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1.
Evolutionary and structural relationships among the group-specific component, albumin and alpha-fetoprotein. 总被引:2,自引:0,他引:2
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F Yang V J Luna R D McAnelly K H Naberhaus R L Cupples B H Bowman 《Nucleic acids research》1985,13(22):8007-8017
The group-specific component (Gc) is a plasma protein that binds vitamin D. Recent characterization of human Gc cDNA demonstrated homology with serum albumin and alpha-fetoprotein. This study compares the sequences of the three proteins and demonstrates a strong evolutionary relationship. Albumin, alpha-fetoprotein and Gc evolved from an ancestral gene containing an intragenic triplication. Comparison of the amino acid sequences and patterns of double disulfide bonds suggests that the Gc gene may have diverged from an ancestral gene earlier in evolution than the genes encoding albumin and alpha-fetoprotein. Analysis of the amino acid and nucleotide sequences of the three internal domains of Gc revealed 19-23% amino acid sequence identity and the localization of three homology blocks with 40-44% nucleotide sequence identity. The deduced amino sequence of Gc furnished data for comparing its molecular configuration based on the predicted secondary structure with those predicted for human albumin and alpha-fetoprotein. Utilization of Gc cDNA has also led to the identification of its genomic DNA and detection of a human DNA polymorphism. 相似文献
2.
M Ogata I Nakasono M Iwasaki S Kubo H Suyama 《Comparative biochemistry and physiology. B, Comparative biochemistry》1988,90(1):193-199
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. 相似文献
3.
M. J. Stanhope D. A. Tagle M. S. Shivji M. Hattori Y. Sakaki J. L. Slightom M. Goodman 《Journal of molecular evolution》1993,37(2):179-189
One of the uncertainties regarding the evolution of L1 elements is whether there are numerous progenitor genes. We present
phylogenetic evidence from ORF1 sequences of slow loris (Nycticebus coucang) and galago (Galago crassicaudatus) that there were at least two distinct progenitors, active at the same time, in the ancestor of this family of prosimian
primates. A maximum parsimony analysis that included representative L1s from human, rabbit, and rodents, along with the prosimian
sequences, revealed that one of the galago L1s (Gc11) grouped very strongly with the slow loris sequences. The remaining galago
elements formed their own unique and strongly supported clade. An analysis of replacement and silent site changes for each
link of the most parsimonious tree indicated that during the descent of the Gc11 sequence approximately two times more synonymous
than nonsynonymous substitutions had occurred, implying that the Gc11 founder was functional for some time after the split
of galago and slow loris. Strong purifying selection was also evident on the galago branch of the tree. These data indicate
that there were two distinct and contemporaneous L1 progenitors in the lorisoid ancestor, evolving under purifying selection,
that were retained as functional L1s in the galago lineage (and presumably also in the slow loris). The prosimian ORF1 sequences
could be further subdivided into subfamilies. ORF1 sequences from both the galago and slow loris have a premature termination
codon near the 3′ end, not shared by the other mammalian sequences, that shortens the open reading frame by 288 bp. An analysis
of synonymous and nonsynonymous substitutions for the 5′ and 3′ portions, that included intra- and inter-subfamily comparisons,
as well as comparisons among the other mammalian sequences, suggested that this premature stop codon is a prosimian acquisition
that has rendered the 3′ portion of ORF1 in these primates noncoding.
Presented at the NATO Advanced Research Workshop onGenome Organization and Evolution, Spetsai, Greece, 16–22 September 1992 相似文献
4.
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. 相似文献
5.
Post-translational heterogeneity of the human vitamin D-binding protein (group-specific component) 总被引:4,自引:0,他引:4
D H Coppenhaver N P Sollenne B H Bowman 《Archives of biochemistry and biophysics》1983,226(1):218-223
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. 相似文献
6.
Brinkman-Van der Linden EC Sjoberg ER Juneja LR Crocker PR Varki N Varki A 《The Journal of biological chemistry》2000,275(12):8633-8640
The common sialic acids of mammalian cells are N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Humans are an exception, because of a mutation in CMP-sialic acid hydroxylase, which occurred after our common ancestor with great apes. We asked if the resulting loss of Neu5Gc and increase in Neu5Ac in humans alters the biology of the siglecs, which are Ig superfamily members that recognize sialic acids. Human siglec-1 (sialoadhesin) strongly prefers Neu5Ac over Neu5Gc. Thus, humans have a higher density of siglec-1 ligands than great apes. Siglec-1-positive macrophages in humans are found primarily in the perifollicular zone, whereas in chimpanzees they also occur in the marginal zone and surrounding the periarteriolar lymphocyte sheaths. Although only a subset of chimpanzee macrophages express siglec-1, most human macrophages are positive. A known evolutionary difference is the strong preference of mouse siglec-2 (CD22) for Neu5Gc, contrasting with human siglec-2, which binds Neu5Ac equally well. To ask when the preference for Neu5Gc was adjusted in the human lineage, we cloned the first three extracellular domains of siglec-2 from all of the great apes and examined their preference. In fact, siglec-2 had evolved a higher degree of recognition flexibility before Neu5Gc was lost in humans. Human siglec-3 (CD33) and siglec-6 (obesity-binding protein 1) also recognize both Neu5Ac and Neu5Gc, and siglec-5 may have some preference for Neu5Gc. Others showed that siglec-4a (myelin-associated glycoprotein) prefers Neu5Ac over Neu5Gc. Thus, the human loss of Neu5Gc may alter biological processes involving siglec-1, and possibly, siglec-4a or -5. 相似文献
7.
