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.     

Population distribution in North and Central America of PGM1 and Gc subtypes as determined by isoelectric focusing (IEF)

Gc subtypes of 20 North and Central American populations and PCM₁ subtypes in 11 populations were analyzed to identify interpopulation variation of the respective gene frequencies for common alleles. A total of 23,304 phenotypings were done. Absolute heterozygosity levels (D) generally increased twofold when phenotyping by isoelectric focusing was compared with conventional electrophoresis. Graphic representation of the Gc subtypes and multivariate analysis to identify genetic affinities of the populations under study reveal genetic clusters consistent with major historical and geographical groupings of man.     

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.     

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.     

Sensitive and Specific Detection of the Non-Human Sialic Acid N-Glycolylneuraminic Acid In Human Tissues and Biotherapeutic Products

Background

Humans are genetically defective in synthesizing the common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc), but can metabolically incorporate it from dietary sources (particularly red meat and milk) into glycoproteins and glycolipids of human tumors, fetuses and some normal tissues. Metabolic incorporation of Neu5Gc from animal-derived cells and medium components also results in variable contamination of molecules and cells intended for human therapies. These Neu5Gc-incorporation phenomena are practically significant, because normal humans can have high levels of circulating anti-Neu5Gc antibodies. Thus, there is need for the sensitive and specific detection of Neu5Gc in human tissues and biotherapeutic products. Unlike monoclonal antibodies that recognize Neu5Gc only in the context of underlying structures, chicken immunoglobulin Y (IgY) polyclonal antibodies can recognize Neu5Gc in broader contexts. However, prior preparations of such antibodies (including our own) suffered from some non-specificity, as well as some cross-reactivity with the human sialic acid N-acetylneuraminic acid (Neu5Ac).

Methodology/Principal Findings

We have developed a novel affinity method utilizing sequential columns of immobilized human and chimpanzee serum sialoglycoproteins, followed by specific elution from the latter column by free Neu5Gc. The resulting mono-specific antibody shows no staining in tissues or cells from mice with a human-like defect in Neu5Gc production. It allows sensitive and specific detection of Neu5Gc in all underlying glycan structural contexts studied, and is applicable to immunohistochemical, enzyme-linked immunosorbent assay (ELISA), Western blot and flow cytometry analyses. Non-immune chicken IgY is used as a reliable negative control. We show that these approaches allow sensitive detection of Neu5Gc in human tissue samples and in some biotherapeutic products, and finally show an example of how Neu5Gc might be eliminated from such products, by using a human cell line grown under defined conditions.

Conclusions

We report a reliable antibody-based method for highly sensitive and specific detection of the non-human sialic acid Neu5Gc in human tissues and biotherapeutic products that has not been previously described.     

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.     

Neisseria gonorrhoeae phagosomes delay fusion with primary granules to enhance bacterial survival inside human neutrophils

Symptomatic infection with Neisseria gonorrhoeae (Gc) promotes inflammation driven by polymorphonuclear leucocytes (PMNs, neutrophils), yet some Gc survive PMN exposure during infection. Here we report a novel mechanism of gonococcal resistance to PMNs: Gc phagosomes avoid maturation into phagolysosomes by delayed fusion with primary (azurophilic) granules, which contain antimicrobial components including serine proteases. Reduced phagosome‐primary granule fusion was observed in gonorrheal exudates and human PMNs infected ex vivo. Delayed phagosome–granule fusion could be overcome by opsonizing Gc with immunoglobulin. Using bacterial viability dyes along with antibodies to primary granules revealed that Gc survival in PMNs correlated with early residence in primary granule‐negative phagosomes. However, when Gc was killed prior to PMN exposure, dead bacteria were also found in primary granule‐negative phagosomes. These results suggest that Gc surface characteristics, rather than active bacterial processes, influence phagosome maturation and that Gc death inside PMNs occurs after phagosome–granule fusion. Ectopically increasing primary granule–phagosome fusion, by immunoglobulin opsonization or PMN treatment with lysophosphatidylcholine, reduced intracellular Gc viability, which was attributed in part to serine protease activity. We conclude that one method for Gc to avoid PMN clearance in acute gonorrhoea is by delaying primary granule–phagosome fusion, thus preventing formation of a degradative phagolysosome.     

