Gc Protein Research Articles

PI, GC, HP, and TF serum protein polymorphisms in Siena, Tuscany, Italy, with a review of data for Italy

The genetic polymorphism of four serum proteins (PI, GC, HP, and TF) is analyzed in a sample of 200 unrelated individuals from the province of Siena, Tuscany, central Italy. Siena is traditionally divided into 17 Contrade, that act as social units. The aim of this report is to ascertain whether this peculiar organization has led to genetic differentiation among the inhabitants of each Contrada and from other populations of Italy. For this purpose, the frequencies of genetic markers found in Siena are also compared with those reported for the same serum proteins in Italy based on provincial samples from the literature. The statistical analyses (heterozygosity estimates, F-statistics, and cluster analysis) suggest that the Contrade represent only a cultural and historical institution without biological consequence and that the Italian population can be considered genetically homogeneous. Am. J. Hum. Biol. 9:629–646, 1997. © 1997 Wiley-Liss, Inc.

Identification and characterization of the feline herpesvirus type 1 glycoprotein C gene.

The feline herpesvirus type 1 (FHV-1) gene encoding glycoprotein C (gC) has been sequenced and identified based on its genomic location and comparative analysis to other alphaherpesvirus gCs, and the expressed gC protein was also identified by using specific monoclonal antibodies. The FHV-1 gC gene was located within a 7.0 kbp EcoRI fragment, and was 1602 bp in length. The amino acid sequence deduced from the nucleotide sequence was predicted to encode a membrane glycoprotein containing a characteristic N-terminal hydrophobic signal sequence, nine potential N-linked glycosylation sites, and C-terminal transmembrane and cytoplasmic domains. The FHV-1 gC was expressed in COS-7 cells. When flowcytometric analysis was carried out, the gC expressed in COS-7 cells reacted with a panel of monoclonal antibodies against gp113: By immunoprecipitation analysis, the gC expressed in COS-7 cells possessed molecular masses of 125-150 kilodalton, and was similar in size to that in FHV-1-infected CRFK cells.

Structural definition of a potent macrophage activating factor derived from vitamin D 3-binding protein with adjuvant activity for antibody production

Incubation of human vitamin D 3-binding protein (Gc protein), with a mixture of immobilized β-galactosidase and sialidase, efficiently generated a potent macrophage activating factor, a protein with N-acetylgalactosamine as the remaining sugar. Stepwise incubation of Gc protein with immobilized β-galactosidase and sialidase, and isolation of the intermediates with immobilized lectins, revealed that either sequence of hydrolysis of Gc glycoprotein by these glycosidases yields the macrophage-activating factor, implying that Gc protein carries a trisaccharide composed of N-acetylgalactosamine and dibranched galactose and sialic acid termini. A 3 hr incubation of mouse peritoneal macrophages with picomolar amounts of the enzymatically generated macrophage-activating factor (GcMAF) resulted in a greatly enhanced phagocytic activity. Administration of a minute amount (10–50 pg/mouse) of GcMAF resulted in a seven- to nine-fold enhanced phagocytic activity of macrophages. Injection of sheep red blood cells (SRBC) along with GcMAF into mice produced a large number of anti-SRBC antibody secreting splenic cells in 2–4 days.

Defective Lymphocyte Glycosidases in the Macrophage Activation Cascade of Juvenile Osteopetrosis

Generation of macrophage-activating factor requires a precursor protein, Gc protein (serum vitamin D3-binding protein), as well as participation of beta-galactosidase of inflammation-primed B lymphocytes and sialidase of T lymphocytes. The treatment of human peripheral blood mononuclear cells with an inflammatory lysophospholipid induced beta-galactosidase and sialidase activity of lymphocytes, leading to the generation of macrophage-activating factor and activation of monocytes/macrophages. However, lysophospholipid treatment of peripheral blood mononuclear cells from three infantile patients with osteopetrosis resulted in no significant activation of monocytes/macrophages. The lysophospholipid-inducible beta-galactosidase activity of B lymphocytes as well as that of the sialidase of T lymphocytes was found to be defective in these patients.

Linkage between the loci for serum albumin and vitamin D binding protein (GC) in the Japanese quail

SummaryVitamin D binding protein (GC) and serum protease inhibitor (PI) have been added to genetic markers in the Japanese quail. Both loci were controlled by autosomal codominant alleles named GCA, GCB and PIA, PIB, PIC, respectively. Close linkage between the loci for serum albumin (ALB) and GC protein is reported. Two recombinants were observed in 145 informative offspring of 14 families. The recombination frequency between the loci was estimated as 0.014±0.006. Thus, GC was assigned to linkage group II in the Japanese quail. No signs of linkage were observed among the loci for the ALB‐GC complex, PI. serum prealbumin 2 (PA2), phosphoglucose isomerase (PG1), 6‐phosphogluconate dehydrogenase (PGD) and esterase‐D (ESD).

