Light Chains Of Immunoglobulin G Research Articles

DMSA-Functionalized Mesoporous Alumina with a High Capacity for Selective Isolation of Immunoglobulin G.

A novel dimercaptosuccinic acid-functionalized mesoporous alumina (DMSA-MA) is synthesized by the dicarboxylic acid groups of dimercaptosuccinic acid molecules coordinating to the Al3+ ions located in the mesostructure. The as-prepared DMSA-MA composites possess a large surface area of 91.17 m2/g as well as a uniform pore size and a high pore volume of 17.22 nm and 0.23 cm3/g, respectively. DMSA coating of mesostructures significantly enhanced their selectivity for glycoprotein adsorption through a powerful hydrophilic binding force, and the maximum adsorption capacity of immunoglobulin G (IgG) can reach 2298.6 mg g-1. The captured IgG could be lightly stripped from the DMSA-MA composites with an elution rate of 98.3% by using 0.5 wt % CTAB solution as the elution reagent. DMSA-MA is further employed as a sorbent for the enrichment of IgG heavy chain and light chain from human serum sample. SDS-PAGE assay results showed the obtained IgG with high purity compared to that of the standard solution of IgG.

Activation of unfolded protein response pathway is important for valproic acid mediated increase in immunoglobulin G productivity in recombinant Chinese hamster ovary cells

Process engineering to improve product quality and titers is gaining importance at late-stage cell culture process development. Valproic acid, a US Food and Drug Administration-approved histone deacetylase (HDAC) inhibitor, has been shown to improve cell culture performance with higher productivities and minimal effect on the product quality. However, the wider physiological impact of valproic acid on recombinant cells has not been investigated till date. In this study, we investigate the role of unfolded protein response pathway when immunoglobulin G (IgG)-secreting Chinese hamster ovary (CHO) cells are treated with valproic acid, resulting in a 3-fold increase in product titers and productivity. It is found that cells undergo an early transient endoplasmic reticulum (ER) stress on treatment with valproic acid, and subsequently adapt to perform as high producers. Induction of chaperones through enhanced XBP1 splicing activity and ATF6 activation suggests an increase in protein processing activity in these cells. We show that in addition to the enhanced recombinant mRNA expression of IgG heavy chain and light chain, the activation of unfolded protein response (UPR) pathway is critical to the increase in productivity of cells on valproic acid treatment. Further, upregulation of the UPR pathway is not through HDAC inhibition alone. To our knowledge, this is the first attempt to arrive at a phenotype-genotype mechanistic understanding of how valproic acid treatment enhances productivity in recombinant CHO cells.

Glycome characterization of immunoglobulin G from buffalo (Bubalus bubalis) colostrum.

Immunoglobulin G (IgG) is a major glycoprotein in ruminant colostrum. First day buffalo colostrum protein was purified on Sephadex G-100 and its mass was determined by MALDI-TOF as 147.848 KDa. The PMF data of protein subunits revealed its homology to IgG, which was supported by the identification of peptide sequences LLIYGATSR and VYNEYLPAPIVR corresponding to light and heavy chains of IgG by CID MS/MS analysis. The N-glycan microheterogeneity was established based on chemoselective glycoblotting technique with the identification of high mannose, neutral complex/hybrid and sialylated complex/hybrid glycans. A complete structural assignment of 54 N-linked oligosaccharides were identified and the ratio of sialyl oligosaccharides was found to be higher compared to neutral saccharides. The fucosylation observed in more than 20 oligosaccharides, high mannose and trisialyl oligosaccharides were present in diminutive amount. The high non-fucosyl and sialyl oligosaccharides in buffalo colostrum IgG provide ample scope for its utilization in targeted therapies to elicit effective ADCC and anti-inflammatory responses.

