Gc Protein Research Articles

Two amino acid pairs in the Gc Glycoprotein of Severe Fever with Thrombocytopenia Syndrome Virus responsible for the enhanced virulence

Severe fever with thrombocytopenia syndrome (SFTS) is a significant public health concern, with a high fatality rate in humans and cats. In this study, we explored the genetic determinants that contribute to the different virulence of SFTS virus (SFTSV) based on Tk-F123 and Ng-F264 strains isolated from cats. Tk-F123 was 100% lethal in type I interferon receptor-knockout mice, whereas Ng-F264 exhibited no fatality. We identified a pair of amino acid residues in the Gc protein, glycine and serine, at residues 581 and 934, respectively, derived from Tk-F123, leading to a fatal infection. Those in Ng-F264 were arginine and asparagine. These results suggest that this pair of residues affects the Gc protein function and regulates SFTSV virulence. Our findings provide useful clues for the elucidation of viral pathogenicity and the development of effective live-attenuated vaccines and antiviral strategies.

Evolution-inspired engineering of nonribosomal peptide synthetases.

Many clinically used drugs are derived from or inspired by bacterial natural products that often are produced through nonribosomal peptide synthetases (NRPSs), megasynthetases that activate and join individual amino acids in an assembly line fashion. In this work, we describe a detailed phylogenetic analysis of several bacterial NRPSs that led to the identification of yet undescribed recombination sites within the thiolation (T) domain that can be used for NRPS engineering. We then developed an evolution-inspired "eXchange Unit between T domains" (XUT) approach, which allows the assembly of NRPS fragments over a broad range of GC contents, protein similarities, and extender unit specificities, as demonstrated for the specific production of a proteasome inhibitor designed and assembled from five different NRPS fragments.

IFITM3 inhibits severe fever with thrombocytopenia syndrome virus entry and interacts with viral Gc protein.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne hemorrhagic fever disease with high fatality rate of 10%-20%. Vaccines or specific therapeutic measures remain lacking. Human interferon inducible transmembrane protein 3 (hIFITM3) is a broad-spectrum antiviral factor targeting viral entry. However, the antiviral activity of hIFITM3 against SFTS virus (SFTSV) and the functional mechanism of IFITM3 remains unclear. Here we demonstrate that endogenous IFITM3 provides protection against SFTSV infection and participates in the anti-SFTSV effect of type Ⅰ and Ⅲ interferons (IFNs). IFITM3 overexpression exhibits anti-SFTSV function by blocking Gn/Gc-mediated viral entry and fusion. Further studies showed that IFITM3 binds SFTSV Gc directly and its intramembrane domain (IMD) is responsible for this interaction and restriction of SFTSV entry. Mutation of two neighboring cysteines on IMD weakens IFITM3-Gc interaction and attenuates the antiviral activity of IFITM3, suggesting that IFITM3-Gc interaction may partly mediate the inhibition of SFTSV entry. Overall, our data demonstrate for the first time that hIFITM3 plays a critical role in the IFNs-mediated anti-SFTSV response, and uncover a novel mechanism of IFITM3 restriction of SFTSV infection, highlighting the potential of clinical intervention on SFTS disease.

Characterization of antibodies targeting severe fever with thrombocytopenia syndrome virus glycoprotein Gc.

Severe fever with thrombocytopenia syndrome (SFTS) is a hemorrhagic fever caused by SFTS virus (SFTSV), which is primarily found in East Asian countries. Despite its high mortality rate and increasing incidence, no vaccines or therapeutics have yet been approved for use against SFTS. Antibody drugs have shown promise in treating lethal infectious diseases that currently have no established treatments. In the case of SFTS, however, only a limited amount of research has been done on SFTSV-neutralizing antibodies targeting the transmembrane proteins Gn and Gc, which play critical roles in viral infection. This study focuses on the production and characterization of antibodies targeting the SFTSV Gc protein. Monoclonal antibodies against Gc were generated through immunization of mice, and their antiviral activity was evaluated. Three out of four anti-Gc antibody clones from this study demonstrated dose-dependent SFTSV neutralization activity, two of which exhibited a synergistic effect on the neutralization activity of the anti-Gn antibody clone Mab4-5. Further studies are necessary to identify key sites on the SFTSV glycoprotein and to develop novel agents as well as antibodies with diverse mechanisms of action against SFTSV.