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.     

Purification and identification of N-glycolylneuraminic acid (Neu5Gc) from the holothuroidea Gumi, Cucumaria echinata.

N-Glycolylneuraminic acid (Neu5Gc), precious sialic acid which could not be synthesized by a chemical method, occurrs in the body of holothuroidea, Gumi Cucumaria echinata. Gumi contains 85% of total sialic acid, as Neu5Gc, in the body. Neu5Gc was purified from dry powder of the body using Dowex 1-x8 (HCOO* form) anion exchange chromatography after mild acid hydrolysis with 0.1 N trifluoroacetic acid. Using GC-MS and 1H-NMR spectroscopy, the purified Neu5Gc was correctly identified to be Neu5Gc. The purity of Neu5Gc was more than 99%. This is the first report of purification and identification of Neu5Gc from holothuroidea by using anion exchange chromatography, GC-MS, and 1H-NMR.     

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.     

N-glycolylneuraminic acid as a carbohydrate cancer biomarker

One of the forms of aberrant glycosylation in human tumors is the expression of N-glycolylneuraminic acid (Neu5Gc). The only known enzyme to biosynthesize Neu5Gc in mammals, cytidine-5′-monophosphate-N-acetylneuraminic acid (CMAH), appears to be genetically inactivated in humans. Regardless, low levels of Neu5Gc have been detected in healthy humans. Therefore, it is proposed that the presence of Neu5Gc in humans is from dietary acquisition, such as red meat. Notably, detection of elevated Neu5Gc levels has been repeatedly found in cancer tissues, cells and serum samples, thereby Neu5Gc-containing antigens may be exploited as a class of cancer biomarkers. Here we review the findings to date on using Neu5Gc-containing tumor glycoconjugates as a class of cancer biomarkers for cancer detection, surveillance, prognosis and therapeutic targets. We review the evidence that supports an emerging hypothesis of de novo Neu5Gc biosynthesis in human cancer cells as a source of Neu5Gc in human tumors, generated under certain metabolic conditions.     

Investigations on the variability of haptoglobin, transferrin and Gc polymorphisms in Assam, India

Ten different population groups of Assam - Brahmins, Kalitas, Kaibartas, Rajbanshis, Muslims, Ahoms, Chutias, Kacharis, Karbis and Sandwals - have been typed for haptoglobin and for transferrin (Tf) and Gc subtype polymorphisms. Tf and Gc allele subtype frequencies show a considerable inter-population heterogeneity. From genetic distance analysis it appears that the populations under study form some distinct clusters, which can be explained by the historical and ethnic affiliations of these populations. Especially the distribution of Gc subtype alleles reveals some Mongoloid admixture among Assamese populations, which is reflected by the presence of Gc1A8 alleles in them.     

Metabolism of Vertebrate Amino Sugars with N-Glycolyl Groups: MECHANISMS UNDERLYING GASTROINTESTINAL INCORPORATION OF THE NON-HUMAN SIALIC ACID XENO-AUTOANTIGEN N-GLYCOLYLNEURAMINIC ACID