The lipooligosaccharide‐modifying enzyme LptA enhances gonococcal defence against human neutrophils
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Infection with Neisseria gonorrhoeae (Gc) is marked by an influx of neutrophils to the site of infection. Despite a robust immune response, viable Gc can be recovered from neutrophil‐rich gonorrhoeal secretions. Gc enzymatically modifies the lipid A portion of lipooligosaccharide by the addition of phosphoethanolamine to the phosphate group at the 4′ position. Loss of lipooligosaccharide phosphoethanolamine transferase A (LptA), the enzyme catalysing this reaction, increases bacterial sensitivity to killing by human complement and cationic antimicrobial peptides. Here, we investigated the importance of LptA for interactions between Gc and human neutrophils. We found that lptA mutant Gc was significantly more sensitive to killing by human neutrophils. Three mechanisms underlie the increased sensitivity of lptA mutant Gc to neutrophils. (i) lptA mutant Gc is more likely to reside in mature phagolysosomes than LptA‐expressing bacteria. (ii) lptA mutant Gc is more sensitive to killing by components found in neutrophil granules, including CAP37/azurocidin, human neutrophil peptide 1 and the serine protease cathepsin G. (iii) lptA mutant Gc is more susceptible to killing by antimicrobial components that are exocytosed from neutrophils, including those decorating neutrophil extracellular traps. By increasing the resistance of Gc to the bactericidal activity of neutrophils, LptA‐catalysed modification of lipooligosaccharide enhances survival of Gc from the human inflammatory response during acute gonorrhoea. 相似文献
8.
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. 相似文献
9.
J Constans S Hazout R M Garruto D C Gajdusek E K Spees 《American journal of physical anthropology》1985,68(1):107-122
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. 相似文献
10.
Isolation and characterization of the O-glycan chain of the human vitamin-D binding protein 总被引:4,自引:0,他引:4
M Viau J Constans H Debray J Montreuil 《Biochemical and biophysical research communications》1983,117(1):324-331
On a highly purified preparation, the structure of the carbohydrate chain of the human vitamin D-binding protein was investigated and two genetic forms of this protein were considered (Gc 2 and Gc 1 proteins). It was found that only the Gc 1 protein (Gc1a isoform) was glycosylated, the glycan moiety representing about 1% of the protein. The structure of this O-glycosidically linked glycan was determined to be: Neu Ac alpha (2 leads to 3) Gal beta (1 leads to 3) GaINAc alpha (1 leads to 0) Ser (or Thr). A tetrasaccharidic O-glycan with two N-acetylneuraminic residues was also characterized. The vitamin D-binding protein is a rare example of a serum protein O-glycosylated only on some genetic forms. 相似文献
11.
M Osawa A R Sabbatini J Erukhimov P A Werner R M Galbraith 《Biochimica et biophysica acta》1992,1117(3):271-278
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. 相似文献
12.
Zusammenfassung Es wurden die Seren 33 anthropoider Affen (Pan 12, Gorilla 6, Pongo 11, Symphalangus 4) mit der Agargel-Immunoelektrophorese auf das Vorkommen von group specific components untersucht.Bei allen anthropoiden Affen konnten wir Gc-Globuline mit Anti-Human-Gc-Pferdeseren nachweisen. Bei Pongo und bei Gorilla sind die Gc-Phänotypen hominid geprägt. Bei Pongo fanden sich die Gc-Typen Gc 1-1, Gc 2-1 und Gc 2-2, bei Gorilla fand sich der Typ Gc 1-1. Der bei Pan und Symphalangus fefundene Gc-Typ steht außerhalb des Gc-Systems von Homo; er wird vermutlich durch ein eigenes Allel kontrolliert, das wir mit Gcape bezeichnen.Mit drei verschiedenen Anti-Human-Gc-Seren erwiesen sich die Gc-Globuline der anthropoiden Affen immunologisch mit denen des Menschen identisch.