Analysis of highly frequent point mutations in glycoprotein C, glycoprotein N, and nucleoprotein of CCHFV.

Crimean-Congo hemorrhagic fever virus (CCHFV) is classified among top 10 priority pathogens by World Health Organization. CCHFV belongs to Bunyaviridae family and negative sense ssRNA genome composed of three RNA segments: L, M, and S. RNA viruses show higher mutation rate as compared to DNA viruses. To gain deeper understanding of impact of point mutations in CCHFV M and S segment, mutation profiling, homology modeling, and molecular dynamic (MD) simulation were performed. Structural glycoproteins (glycoprotein C [Gc] and glycoprotein N [Gn]) of CCHFV are important for host-virus interaction and genome packaging, whereas CCHFV nucleoprotein (NP) is crucial for viral replication. Hence, current study is focused on evaluation of eight mutations in structural glycoproteins (Gc: 7 and Gn: 1) of M segment and seven mutations in NP of S segment. All these mutations were highly frequent, with mutation frequency between 0.81 and 1.0 and found to be persistent in the recent strains of CCHFV. Solubility analysis predicted that selected point mutations reduce solubility of Gc protein and increase solubility of Gn and NP proteins. MD simulation study deciphered that A1046V and G1158E in Gc protein, I778T in Gn protein, and H195R in NP protein displayed large deviation and fluctuation, and affected intramolecular interactions. In conclusion, we observed that point mutations could impact structure, stability, and host-virus interaction of protein, and might lead to evolution of new strains for better survival and drug resistance.

A bacterium-like particle vaccine displaying protective feline herpesvirus 1 antigens can induce an immune response in mice and cats

Feline herpesvirus 1 (FHV-1) is a highly transmissible virus that mainly causes ocular and upper respiratory infections in cats and seriously threatens the health of domestic cats and captive or wild cats (such as tigers, cheetahs, and lions). Vaccination is crucial to reduce the incidence rate and mortality of cats infected with FHV-1. In this study, three bacterium-like particles (BLPs) displaying the gB, gC, and gD proteins of FHV-1 were constructed based on a gram-positive enhancer matrix-protein anchor (GEM-PA) surface display system. Indirect immunofluorescence assay, western blot, and electron microscopy results showed that gB, gC or gD protein of FHV-1 was successfully displayed on the surface of GEM particles. Additionally, we designed one more BLPs, designated gB&gC&gD-GEM, which consisted of a mixture of gB-GEM, gC-GEM, and gD-GEM at a protein content ratio of 1:1:1. Mice were immunized with the four BLPs mixed with Gel02 adjuvant, and the results indicated that neutralizing antibody level in the gB&gC&gD-GEM group was superior than those in the other groups. Moreover, gB&gC&gD-GEM significantly increased the secretion of cytokines, as well as the activation and maturation of B cells. It also boosted the production of central memory T cells among CD4 + and CD8 + T cells. Moreover, gB&gC&gD-GEM mixed with Gel02 adjuvant provoked an antibody response in cats. In conclusion, the BLPs vaccine prepared from gB&gC&gD-GEM induced specific humoral and cellular immune responses to FHV-1 and be used as a potential vaccine candidate for the control of FHV-1 infection in cats.

