Group-specific Component Protein Research Articles

Simple method for large-scale production of macrophage activating factor GcMAF

Human group-specific component protein (Gc protein) is a multifunctional serum protein which has three common allelic variants, Gc1F, Gc1S and Gc2 in humans. Gc1 contains an O-linked trisaccharide [sialic acid-galactose-N-acetylgalactosamine (GalNAc)] on the threonine420 (Thr420) residue and can be converted to a potent macrophage activating factor (GcMAF) by selective removal of sialic acid and galactose, leaving GalNAc at Thr420. In contrast, Gc2 is not glycosylated. GcMAF is considered a promising candidate for immunotherapy and antiangiogenic therapy of cancers and has attracted great interest, but it remains difficult to compare findings among research groups because different procedures have been used to prepare GcMAF. Here, we present a simple, practical method to prepare high-quality GcMAF by overexpressing Gc-protein in a serum-free suspension culture of ExpiCHO-S cells, without the need for a de-glycosylation step. We believe this protocol is suitable for large-scale production of GcMAF for functional analysis and clinical testing.

Group-specific component exon 11 haplotypes (D432E and T436K) and risk of albuminuria in type 2 diabetes mellitus patients

Background Emerging evidence indicates group-specific component (GC) variants are associated with ethnicity. We aimed to investigate the association of GC variants and protein expression level with T2DM and diabetic nephropathy (DN) in Saudi patients. Subjects and methods A total of 200 participants (120 T2DM/80 controls) were genotyped for GC-rs7041/GC-rs4588 by real-time polymerase chain reaction. Serum GC was assessed by ELISA and in silico analysis was executed. Results GC-rs7041 frequency distribution showed no difference between the study groups, while GC-rs4588 showed association with T2DM under all genetic models. rs4588*AA variant was correlated with higher serum GC globulin, albuminuria, and poor glycaemic control. A higher frequency of rs7041*TT and rs4588*AA was evident in macroalbuminuria vs. normoalbuminuria group. Carrying GC-2 haplotype was 2.5 more likely to develop diabetes and correlated with the levels of albuminuria. Conclusions GC variants could have independent effects on the risk of T2DM and DN in the study population.

Fine mapping of a QTL on bovine chromosome 6 using imputed full sequence data suggests a key role for the group-specific component (GC) gene in clinical mastitis and milk production.

BackgroundClinical mastitis is an inflammation of the mammary gland and causes significant costs to dairy production. It is unfavourably genetically correlated to milk production, and, thus, knowledge of the mechanisms that underlie these traits would be valuable to improve both of them simultaneously through breeding. A quantitative trait locus (QTL) that affects both clinical mastitis and milk production has recently been fine-mapped to around 89 Mb on bovine chromosome 6 (BTA6), but identification of the gene that underlies this QTL was not possible due to the strong linkage disequilibrium between single nucleotide polymorphisms (SNPs) within this region. Our aim was to identify the gene and, if possible, the causal polymorphism(s) responsible for this QTL through association analysis of high-density SNPs and imputed full sequence data in combination with analyses of transcript and protein levels of the identified candidate gene.ResultsAssociations between SNPs and the studied traits were strongest for SNPs that were located within and immediately upstream of the group-specific component (GC) gene. This gene encodes the vitamin D-binding protein (DBP) and has multiple roles in immune defense and milk production. A 12-kb duplication that was identified downstream of this gene covered its last exon and segregated with the QTL allele that is associated with increased mastitis susceptibility and milk production. However, analyses of GC mRNA levels on the available samples revealed no differences in expression between animals having or lacking this duplication. Moreover, we detected no differences in the concentrations of DBP and its ligand vitamin D between the animals with different GC genotypes that were available for this study.ConclusionsOur results suggest GC as the gene that underlies the QTL for clinical mastitis and milk production. However, since only healthy animals were sampled for transcription and expression analyses, we could not draw any final conclusion on the absence of quantitative differences between animals with different genotypes. Future studies should investigate GC RNA expression and protein levels in cows with different genotypes during an infection.Electronic supplementary materialThe online version of this article (doi:10.1186/s12711-016-0257-2) contains supplementary material, which is available to authorized users.

