Protein modification and maintenance systems as biomarkers of ageing

Changes in the abundance and post-translational modification of proteins and accumulation of some covalently modified proteins have been proposed to represent hallmarks of biological ageing. Within the frame of the Mark-Age project, the workpackage dedicated to "markers based on proteins and their modifications" has been firstly focused on enzymatic and non-enzymatic post-translational modifications of serum proteins by carbohydrates. The second focus of the workpackage has been directed towards protein maintenance systems that are involved either in protein quality control (ApoJ/Clusterin) or in the removal of oxidatively damaged proteins through degradation and repair (proteasome and methionine sulfoxide reductase systems). This review describes the most relevant features of these protein modifications and maintenance systems, their fate during ageing and/or their implication in ageing and longevity

Alteration of liver N-glycome in patients with hepatocellular carcinoma

Purpose: Alteration of liver function during progression of hepatocellular carcinoma (HCC) and cirrhosis affects the serum glycoprotein pattern. In this study, the changes in the N-glycome in liver tissue from patients with hepatocellular carcinoma and cirrhosis caused by hepatitis B virus infection were investigated to find out the relationship between this maker and liver disease. Methods: Twenty patients, 11 with cirrhosis and 9 with hepatocellular carcinoma, and 15 healthy donors were involved in this study. Liver protein N-glycans were profiled using the DSA-FACE technique developed in our laboratory. To further analyze the fucosylation status of these liver glycans Western lectin blots of total liver proteins were performed using Aspergillus oryzae lectin (AOL) as probe, which is a carbohydrate-binding protein that recognizes specifically α-1,6-fucosylated glycans. Results: The N-glycome of liver proteins in patients with HBV related HCC and cirrhosis was analyzed. Compared with healthy donors, the N-glycome had significantly less (p < 0.05) high mannose (M8) in both groups of patients. The total core α-1,6-fucosylation in total liver glycopro-teins was dramatically increased during the progress of hepatocellular carcinoma and cirrhosis compared to the controls. Conclusion: These results show that fucosylation not only increases in serum proteins but also in liver tissue itself of patients with HBV related HCC and cirrhosis

N-glycome profile levels relate to silent brain infarcts in a cohort of hypertensives

Background: Silent brain infarcts (SBIs) are highly prevalent in the aged population and relate to the occurrence of further stroke and dementia. Serum N-glycome levels have been previously associated with aging and they might be related as well to the presence of SBIs and age-related white matter hyperintensities. Methods and Results: We determined the serum N-glycome profile in a cohort study comprising 972 subjects and evaluated the relationship between N-glycome levels and the presence and number of SBIs and with age-related white matter hyperintensities grades, assessed by brain magnetic resonance imaging. Decreasing concentrations of bigalacto core-alpha-1,6-fucosylated biantennary glycan and increasing concentrations of branching alpha-1,3-fucosylated triantennary glycan remained as independent predictors of SBIs (odds ratio 0.4, 95% CI 0.3-0.7 and odds ratio 1.8, 95% CI 1-3.2, respectively), after controlling for the presence of age and classic vascular risk factors. A similar pattern was found to be related to an increasing number of SBIs and white matter hyperintensities grade. Conclusions: N-glycome levels might be potentially useful as biomarkers for the presence of silent cerebrovascular disease

Identification of a novel mechanism of blood-brain communication during peripheral inflammation via choroid plexus-derived extracellular vesicles

Here, we identified release of extracellular vesicles (EVs) by the choroid plexus epithelium (CPE) as a new mechanism of blood-brain communication. Systemic inflammation induced an increase in EVs and associated pro-inflammatory miRNAs, including miR-146a and miR-155, in the CSF. Interestingly, this was associated with an increase in amount of multivesicular bodies (MVBs) and exosomes per MVB in the CPE cells. Additionally, we could mimic this using LPS-stimulated primary CPE cells and choroid plexus explants. These choroid plexus-derived EVs can enter the brain parenchyma and are taken up by astrocytes and microglia, inducing miRNA target repression and inflammatory gene up-regulation. Interestingly, this could be blocked in vivo by intracerebroventricular (icv) injection of an inhibitor of exosome production. Our data show that CPE cells sense and transmit information about the peripheral inflammatory status to the central nervous system (CNS) via the release of EVs into the CSF, which transfer this pro-inflammatory message to recipient brain cells. Additionally, we revealed that blockage of EV secretion decreases brain inflammation, which opens up new avenues to treat systemic inflammatory diseases such as sepsis

