseria gonorrhoeae to non-oxidative killing by adherent human polymorphonuclear leucocytes

Symptomatic infection with Neisseria gonorrhoeae (Gc) is characterized by abundant neutrophil (PMN, polymorphonuclear leucocyte) influx, but PMNs cannot clear initial infection, indicating that Gc possess defences against PMN challenge. In this study, survival of liquid-grown Gc was monitored after synchronous infection of adherent, interleukin 8-treated human PMNs. 40–70% of FA1090 Gc survived 1 h of PMN exposure, after which bacterial numbers increased. Assays with bacterial viability dyes along with soybean lectin to detect extracellular Gc revealed that a subset of both intracellular and extracellular PMN-associated Gc were viable. Gc survival was unaffected in PMNs chemically or genetically deficient for producing reactive oxygen species (ROS). This result held true even for OpaB+ Gc, which stimulate neutrophil ROS production. Catalase- and RecA-deficient Gc, which are more sensitive to ROS in vitro , had no PMN survival defect. recN and ngo1686 mutant Gc also exhibit increased sensitivity to ROS and PMNs, but survival of these mutants was not rescued in ROS-deficient cells. The ngo1686 mutant showed increased sensitivity to extracellular but not intracellular PMN killing. We conclude that Gc are remarkably resistant to PMN killing, killing occurs independently of neutrophil ROS production and Ngo1686 and RecN defend Gc from non-oxidative PMN antimicrobial factors.     

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.     

Binding of a monoclonal antibody E12 to Gc globulin (vitamin D-binding protein) is inhibited by actin.

A monoclonal antibody, E12, to human Gc globulin was raised in murine somatic cell using purified Gc. The antibody was subtyped IgG2b kappa and had a kd of 3.0 x 10(-8) M for antigen Gc. Monospecificity for Gc was demonstrated by Western blotting of normal human serum using nondenaturing polyacrylamide gel electrophoresis. As judged by ELISA, actin inhibited binding of E12 to Gc in dose-dependent fashion. Affinity chromatography studies further showed that ternary complexes of actin-Gc-E12 were not formed, and actin displaced Gc from Gc-E12 complexes. Proteolytic digestion of Gc with trypsin showed that the monoclonal antibody E12 reacted with the major 30-kDa tryptic fragment containing the amino terminal fragment of Gc, but actin did not react with this fragment. These results indicate that interaction of actin with Gc causes conformational changes which inhibit binding of E12.     

Population distribution of the human vitamin D binding protein: anthropological considerations

The polymorphism of the serum vitamin D binding protein (DBP) in humans is based on the existence of three common alleles, Gc1F, Gc1S, and Gc2, and 84 rare alleles. The geographical distribution of Gc1F, Gc1S, and Gc2 alleles shows north to south clines, together with a balanced equilibrium between the Gc1F or Gc1S allele frequency and the Gc2 frequency. The distribution of the FST values shows high variability within a geographical area. For European and North Asiatic groups, the FST values are the lowest observed, and the reason may be a long process of homogenization. Aboriginal populations from Australia and New Guinea and groups from both North Africa and South America show the greatest heterogeneity of their allele frequencies. Systematic factors such as genetic drift and selection may account for this distribution. In contrast with the three main DBP alleles, the distribution of the rare alleles corresponds to patterns of human migrations that occurred during prehistoric and historic periods. Thus, the rare mutants are of particular relevance to anthropological and genetical investigations.     

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.     

Genetic polymorphism of the vitamin D binding protein and another post-albumin protein in horse serum

Horizontal polyacrylamide gel electrophoresis, on 10% separation gel, of horse serum revealed polymorphism of the vitamin D binding protein (Gc protein) and another post-albumin protein (Pa). Family data supported the hypothesis that Gc and Pa types were controlled by autosomal codominant alleles. For both Gc and Pa proteins, the homozygous types showed a single fraction while the heterozygous type had two fractions. Pa types were found to be identical to the post-albumin types reported earlier by starch gel electrophoresis. Two Gc alleles, Gc F and Gc S , and three Pa alleles, Pa D, Pa F and Pa S , were observed in samples from Swedish (four breeds), Lipizzaner and Arab horses. The frequency of the more common allele at the two loci, i.e. Gc F and Pa F , ranged from 0.72–0.93 and from 0.58–0.99, respectively, in the different breeds studied. Plasma samples showed an extra protein fraction near the Gc S fraction and thus were found unsuitable for Gc typing.