The upregulation of zinc finger protein 670 and prostaglandin D2 synthase in proliferative vitreoretinopathy

Proteins in the vitreous play an important role on the induction of proliferative vitreoretinopathy (PVR) after retinal detachment. The aim of this study was to investigate the variation of protein patterns in the vitreous of PVR eyes and examine whether differentially expressed protein levels were expressed in experimental PVR retina. Vitreous samples from PVR and macular hole patients were selected for proteomic analysis. The vitreous protein samples were separated by two-dimensional electrophoresis (2-DE). The differentially expressed protein spots in the two groups were excised and subjected to in-gel digestion and identification by electrospray ionization mass spectrometry (ESI-MS) analysis. Two differentially expressed proteins, zinc finger protein 670 (ZFP 670) and prostaglandin D2 synthase (PGD2S), were further validated by immunohistochemical staining and western blotting analysis in the retina of the experimental rabbit PVR model. In proteome analysis of human vitreous samples, five proteins had increased expression in PVR, including zinc finger protein 670 (ZFP 670), prostaglandin D2 synthase (PGD2S), IgG (Immunoglobulin G) light chain, transthyretin precursor, and haptoglobin precursor. ZFP 670 and PGD2S levels were expressed significantly higher in the experimental PVR retinas than in the control group. Levels of ZFP 670 and PGD2S were elevated in the vitreous fluid of patients with PVR. In addition, there were higher expressions of ZFP 670 and PGD2S in the experimental PVR retina. This result will expand our knowledge of pathophysiologic characteristics of PVR, and might be helpful for further developing possible treatment on this disorder.

Establishment of peritoneal liquid electrophoretogram from healthy horses and horses submitted to experimentally induced intestinal obstruction

The initial inflammatory stages of the colic syndrome include changes known as acute phase response. The aim of this study was to contribute with the establishment of reference values concerning the electrophoretogram of peritoneal liquid from healthy horses and horses submitted to experimentally induced intestinal obstruction. Twenty-one horses were allotted in four groups: duodenal obstruction (DG), ileum obstruction (IG), left-dorsal colon obstruction (MG), and control group (CG). Peritoneal liquid was sampled before obtruction (T0), with 3 hours of obstruction (T3) and 6, 30, 102 and 174 hours after desobstructing (T6, T30, T102 and T174, respectively). Total protein levels were determined by the biuret method and protein fractions were obtained by SDS-PAGE electrophoresis. The acute phase proteins (APP) identified were Immunoglobulin-A, ceruloplasmin, transferrin, albumin, α1-antitrypsin, heavy and light chains of immunoglobulin-G, haptoglobin, α1-acid glycoprotein and a still unnamed protein, which was called P24. There was no difference (P>0.3) in protein levels among groups, although a significant difference (P>0.05) was observed between distinct experimental moments in each group evidencing a higher response of the APP in the obstructed groups. The APP fractioning of the peritoneal liquid was standardized to establish a standard curve for healthy equines and those submitted to induced intestinal obstruction. Moreover, it was verified that the SDS-PAGE electrophoresis was sensitive and effective to help diagnose abdominal inflammatory processes.

Evidence that calf bronchopneumonia may be accompanied by increased sialylation of circulating immune complexes’ IgG