N-Glycomic changes in serum proteins in type 2 diabetes mellitus correlate with complications and with metabolic syndrome parameters

Background: Glycosylation, i.e the enzymatic addition of oligosaccharides (or glycans) to proteins and lipids, known as glycosylation, is one of the most common co-/posttranslational modifications of proteins. Many important biological roles of glycoproteins are modulated by N-linked oligosaccharides. As glucose levels can affect the pathways leading to glycosylation of proteins, we investigated whether metabolic syndrome (MS) and type 2 diabetes mellitus (T2DM), pathological conditions characterized by altered glucose levels, are associated with specific modifications in serum N-glycome. Methods: We enrolled in the study 562 patients with Type 2 Diabetes Mellitus (T2DM) (mean age 65.6 +/- 8.2 years) and 599 healthy control subjects (CTRs) (mean age, 58.5 +/- 12.4 years). N-glycome was evaluated in serum glycoproteins. Results: We found significant changes in N-glycan composition in the sera of T2DM patients. In particular, alpha(1,6)-linked arm monogalactosylated, core-fucosylated diantennary N-glycans (NG1(6)A2F) were significantly reduced in T2DM compared with CTR subjects. Importantly, they were equally reduced in diabetic patients with and without complications (P<0.001) compared with CTRs. Macro vascular-complications were found to be related with decreased levels of NG1(6) A2F. In addition, NG1(6) A2F and NG1(3) A2F, identifying, respectively, monogalactosylated N-glycans with alpha(1,6)- and alpha(1,3)-antennary galactosylation, resulted strongly correlated with most MS parameters. The plasmatic levels of these two glycans were lower in T2DM as compared to healthy controls, and even lower in patients with complications and MS, that is the extreme "unhealthy" phenotype (T2DM+ with MS). Conclusions: Imbalance of glycosyltransferases, glycosidases and sugar nucleotide donor levels is able to cause the structural changes evidenced by our findings. Serum N-glycan profiles are thus sensitive to the presence of diabetes and MS. Serum N-glycan levels could therefore provide a non-invasive alternative marker for T2DM and MS

Serum N-glycome biomarker for monitoring development of DENA-induced hepatocellular carcinoma in rat

<p>Abstract</p> <p>Background</p> <p>There is a demand for serum markers for the routine assessment of the progression of liver cancer. We previously found that serum N-linked sugar chains are altered in hepatocellular carcinoma (HCC). Here, we studied glycomic alterations during development of HCC in a rat model.</p> <p>Results</p> <p>Rat HCC was induced by the hepatocarcinogen, diethylnitrosamine (DENA). N-glycans were profiled using the DSA-FACE technique developed in our laboratory.</p> <p>In comparison with control rats, DENA rats showed a gradual but significant increase in two glycans (R5a and R5b) in serum total N-glycans during progression of liver cirrhosis and cancer, and a decrease in a biantennary glycan (P5). The log of the ratio of R5a to P1 (NGA2F) and R5b to P1 [log(R5a/P1) and log(R5b/P1)] were significantly (p < 0.0001) elevated in HCC rats, but not in rats with cirrhosis or fibrosis or in control rats. We thus propose a GlycoTest model using the above-mentioned serum glycan markers to monitor the progression of cirrhosis and HCC in the DENA-treated rat model. When DENA-treated rats were subsequently treated with farnesylthiosalicyclic acid, an anticancer drug, progression to HCC was prevented and GlycoTest markers (P5, R5a and R5b) reverted towards non-DENA levels, and the HCC-specific markers, log(R5a/P1) and log(R5b/P1), normalized completely. <b>Conclusions</b>: We found an increase in core-α-1,6-fucosylated glycoproteins in serum and liver of rats with HCC, which demonstrates that fucosylation is altered during progression of HCC. Our GlycoTest model can be used to monitor progression of HCC and to follow up treatment of liver tumors in the DENA rat. This GlycoTest model is particularly important because a rapid non-invasive diagnostic procedure for tumour progression in this rat model would greatly facilitate the search for anticancer drugs.</p

