Dataset Information

Protein-Specific Differential Glycosylation of Immunoglobulins in Serum of Ovarian Cancer Patients.

ABSTRACT: Previous studies indicated that glycans in serum may serve as biomarkers for diagnosis of ovarian cancer; however, it was unclear to which proteins these glycans belong. We hypothesize that protein-specific glycosylation profiles of the glycans may be more informative of ovarian cancer and can provide insight into biological mechanisms underlying glycan aberration in serum of diseased individuals. Serum samples from women diagnosed with epithelial ovarian cancer (EOC, n = 84) and matched healthy controls (n = 84) were obtained from the Gynecologic Oncology Group. Immunoglobulin (IgG, IgA, and IgM) concentrations and glycosylation profiles were quantified using multiple reaction monitoring mass spectrometry. Differential and classification analyses were performed to identify aberrant protein-specific glycopeptides using a training set. All findings were validated in an independent test set. Multiple glycopeptides from immunoglubins IgA, IgG, and IgM were found to be differentially expressed in serum of EOC patients compared with controls. The protein-specific glycosylation profiles showed their potential in the diagnosis of EOC. In particular, IgG-specific glycosylation profiles are the most powerful in discriminating between EOC case and controls. Additional studies of protein- and site-specific glycosylation profiles of immunoglobulins and other proteins will allow further elaboration on the characteristics of biological functionality and causality of the differential glycosylation in ovarian cancer and thus ultimately lead to increased sensitivity and specificity of diagnosis.

SUBMITTER: Ruhaak LR

PROVIDER: S-EPMC5637400 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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Protein-Specific Differential Glycosylation of Immunoglobulins in Serum of Ovarian Cancer Patients.

Ruhaak L Renee LR Kim Kyoungmi K Stroble Carol C Taylor Sandra L SL Hong Qiuting Q Miyamoto Suzanne S Lebrilla Carlito B CB Leiserowitz Gary G

Journal of proteome research 20160224 3

Previous studies indicated that glycans in serum may serve as biomarkers for diagnosis of ovarian cancer; however, it was unclear to which proteins these glycans belong. We hypothesize that protein-specific glycosylation profiles of the glycans may be more informative of ovarian cancer and can provide insight into biological mechanisms underlying glycan aberration in serum of diseased individuals. Serum samples from women diagnosed with epithelial ovarian cancer (EOC, n = 84) and matched healthy ...[more]

PMID: 26813784

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Project description:Epithelial ovarian cancer (EOC) was previously shown to be associated with glycosylation changes of total serum and total IgG proteins. However, as a majority of previous studies analyzed released glycan profiles, still little is known about IgG subclass-specific alterations in ovarian cancer. Hence, in this study, we investigated EOC-related glycosylation changes of the three most abundant IgG subclasses, namely, IgG1, IgG2 and IgG3 isolated from sera of 87 EOC patients and 74 age-matched healthy controls. In order to separate IgG2 and IgG3, we performed a two-step affinity purification employing Protein A and Protein G Sepharose. After tryptic digestion, IgG glycopeptides were enriched and measured by MALDI-TOF-MS. Finally, EOC-related glycosylation changes were monitored at the level of total agalactosylation, monogalactosylation, digalactosylation, sialylation, bisection and fucosylation, which were calculated separately for each IgG subclass. Interestingly, aside from an EOC-related increase in agalactosylation/decrease in monogalactosylation and digalactosylation observed in all IgG subclasses, some subclass-specific trends were detected. Glycosylation of IgG1 was found to be most strongly affected in EOC, as it exhibited the highest number of significant differences between healthy controls and EOC patients. Specifically, IgG1 was the only subclass that showed a significant decrease in sialylation and a significant increase in fucosylation in EOC patients. Interestingly, IgG2 and IgG3 that were often investigated collectively in previous studies, were found to have distinct glycosylation patterns. IgG3 displayed stronger EOC-related increase in agalactosylation/decrease in digalactosylation and was characterized by notably higher sialylation, which consequently decreased in EOC patients. In conclusion, our study indicates that IgG subclasses exhibit subtly distinct glycosylation patterns of EOC-related alterations and that IgG1 and IgG3 agalactosylation show the strongest association with CA125, the routine diagnostic marker. Additionally, our results show that simultaneous analyses of IgG2 and IgG3 might lead to wrong conclusions as these two subclasses exhibit noticeably different glycosylation phenotypes.

