Altered Glycosylation of Human Alpha-1-Acid Glycoprotein as a Biomarker for Malignant Melanoma.
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ABSTRACT:
SUBMITTER: Virag D
PROVIDER: S-EPMC8513036 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Alpha-1-acid glycoprotein (AGP) is an acute phase glycoprotein in blood, which is primarily synthetized in the liver and whose biological role is not completely understood. It consists of 45% carbohydrates that are present in the form of five N-linked complex glycans. AGP N-glycosylation was shown to be changed in many different diseases, and some changes appear to be disease-specific; thus, it has a great diagnostic and prognostic potential. However, AGP glycosylation was mainly analyzed in small cohorts and without detailed site-specific glycan information. Here, we developed a cost-effective method for a high-throughput and site-specific N-glycosylation LC-MS analysis of AGP which can be applied on large cohorts, aid in search for novel disease biomarkers, and enable better understanding of AGP's role and function in health and disease. The method does not require isolation of AGP with antibodies and affinity chromatography, but AGP is enriched by acid precipitation from 5 μl of bloodplasma in a 96-well format. After trypsinization, AGP glycopeptides are purified using a hydrophilic interaction chromatography-based solid-phase extraction and analyzed by reversed-phase-liquid chromatography-electrospray ionization-MS. We used our method to show for the first time that AGP N-glycan profile is stable in healthy individuals (14 individuals in three time points), which is a requirement for evaluation of its diagnostic potential. Furthermore, we tested our method on a population including individuals with registered hyperglycemia in critical illness (59 cases and 49 controls), which represents a significantly increased risk of developing type 2 diabetes. Individuals at higher risk of diabetes presented increased N-glycan branching on AGP's second glycosylation site and lower sialylation of N-glycans on AGP's third and AGP1's fourth glycosylation site. Although this should be confirmed on a larger prospective cohort, it indicates that site-specific AGP N-glycan profile could help distinguish individuals who are at risk of type 2 diabetes.
| S-EPMC7950198 | biostudies-literature
Project description:Orosomucoid (OMD) contains complex bi-, tri- and tetra-antennary glycan chains. Subfractionation of OMD into three molecular variants using concanavalin A lectin chromatography is based on variations in these complex structures. Standard h.p.l.c. profiles have been developed to analyse the percentage and distribution of the glycoforms present at each glycosylation site in OMD and its molecular variants. The ability to quantify the glycoforms present at each site allows us to extend the earlier results of others and resolve the remaining questions concerning the glycan structures of these variants. Most significantly, the proportions of bi-, tri- and tetra-antennary chains differ at each site for the three molecular variants. The most strongly retained variant from concanavalin A is uniquely capable of possessing biantennary chains at all five sites, whereas the unretained variant is completely devoid of biantennary chains. Only glycosylation site II of the five present is 100% biantennary in the retained and weakly retained variants. In addition, the two gene products of OMD were differentially glycosylated. Molecular masses of the glycoforms were verified by matrix-assisted u.v. laser desorption mass spectrometry. On the basis of the site distribution of oligosaccharides in the variants, efforts were made to understand the factors that control the processing of the carbohydrate chains in OMD. The results indicate that the 'site-directed' model of processing offers the most consistent explanation for the structures seen at the individual glycosylation sites of OMD.
| S-EPMC1131000 | biostudies-other
Project description:Alpha-1-acid glycoprotein (AGP) is an acute phase glycoprotein in blood, which is primarily synthetized in the liver and whose biological role is not completely understood. It consists 45% of carbohydrates attached in the form of five N-linked complex glycans. AGP N-glycosylation was shown to be changed in many different diseases and some changes appear to be disease-specific, thus it has a great diagnostic and prognostic potential. However, AGP glycosylation was mainly analyzed in small cohorts and without detailed site-specific glycan information. Here, we developed a cost-effective method for a high-throughput and site-specific N-glycosylation LC-MS analysis of AGP which can be applied on large cohorts, aid in search for novel disease biomarkers and enable better understanding of AGP’s role and function in health and disease.