Structural modification of serum vitamin D3-binding protein and immunosuppression in AIDS patients.

A serum glycoprotein, vitamin D3-binding protein (Gc protein), can be converted by beta-galactosidase of stimulated B lymphocytes and sialidase of T lymphocytes to a potent macrophage-activating factor (MAF), a protein with N-acetylgalactosamine as the remaining sugar moiety. Thus, Gc protein is a precursor for MAF. Treatment of purified Gc protein with immobilized beta-galactosidase and sialidase generates an extremely high-titered MAF (GcMAF). When peripheral blood monocytes/macrophages of 46 HIV-infected patients were treated with GcMAF (100 pg/ml), the monocytes/macrophages of all patients were efficiently activated. However, the MAF precursor activity of plasma Gc protein was low in 16 (35%) of of these patients. Loss of the MAF precursor activity appeared to be due to deglycosylation of plasma Gc protein by alpha-N-acetylgalactosaminidase found in the patient blood stream. Levels of plasma alpha-N-acetylgalactosaminidase activity in individual patients had an inverse correlation with the MAF precursor activity of their plasma Gc protein. Thus, precursor activity of Gc protein and alpha-N-acetylgalactosaminidase activity in patient blood can serve as diagnostic and prognostic indices.

Measurement of serum Gc protein levels by nephelometry: A rapid, accurate technique

Measurement of serum Gc protein levels by nephelometry: A rapid, accurate technique

Alpha-fetoprotein signal sequences: a proposed mechanism for subcellular localization and organelle targeting

Alpha-fetoprotein (AFP) is an oncofetal protein, classified in an “albuminoid” superfamily (with albumin and Vitamin-D binding (Gc) protein) comprising molecules with three characteristic globular domains. The cellular uptake and internalization of AFP and its subcellular compartmentalization has previously been reported in a multitude of cell types. Studies have also emerged which have detected and characterized binding proteins complexed to AFP in various cell membranes and intracellular compartments. However, the literature is devoid of any attempts to relate these binding proteins to possible intracellular trafficking interactions of AFP. Recombinant DNA mutation/deletion technology has provided a means to pinpoint the amino acid sequence location of organelle localization signals on various transcription factors and/or receptors. Several subdomain regions on AFP have been reported to mimic heptad dimerization regions of the steroid/thyroid nuclear receptors. In light of these transcription factor-like docking motifs reported for AFP, the present report purposes various subdomain regions which might constitute basic amino acid sequences resembling recognition signals for binding (dimerizing) proteins. AFP appears to possess multiple prototypic amino acid sequence cassettes on each domain which consist of (i) classical short, compact sequences found on both steroid and thyroid receptors; (ii) proto-signals resembling a steroid receptor type; and (iii) degenerative signal sequence similar to the thyroid/retinoic acid receptor type. The concepts identifying binding or escort proteins for AFP together with organelle signal sequences on AFP have been reconciled in a composite hypothesis to formulate a mechanism which could account for some of the growth-regulating properties described for AFP during the last decade.

Glucocorticoid-mediated regulation of protein phosphorylation in primary human T cells. Evidence for induction of phosphatase activity.

Glucocorticoid hormones (GC) have profound effects on the development and homeostasis of the immune system. In this communication we present evidence that GC regulate Ca(2+)-mediated pathways of T cell activation by a mechanism that involves abrogation of the autophosphorylation of the multifunctional Ca2+/calmodulin kinase (CaM kinase II) and induction of protein phosphatase activity. Primary human T cells were stimulated with the combination of ionomycin and phorbol ester in the presence or absence of dexamethasone (Dex) (10(-6)-10(-12) M). Stimulation of T cells resulted in a rapid activation of CaM kinase II and protein kinase C (PKC) activity as determined by the phosphorylation of synthetic peptide substrates recognized by these enzymes. Dex inhibited the activity of CaM kinase II but not PKC activity in a dose-dependent fashion (minimum effective dose 10(-10) M). Stimulation of 32P-labeled T cells induced a rapid increase in the phosphorylation level of CaM kinase II which was inhibited by Dex. The inhibitory effect of Dex on this enzyme was fully reversed in the presence of the phosphatase inhibitor okadaic acid (250 nM) or RU 486, a glucocorticoid antagonist. These results suggest that GC inhibit the activation of CaM kinase during T cell activation through a mechanism that involves both the GC receptor and protein phosphatases 2A and/or 1. Inhibition of protein phosphorylation through the induction of protein phosphatase activity may represent a novel mechanism for the diverse effects of GC on eukaryotic cells.