Mit 5 Textabbildungen
Mit Unterstützung durch die Deutsche Forschungsgemeinschaft. 相似文献
Sera of 33 hominide primates (Pan 12, Gorilla 6, Pongo 11, Symphalangus 4) have been examined for the presence of the group specific components (Gc) by agar-gel immunoelectrophoresis.With the use of anti-human-Gc-horsesera Gc-globulins were demonstrated in all 33 sera of the four hominide primate species. Gc phenotypes of Pongo and of Gorilla were indistinguishable from human Gc phenotypes. In Pongo the types Gc 1-1, Gc 2-1 and Gc 2-2 have been observed, in Gorilla only the type Gc 1-1 has been found. The Gc phenotype in sera of Pan and Symphalangus was found to be different from the Gc phenotypes in Man. This Gc-type is probably determined by a specific Gc allele, for which the notation Gcape has been given.With three different anti-human-Gc-sera reaction of immunologic identity has been demonstrated between the human Gc and the Gc of the four hominide primate species.
Mit 5 Textabbildungen
Mit Unterstützung durch die Deutsche Forschungsgemeinschaft. 相似文献
13.
P J Goldschmidt-Clermont R C Allen A E Nel D L Emerson J R Day R M Galbraith 《Life sciences》1986,38(8):735-742
Limited proteolysis of G-actin was performed with trypsin and chymotrypsin to compare the binding sites for Gc and DNase. DNase I bound to the N-terminal area corresponding to the major cleavage site on G-actin (residues 62-68) and inhibited proteolysis, but did not bind the 33.5K C-terminal fragment (G-actin33.5) generated. In contrast, Gc did not exert any inhibitory effect upon proteolysis of the intact native G-actin42.0 molecule, although its presence protected G-actin33.5 from further proteolysis. This was shown by gel filtration to be due to the formation of complexes between Gc and G-actin33.5. 相似文献
14.
Xiaohong Shi Jo?l T. van Mierlo Andrew French Richard M. Elliott 《Journal of virology》2010,84(17):8460-8469
The virion glycoproteins Gn and Gc of Bunyamwera virus (BUNV), the prototype of the Bunyaviridae family and also of the Orthobunyavirus genus, are encoded by the medium (M) RNA genome segment and are involved in both viral attachment and entry. After their synthesis Gn and Gc form a heterodimer in the endoplasmic reticulum (ER) and transit to the Golgi compartment for virus assembly. The N-terminal half of the Gc ectodomain was previously shown to be dispensable for virus replication in cell culture (X. Shi, J. Goli, G. Clark, K. Brauburger, and R. M. Elliott, J. Gen. Virol. 90:2483-2492, 2009.). In this study, the coding sequence for a fluorescent protein, either enhanced green fluorescent protein (eGFP) or mCherry fluorescent protein, was fused to the N terminus of truncated Gc, and two recombinant BUNVs (rBUNGc-eGFP and rBUNGc-mCherry) were rescued by reverse genetics. The recombinant viruses showed bright autofluorescence under UV light and were competent for replication in various mammalian cell lines. rBUNGc-mCherry was completely stable over 10 passages, whereas internal, in-frame deletions occurred in the chimeric Gc-eGFP protein of rBUNGc-eGFP, resulting in loss of fluorescence between passages 5 and 7. Autofluorescence of the recombinant viruses allowed visualization of different stages of the infection cycle, including virus attachment to the cell surface, budding of virus particles in Golgi membranes, and virus-induced morphological changes to the Golgi compartment at later stages of infection. The fluorescent protein-tagged viruses will be valuable reagents for live-cell imaging studies to investigate virus entry, budding, and morphogenesis in real time.Bunyamwera virus (BUNV) is the prototype of both the family Bunyaviridae and the genus Orthobunyavirus. The characteristic features shared by all viruses in the family (known as bunyaviruses) include spherical virion morphology, possession of a tripartite, single-stranded RNA genome of negative or ambisense polarity, cytoplasmic site of virus replication, and assembly and budding of progeny particles at membranes of the Golgi complex (6, 27). The family includes a number of significant human pathogens such as La Crosse virus (LACV), Hantaan virus (HTNV), Sin Nombre virus (SNV), Rift Valley fever virus (RVFV), and Crimean-Congo hemorrhagic fever virus (CCHFV) (7). All bunyaviruses encode four structural proteins, two surface glycoproteins called Gn and Gc, and two internal proteins, N (nucleocapsid protein that encapsidates the genomic RNA segments) and L (RNA-dependent RNA polymerase). In addition, the majority of bunyaviruses also encode nonstructural proteins. The sizes of the viral proteins vary considerably across the family though they are relatively well conserved between viruses within a particular genus. The glycoproteins form spikes on the virion surface and are involved in viral attachment and cell fusion (35). They are encoded by