CD8+ T-cells target the Crimean-Congo haemorrhagic fever virus Gc protein to control the infection in wild-type mice

Crimean-Congo haemorrhagic fever (CCHF) is a serious viral hemorrhagic fever caused by the CCHF virus (CCHFV). Spread by the bites of infected ticks or handling of viremic livestock, human disease is characterized by a non-specific febrile illness that can rapidly progress to fatal hemorrhagic disease. No vaccines or antivirals are available. Case fatality rates can vary but can be higher than 30%, although sub-clinical infections are often unrecognized and unreported. Yet, while most humans infected with CCHFV will survive the infection, often with little-to-no symptoms, the host responses that control the infection are unknown. Here we investigated the role of cellular immunity in control of CCHFV infection in an immunocompetent mouse model. We found that CD8+ T-cells are crucial for efficient control of the acute infection and rapidly acquired CCHFV-specific antiviral effector functions such as production of antiviral cytokines and degranulating in response to CCHFV peptides. We further identified the minimal CD8+ T-cell epitopes in the viral Gc proteins and that infection of mice lacking IFNγ resulted in worsened disease and higher viral loads. Together our data suggest that CD8+ T-cells are important for control of acute CCHFV infection likely through production of antiviral cytokines. This work was supported by the Intramural Research Program of the NIH.

Exploring Quercetin Hydrate’s Potential as an Antiviral Treatment for Oropouche Virus

The Oropouche virus is an orthobunyavirus responsible for causing Oropouche fever, a disease that primarily affects thousands of people in South and Central America. Currently, no specific antiviral treatments or vaccines are available against this virus, highlighting the urgent need for safe, affordable, and effective therapies. Natural products serve as an important source of bioactive compounds, and there is growing interest in identifying natural bioactive molecules that could be used for treating viral diseases. Quercetin hydrate is a compound classified as a flavonoid, which has garnered scientific attention due to its potential health benefits and its presence in various plant-based foods. In this study, we aim to evaluate the in vitro antiviral activity of quercetin hydrate against the Oropouche virus (OROV). Furthermore, we intend to explore its mode of action through in silico approaches. The cytotoxicity and antiviral activity of the compound were assessed using Vero cells. In addition, in silico studies were also performed through molecular docking, molecular dynamics simulations, Molecular Mechanics Poisson–Boltzmann surface area (MM/PBSA), and quantum-mechanical analysis in order to evaluate the interaction with the Gc protein of OROV. The assay revealed that the compound was highly active against the virus, inhibiting OROV with an EC50 value of 53.5 ± 26.5 µM under post-infection treatment conditions. The present study demonstrates that the compound is a promising antiviral agent; however, the mechanisms of action proposed in this study need to be experimentally verified by future assays.

In Silico Physicochemical Characterization of Fusion Proteins from Emerging Amazonian Arboviruses.

Mayaro (MAYV), Saint Louis encephalitis (SLEV), and Oropouche (OROV) viruses are neglected members of the three main families of arboviruses with medical relevance that circulate in the Amazon region as etiological agents of outbreaks of febrile illnesses in humans. As enveloped viruses, MAYV, SLEV, and OROV largely depend on their class II fusion proteins (E1, E, and Gc, respectively) for entry into the host cell. Since many aspects of the structural biology of such proteins remain unclear, the present study aimed at physicochemically characterizing them by an in silico approach. The complete amino acid sequences of MAYV E1, SLEV E, and OROV Gc proteins derived by conceptual translation from annotated coding regions in the reference sequence genome of the respective viruses were obtained from the NCBI Protein database in the FASTA format and then submitted to the ClustalO, Protcalc, Pepstats, Predator, Proscan, PCprof, Phyre2, and 3Drefine web servers for the determination of sequence identities, the estimation of residual properties, the prediction of secondary structures, the identification of potential post-translational modifications, the recognition of antigenic propensities, and the modeling/refinement of three-dimensional structures. Sequence identities were 20.44%, 18.82%, and 13.70% between MAYV/SLEV, SLEV/OROV, and MAYV/OROV fusion proteins, respectively. As for the residual properties, MAYV E1 and SLEV E proteins showed a predominance of the non-polar profile (56% and 55% of the residues, respectively), whereas the OROV Gc protein showed a predominance of the polar profile (52% of the residues). Regarding predicted secondary structures, MAYV E1 and SLEV E proteins showed fewer alpha-helices (16.51% and 15.17%, respectively) than beta-sheets (21.79% and 25.15%, respectively), while the opposite was observed in the OROV Gc protein (20.39% alpha-helices and 12.14% beta-sheets). Regarding post-translational modifications, MAYV E1, SLEV E, and OROV Gc proteins showed greater relative potential for protein kinase C phosphorylation, N-myristoylation, and casein kinase II phosphorylation, respectively. Finally, antigenic propensities were higher in the N-terminus half than in the C-terminus half of these three proteins, whose three-dimensional structures revealed three distinctive domains. In conclusion, MAYV E1 and SLEV E proteins were found to share more physicochemical characteristics with each other than the OROV Gc protein, although they are all grouped under the same class of viral fusion proteins.

