Serum and Plasma Immunoglobulin G Fc N-Glycosylation Is Stable during Storage.
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ABSTRACT:
SUBMITTER: Amez Martin M
PROVIDER: S-EPMC8155565 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Intravenous immunoglobulin (IVIg) products from different pharmaceutical companies vary in composition, in part because of the selected blood donors and production process. N-glycosylation of the Fc-portion of IgG varies between blood donors and may influence both the side-effects and therapeutic effectiveness of IVIg. At present, the variation in Fc N-glycosylation between IVIg products has not been defined. Utilizing mass spectrometry, we performed relative quantitation of the Fc N-glycosylation of IgG, assessing a total of 154 unique lot numbers of IVIg. Seven products showed comparable Fc N-glycosylation, with only one product differing from the others in all glycosylation features (galactosylation, sialylation, fucosylation and bisecting N-acetylglucosamine). However, the mean difference did not exceed 3%. Within product variation was present to a minor degree, but largely indistinguishable from analytical variation. In conclusion, we expect that the minor variation in Fc N-glycosylation between IVIg products has a small effect, if any, on the biological activity.
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Project description:Biomarkers may facilitate detection of gastric cancer at an earlier stage and reduce mortality. Here we sought to determine if the glycosylation profile of serum immunoglobulin G (IgG) could distinguish patients with non-atrophic gastritis (NAG), duodenal ulcer (DU) and gastric cancer (GC). Serum IgG was released and analyzed using nano-LC-TOF mass spectrometry. Statistically significant false discovery rate (FDR)-adjusted p-values were observed for 18 glycans, eight that differed significantly between NAG and GC, three that distinguished NAG from DU, and eight that differed between DU and GC. The IgG glycosylation signature may be useful as a predictive marker for gastric cancer.
| S-EPMC4322429 | biostudies-literature
Project description:Occurrences of high values in patients with benign prostate disease and low values in patients with highly suspicious cancer have diminished the trustworthiness of prostate-specific antigen as an early diagnostic marker of prostate cancer. In the search for other complimentary markers, we focused on serum IgG from patients with prostate diseases as well as normal subjects. IgG purified from the sera of normal control subjects and patients with prostate diseases, was digested with peptide N-glycanase. Released glycans were quantified using MALDI-time of flight mass spectrometry. We report that N-linked (N-acetylhexosamine)2 (deoxyhexose)(mannose)3 (N-acetylglucosamine)2 was significantly increased in the IgG heavy chains of patients with prostate cancer compared with that of either benign prostatic disease patients or healthy subjects, whereas (hexose)(N-acetylhexosamine)2 (deoxyhexose)(mannose)3 (N-acetylglucosamine)2 was more abundant in the heavy chains of healthy subjects and benign prostatic disease patients. Thus, an absence of the terminal hexose of N-linked glycans has been closely connected to the progression of prostate cancer. Furthermore, surface plasmon resonance analyses have revealed that IgG from patients with prostate cancer has a decreased binding for Sambucus nigra lectin, compared with that from the benign prostatic disease patients or from normal subjects, suggesting lower levels of (N-acetylneuraminic acid)(?2-6)galactose/N-acetylgalactosamine groups in the N-linked glycans of patient IgG. Meanwhile, wheat germ agglutinin binding to IgG of the cancer group was significantly larger than that for the benign prostatic disease group but smaller than that for normal subjects. Our study indicates that the glycosylation changes in IgG can become useful diagnostic parameters for prostate cancer.
| S-EPMC4924372 | biostudies-literature
Project description:UnlabelledVisceral leishmaniasis (VL) has a high fatality rate if not treated; nevertheless, the majority of human infections with the causative agent, Leishmania infantum chagasi, are asymptomatic. Although VL patients often present with increased levels of serum immunoglobulins, the contribution of antibodies to resistance or progression to disease remains unknown. Effector and regulatory functions of antibodies rely on their interactions with type I and II Fc receptors, and these interactions are tuned by the patterns of antibody Fc N-glycosylation. In view of these facts, we applied a robust method of IgG Fc N-glycopeptide profiling of serum samples from 187 patients with VL, 177 asymptomatic individuals, 116 endemic controls (individuals residing in areas where VL is endemic) and 43 nonendemic controls (individuals living in an area where VL is not endemic). We show that, in comparison to the overall IgG Fc N-glycan profiles of asymptomatic or uninfected healthy individuals, those of patients with VL are profoundly altered. These changes correlate with levels of serum cytokines and the inflammation marker C-reactive protein. We also fitted univariate and multivariate ordinal logistic regression models to demonstrate the ability of IgG Fc N-glycosylation features and immunity regulators present in serum to predict disease severity in VL patients. Importantly, we show that Fc N-glycosylation profiles change after treatment of VL. This study introduces important concepts contributing to the understanding of antibody responses in infections with Leishmania parasites and provides new insights into the pathology of human VL.ImportanceImmunoglobulins (Ig) have been shown to present pro- and anti-inflammatory functions according to the profile of carbohydrates attached to their Fc region. Glycosylation features of serum IgG have been examined in relation to several autoimmune and infectious diseases and provide a mechanistic basis for the protective or pathogenic role of antibodies. Leishmania infantum chagasi is the causative agent of visceral leishmaniasis (VL) in South America, and we show that VL patients produce IgG with patterns of Fc glycans similar to those found in other inflammatory conditions. Specific Fc N-glycosylation features and levels of serum cytokines and C-reactive protein are significantly associated with the development of severe clinical symptoms and, notably, Fc glycosylation changes after treatment. The modifications detected in the N-glycosylation features of IgG Fc from VL patients raise new perspectives on the effector or regulatory role of antibodies in immune responses elicited by infection with Leishmania parasites.