the medium (M) RNA genome segment as a polyprotein precursor (Gn at the N terminus and Gc at the C terminus) that is cleaved cotranslationally to yield the mature virion glycoproteins. Both glycoproteins are type I integral transmembrane (TM) proteins and are modified by N-linked glycosylation. Gn and Gc form a heterodimer in the endoplasmic reticulum (ER) prior to trafficking and retention in the Golgi compartment for virus assembly (31, 35). The BUNV M segment additionally encodes a nonstructural protein termed NSm that is sandwiched between Gn and Gc (19). BUNV NSm is also an integral membrane protein, and the N-terminal domain, at least, of NSm is required for virus assembly (42). BUNV Gn is able to target to the Golgi complex alone, whereas correct folding, maturation, and Golgi complex targeting of the Gc protein depends on the chaperone-like assistance of Gn (18, 38, 44).The BUNV Gn protein consists of 302 residues with a rather long predicted cytoplasmic tail (CT) of 78 residues, while the larger Gc protein comprises 957 residues with a CT of only 25 residues (Fig. (Fig.1)1) (8, 19). The CT domains of both Gn and Gc play crucial roles in BUNV-mediated membrane fusion, virus assembly, and morphogenesis (43). Functional analysis of deletion mutants of BUNV Gc indicated that nearly half of its N-terminal ectodomain (453 residues out of 909 residues) is dispensable for Golgi trafficking, cell fusion, and virus replication in cell culture (41). Similarly, characterization of mutants of the related Maguari virus (MAGV) also showed that the N-terminal domain of Gc was not essential for growth in cell culture (33). These data suggested that it might be possible to insert foreign sequences, e.g., those encoding an autofluorescent protein, in place of the N-terminal domain to generate recombinant viruses expressing a tagged Gc protein (41).Open in a separate windowFIG. 1.Schematic diagrams of eGFP- and mCherry-tagged BUNV glycoproteins. The layout of the wt BUNV glycoprotein precursor (Gn, NSm, and Gc) is shown at the top, with positions of amino acid residues marking the protein boundaries indicated. Below is shown the structure of the chimeric Gc protein, with substitution of the N terminus of Gc (residues 500 to 826) with the coding sequence of either enhanced green fluorescent protein (eGFP) or mCherry fluorescent protein (mC) attached to truncated Gc. The predicted topology of Gn and eGFP/mCherry-tagged Gc on the viral envelope is shown at the bottom. SS, signal peptide; TMD, transmembrane domain. The filled diamonds indicate glycosylation sites.In this paper we report the successful insertion of the coding region of either the enhanced green fluorescent protein (eGFP) or mCherry red fluorescent protein into the BUNV glycoprotein precursor to replace the dispensable region at the N terminus of the Gc ectodomain. Viable viruses with Gc tagged by either eGFP or mCherry were rescued by reverse genetics, and fluorescent extracellular virions were detectable by conventional fluorescence or confocal microscopy. The processes of virus attachment and internalization and budding of progeny virions could be visualized in infected cells. 相似文献
15.
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. 相似文献
16.
Sandra L. Diaz Vered Padler-Karavani Darius Ghaderi Nancy Hurtado-Ziola Hai Yu Xi Chen Els C. M. Brinkman-Van der Linden Ajit Varki Nissi M. Varki 《PloS one》2009,4(1)
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. 相似文献17.
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. 相似文献
18.
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. 相似文献
19.
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. 相似文献
20.
Tina Ravnsborg Dorthe T. Olsen Anna Hammerich Thysen Maja Christiansen Gunnar Houen Peter Højrup 《Biochimica et Biophysica Acta - Proteins and Proteomics》2010,1804(4):909-917
The vitamin D binding protein, Gc globulin, has in recent years received some attention for its role as precursor for the extremely potent macrophage activating factor (GcMAF). An O-linked trisaccharide has been allocated to the threonine residue at position 420 in two of the three most common isoforms of Gc globulin (Gc1s and Gc1f). A substitution for a lysine residue at position 420 in Gc2 prevents this isoform from being glycosylated at that position. It has been suggested that Gc globulin subjected sequentially to sialidase and galactosidase treatment generates GcMAF in the form of Gc globulin with only a single GalNAc attached to T420. In this study we confirm the location of a linear trisaccharide on T420. Furthermore, we provide the first structural evidence of the generation of the proposed GcMAF by use of glycosidase treatment and mass spectrometry. Additionally the generated GcMAF candidate was tested for its effect on cytokine release from macrophages in human whole blood. 相似文献