Regulation of human Gc (vitamin d—binding) protein levels: Hormonal and cytokine control of gene expression in vitro

Studies were performed in Hep3B hepatocytes to better elucidate the mechanisms regulating circulating levels of human group—specific component (Gc). We measured changes in Gc messenger RNA (mRNA) synthesis and levels of secreted protein resulting from treatment of hepatocytes with cytokines and hormones known to influence synthesis of other proteins of hepatic origin. We particularly focused on compounds known to be prototypic stimulants during the acute phase response. Interleukin-6 (IL-6) and dexamethasone were shown to increase Gc mRNA approximately twofold while transforming growth factor beta (TGFβ) decreased Gc mRNA in a dose-dependent fashion by up to fivefold. The effects on secreted Gc protein levels were similar. These results indicate that Gc protein appears to be regulated differently than the other members of this gene family, albumin and alpha-fetoprotein (AFP), which are negative acute phase reactants. In addition, these contrasting effects on Gc synthesis of IL-6 and dexamethasone and of TGFβ suggest that high basal levels of Gc synthesis may be maintained during the acute phase response.

Regulation of human Gc (vitamin D — binding) protein levels: Hormonal and cytokine control of gene expression in vitro

Studies were performed in Hep3B hepatocytes to better elucidate the mechanisms regulating circulating levels of human group-specific component (Gc). We measured changes in Gc messenger RNA (mRNA) synthesis and levels of secreted protein resulting from treatment of hepatocytes with cytokines and hormones known to influence synthesis of other proteins of hepatic origin. We particularly focused on compounds known to be prototypic stimulants during the acute phase response. Interleukin-6 (IL-6) and dexamethasone were shown to increase Gc mRNA approximately twofold while transforming growth factor beta (TGF beta) decreased Gc mRNA in a dose-dependent fashion by up to fivefold. The effects on secreted Gc protein levels were similar. These results indicate that Gc protein appears to be regulated differently than the other members of this gene family, albumin and alpha-fetoprotein (AFP), which are negative acute phase reactants. In addition, these contrasting effects on Gc synthesis of IL-6 and dexamethasone and of TGF beta suggest that high basal levels of Gc synthesis may be maintained during the acute phase response.

Predicting survival in fulminant hepatic failure using serum Gc protein concentrations

Plasma Gc protein sequesters actin released into the circulation after massive hepatocyte necrosis, but is greatly depleted in the process. In fulminant hepatic failure (FHF), Gc is present in serum both as a complex with actin and as unbound protein, the latter becoming completely exhausted in those patients with the most severe FHF. In the present study, 47 consecutive patients with FHF, 39 of whom were the result of acetaminophen (AC) overdose, were evaluated to determine whether measurement of Gc protein levels could be used to predict survival. Using serum samples obtained shortly after admission as well as later samples, levels for total Gc protein, percentage of Gc complexed with actin, and calculated unbound Gc remaining in serum were compared for survivors and those who died of their illness. The most marked changes were present in unbound Gc levels in nonsurvivors, the mean of which for follow-up samples was 10% of normal mean values, as compared with 23% of normal mean values in those who survived ( P < .01). Using a cutoff value for unbound Gc protein of ≥34 μg/mL to predict survival, outcome was correctly predicted in 32 of 47 (68%) patients using early samples, and in 24 of 27 (89%) patients using later sera. No differences were observed between values and/or outcome in AC and non-AC cases. Measurement of Gc protein level correctly predicted all patients dying of hepatic failure. This single measurement compares favorably with multifactorial predictive models, such as the King's College model, and might be a useful test for patients being considered for transplantation.

Predicting survival in fulminant hepatic failure using serum gc protein concentrations