Although N-acetyl groups are common in nature, N-glycolyl groups are rare. Mammals express two major sialic acids, N-acetylneuraminic acid and N-glycolylneuraminic acid (Neu5Gc). Although humans cannot produce Neu5Gc, it is detected in the epithelial lining of hollow organs, endothelial lining of the vasculature, fetal tissues, and carcinomas. This unexpected expression is hypothesized to result via metabolic incorporation of Neu5Gc from mammalian foods. This accumulation has relevance for diseases associated with such nutrients, via interaction with Neu5Gc-specific antibodies. Little is known about how ingested sialic acids in general and Neu5Gc in particular are metabolized in the gastrointestinal tract. We studied the gastrointestinal and systemic fate of Neu5Gc-containing glycoproteins (Neu5Gc-glycoproteins) or free Neu5Gc in the Neu5Gc-free Cmah(-/-) mouse model. Ingested free Neu5Gc showed rapid absorption into the circulation and urinary excretion. In contrast, ingestion of Neu5Gc-glycoproteins led to Neu5Gc incorporation into the small intestinal wall, appearance in circulation at a steady-state level for several hours, and metabolic incorporation into multiple peripheral tissue glycoproteins and glycolipids, thus conclusively proving that Neu5Gc can be metabolically incorporated from food. Feeding Neu5Gc-glycoproteins but not free Neu5Gc mimics the human condition, causing tissue incorporation into human-like sites in Cmah(-/-) fetal and adult tissues, as well as developing tumors. Thus, glycoproteins containing glycosidically linked Neu5Gc are the likely dietary source for human tissue accumulation, and not the free monosaccharide. This human-like model can be used to elucidate specific mechanisms of Neu5Gc delivery from the gut to tissues, as well as general mechanisms of metabolism of ingested sialic acids.     

Analysis of the N-acetylneuraminic acid and N-glycolylneuraminic acid contents of glycoproteins by high-pH anion-exchange chromatography with pulsed amperometric detection

Presence or absence of N-acetylneuraminic acid (Neu5Ac) can change a sialylated glycoprotein's serum half-life and possibly its function. We evaluated the linearity, sensitivity, reproducibility, and accuracy of a HPAEC/PAD method to determine its suitability for routine simultaneous analysis of Neu5Ac and N-glycolylneuraminic acid (Neu5Gc). An effective internal standard for this analysis is 3-deoxy-d-glycero-d- galacto-2-nonulosonic acid (KDN). We investigated the effect of the Au working electrode recession and determined that linear range and sensitivity were dependent on electrode recession. Using an electrode that was 350 &mgr;m recessed from the electrode block, the minimum detection limits of Neu5Ac, KDN, and Neu5Gc were 2, 5, and 2 pmol, respectively, and were reduced to 1, 2, and 0.5 pmol using a new electrode. The response of standards was linear from 10 to 500 pmol (r2>0.99) regardless of electrode recession. When Neu5Ac, KDN, and Neu5Gc (200 pmol each) were analyzed repetitively for 48 h, area RSDs were <3%. Reproducibility was unaffected when injections of glycoprotein neuraminidase and acid digestions were interspersed with standard injections. Area RSDs of Neu5Ac and Neu5Gc improved when the internal standard was used. We determined the precision and accuracy of this method for both a recessed and a new working electrode by analyzing Neu5Ac and Neu5Gc contents of bovine fetuin and bovine and human transferrins. Results were consistent with published values and independent of the working electrode. The sensitivity, reproducibility, and accuracy of this method make it suitable for direct routine analysis of glycoprotein Neu5Ac and Neu5Gc contents.      

N-Glycolylneuraminic acid in human tumours

N-Glycolylneuraminic acid (Neu5Gc) is an abundant sialic acid, occurring in the glycoconjugates of most deuterostome animals. Homo sapiens is a notable exception, since Neu5Gc is effectively absent from normal human tissues. This is due to a deletion in the human gene coding for CMP-Neu5Ac hydroxylase, the enzyme usually responsible for Neu5Gc biosynthesis. Despite this mutation, persistent reports in the literature suggest that Neu5Gc occurs in the glycoconjugates of many human tumours, where it might be responsible for the formation of so-called Hanganutziu-Deicher antibodies. However, the variety of systems studied and the various experimental approaches adopted have yielded a complex picture of Neu5Gc occurrence in human neoplasias. The aim of this paper is therefore to provide a critical review of the evidence for Neu5Gc in human tumours, paying particular attention to the analytical methods employed. The possible clinical applications of Neu5Gc-containing glycoconjugates and Hanganutziu-Deicher antibodies in the diagnosis and treatment of breast cancer and melanoma are also discussed. In view of the lack of CMP-Neu5Ac hydroxylase in human cells, alternative metabolic pathways for the biosynthesis of glycoconjugate-bound Neu5Gc are considered.     