Glucosylceramide is essential for Heartland and Dabie bandavirus glycoprotein-induced membrane fusion.

Due to climate changes, there has been a large expansion of emerging tick-borne zoonotic viruses, including Heartland bandavirus (HRTV) and Dabie bandavirus (DBV). As etiologic agents of hemorrhagic fever with high fatality, HRTV and DBV have been recognized as dangerous viral pathogens that likely cause future wide epidemics. Despite serious health concerns, the mechanisms underlying viral infection are largely unknown. HRTV and DBV Gn and Gc are viral surface glycoproteins required for early entry events during infection. Glycosphingolipids, including galactosylceramide (GalCer), glucosylceramide (GlcCer) and lactosylceramide (LacCer), are a class of membrane lipids that play essential roles in membrane structure and viral lifecycle. Here, our genome-wide CRISPR/Cas9 knockout screen identifies that glycosphingolipid biosynthesis pathway is essential for HRTV and DBV infection. The deficiency of UDP-glucose ceramide glucosyltransferase (UGCG) that produces GlcCer resulted in the loss of infectivity of recombinant viruses pseudotyped with HRTV or DBV Gn/Gc glycoproteins. Conversely, exogenous supplement of GlcCer, but not GalCer or LacCer, recovered viral entry of UGCG-deficient cells in a dose-dependent manner. Biophysical analyses showed that GlcCer targeted the lipid-head-group binding pocket of Gc to form a stable protein-lipid complex, which allowed the insertion of Gc protein into host lysosomal membrane lipid bilayers for viral fusion. Mutagenesis showed that D841 residue at the Gc lipid binding pocket was critical for GlcCer interaction and thereby, viral entry. These findings reveal detailed mechanism of GlcCer glycosphingolipid in HRTV and DBV Gc-mediated membrane fusion and provide a potential therapeutic target for tickborne virus infection.

Investigation of the Protective Effect for GcMAF by a Glycosidase Inhibitor and the Glycan Structure of Gc Protein

O-linked α-N-acetylgalactosamine (α-GalNAc) in the Gc protein is essential for macrophage activation; thus, the GalNAc-attached form of Gc protein is called Gc macrophage activating factor (GcMAF). O-linked glycans in Gc proteins from human plasma mainly consist of trisaccharides. GcMAF is produced when glycans on the Gc protein are hydrolyzed by α-Sia-ase and β-Gal-ase, leaving an α-GalNAc. Upon hydrolysis of α-GalNAc present on GcMAF, the protein loses the macrophage-activating effect. In contrast, our synthesized pyrrolidine-type iminocyclitol possessed strong in vitro α-GalNAc-ase inhibitory activity. In this study, we examined the protective effects of iminocyclitol against GcMAF via inhibition of α-GalNAc-ase activity. Detailed mass spectrometric analyses revealed the protective effect of the inhibitor on GcMAF. Furthermore, structural information regarding the glycosylation site and glycan structure was obtained using tandem mass spectrometric (MS/MS) analysis of the glycosylated peptides after tryptic digestion.