Plasma Gc protein sequesters actin released into the circulation after massive hepatocyte necrosis, but is greatly depleted in the process. In fulminant hepatic failure (FHF), Gc is present in serum both as a complex with actin and as unbound protein, the latter becoming completely exhausted in those patients with the most severe FHF. In the present study, 47 consecutive patients with FHF, 39 of whom were the result of acetaminophen (AC) overdose, were evaluated to determine whether measurement of Gc protein levels could be used to predict survival. Using serum samples obtained shortly after admission as well as later samples, levels for total Gc protein, percentage of Gc complexed with actin, and calculated unbound Gc remaining in serum were compared for survivors and those who died of their illness. The most marked changes were present in unbound Gc levels in nonsurvivors, the mean of which for follow-up samples was 10% of normal mean values, as compared with 23% of normal mean values in those who survived (P&lt;.01). Using a cutoff value for unbound Gc protein of ±34 μg/mL to predict survival, outcome was correctly predicted in 32 of 47 (68%) patients using early samples, and in 24 of 27 (89%) patients using later sera. No differences were observed between values and/or outcome in AC and non-AC cases. Measurement of Gc protein level correctly predicted all patients dying of hepatic failure. This single measurement compares favorably with multifactorial predictive models, such as the King's College model, and might be a useful test for patients being considered for transplantation. (Hepathology 1995;21:101-105).

Polymorphic gene markers in Mexican-Americans residing in southern California.

The gene frequencies of nine different genetic polymorphic markers [ABO, MNS and P blood groups; haptoglobin, transferrin, Gc protein, complement (C3), properdin factor B and alpha 1-antitrypsin] were determined in 94 Mexican-Americans residing in the Los Angeles, California area. Comparisons with published data on Mexican-Americans living in other areas of the United States or in Mexico itself revealed no significant differences in the gene frequencies between this and previous studies. However, data from the current study demonstrated significant differences in ABO and haptoglobin allele frequencies compared to published non-Hispanic Caucasian data. These data suggest a large degree of genetic homogeneity in the Mexican-American population residing in the United States. Additional gene marker studies will be important to test this hypothesis and further define the degree of non-Hispanic Caucasian admixture in this population.

Roles of β-galactosidase of B lymphocytes and sialidase of T lymphocytes in inflammation-primed activation of macrophages

The outer surface of mouse B lymphocytes carries constitutive and inducible β-galactosidase isozymes. A brief (30 min) treatment of B lymphocytes with lysophosphatidylcholine (lyso-Pc) immediately induced an approximate 3-fold higher β-galactosidase activity than the constitutive isozyme of untreated B lymphocytes. Thus, the lyso-Pc-inducible isozyme is not a de novo enzyme. Outer surface of mouse T lymphocytes carries constitutive (non- Neu-1) and inducible ( Neu-1) sialidase isozymes. The lyso-Pc-inducible β-galactosidase of B lymphocytes and the Neu-1 sialidase of T lymphocytes were required for conversion of vitamin D 3-binding protein (Gc protein) to a potent macrophage activating factor. This enzymatic generation of the macrophage activating factor was mediated via enzyme-associated receptors.

Alpha-fetoprotein binding proteins: implications for transmembrane passage and subcellular localization.

Alpha-fetoprotein (AFP) is an oncofetal protein classified in a super-family together with albumin and Vitamin-D binding (Gc) protein which present as globular proteins comprised of three domains. Several subdomain regions on AFP have been previously proposed to serve as dimerization interfaces for nuclear receptors or perhaps other co-factors/inhibitors. The cellular uptake and internalization of AFP together with its subcellular compartmentalization is now well documented in a variety of cell types. A myriad of reports have emerged which have detected, identified, and characterized various binding proteins associated with AFP in different cellular compartments. However, the literature is devoid of any attempts to summarize, categorize, and relate these proteins to the various physiological activities attributed to this fetal protein. It is conceivable that AFP could interact and/or bind cytoplasmic chaperone proteins that normally escort nuclear factors or transcription co-factors through the cytoplasm toward organelle interfaces. A dual concept proposing binding or escort proteins for AFP together with subdomain dimerization interfaces on the AFP molecule can be reconciled into a composite hypothesis to formulate a rationale for the growth regulating properties ascribed to AFP during the last decade. Thus, AFP might serve as a modulator/modifier of various cell growth regulatory pathways during embryonic and fetal development in vertebrates.

Detection of the actin scavenger system in burn wound fluid

The intravascular actin scavenger system depolymerizes and sequesters actin released after tissue injury. Studies were carried out to determine if this system is active in the extracellular space during wound repair. Using burn wound fluid as a noninvasive means for analyzing the wound environment, we measured actin accumulation and actin complex formation with the plasma proteins responsible for scavenger function. Actin at concentrations as high as 0.25 mg/ml ( approximately 5 micromol/L) was found in burn wound fluid samples from 9 of 11 patients. Wound fluid also contained the two plasma proteins that bind actin-gelsolin (both plasma and cytoplasmic forms) and Gc protein. Because actin in wound fluid was complexed with gelsolin and Gc protein, we conclude that the components of the actin scavenger system are functional in wound tissue. In addition, proteolysis of gelsolin, but not actin or Gc protein, appeared to occur at the wound site. Gelsolin proteolysis was accompanied by the appearance of 49 kd gelsolin fragments, and wound fluid samples lacking intact gelsolin also contained high metalloproteinase levels.