2021-01-04 | PXD020387 | Pride
Project description:Current interest in the MUC1/EMA mucin relates to its role in malignancy, and its potential as a therapeutic target. MUC1/EMA expression has been observed in the majority of epithelioid mesotheliomas. However, little is known of the characteristics of MUC1/EMA in mesothelioma. Herein, we studied the cell surface and soluble expression of the MUC1/EMA glycoprotein, and determined the mRNA and genomic expression profiles in mesothelioma. We found that the anti-MUC1 antibody, E29, was the most diagnostically useful of seven antibody clones examined with a sensitivity of 84% (16 out of 19 cases) and no false positive results. MUC1 mRNA expression was significantly higher in mesothelioma samples than in benign mesothelial cells. No amplification of the MUC1 gene was observed by FISH. Seven of 9 mesothelioma samples expressed MUC1-secreted mRNA isoform in addition to the archetypal MUC1/transmembrane form. CA15.3 (soluble MUC1) levels were significantly higher in the serum of mesothelioma patients than in healthy controls but were not significantly different to levels in patients with benign asbestos-related disease. CA15-3 in effusions could differentiate malignant from benign effusions but were not specific for mesothelioma. Thus, as in other cancers, alterations in MUC1 biology occur in mesothelioma and these results suggest that specific MUC1 characteristics may be useful for mesothelioma diagnosis and should also be investigated as a potential therapeutic target.
| S-EPMC2391110 | biostudies-literature
Project description:The ability of AGP to bind circulating lipopolysaccharide (LPS) in plasma is believed to help reduce the proinflammatory effect of bacterial lipid A molecules. Here, for the first time we have characterized human AGP binding characteristics of the LPS from a number of pathogenic Gram-negative bacteria: Escherichia coli, Salmonella typhimurium, Klebsiella pneumonia, Pseudomonas aeruginosa, and Serratia marcescens. The binding affinity and structure activity relationships (SAR) of the AGP-LPS interactions were characterized by surface plasma resonance (SPR). In order to dissect the contribution of the lipid A, core oligosaccharide and O-antigen polysaccharide components of LPS, the AGP binding affinity of LPS from smooth strains, were compared to lipid A, Kdo2-lipid A, R(a), R(d), and R(e) rough LPS mutants. The SAR analysis enabled by the binding data suggested that, in addition to the important role played by the lipid A and core components of LPS, it is predominately the unique species- and strain-specific carbohydrate structure of the O-antigen polysaccharide that largely determines the binding affinity for AGP. Together, these data are consistent with the role of AGP in the binding and transport of LPS in plasma during acute-phase inflammatory responses to invading Gram-negative bacteria.
| S-EPMC3539403 | biostudies-literature
Project description:Fetuin, also known as α-2-HS-glycoprotein (gene name: AHSG), is one of the more abundant glycoproteins secreted into the bloodstream. There are two frequently occurring alleles of human AHSG, resulting in three genotypes (AHSG*1, AHSG*2, and heterozygous AHSG1/2). The backbone amino acid sequences of fetuin coded by the AHSG*1 and AHSG*2 genes differ in two amino acids including one known O-glycosylation site (aa position 256). Although fetuin levels have been extensively studied, the originating genotype is often ignored in such analysis. As fetuin has been suggested repeatedly as a potential biomarker for several disorders, the question whether the gene polymorphism affects the fetuin profile is of great interest. In this work, we describe detailed proteoform profiles of fetuin, isolated from serum of 10 healthy and 10 septic patient individuals and investigate potential glycoproteogenomics correlations, e.g. how gene polymorphisms affect glycosylation. We established an efficient method for fetuin purification from individuals' serum using ion-exchange chromatography. Subsequently, we performed hybrid mass spectrometric approaches integrating data from native mass spectra and peptide-centric MS analysis. Our data reveal a crucial effect of the gene polymorphism on the glycosylation pattern of fetuin. Moreover, we clearly observed increased fucosylation in the samples derived from the septic patients. Our serum proteoform analysis, targeted at one protein obtained from 20 individuals, exposes the wide variability in proteoform profiles, which should be taken into consideration when using fetuin as biomarker. Importantly, focusing on a single or few proteins, the quantitative proteoform profiles can provide, as shown here, already ample data to classify individuals by genotype and disease state.