Association between liver-specific gene polymorphisms and their expression levels with nonalcoholic fatty liver disease

Genetic factors account for a significant proportion of the phenotypic variance of nonalcoholic fatty liver disease (NAFLD); however, very few predisposing genes have been identified. We aimed to (1) identify novel genetic associations with NAFLD by performing a genome-wide association study (GWAS), and (2) examine the biological expression of the strongest genetic associations in a separate cohort. We performed GWAS of a population-based cohort (Raine Study) of 928 adolescents assessed for NAFLD by ultrasound at age 17. Expression of genes with single nucleotide polymorphisms (SNPs) that were associated with NAFLD at a significance level of P < 10(-5) was examined in adults with NAFLD and controls by quantifying hepatic messenger RNA (mRNA) expression and serum levels of protein. After adjustment for sex and degree of adiposity, SNPs in two genes expressed in liver were associated with NAFLD adolescents: group-specific component (GC) (odds ratio [OR], 2.54; P = 1.20 × 10(-6)) and lymphocyte cytosolic protein-1 (LCP1) (OR, 3.29; P = 2.96 × 10(-6)). SNPs in two genes expressed in neurons were also associated with NAFLD: lipid phosphate phosphatase-related protein type 4 (LPPR4) (OR, 2.30; P = 4.82 × 10(-6)) and solute carrier family 38 member 8 (SLC38A8) (OR, 3.14; P = 1.86 × 10(-6) ). Hepatic GC mRNA was significantly reduced (by 83%) and LCP1 mRNA was increased (by 300%) in liver biopsy samples from patients with NAFLD compared to controls (P < 0.05). Mean serum levels of GC protein were significantly lower in patients with NAFLD than controls (250 ± 90 versus 298 ± 90, respectively; P = 0.004); GC protein levels decreased with increasing severity of hepatic steatosis (P < 0.01). The association between GC and LCP1 SNPs and NAFLD as well as altered biological expression implicate these genes in the pathogenesis of NAFLD.

Role of vitamin D on cancer immunotherapy

Dear Editor in Chief, With a great interest, we read a recently published paper in Frontier in Endocrinology journal by Vuolo et al., entitled “Vitamin D and Cancer” (Vuolo et al., 2012). They skillfully reviewed the role of vitamin D in all aspects of cancer such as cancer prevention, immune system and cancer, reverse effect of cancer on vitamin D, and vitamin D status in some important type of malignancy. This issue is a hot topic in oncology, but in this study there are some concerns which undermine the results to make a definite conclusion. Although they concluded that “The immune system seems to represent a relevant target for the antineoplastic effects of vitamin D,” (Vuolo et al., 2012) they did not consider anti-neoplastic role of activated form of vitamin D using for immunotherapy of cancer. It was recently shown that a group of vitamin D-binding protein (DBP) so called Gc protein is an important precursor for activating macrophage as one of the main innate immune cell for cancer immunotherapy (Nagasawa et al., 2005). Although the aim of the review by Vuolo et al. was the role of Vitamin D in cancer, it was confirmed that the level of vitamin D is related to Vitamin D binding proteins (Carpenter et al., 2012). In normal tissue, when a neoplastic tissue is arise, DBP is naturally activated by glycosylation and transform to DBP-derived macrophage-activating factor (Gc-MAF) by b-galactosidase and sialidase of B and T cells, respectively, which can activate macrophage to phagocyte the neoplastic cells. Group-specific component protein (Gc) as DBP preserve vitamin D in body fluids and put it available for tissues. But an enzyme (a-N-acetylgalactosaminidase) produced by neoplastic cells deactivate and denaturize this factor and provide neoplastic cells to spread (Yamamoto and Naraparaju, 1998; Ghanei et al., 2012). In some clinical studies, Gc-MAF has successfully been used for prostate, colorectal, and breast cancer (Yamamoto and Suyama, 2008; Yamamoto et al., 2008a,b). Therefore, vitamin D has a prominent role in natural tumorocidal activity of immune system as an initial precursor.