Immune complexes (IC) could have an important role in the pathogenesis of pre-ruminant calves’ bronchopneumonia. IC are potent activators of complement and neutrophils and they might be responsible for immune protection, as well as for pulmonary damage. Immunoglobulin G (IgG), as constituents of IC, initiates the effector phase of immune response through binding of Fcγ and complement receptors. The oligosaccharide moieties expressed on IgG can modulate their antigen affinity and effector function. Structural characteristics of IgG molecules from IC in the pre-ruminant calves have not been studied in detail. The aim of our study was to determine if the glycosylation profile of IgG from circulating IC (CIC) in calves with bronchopneumonia differed from those of healthy control calves.A total number of 13 Holstein-Friesian calves, at the age of three months were included in the study. All calves were clinically examined by a veterinarian. Calves were classified by signs of respiratory disease in two groups: healthy (n=6) and diseased (n=7) calves. The CIC from calves’ sera were isolated by the polyethylene glycol precipitation (PEG) method. IgG molecules were isolated from PEG precipitates by Protein G affinity method. The level of expression and localization N-acetylglucosamine, galactose, sialic acid, and fucose within the isolated IgG was determined by lectin blot assay.Calves with bronchopneumonia had a statistically significantly increased level of CIC. IgG molecules were isolated from CIC of both healthy and diseased calves. Several other proteins in complex with IgG were detected in both groups of animals. The isolated IgG heavy chains of healthy calves expressed N-acetylglucosamine, galactose, sialic acid, and fucose. The light chains of IgG expressed N-acetylglucosamine, sialic acid, and fucose whereas galactose was not detected in healthy calves. In diseased animals, galactose was detected on light chains, and both heavy and light IgG chains were more sialylated.Proteins in complex with IgG were also lectin reactive, and their glycosylation in diseased animals was different compared to healthy controls.Increased sialylation is a characteristic of anti-inflammatory IgG. The increased sialylation of IgG from CIC in bronchopneumonia might be an attempt of immune system of calves to protect lung tissues against damages provoked by activated cells and secreted pro-inflammatory cytokines. At the same time, increased IgG sialylation could explain the inability of calves’ immune system to initiate the process of antigen elimination by activation of Fcγ receptors.

Immunoglobulin G expression and its colocalization with complement proteins in papillary thyroid cancer

Except for the well-known immunoglobulin G (IgG) producing cell types, ie, mature B lymphocytes and plasma cells, various non-lymphoid cell types, including human cancer cells, neurons, and some specified epithelial cells, have been found to express IgG. In this study, we detected the expression of the heavy chain of IgG (IgGγ) and kappa light chain (Igκ) in papillary thyroid cancer cells. Using in situ hybridization, we detected the constant region of human IgG1 (IGHG1) in papillary thyroid cancer cells. With laser capture microdissection followed by RT-PCR, mRNA transcripts of IGHG1, Igκ, recombination activating gene 1 (RAG1), RAG2, and activation-induced cytidine deaminase genes were successfully amplified from isolated papillary thyroid cancer cells. We further confirmed IgG protein expression with immunohistochemistry and found that none of the IgG receptors was expressed in papillary thyroid cancer. Differences in the level of IgGγ expression between tumor size, between papillary thyroid cancer and normal thyroid tissue, as well as between papillary thyroid cancer with and without lymph node metastasis were significant. Taken together, these results indicate that IgG is produced by papillary thyroid cancer cells and that it might be positively related to the growth and metastasis of papillary thyroid cancer cells. Furthermore, it was demonstrated that IgGγ colocalized with complement proteins in the same cancer cells, which could indicate that immune complexes were formed. Such immune complexes might consist of IgG synthesized by the host against tumor surface antigens and locally produced anti-idiotypic IgG with specificity for the variable region of these ‘primary' antibodies. The cancer cells might thus escape the host tumor-antigen-specific immune responses, hence promoting tumor progression.

Global assessment of protein turnover in recombinant antibody producing myeloma cells

The global turnover rates of cellular proteins and the secretion rate of a recombinant immunoglobulin G (IgG) in a myeloma cell line, NS0, were determined using SILAC proteomic analysis. After complete labeling of cellular proteins with 13C 6, 15N 4-arginine, cells were transferred to unlabeled medium and the decay of the labeled arginine in proteins was monitored during exponential cell growth. After PAGE separation and mass-spectrometric identification of proteins, those detected with high confidence over at least three time points were used for the determination of turnover rates. Among the 224 proteins quantified with a protein half-life, about 15% have a degradation rate constant lower than one-tenth of specific growth rate. For most proteins, the turnover rate is insignificant in its overall dynamics. Only 6.3% of proteins have a half-life shorter than the cell doubling time. For IgG secretion, both heavy and light chain molecules follow the same kinetic behavior with a half-life estimated to be 2 h. The label decay curve appears to show a second region with very slow kinetics, raising the possibility of two populations of IgG molecules with different secretion characteristics.