Characterization of new strains of Pseudorabies virus in Argentina: Detection of interspecies transmission.

Aujeszky's disease is mainly a swine disease, still endemic worldwide. It can infect other mammalians, including human beings, and it is usually fatal with nervous symptoms. Ever since the disease was detected in 1988 in Argentina, many outbreaks have been reported involving both feral swine and dogs. At present, in Argentina, Pseudorabies virus (PRV) cases are sporadically reported; however, clinical cases are informed. This study aims to obtain information about the seroprevalence of PRV in wild boars and to isolate and characterize PRV from clinical samples. From 2018 to 2019, 78 wild boars' serum samples from Bahía de Samborombón natural reserve were analyzed for antibodies to PRV using a virus neutralization test. Clinical samples from 17 pigs, 2 wild boars, 1 dog, and 1 cat were collected from 2013 to 2019 for viral isolation and detection of the presence of the gD gene by PCR. For sequence analysis, the gC partial gene was amplified. Five strains were isolated from the dog, cat, and swine samples. The new PRV strains identified were confirmed by BLAST analysis, which revealed between 99.74% and 100% of similarity to the NIA-3 strain and phylogenetic analysis of the partial gene encoding the gC protein revealed that the PRV strains have divided into two main clades, clade 1 and clade 2. This report informed that most new cases of PRV were detected in the central regions of Argentina, where pig production is concentrated. The study in Bahía de Samborombón revealed a high percentage of detection but, the sampling is not representative of that of the rest of the country. Therefore, a systematic sampling effort of wild boar throughout the country should be included in the national program control. Although in Argentina only the inactivated Bartha vaccine is allowed, recombination risk should not be ignored if attenuated vaccines are incorporated into the National control plan. The two strains, one from the cat and one from the dog sample, are directly related to infected swine. The information about clinical cases and molecular characterization of new strains is important for a better understanding of the dynamics of PRV and to promote preventive measures.

CGMP signaling: a potential therapeutic target for neurodegeneration in glaucoma?

cGMP signaling: a potential therapeutic target for neurodegeneration in glaucoma?

Fine mapping of the antigenic epitopes of the Gc protein of Guertu virus.

Guertu virus (GTV), a newly discovered member of the genus Banyangvirus in the family Phenuiviridae, poses a potential health threat to humans and animals. The viral glycoprotein (GP) binds to host cell receptors to induce a neutralizing immune response in the host. Therefore, identification of the B-cell epitopes (BCEs) in the immunodominant region of the GTV Gc protein is important for the elucidation of the virus-host cell interactions and the development of GTV epitope assays and vaccines. In this study, an improved overlapping biosynthetic peptide method and rabbit anti-GTV Gc polyclonal antibodies were used for fine mapping of the minimal motifs of linear BCEs of the GTV Gc protein. Thirteen BCE motifs were identified from eleven positive 16mer-peptides, namely EGc1 (19KVCATTGRA27), EGc2 (58KKINLKCKK66), EGc3 (68SSYYVPDA75), EGc4 (75ARSRCTSVRR84), EGc5 (79CTSVRRCRWA88), EGc6 (90DCQSGCPS97), EGc7 (96PSHFTSNS103), EGc8 (115AGLGFSG121), EGc9 (148ENPHGVI154), EGc10 (179KVFHPMS185), EGc11 (230QAGMGVVG237), EGc12 (303RSHDSQGKIS312), and EGc13 (430DIPRFV435). Of these, 7 could be recognized by GTV IgG-positive sheep sera. Three-dimensional structural analysis revealed that all 13 BCEs were present on the surface of the Gc protein. Sequence alignment of the 13 BCEs against homologous proteins from 10 closely related strains of severe fever with thrombocytopenia syndrome virus from different geographical regions revealed that the amino acid sequences of EGc4, EGc5, EGc8, EGc11, and EGc12 were highly conserved, with 100% similarity. The remaining 8 epitopes (EGc1, EGc2, EGc3, EGc6, EGc7, EGc9, EGc10, and EGc13) showed high sequence similarity in the range of 71.43%-87.50%. These 13 BCEs of the GTV Gc protein provide a molecular foundation for future studies of the immunological properties of GTV glycoproteins and the development of GTV multi-epitope assays and vaccines.