Conversion of vitamin D3 binding protein (group-specific component) to a macrophage activating factor by the stepwise action of beta-galactosidase of B cells and sialidase of T cells.

Highly conserved DBP (human DBP is known as Gc) of serum alpha 2-globulin fraction can be converted to a potent macrophage activating factor by stepwise modification of Gc glycoprotein with beta-galactosidase of B cells and sialidase of T cells. These glycosidases, beta-galactosidase and sialidase, are membrane bound and not soluble in culture medium. Thus, consecutive contact of Gc protein with B cells and T cells, presumably via specific receptors, is required for conversion of Gc glycoprotein to the macrophage activating factor. The essential role of T cell sialidase in macrophage activation was confirmed by the finding that peritoneal nonadherent cells of SM/J mouse, whose T cells are deficient in sialidase activity, were unable to convert Gc protein to the macrophage activating factor and thus did not activate macrophages. Treatment with sialidase of a conditioned medium of lipid metabolite-treated SM/J mouse nonadherent cells efficiently generated the macrophage activating factor. When Gc protein was first treated with soluble or immobilized sialidase and used in a medium for 2 h cultivation of lipid metabolite-treated SM/J mouse nonadherent cells or BALB/c mouse B cells, the resultant conditioned media contained a large amount of the macrophage activating factor. These results support the hypothesis that Gc protein carries a dibranched trisaccharide with galactose and sialic acid termini.

Vitamin D‐binding protein (group‐specific component) is the sole serum protein required for macrophage activation after treatment of peritoneal cells with lysophosphatidylcholine

In vitro treatment of mouse peritoneal cells with 1 micrograms lysophosphatidylcholine (lyso-Pc)/mL in serum free-0.1% egg albumin-supplemented RPMI 1640 medium for 30 min, followed by 3 h cultivation in a medium supplemented with human serum, resulted in a greatly enhanced Fc-receptor-mediated phagocytic activity of macrophages. Vitamin D-binding protein (group-specific component [Gc]) of alpha 2-globulin fraction was shown to be the sole serum glycoprotein required for the generation of a potent macrophage-activating factor. When a mixture of lysophosphatidylcholine (lyso-Pc)-treated nonadherent and adherent cells were cultured in a medium supplemented with a small amount of purified Gc protein (1 ng/mL), a greatly enhanced activation of macrophages was demonstrated. The generation of macrophage-activating factor from purified Gc protein was far more efficient than that from whole serum, indicating that a serum component is inhibitory to the activation process of macrophages. While three other major serum glycoproteins (alpha 2-macroglobulin, alpha 2-HS-glycoprotein and haptoglobin) were neither stimulatory nor inhibitory to lyso-Pc-primed macrophage activation, serum albumin (competitively with Gc protein) appeared to be inhibitory to the process of macrophage activation.

Complete Structure of the Human Gc Gene: Differences and Similarities between Members of the Albumin Gene Family

The sequence of the human Gc gene, including 4228 base pairs of the 5′-flanking region and 8514 base pairs of the 3′ flanking region (55,136 in total), was determined from five overlapping λ phage clones. The sequence spans 42,394 base pairs from the cap site to the polyadenylation site, and it reveals that the gene is composed of 13 exons, which are symmetrically placed within the three domains of the Gc protein. The first exon is partially untranslated, as is exon 12, which contains the termination codon TAG. Exon 13 is entirely untranslated, but contains the polyadenylation signal AATAAA. Ten central introns split the coding sequence between codon positions 2 and 3 and between codon positions 3 and 1 in an alternating pattern, exactly as has been observed in the structure of the albumin and α-fetoprotein genes. The Gc gene has several distinctive features which set it apart from the other members of the family. First, the gene is smaller by two exons, which results in a protein some 130 amino acids shorter than albumin or AFP. This decrease in size may result from the loss of two internal exons during the evolutionary history of the Gc gene. Second, exons 6, 8, 9, and 11 are smaller than their counterparts in albumin or AFP by a total of 8 codons (1, 4, 1, and 2, respectively). Although the mRNA and protein expressed from the Gc gene are significantly smaller, the gene itself is about 2.5 times larger than the other genes of the family. There are 13 interspersed DNA repeats within the human Gc gene which are absent from the same positions in the albumin or AFP genes, and hence must have been inserted after the triplication event(s) that gave rise to the gene family. Despite the differences, the Gc gene is nonetheless recognizable as a member of the albumin family.