| S-EPMC6683009 | biostudies-literature
Project description:The ever-increasing complexity of biological samples to be analysed by mass spectrometry has led to the necessity of sophisticated separation techniques, including multidimensional separation. Despite a high degree of orthogonality, the coupling of liquid chromatography (LC) and capillary zone electrophoresis (CZE) has not gained notable attention in research. Here, we present a heart-cut nanoLC-CZE-ESI-MS platform to analyse intact proteins. NanoLC and CZE-MS are coupled using a four-port valve with an internal nanoliter loop. NanoLC and CZE-MS conditions were optimised independently to find ideal conditions for the combined setup. The valve setup enables an ideal transfer efficiency between the dimensions while maintaining good separation conditions in both dimensions. Due to the higher loadability, the nanoLC-CZE-MS setup exhibits a 280-fold increased concentration sensitivity compared to CZE-MS. The platform was used to characterise intact human alpha-1-acid glycoprotein (AGP), an extremely heterogeneous N-glycosylated protein. With the nanoLC-CZE-MS approach, 368 glycoforms can be assigned at a concentration of 50 μg/mL as opposed to the assignment of only 186 glycoforms from 1 mg/mL by CZE-MS. Additionally, we demonstrate that glycosylation profiling is accessible for dried blood spot analysis (25 μg/mL AGP spiked), indicating the general applicability of our setup to biological matrices. The combination of high sensitivity and orthogonal selectivity in both dimensions makes the here-presented nanoLC-CZE-MS approach capable of detailed characterisation of intact proteins and their proteoforms from complex biological samples and in physiologically relevant concentrations.
| S-EPMC8791864 | biostudies-literature
Project description:BackgroundIt has been shown that proteasome inhibition leads to growth arrest in the G1 phase of the cell cycle and/or induction of apoptosis. However, it was found that some of these inhibitors do not induce apoptosis in several human normal cell lines. This selective activity makes proteasome inhibition a promising target for new generation of anticancer drugs. Clinical validation of the proteasome, as a therapeutic target in oncology, has been provided by the dipeptide boronic acid derivative; bortezomib. Bortezomib has proven to be effective as a single agent in multiple myeloma and some forms of non-Hodgkin's lymphoma. Syringic acid (4-hydroxy-3,5-dimethoxybenzoic acid, 1), a known phenolic acid, was isolated from the methanol extract of Tamarix aucheriana and was shown to possess proteasome inhibitory activity.MethodsUsing Surflex-Dock program interfaced with SYBYL, the docking affinities of syringic acid and its proposed derivatives to 20S proteasome were studied. Several derivatives were virtually proposed, however, five derivatives: benzyl 4-hydroxy-3,5-dimethoxybenzoate (2), benzyl 4-(benzyloxy)-3,5-dimethoxybenzoate (3), 3'-methoxybenzyl 3,5-dimethoxy-4-(3'-methoxybenzyloxy)benzoate (4), 3'-methoxybenzyl 4-hydroxy-3,5-dimethoxybenzoate (5) and 3',5'-dimethoxybenzyl 4-hydroxy-3,5-dimethoxybenzoate (6), were selected based on high docking scores, synthesized, and tested for their anti-mitogenic activity against human colorectal, breast and malignant melanoma cells as well as normal human fibroblast cells.ResultsDerivatives 2, 5, and 6 showed selective dose-dependent anti-mitogenic effect against human malignant melanoma cell lines HTB66 and HTB68 with minimal cytotoxicity on colorectal and breast cancer cells as well as normal human fibroblast cells. Derivatives 2, 5 and 6 significantly (p ≤ 0.0001) inhibited the various proteasomal chymotrypsin, PGPH, and trypsin like activities. They growth arrested the growth of HTB66 cells at G1 and G2-phases. They also arrested the growth of HTB68 cells at S- and G2-phase, respectively. Moreover, derivatives 2, 5, and 6 markedly induced apoptosis (≥ 90%) in both HTB66 and HTB68.ConclusionsComputer-derived syringic acid derivatives possess selective anti-mitogenic activity on human malignant melanoma cells that may be attributed to perturbation of cell cycle, induction of apoptosis and inhibition of various 26S proteasomal activities.