Co-circulation of Orthobunyaviruses and Rift Valley Fever Virus in Mauritania, 2015.

Ngari virus (NRIV) has been mostly detected during concurrent outbreaks of Rift Valley fever virus (RVFV). NRIV is grouped in the genus Orthobunyavirus within the Bunyaviridae family and RVFV in the genus Phlebovirus in the family Phenuiviridae. Both are zoonotic arboviruses and can induce hemorrhagic fever displaying the same clinical picture in humans and small ruminants. To investigate if NRIV and its parental viruses, Bunyamwera virus (BUNV) and Batai virus (BATV), played a role during the Mauritanian RVF outbreak in 2015/16, we analyzed serum samples of sheep and goats from central and southern regions in Mauritania by quantitative real-time RT-PCR, serum neutralization test (SNT) and ELISA. 41 of 458 samples exhibited neutralizing reactivity against NRIV, nine against BATV and three against BUNV. Moreover, complete virus genomes from BUNV could be recovered from two sheep as well as two NRIV isolates from a goat and a sheep. No RVFV-derived viral RNA was detected, but 81 seropositive animals including 22 IgM-positive individuals were found. Of these specimens, 61 samples revealed antibodies against RVFV and at least against one of the three orthobunyaviruses. An indirect ELISA based on NRIV/BATV and BUNV derived Gc protein was established as complement to SNT, which showed high performance regarding NRIV, but decreased sensitivity and specificity regarding BATV and BUNV. Moreover, we observed high cross-reactivity among NRIV and BATV serological assays. Taken together, the data indicate the co-circulation of at least BUNV and NRIV in the Mauritanian sheep and goat populations.

Isolation and Phylogenetic Analysis of Reemerging Pseudorabies Virus Within Pig Populations in Central China During 2012 to 2019.

To understand the biological characteristics of the reemerging pseudorabies virus (PRV) strains, a total of 392 tissue samples were collected from diseased pigs during reemerging PR outbreaks between 2012 and 2019 on farms in central China where swine had been immunized with Bartha-K61 and 51 (13. 01%) were positive for the gE gene by PCR. Sixteen PRV strains were isolated and caused clinical symptoms and death in mice. Subsequently, gE, gC, gB, and gD complete genes were amplified from the 16 PRV isolates and sequenced. Phylogenetic analysis based on these four gene sequences shows that the 16 PRV isolates were more closely related to the Chinese PRV variants (after 2012) but genetically differed from early Chinese PRV isolates (before 2012). Sequence analysis reveals that PRV isolates exhibited amino acid insertions, substitutions, or deletions compared with early Chinese PRV isolates and European–American PRV strains. In addition, this is the first report that eight isolates (8/16) in this study harbor a unique amino acid substitution at position 280 (F to L) of the gC protein, and six isolates have an amino acid substitution at position 338 (A to V) of the gD protein compared with the Chinese PRV variants. The emulsion containing inactivated PRV NY isolate could provide complete protection against the NY isolate. This study might enrich our understanding of the evolution of reemerging PRV strains as well as pave the way for finding a model virus to develop a novel vaccine based on reemerging PRV strains.