Glycomics of mammalian aging

N-glycosylation is the mirror of the status of the cell. N-glycosylation changes in relation to cellular activation, embryonic development, organogenesis and differentiation have been thoroughly studied. But little is known about the involvement of N-glycosylation in the aging process. Therefore, we analyzed how the N-glycosylation status of serum proteins is altered during aging in humans. We questioned whether N-glycosylation of serum proteins could be a suitable aging biomarker because no effective and easy to measure parameter for physiological age has yet been found (see I.3). After studying age-related N-glycan alterations in humans, we investigated whether this phenomenon also occurs in other species, such as mice. If alterations in N-glycosylation in mice can also be used as an aging biomarker, different anti-aging interventions can be monitored in mice by determining the N-glycosylation status. An advantage of using mice is the ability to control their housing environment. To determine whether age-related glycosylation alteration is inherent to the organism, we can study whether mice isolated from environmental influences (i.e. in specific pathogen free conditions; SPF) also exhibit the same N-glycosylation changes. Another advantage is that wile the influence of genetic background cannot be controlled in humans, genetically identical inbred mouse strains are available. Therefore, we analyzed the age-related N-glycosylation alterations in genetically identical mice, namely C57BL/6, housed in SPF conditions. Moreover, to further confirm the age-related nature of the N-glycosylation changes, the N-glycosylation status was analyzed in short-lived and long-lived mice (see I.1.6.2). Next, we explored the mechanism behind these N-glycosylation alterations. Are N-glycosylation changes the cause of aging (since N-glycosylation regulates important pathways in the organism) or are they the consequence of aging? We hypothesized that structural changes in N-glycosylation could be caused by alterations in the biosynthesis pathway, degradation in the blood, changes in clearance capacity, or a combination of all. Age-related alteration in gene expression might affect the synthesis of N-glycans in the ER and Golgi. Some glycosyltransferases and glycosidases might be up or down regulated, which could lead to incomplete or aberrant N-glycan structures. If the structure of a certain glycan changes with age, it could be easy to predict which gene is responsible for the fluctuation because biosynthesis of glycans occurs through the consecutive actions of well defined glycosyltransferases. Therefore, gene expression levels and protein levels of different glyco-genes were measured. If a certain glyco-gene is suspected of being involved, a transgenic mouse overexpressing that glyco-gene can be made and studied. Also the regulation of interesting glyco-genes can be analyzed by promoter studies. We also investigated if there is a link between these glyco-genes and essential “aging” molecules, e.g. GH and IGF-I (see I.1.5.3). Another mechanism that could explain the observed N-glycosylation differences is degradation. While circulating in the blood, the N-glycan of an N-glycoprotein can be attacked by glycosidases. These enzymes are normally present in the lysosomes but some leakage could occur. To tackle this possibility, we measured the amounts and activities of glycosidases in serum. A third mechanism that was investigated is clearance. Proteins in the blood are cleared mostly by the kidneys and the liver. Proteins smaller than 45 kDa are cleared by the kidneys but most serum N-glycoproteins are larger. These glycoproteins can be cleared from the circulation by lectins that recognize a certain glycan structure (see I.2.3.3). We explored the possibility that clearance capacity is decreased during the aging process. The gene expression level of different clearance receptors was measured and an in vivo clearance tests was performed. Finally, we aimed to investigate possible longevity of candidate long-living mice. Studies performed in lower organisms have shown that overexpressing chaperones extends lifespan (see I.1.5.4). We analyzed the N-glycan profile of chaperone-overexpressing mice to see if we could find any retardation of the age-related N-glycosylation changes that could predict the extension of life in these mice. We also followed the lifespan of these mice and investigated gene expression levels and protein activity of candidate molecules