| S-EPMC3765228 | biostudies-literature
Project description:Glyoxalase I [lactoylglutathione lyase (EC 4.4.1.5) encoded by GLO1] is a ubiquitous cellular defense enzyme involved in the detoxification of methylglyoxal, a cytotoxic byproduct of glycolysis. Accumulative evidence suggests an important role of GLO1 expression in protection against methylglyoxal-dependent protein adduction and cellular damage associated with diabetes, cancer, and chronological aging. On the basis of the hypothesis that GLO1 upregulation may play a functional role in glycolytic adaptations of cancer cells, we examined GLO1 expression status in human melanoma tissue. Quantitative reverse transcription polymerase chain reaction analysis of a cDNA tissue array containing 40 human melanoma tissues (stages III and IV) and 13 healthy controls revealed pronounced upregulation of GLO1 expression at the mRNA level. Immunohistochemical analysis of a melanoma tissue microarray confirmed upregulation of glyoxalase I protein levels in malignant melanoma tissue versus healthy human skin. Consistent with an essential role of GLO1 in melanoma cell defense against methylglyoxal cytotoxicity, siRNA interference targeting GLO1-expression (siGLO1) sensitized A375 and G361 human metastatic melanoma cells towards the antiproliferative, apoptogenic, and oxidative stress-inducing activity of exogenous methylglyoxal. Protein adduction by methylglyoxal was increased in siGLO1-transfected cells as revealed by immunodetection using a monoclonal antibody directed against the major methylglyoxal-derived epitope argpyrimidine that detected a single band of methylglyoxal-adducted protein in human LOX, G361, and A375 total cell lysates. Using two-dimensional proteomics followed by mass spectrometry the methylglyoxal-adducted protein was identified as heat shock protein 27 (Hsp27; HSPB1). Taken together, our data suggest a function of GLO1 in the regulation of detoxification and target adduction by the glycolytic byproduct methylglyoxal in malignant melanoma.
| S-EPMC2891514 | biostudies-literature
Project description:Endoplasmic reticulum (ER) α-glucosidase I is an enzyme that trims the distal α1,2-linked glucose (Glc) residue from the Glc3Man9GlcNAc2 oligosaccharide following its addition to nascent glycoproteins in the initial step of processing. This reaction is critical to the subsequent processing of N-glycans and thus defects in α-glucosidase I gene in human cause congenital disorder of glycosylation (CDG) type IIb. We identified the Caenorhabditis elegans α-glucosidase I gene (F13H10.4, designated agl-1) that encodes a polypeptide with 36% identity to human α-glucosidase I. The agl-1 cDNA restored the expression of complex-type N-glycans on the cell surface of α-glucosidase I-defective Chinese hamster ovary Lec23 cells. RNAi knockdown of agl-1 [agl-1(RNAi)] produced worms that were visibly similar to wild-type, but lifespan was reduced to about half of the control. Analyses of N-glycosylation in agl-1(RNAi) animals by western blotting and mass spectrometry showed reduction of paucimannose and complex-type glycans and dramatic increase of glucosylated oligomannose glycans. In addition, a significant amount of unusual terminally fucosylated N-glycans were found in agl-1(RNAi) animals. ER stress response was also provoked, leading to the accumulation of large amounts of triglucosylated free oligosaccharides (FOSs) (Glc3Man4-5GlcNAc1-2) in agl-1(RNAi) animals. Acceleration of ER-associated degradation in response to the accumulation of unfolded glycoproteins and insufficient interaction with calnexin/calreticulin in the ER lumen likely accounts for the increase of FOSs. Taken together, these studies in C. elegans demonstrate that decreased ER α-glucosidase I affects the entire N-glycan profile and induces chronic ER stress, which may contribute to the pathophysiology of CDG-IIb in humans.
| S-EPMC3766279 | biostudies-literature