Erratum: Relationship between Group-Specific Component Protein and the Development of Asthma

Erratum: Relationship between Group-Specific Component Protein and the Development of Asthma

Association and expression quantitative trait loci (eQTL) analysis of porcine AMBP, GC and PPP1R3B genes with meat quality traits

The aim of this research was to screen polymorphism and to perform association study of porcine AMBP (alpha-1-microglobulin/bikunin precursor), GC (group-specific component protein) and PPP1R3B (protein phosphatase 1, regulatory (inhibitor) subunit 3B) genes with meat quality traits as well as to unravel the transcriptional regulation of these genes by expression QTL (eQTL) study. For this purpose, Duroc×Pietrain F2 resource population (DuPi; n=313) and a commercial breed Pietrain (Pi; n=110) were used for association and only DuPi for expression and eQTL study. A SNP was identified in the genes AMBP (g.22229C>T), GC (g.398C>T) and PPP1R3B (c.479A>G), respectively. In DuPi SNP of AMBP was associated (P<0.05) with meat colour, pH(1L), pH(24L), pH(24H) and conductivity(24L); SNP of GC showed tendency to association (P<0.10) with pH24H, conductivity(1L) and thawing loss, and SNP of PPP1R3B was associated (P<0.05) with meat colour, pH(1L), pH(24L), pH(24H) and shear force. In Pi SNPs of AMBP and GC was associated with pH(24H) and PPP1R3B SNP was associated with pH(24L). The mRNA levels in Longissimus dorsi muscle tissue of these three genes were evaluated by using qRT-PCR to identify association between gene expression and meat quality traits as well as to analyse eQTL. The mRNA expression of PPP1R3B associated with pH(24L) (P<0.05). Expression of these three genes was higher in animals with low pH of muscle. Linkage analysis using QTL Express revealed ten trans-regulated eQTL on seven porcine autosomes. Suggestive eQTL [P<0.05, CW (chromosome-wide)] were found for PPP1R3B on SSC3 and 13. These results revealed that genetic variation and gene expression of these genes are associated with the meat quality traits. These three genes could influence meat quality and could be potential positional, physiological and functional candidate gene for meat quality traits in pigs. However, the analysis of eQTL also suggested that we need to consider additional genes encoding for transcription factors (TF), via fine-mapping underlying the eQTL peaks, in order to understand interaction among these genes.

Relationship between Group-Specific Component Protein and the Development of Asthma

Airway inflammation and remodeling during asthma are attributed to the altered expression of biologically relevant proteins. To search for asthma-specific proteins in bronchoalveolar lavage fluid (BAL) from individuals with asthma and to validate the identified proteins in an experimental model of asthma. Liquid chromatography-tandem mass spectrometry was performed to identify proteins in BAL fluid found by two dimensional electrophoresis (2DE) to be differentially expressed in subjects with asthma versus control subjects. Group-specific component (Gc) and mRNA levels were measured using an ELISA, Western blots, and PCR. A neutralization study using an antibody against Gc protein was performed in an experimental asthma model. Based on 2DE, 15 proteins were significantly up-regulated or down-regulated in eight subjects with asthma compared with eight control subjects. The protein levels of Gc, hemopexin, and haptoglobin-b were increased, whereas the a1- antitrypsin and glutathione S-transferase levels were decreased in subjects with asthma. The Gc concentration in BAL fluid was significantly elevated in 67 subjects with asthma compared with that in 22 control subjects (P < 0.009). The Gc was significantly correlated with the neutrophil percentage in BAL fluid of subjects with asthma (P = 0.001). Gc mRNA and protein levels were higher in ovalbumin-sensitized/ challenged asthma mice than in sham-treated mice. Gc protein were expressed on alveolar macrophages and on epithelial cells. Treatment with an anti-Gc antibody dose-dependently reduced the ovalbumin sensitization/challenge-induced enhancement of airway hyperreactivity, airway inflammation, goblet cell hyperplasia,and levels of eotaxin, interleukin-4, -5, and -13, and interferon-g. Gc may be involved in the development of asthma, and the neutralization of Gc protein could be a therapeutic strategy for asthma.