On the variability of Gc subtypes in Italy

909 individuals from different places of Italy were analyzed for the distribution of Gc subtypes. The observed heterogeneity in the distribution of the allele frequencies was found to be statistically significant. Comparing our results with those reported by other authors it is seen that within Italy a considerable regional variation in the frequencies of the Gc subtype alleles is present. However, there are no indications for any particular distribution patterns or gradients. In one of our samples (Bari district), one case of Gc 1S-1C3 was found.     

Heritability of quantitative variation at the group-specific component (Gc) locus

Human group-specific component (Gc) is the plasma transport protein for vitamin D; in addition, polymorphic electrophoretic variants of Gc are found in all human populations. Because of its physiologic importance and in view of the extensive genetic variation at the Gc locus, we have determined the heritability of quantitative variation in Gc by comparing a series of monozygotic (MZ) and dizygotic (DZ) twins of known Gc genotype. The series included 31 MZ twin pairs, 13 DZ twin pairs, and 45 unrelated controls. Since Gc concentration is increased by estrogens, pregnant women and women taking oral contraceptives were excluded. We found no age-related differences in Gc concentration or differences between males and females, but the concentrations of Gc in the three electrophoretically determined genotypes were significantly different from each other. Using classical methods of heritability analysis, the overall heritability of variation in Gc concentration is approximately 70%. Heritability in males is greater than in females, probably reflecting the additional environmental effect of estrogens in women. To determine if the differences in Gc concentration between the three genotypes explain the high heritability, a new variance decomposition procedure was developed following classical methods in quantitative genetics. Application of this method suggests that 19% of the total variation in Gc concentration, combining both sexes, is due to electrophoretic differences between individuals (30% in females and 20% in males). Thus, the genetic component of variation in Gc concentration can be decomposed into a major gene component--the result of electrophoretic variation at the structural locus--and a second, unexplained, polygenic component.     

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.     

PCR and sequence analysis of barley chromosome 2H subjected to the gametocidal action of chromosome 2C

Gametocidal (Gc) chromosomes induce various types of chromosomal mutations during gametogenesis in the chromosomes of common wheat and alien chromosomes added to common wheat. However, it is not yet known whether the Gc chromosome causes aberrations at the nucleotide level because mutations caused by Gc chromosomes have been studied only by cytological screening. In order to know whether the Gc chromosome induces point mutations, we conducted PCR analysis and sequencing with the progeny of a common wheat line that is disomic for barley chromosome 2H and monosomic for Gc chromosome 2C. We analyzed 18 2H-specific EST sequences using 81 progeny plants carrying a cytologically normal-appearing 2H chromosome and found no nucleotide changes in the analyzed 1,419 sequences (in total 647,075 bp). During this analysis, we found six plants for which some ESTs could not be PCR amplified, suggesting the presence of chromosomal mutations in these plants. The cytological and PCR analyses of the progeny of the six plants confirmed the occurrence of chromosomal mutations in the parental plants. These results suggested that the Gc chromosome mostly induced chromosomal aberrations, not nucleotide changes, and that the Gc-induced chromosomal mutations in the six plants occurred after fertilization.     

N-glycolylneuraminic acid deficiency in humans

Classic studies suggested that the common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc) is an oncofetal antigen in humans, being immunogenic in adult humans and yet apparently expressed in human fetuses and tumors. We and others have recently found that the human deficiency of Neu5Gc can be explained by an inactivating mutation in the gene encoding CMP-N-acetylneuraminic acid hydroxylase. Thus, Neu5Gc is not an oncofetal antigen in the classical sense, and other explanations must be found for the observed expression pattern. This review provides an update on this matter, and considers a variety of other old and new questions that arise from it.