Project description:Glycoproteins perform extra- and intracellular functions in innate and adaptive immunity by lectin-based interactions to exposed glyco-determinants. Herein, we document and mechanistically explain the formation of subcellular-specific N-glycosylation determinants on glycoproteins trafficking through the shared biosynthetic machinery of human cells. LC-MS/MS-based quantitative glycomics showed that the secreted glycoproteins of eight human breast epithelial cells displaying diverse geno- and phenotypes consistently displayed more processed, primarily complex type, N-glycans than the high-mannose-rich microsomal glycoproteins. Detailed subcellular glycome profiling of proteins derived from three breast cell lines (MCF7/MDA468/MCF10A) demonstrated that secreted glycoproteins displayed significantly more α-sialylation and α1,6-fucosylation, but less α-mannosylation, than both the intermediately glycan-processed cell-surface glycoproteomes and the under-processed microsomal glycoproteomes. Subcellular proteomics and gene ontology revealed substantial presence of endoplasmic reticulum resident glycoproteins in the microsomes and confirmed significant enrichment of secreted and cell-surface glycoproteins in the respective subcellular fractions. The solvent accessibility of the glycosylation sites on maturely folded proteins of the 100 most abundant putative N-glycoproteins observed uniquely in the three subcellular glycoproteomes correlated with the glycan type processing thereby mechanistically explaining the formation of subcellular-specific N-glycosylation. In conclusion, human cells have developed mechanisms to simultaneously and reproducibly generate subcellular-specific N-glycosylation using a shared biosynthetic machinery. This aspect of protein-specific glycosylation is important for structural and functional glycobiology and discussed here in the context of the spatio-temporal interaction of glyco-determinants with lectins central to infection and immunity.

Project description:Given the growing incidence and prevalence of life-threatening food allergies, health concerns have raised new perspectives for in vivo and in vitro diagnostic methodologies, pointing to saliva as a promising material, already used to diagnose other pathologies. Based on the above considerations, this study aimed to verify the possible use of saliva for the detection of IgE and IgG1 in the diagnosis of food allergy. This was a randomized, cross-sectional clinical study with a quantitative approach, developed at a hospital referral center in allergy in the state of Ceará, from January to July 2015. The sample consisted of 36 children of both sexes, aged between 1 and 60 months, with a diagnosis of cow's milk protein allergy (CMPA) by the RAST test. Children hospitalized or under immunosuppressive drugs were excluded from the study. Serum and saliva samples of the participants were collected and subsequently subjected to the indirect immunoenzymatic assay (ELISA) for the detection of specific serum and salivary immunoglobulins for food: corn, papaya, cow's milk, egg white, wheat, soybeans, peanuts, nuts, kiwi, cacao, fish, shrimp, bananas and tomatoes. For comparison of serum and saliva results, the T-test of independent samples and Mann-Whitney were adopted, for samples with normal and non-normal distribution respectively. A confidence interval of 95% was adopted for significant results. It was observed that 100% (n = 36) of the participants presented cow's milk allergy through the indirect ELISA, detecting IgE or IgG1 in serum and saliva. When serum IgE and IgG1 concentrations were compared, there was no statistical difference (p > 0.05) in 12 of the 14 foods evaluated. The same amount (n = 12) of non-significant differences (p > 0.05) was observed in the comparison of the 14 foods under IgE and IgG1 contractions in saliva. In the verification of the average values of IgE present in the serum and saliva of the foods, only cow's milk, fish and papaya showed statistically significant differences (p < 0.05). Of the total food evaluated, only the average levels of IgG1 present in serum and saliva showed a significant value (p < 0.05) in banana and tomato. These findings indicate that the detection of IgE and IgG1 in saliva proves to be as efficient as in the serum. The use of the salivary technique for use in the diagnosis of food allergy is suggested.

Dataset Information

Protein-Specific Differential Glycosylation of Immunoglobulins in Serum of Ovarian Cancer Patients.

Publications

Protein-Specific Differential Glycosylation of Immunoglobulins in Serum of Ovarian Cancer Patients.

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