Variations in vitamin D-binding protein (group-specific component protein) are associated with fasting plasma insulin levels in Japanese with normal glucose tolerance.

The locus of the vitamin D-binding protein (DBP; also known as group-specific component protein or Gc) gene, chromosome 4q12, has been reported to be associated with glucose metabolism in several ethnic groups, including Pima Indians. We have recently reported the association of the DBP genotype with type 2 diabetes mellitus in Japan. The aim of this study was to investigate whether genetic variations of DBP have any influence on glucose metabolism without secondary effects of hyperglycemia or diabetes mellitus using 82 Japanese with normal glucose tolerance. The variations of the DBP gene (Gc 1F, 1S, and 2) were determined by PCR-restriction fragment length polymorphism. Fasting plasma insulin concentration and homeostasis model assessment, an index of insulin resistance, were significantly different based on the DBP genotype (P < 0.01 and P < 0.05, respectively). The people with Gc 1S-2 (5.73 +/- 2.57 microU/mL) and 1S-1S (5.30 +/- 3.46 microU/mL) had significantly higher fasting plasma concentrations than those with 1F-1F (2.84 +/- 1.67 microU/mL) (P < 0.01 and P < 0.03, respectively). There was no significant difference in plasma glucose concentration, body mass index, total cholesterol, triglyceride, and blood pressure. In conclusion, genetic variations of DBP are associated with insulin resistance in Japanese with normal glucose tolerance, which might contribute to the development of type 2 diabetes.

Variations in Vitamin D-Binding Protein (Group-Specific Component Protein) Are Associated with Fasting Plasma Insulin Levels in Japanese with Normal Glucose Tolerance

Variations in Vitamin D-Binding Protein (Group-Specific Component Protein) Are Associated with Fasting Plasma Insulin Levels in Japanese with Normal Glucose Tolerance

Immunonephelometric quantification of group-specific component protein in patients with acute liver failure

This chapter discusses the immunonephelometric assay for immunoglobulins released by cultured lymphocytes. The human immune system, like those of many other mammals and vertebrates, is a complex conglomerate of specific and nonspecific components, among which the immunologists have paid particular attention to two groups of immunocompetent cells that include T lymphocytes and B lymphocyte. Immunonephelometry was introduced for the assay of serum proteins, particularly for diagnostic purposes. The influence of culture conditions, such as cell concentration, culture volume, stationary vs rocking cultures, and addition of glutamine to the cultures every other day, have been analyzed separately, using unfractionated purified B lymphocytes (PBL). The immunoglobulin production was studied by the determination of levels in day 7 supernatants of pokeweed mitogen-stimulated cultures of PBL from 12 healthy donors and S. aureus-stimulated cultures of PBL from 7 healthy donors. Immunonephelometric assays could be devised for any other proteins released from cell cultures, providing that suitable antisera are available, which do not cross-react, with the exogenous proteins used to supplement the cultures.

Immunonephelometric quantification of group-specific component protein in patients with acute liver failure.

Serum levels of group-specific component (Gc) protein are useful in evaluating the likelihood of survival in patients with acute liver failure (ALF) who may be candidates for liver transplant surgery. Most methods for quantifying Gc protein concentration are either isotopic, manual, technically demanding, and/or time consuming to perform, and thus are not well suited for routine clinical use in a hospital setting. We modified and evaluated a recently described nonisotopic, fully automated, immunonephelometric method for quantifying serum Gc protein concentration and compared it to our previous immunoblotting method. In addition, we evaluated the effect of G-actin on the immunonephelometric measurement of Gc protein. Serum samples from 20 patients with ALF and from 20 age- and sex-matched clinic patients without liver disease were quantified by both immunoblotting and immunonephelometry. We assessed the intra-assay precision, correlation, and diagnostic accuracy of these methods in discriminating between individuals with no preexisting liver disease and those with ALF. Actin in 1.3- to 4-fold excess of Gc protein levels demonstrated minimal to no interference in the quantification of Gc protein by immunonephelometry. Immunonephelometry was more precise than immunoblotting. Gc protein values by immunonephelometry were similar to those obtained by immunoblotting, and the diagnostic accuracy of Gc protein concentration by immunonephelometry was similar to that observed by immunoblotting. Immunonephelometry provides a nonisotopic, fully automated, rapid, precise, accurate, and cost-effective method for quantifying serum levels of total Gc protein that is well suited for routine use in a hospital-based clinical laboratory.