Immunoglobulin G bound to ovine placenta is eluted by surgical cannulation and acid perfusion <i>in situ</i>

To elute placental bound immunoglobulin G (IgG) in situ. Laboratory based experimentation. Biological Sciences Department, The University of Newcastle Australia and the Department of Biochemistry, University of Nairobi, Kenya. Twelve pregnant ewes 10 to 15 days before the onset of natural parturition. Placental eluates were rich in IgG, and IgG2. The relative molecular weight of placental IgG was estimated at 158kDa by gel filtration chromatography. Analysis of eluate by SDS PAGE revealed the heavy and light chains of IgG at 57 and 27kDa respectively together giving a relative molecular weight of 168kDa. Placental bound IgG may be crucial in immunology of pregnancy and together with the cognate antigen thereof may be useful as models for the study of maternal-fetal interaction in human pregnancy and in the development of experimental immunotherapy to immunologically compromised pregnancies in humans and livestock.

Alterations of the sugar moiety of Tamm-Horsfall protein in children with malignancies of lymphoid cells.

The aim of this study was to examine whether the sugar moiety of Tamm-Horsfall protein (THP) is affected by pathological processes caused by acute lymphoblastic leukemia (ALL) or non-Hodgkin's lymphoma (NHL). The carbohydrate part of THP was studied by monosaccharide analysis, N-glycan profiling, and reactivity with specific lectins. Our results have shown that THP of ALL or NHL patients, in comparison with healthy subjects, have modified sugar chains. This is expressed in lower contents of N-acetylgalactosamine, alpha2,6-linked sialic acid and alpha1,6-linked fucose as well as in altered proportions of various N-glycans. We have shown that pathological processes also affect the carbohydrate unit of human immunoglobulin G (IgG) isolated from sera of ALL or NHL patients. As compared with healthy subjects, in IgG of the patient group, lower amounts of sialic acid and fucose were observed. These changes did not influence the biological properties of THP as judged by their unaltered ability to bind with interleukin-1alpha, alpha1-acid glycoprotein, serum albumin, transferrin, IgG1 and the light and heavy chains of IgG. Neither the in vivo alterations of IgG caused by ALL or NHL nor its in vitro modifications influence the interaction between IgG and THP.

Sequencing of N-Linked Oligosaccharides Directly from Protein Gels: In-Gel Deglycosylation Followed by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry and Normal-Phase High-Performance Liquid Chromatography

A generally applicable, rapid, and sensitive method for profiling and sequencing of glycoprotein-associated N-linked oligosaccharides from protein gels was developed. The method employed sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) for protein separation and purification and in-gel deglycosylation using PNGase F for glycan release. Profiles of the neutral glycans from bovine ribonuclease B, chicken ovalbumin, and human immunoglobulin G (IgG), as well as sialic acid-containing sugars (following esterification of the acidic groups) of bovine fetuin and bovine α1-acid glycoprotein, were obtained by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and by normal-phase high-performance liquid chromatography following fluorescent labeling. Oligosaccharides were sequenced using specific exoglycosidases, and digestion products were analyzed by MALDI MS. Between 50 and 100 pmol (1.5 to 15 μg) of glycoprotein applied to the gel was sufficient to characterize its oligosaccharide contents. The identity of all glycoproteins investigated could be confirmed after deglycosylation by in-gel trypsin treatment followed by MALDI MS mass mapping and matching the measured molecular weights to a sequence database. The technique was used for the characterization of the glycan moieties of human immunodeficiency virus recombinant gp120 (Chinese hamster ovary cells) and to monitor changes in the glycosylation of this glycoprotein when produced in the presence of a glucosidase I inhibitor. Furthermore, since heavy and light chains of IgG became separated by SDS–PAGE, it could be established that most glycans were associated with the heavy chains.