| S-EPMC4324239 | biostudies-literature
Project description:Fc-receptors for immunoglobulin G (Fc?Rs) mediate a variety of effector and regulatory mechanisms in the immune system. N-glycosylation of Fc?Rs critically affects their functions which is well exemplified by antibody-dependent cell-mediated cytotoxicity (ADCC) and phagocytosis mediated by homologous Fc?RIIIa and Fc?RIIIb, respectively. Although several reports describe N-glycosylation profiles of recombinant Fc?RIII glycoproteins, much remains unknown regarding their native glycoforms. Here we performed site-specific N-glycosylation profiling of a soluble form of Fc?RIIIb purified from human serum based on mass spectrometric analysis. Our data indicate a distinct and common tendency of the glycoforms exhibited at each N-glycosylation site between the native and the previously reported recombinant Fc?RIII glycoproteins. Among the six N-glycosylation sites of serum soluble Fc?RIIIb, Asn45 was shown to be exclusively occupied by high-mannose-type oligosaccharides, whereas the remaining sites were solely modified by the complex-type oligosaccharides with sialic acid and fucose residues. The results of our endogenous Fc?RIII glycoform analyses are important for the optimization of therapeutic antibody efficacy.
| S-EPMC5807427 | biostudies-literature
Project description:BACKGROUND:Serum folate forms, and particularly tetrahydrofolate, are sensitive to oxidation. METHODS:Using a repeated measures design, we investigated the stability of folate forms in convenience samples with added ascorbic acid (AA; 5 g/L) analyzed initially and after variable (approximately 1-33 weeks) storage time at -70 °C. We examined the recovery of tetrahydrofolate added at different spiking levels to serum with and without AA (5 g/L). We also assessed the long-term frozen storage stability of folate forms. RESULTS:Repeat analysis produced consistent results with the initial analysis; the mean relative change (95% CI; Lin's concordance correlation between initial and repeat result; sample size) was 0.08% (-0.24% to 0.39%; r c = 0.999; n = 301) for 5-methyltetrahydrofolate, 4.23% (2.44%-6.05%; r c = 0.984; n = 211) for pyrazino-s-triazine derivative of 4?-hydroxy-5-methyltetrahydrofolate (MeFox), -0.22% (-1.90% to 1.49%; r c = 0.986; n = 214) for folic acid, and 1.49% (-2.71% to 5.88%; r c = 0.889; n = 81) for tetrahydrofolate. Linear regression testing for a time trend indicated an estimated average percent change of less than ±5% for samples retested after 4 months: 5-methyltetrahydrofolate P trend = 0.0007, folic acid P trend < 0.0001, MeFox P trend = 0.38, and tetrahydrofolate P trend = 0.0256. The mean ± SD tetrahydrofolate spiking recovery was 96.7% ± 9.4% for serum with added AA, but <50% for serum without added AA. We observed ?10% loss for most serum folate forms during 4 years of storage at -70 °C. CONCLUSIONS:Serum containing added AA showed acceptable stability of folate forms during repeat analysis from the same vial within 4 months, complete spiking recovery of tetrahydrofolate during sample processing, and long-term frozen storage stability of folate forms.
| S-EPMC7263443 | biostudies-literature
Project description:The fragment crystallizable (Fc) region links the key pathogen identification and destruction properties of immunoglobulin G (IgG). Pathogen opsonization positions Fcs to activate pro-inflammatory Fc? receptors (Fc?Rs) on immune cells. The cellular response and committal to a damaging, though protective, immune response are tightly controlled at multiple levels. Control mechanisms are diverse and in many cases unclear, but one frequently suggested contribution originates in Fc?R affinity being modulated through shifts in Fc conformational sampling. Here, we report a previously unseen IgG1 Fc conformation. This observation motivated an extensive molecular dynamics investigation of polypeptide and glycan motions that revealed greater amplitude of motion for the N-terminal C?2 domains and N-glycan than previously observed. Residues in the C?2/C?3 interface and disulfide-bonded hinge were identified as influencing the C?2 motion. Our results are consistent with a model of Fc that is structurally dynamic. Conformational states that are competent to bind immune-stimulating Fc?Rs interconverted with Fc conformations distinct from those observed in Fc?R complexes, which may represent a transient, nonbinding population.