Immunonephelometric quantification of group-specific component protein in patients with acute liver failure

Serum levels of group-specific component (Gc) protein are useful in evaluating the likelihood of survival in patients with acute liver failure (ALF) who may be candidates for liver transplant surgery. Most methods for quantifying Gc protein concentration are either isotopic, manual, technically demanding, and/or time consuming to perform, and thus are not well suited for routine clinical use in a hospital setting. We modified and evaluated a recently described nonisotopic, fully automated, immunonephelometric method for quantifying serum Gc protein concentration and compared it to our previous immunoblotting method. In addition, we evaluated the effect of G-actin on the immunonephelometric measurement of Gc protein. Serum samples from 20 patients with ALF and from 20 age- and sex-matched clinic patients without liver disease were quantified by both immunoblotting and immunonephelometry. We assessed the intra-assay precision, correlation, and diagnostic accuracy of these methods in discriminating between individuals with no preexisting liver disease and those with ALF. Actin in 1.3- to 4-fold excess of Gc protein levels demonstrated minimal to no interference in the quantification of Gc protein by immunonephelometry. Immunonephelometry was more precise than immunoblotting. Gc protein values by immunonephelometry were similar to those obtained by immunoblotting, and the diagnostic accuracy of Gc protein concentration by immunonephelometry was similar to that observed by immunoblotting. Immunonephelometry provides a nonisotopic, fully automated, rapid, precise, accurate, and cost-effective method for quantifying serum levels of total Gc protein that is well suited for routine use in a hospital-based clinical laboratory. (Liver Transpl Surg 1997 Jan;3(1):28-33)

Non-genetic group specific component (GC) observed in a patient with acute non-lymphocytic leukemia

This is the case of a male non-lymphocytic leukemia patient who continuously exhibited non-genetic group specific component (GC). The patient was hospitalized 10 months for treatment. The patient's GC type was GC 2-1A2 upon his admission to the hospital, which was consistent with his parents' type. The additional GC protein isoform appeared during treatment and was detected by polyacrylamide gel isoelectric focusing (PAGIEF). This acquired GC component corresponded to GC 1F in PAGIEF and was thought to have originated from GC 1A2 protein. This phenomenon most probably occurred due to a metabolic abnormality or modification of the GC molecule caused by the disease itself, or by chemotherapy.

血清ｇｒｏｕｐ‐ｓｐｅｃｉｆｉｃ ｃｏｍｐｏｎｅｎｔ蛋白と頭皮組織内汗腺にｍｏｓａｉｃｉｓｍを立証した先天性母斑児についての免疫遺伝学的検討

There are two broad categories of chimera: twin and dispermic. Here we report a case of a young girl who was a dispermic chimera and had both group A1 and B red cells in her serum simultaneously.The patient (H. B.) was a five-year old girl with congenital nevus. She was born by normal delivery as the first child of healthy parents. She had nerve received a transfusion, and there was no evidence of a twin. Her blood contained 96% group B, type MNss red cells and 4% group A1, type NNss red cells. The patient's saliva also contained A and B substances at normal secretor ranges. The level of D-galactosaminyltransferase activity in her serum was within the normal ranges but the level of N-acetyl-d-galactosaminyltransferase activity was at the extreme lower end of the normal range for group A1B persons. One hundred metaphase cells from a culture of the patient's peripheral lymphocytes were all 46, XX. The phenotype of the serum group-specific component (GC) protein of the father was GC2-1S, and of the mother, GC2-1F, but the patient's GC protein was GC1S-1F-(-2). This suggests that the patient's serum contained two populations of GC protein. The sweat gland cells of her scalp tissue stained with both anti-A and anti-B reagents.These findings demonstrate this child to be a dispermic chimera according to Race and Sanger's classification system.