Circulating fibroblast growth factor-like autoantibodies in two patients with multiple endocrine neoplasia type 1 and prolactinoma.

Basic fibroblast growth factor (bFGF) is a potent endothelial cell mitogen found in a variety of normal and tumor tissues. bFGF lacks a classical amino-terminal signal sequence and is not readily detectable in plasma from normal subjects. In earlier studies we showed increased bFGF-like mitogenic activity for parathyroid-derived endothelial cells and (increased) bFGF immunoreactivity (0.24-1.28 ng/mL) in plasma of subjects with multiple endocrine neoplasia type 1 (MEN-1). In the present study we examined the proliferative activity of MEN-1 and normal plasmas (applied to protein-A columns) in calf pulmonary artery endothelial cells. Protein-A-eluted activity in plasma from MEN-1 prolactinoma plasma exceeded activity from normal and MEN-1 nonprolactinoma plasma in three of eight MEN-1 subjects with untreated or recurrent prolactinoma. Protein-A-eluted active fractions from MEN-1 prolactinoma plasma had several properties of an immunoglobulin G, including affinity for antihuman immunoglobulin G (IgG) agarose, sensitivity to thiols, and (prepared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions) apparent mol wt corresponding to those of the heavy and light chains of IgG. The IgG fraction of MEN-1 prolactinoma plasma had far more activity in endothelial cells than did optimal concentrations of known growth factors or conditioned medium from prolactinoma cells. Endothelial cell bioactivity in protein-A-eluted fractions from MEN-1 prolactinoma plasma was neutralized 70% by rabbit antibodies to intact bFGF. These results imply novel growth stimulatory bFGF-like autoantibodies in a subset of MEN-1 patients with prolactinoma.

Characterization of HBeAg by physicochemical and immunochemical methods.

HBeAg, which is a hepatitis B-associated antigen, was purified and characterized. The end product from ion exchange, gel filtration and immunoadsorbent columns consisted mainly of immunoglobulin G (IgG) molecules, identified by immunoelectrophoresis and polyacrylamide gel electrophoresis. At least four polypeptides with molecular weights of 55,000, 38,000, 25,000 and 20,000 daltons were detected in four different preparations of HBeAg. Two of these have molecular weights similar to the heavy and light chains of IgG (55,000 and 25,000 daltons). It is postulated that HBeAg may be a small ligand of 20,000 daltons with an affinity for IgG. This affinity, especially if it involves the light chain of IgG, could account for the 38,000 or 42,000 molecular weight polypeptides seen by autoradiography.

The spontaneous release of a high-molecular-weight aggregate containing immunoglobulin G from the surface of Ehrlich ascites tumor cells.

The spontaneous release of tumor cell antigens from the cell surface into the circulation has been proposed as a mechanism whereby tumors may escape the immune response of the host. In this study we have found that Ehrlich ascites tumor cells after removal from the host (mouse) spontaneously release significant amounts of cell surface components during incubation for 1 h in cold isotonic buffer. Immunodiffusion studies revealed that immunoglobulin G (IgG) and a complement component (C3) are included in this spontaneously released material. These surface-bound humoral immune components are apparently released in the form of a high-molecular-weight aggregate (cell coat particle) as shown by ultracentrifugation and ultrafiltration experiments. Precipitation of IgG from the cell coat particle preparation with antibodies directed against mouse IgG followed by detergent gel electrophoresis of the immune precipitate revealed five major bands in addition to the heavy and light chains of IgG. These results suggest that host IgG is tightly bound to several other components at the cell surface, perhaps in the form of immune complexes. IgG is localized on the tumor cell surface in a highly heterogenous pattern with the appearance of patches and caps in some cells as shown by immunofluorescence analysis. The possibility that humoral immune components bind to the tumor cell surface and result in the shedding of high-molecular-weight aggregates of cell surface antigens into extracellular fluids is discussed.