Proteomic analysis of host cellular proteins co-immunoprecipitated with duck enteritis virus gC

Duck enteritis virus (DEV), the causative agent of duck viral enteritis, causes a contagious, lethal viral disease in Anseriformes (waterfowls). In virus infection, host-virus interaction plays a crucial role in virus replication and pathogenesis. In our previous study, mRFP was fused with the C-terminus of DEV glycoprotein C (gC) to construct a fluorescent-tag DEV virus rgCRFP. In the current study, fluorescent fusion protein (gC-mRFP) was used as the proteomic probe. Co-immunoprecipitation and mass spectrometric analysis of proteins from rgCRFP-infected chicken embryo fibroblasts using commercial anti-RFP antibody led to the identification of a total of 21 gC interacting host proteins. Out of these 21 proteins, the interaction of seven host proteins (GNG2, AR1H1, PPP2CA, UBE2I, MCM5, NUBP1, HN1) with DEV gC protein was validated using membrane-bound split-ubiquitin yeast two-hybrid system (MbYTH) and bimolecular fluorescence complementation (BiFC) analyses. It indicated direct interaction between these proteins with DEV gC protein. This study has furthered the current understanding of DEV virus infection and pathogenesis. SignificancegC is an crucial glycoprotein of duck enteritis virus that plays an important role in the viral life cycle. Uncovering the interaction between virus-host is very important to elucidate the pathogenic mechanism of the virus. In this study, host factors interacting with DEV gC have been discerned. And seven host proteins (GNG2, AR1H1, PPP2CA, UBE2I, MCM5, NUBP1, HN1) have been further validated to interact with DEV gC using MbYTH and BiFC analyses. These outcomes could shed light on how DEV manipulates the cellular machinery, which could further our understanding of DEV pathogenesis.

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.

Improved preparation of group-specific component (Gc) protein to derive macrophage activating factor

Cancer immunotherapy has recently attracted attention as an approach for cancer treatment through the activation of the immune system. Group-specific component (Gc) protein is a precursor for macrophage activating factor (GcMAF), which has a promising immunomodulatory effect on the suppression of tumor growth and angiogenesis. In this study, we successfully purified Gc protein from human serum using anion-exchange chromatography combined with affinity chromatography using a 25-OH-D3-immobilized column. The purity of Gc protein reached 95.0% after anion-exchange chromatography. The known allelic variants of Gc protein are classified into three subtypes—Gc1F, Gc1S and Gc2. The fragment sequence of residues 412–424 determined according to their MS/MS spectra is available to evaluate the subtypes of Gc protein. The data showed that the Gc protein purified in this study consisted of the Gc1F and Gc2 subtypes. Our method improved the purity of Gc protein, which was not affected by the treatment to convert it into GcMAF using β-galactosidase- or neuraminidase-immobilized resin, and will be useful for biological studies and/or advanced clinical uses of GcMAF, such as cancer immunotherapy.

Differential Frequency of CYP2R1 Variants Across Populations Reveals Pathway Selection for Vitamin D Homeostasis.

Normal vitamin D homeostasis is necessary to ensure optimal mineral metabolism. Dietary insufficiency of vitamin D and the lack of sunlight each have well understood roles in vitamin D deficiency; however, the extent to which common genetic variations in vitamin D metabolizing enzymes contribute to alterations in vitamin D homeostasis remains uncertain. To examine the possibility that common coding variation in vitamin D metabolizing enzymes alters vitamin D homeostasis we determined the effect of 44 nonsynonymous polymorphisms in CYP2R1, the vitamin D 25-hydroxylase, on enzyme function. Twenty-one of these polymorphisms decreased activity, while 2 variants increased activity. The frequency of CYP2R1 alleles with decreased 25-hydroxylase activity is 3 in every 1000 Caucasians and 7 in every 1000 African Americans. In populations where exposure to sunlight is high, alleles with decreased function occur at a frequency as high as 8%. The pattern of selected variation as compared to nonselected variation is consistent with it being the result of positive selection for nonfunctional alleles closer to the equator. To examine this possibility, we examined the variation pattern in another protein in the vitamin D pathway, the vitamin D binding protein (GC protein). The pattern of selected variation in the GC protein as compared to nonselected variation is also consistent with it being the result of positive selection for nonfunctional alleles closer to the equator. CYP2R1 polymorphisms have important effects on vitamin D homeostasis, and the geographic variability of CYP2R1 alleles represents an adaptation to differential exposures to UVB irradiation from sunlight.