Typing of human red cell and serum mixtures in three electrophoretic systems

The isoenzymes erythrocyte acid phosphatase and phosphoglucomutase were typed in mixed red cell samples which had been derived from two individuals; the protein group specific component was typed in mixed serum samples. Typing was performed by isoelectric focusing on ultrathin polyacrylamide gels. Depending upon the mixture, from 2 to 20% (but typically 5-10%) by volume of a second blood or serum needed to be present in a mixture before it could be detected. In the majority of cases when there was significant mixing, samples were readily identified as a mixture when the results consisted of unusual band patterns or unusual band intensities. There would be a few instances when blood or serum could not be identified as a mixture when masking effects occurred or when the mixture produced a combined, apparently normal, pattern.

Group specific component protein (Gc) in patients with acute hepatic necrosis

Group specific component protein (Gc) in patients with acute hepatic necrosis

Decreased serum group-specific component protein levels and complexes with actin in fulminant hepatic necrosis

Monomeric G-actin has recently been shown to form high-affinity complexes with group-specific component protein (Gc), and this process might be expected to occur in vivo when hepatocyte necrosis with release of actin takes place. We therefore measured serum Gc levels and searched for evidence of actin-containing complexes of Gc in 147 sera from 21 normal subjects and 126 patients with acute and chronic liver diseases. Sera were examined for Gc concentration using an ELISA with purified Gc as standard. Gc levels were greatly diminished (less than 10 mg per dl) in 5 of 37 patients with acute hepatitis and 1 of 3 patients with subacute hepatic necrosis, and the mean values for these groups overall as well as those for chronic active hepatitis patients were significantly less than that observed in the normal controls (mean, 35.5 mg per dl). No significant alteration in Gc level was observed in the chronic disease categories studied. In contrast, Gc concentrations less than 10 mg per dl were found in 12 of 14 patients with fulminant hepatic necrosis, and the mean for this group (9.7) was significantly reduced (p less than 0.001). Sequential studies in 12 patients with acute hepatitis of moderate to marked severity but without a fulminant phase showed significant diminutions in initial Gc levels when these were compared with Gc levels obtained during recovery. Using analytical isoelectric focusing with print immunofixation, variable amounts of Gc:G-actin complexes could be demonstrated in virtually all sera examined, but the proportion of total Gc present as complex with G-actin appeared greater in fulminant hepatic necrosis sera.(ABSTRACT TRUNCATED AT 250 WORDS)

Assignment of the structural gene coding for albumin to human chromosome 4.

Albumin is a developmentally regulated serum protein synthesized in the liver mainly during adulthood. Family studies using variant forms of albumin established autosomal linkage between albumin and group-specific component protein (GS). Since GC has been assigned to human chromosome 4, albumin can be indirectly assigned to the same chromosome; however no direct assignment has been made. Recently, the human albumin cDNA probe has been isolated and characterized. It thus permits a direct chromosomal assignment of the albumin gene in the human genome. When the cDNA probe was hybridized to the HindIII digested total human DNA, an intense band at 6.8 kb was present. When the probe was hybridized to the HindIII digested Chinese hamster CHO-K1 DNA, a less intense band at 3.5 kb was found, plus three other faint bands. When the probe was hybridized to a series of human/CHO-K1 cell hybrids retaining a complete hamster genome and various combinations of human chromosomes, it was evident that hybrids containing human albumin gene sequences could be readily distinguished from hybrids containing no human albumin gene. Analysis of 22 primary cell hybrids for the presence or absence of human albumin sequences has assigned the albumin gene to human chromosome 4. Similar results were obtained using another restriction endonuclease EcoR1. Thus, by direct assay of the genomic albumin gene sequences in the cell hybrids, we provide evidence for a direct assignment of the structural gene for human albumin to chromosome 4.