| S-EPMC4041121 | biostudies-literature
Project description:To monitor the Fc glycosylation of therapeutic immunoglobulin G in bioprocess development, product characterization and release analytics, reliable techniques for glycosylation analysis are needed. Several analytical methods are suitable for this application. We recently presented results comparing detection methods for glycan analysis that are separation-based, but did not include mass spectrometry (MS). In the study reported here, we comprehensively compared MS-based methods for Fc glycosylation profiling of an IgG biopharmaceutical. A therapeutic antibody reference material was analyzed 6-fold on 2 different days, and the methods investigated were compared with respect to precision, accuracy, throughput and analysis time. Emphasis was put on the detection and quantitation of sialic acid-containing glycans. Eleven MS methods were compared to hydrophilic interaction liquid chromatography of 2-aminobenzamide labeled glycans with fluorescence detection, which served as a reference method and was also used in the first part of the study. The methods compared include electrospray MS of the heavy chain and Fc part after limited digestion, liquid chromatography MS of a tryptic digest, porous graphitized carbon chromatography MS of released glycans, electrospray MS of glycopeptides, as well as matrix assisted laser desorption ionization MS of glycans and glycopeptides. Most methods showed excellent precision and accuracy. Some differences were observed with regard to the detection and quantitation of low abundant glycan species like the sialylated glycans and the amount of artefacts due to in-source decay.
| S-EPMC4622708 | biostudies-literature
Project description:Background and objectiveMigraine is a common neurological disease, but its pathogenesis is still unclear. Previous studies suggested that migraine was related to immunoglobulin G (IgG). We intended to analyze the immune characteristics of migraine from the perspective of IgG glycosylation and provide theoretical assistance for exploring its pathogenesis.MethodsThe differences in the serum level of IgG glycosylation and glycopeptides between patients with episodic migraine and healthy controls were analyzed by applying the poly(glycerol methacrylate)@chitosan (PGMA@CS) nanomaterial in combination with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). We constructed a binary classification model with a feedforward neural network using PyTorch 1.6.0 in Python 3.8.3 to classify the episodic migraine and healthy control groups.ResultsTwenty patients with migraine and 20 healthy controls were enrolled and the blood samples and clinical information were collected. Forty-nine IgG N-glycopeptides were detected in the serum of the subjects. The serum level of N-glycopeptide IgG1 G0-NF (p = 0.012) was increased in patients with migraine. The serum level of N-glycopeptide IgG3/4 G2FS (p = 0.041) was decreased in patients with migraine with family history of headache. It was found that the serum level of the IgG1 G1 (p = 0.004) and IgG2 G0 (p = 0.045) was increased in patients with migraine with aura, while the serum level of IgG2 G0N (p = 0.043) in patients with migraine with aura was significantly lower than that in patients with migraine without aura. In addition, a linear feedforward neural network (FFNN) was used to construct a binary classification model by detected IgG N-glycopeptides. The area under the curve (AUC) value of the binary classification model, which was constructed with 7 IgG N-glycopeptides, was 0.857, suggesting a good prediction performance. Among these IgG N-glycopeptides that were constructed the model, IgG1 G0-NF was overlapped with the differential IgG N-glycopeptide between patients with migraine and healthy controls detected with MALDI-TOF-MS.ConclusionOur results indicated that the serum level of N-glycopeptides IgG1 G0-NF might be one of the important biomarkers for the diagnosis of migraine. To the best of our knowledge, this is the first study about the changes of IgG N-glycosylation in patients with migraine by the method of MALDI-TOF-MS. The results indicated a relationship between the migraine and immune response.
| S-EPMC9168990 | biostudies-literature
Project description:BackgroundChronic obstructive pulmonary disease (COPD) is a complex condition, whose diagnosis requires spirometric assessment. However, considering its heterogeneity, subjects with similar spirometric parameters do not necessarily have the same functional status. To overcome this limitation novel biomarkers for COPD have been investigated. Therefore, we aimed to explore the potential value of N-glycans as COPD biomarkers and to examine the individual variation of plasma protein and immunoglobulin G (IgG) glycosylation profiles in subjects with COPD and healthy controls.MethodsBoth the total plasma protein and IgG N-glycome have been profiled in the total of 137 patients with COPD and 95 matching controls from Croatia. Replication cohort consisted of 61 subjects with COPD and 148 controls recruited at another Croatian medical centre.ResultsPlasma protein N-glycome in COPD subjects exhibited significant decrease in low branched and conversely, an increase in more complex glycan structures (tetragalactosylated, trisialylated, tetrasialylated and antennary fucosylated glycoforms). We also observed a significant decline in plasma monogalactosylated species, and the same change replicated in IgG glycome. N-glycans also showed value in distinguishing subjects in different COPD GOLD stages, where the relative abundance of more complex glycan structures increased as the disease progressed. Glycans also showed statistically significant associations with the frequency of exacerbations and demonstrated to be affected by smoking, which is the major risk factor for COPD development.ConclusionsThis study showed that complexity of glycans associates with COPD, mirroring also the disease severity. Moreover, changes in N-glycome associate with exacerbation frequency and are affected by smoking. In general, this study provided new insights into plasma protein and IgG N-glycome changes occurring in COPD and pointed out potential novel markers of the disease progression and severity.
| S-EPMC6249776 | biostudies-literature