N-glycosylation as biomarker of disease.
Aberrant N-glycosylation is associated to cancer, autoimmune and infectious diseases, as well as congenital disorders, ageing and degenerative processes
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- Protocol optimization for sample treatment and qualitative glycan biomarker-profiling
- Application of a customized mAbsolute kit for quantitative N-glycan biomarker analysis
Changes in glycosylation are a hallmark of many cancers and inflammatory diseases and show great potential as clinical disease markers.
The interaction of cell surface glycans with complementary glycan binding proteins (lectins) located on neighboring cells, other cell types, or pathogens like virus, bacteria or parasitesmediates is crucial in biologically and biomedically important processes like cell–cell adhesion, cell migration, development, pathogen recognition and infection. Their implication in nearly every pathological condition, consequently suggests an increasing role for glycans as disease markers.
Their implication in nearly every pathological condition, consequently suggests an increasing role for glycans as disease markers. The majority of mammalian proteins are N-glycosylated, a common post-translational modification of the asparagine residue within the consensus sequence Asn-X-Ser/ Thr. N-glycans are large complex and branched glycans that share a common pentasaccharide core and present considerable microheterogeneity due to variations in the number of antennae, terminal glycan residues and core modifications.
Glycan Biomarker Research
Although previous glycan biomarker research has also included analysis of glycosphingolipid and glycosaminoglycan levels by mass spectrometry (MS), immunostaining or lectin arrays the focus of most studies has been on the plasma N-glycome followed by the analysis of O-glycans on mucins which are highly over-expressed in carcinomas. This preference can be explained by the high abundance of N-glycans in serumand other body fluids, a manageable number of structures and more mature techniques for sample preparation and analysis of N-glycans compared to those available for other glycan classes. Human N-glycans, that are present on the majority of secreted proteins are easily obtained from a larger number of body fluids including serum/plasma, urine, saliva, tears, milk, semen or amniotic fluid by enzymatic or chemical removal.
Although estimates for the human N-glycan repertoire go as far as 2000 different structures, the currently experimentally accessible human N-glycome is far smaller and very dependent on the employed analytical method. The plasma N-glycome has been reported to contain more than 100 different glycan structures but depending on the analytical method used only a fraction is routinely quantified due to either intense peak overlap during the chromatographic separation or a lack of sensitivity for detecting less abundant species by direct injection methods or MALDI-TOF (matrix assisted laser desorption/ionization coupled to time-of-flight detection) analysis. By nano-liquid chromatography coupled to tandem mass spectrometry (nano-LC MS/MS) recently over 170 distinct N-glycan structures where registered in the plasma glycome and partially assigned by exoglycosidase digestion and diagnostic fragment ions. The 20 most abundant plasma Nglycans that account for over half of the plasma glycome are mono and bis-sialylated or neutral core-fucosylated bi-antennary structures.
These are major structures present on the most abundant plasma glycoproteins like the immunoglobulin isotypes IgG, IgMand IgA, transferrin, alpha-2-macroglobin, C3-complement or haptoglobin. Alpha-2-macroglobin, C3-complement and haptoglobin are acute phase proteins (APPs) which are part of the innate immune system and that show expression levels that are sensitive to inflammatory processes. The high basal concentration of some APPs in plasma together with their significant changes in expression levels during inflammation ranging from 50% of ceruloplasmin to over 1000-fold for the C-reactive protein, can have an impact even on the total serum glycan levels which is measurable as an increase of mobile hexosamine N-acetyl methyl groups by nuclear magnetic resonance (NMR).
Only absolute glycan quantification can provide a complete picture of the disease related changes and will provide the method robustness required by clinical applications.
The entry into the clinical practice of glycan markers is delayed in large part due to a lack of adequate methodology for the precise and robust quantification of protein glycosylation. Probably the most important area for absolute quantification of specific glycan levels is clinical diagnostics, which requires robust, reliable and precise methods for the quantification of disease markers. A large number of glycan biomarker studies have revealed the perturbation of glycan profiles, but methods for quantifying singlemarkers in the context of a complexmatrix are still underdeveloped. As a result, the decade-long exploration of glycans as biomarkers has not been matched by their introduction as disease markers into clinical practice. An important reason for this could be the lack of robust, transferable and quantitative methods for themeasurement of single glycans in the clinical practice.
Glycosylation not only changes qualitatively but is also increased in absolute terms as several pioneering studies suggest, allowing the definition of clinically relevant cut-off values for individual serumglycan levels for the clinical practice. Elevated serum glycan levels for a large number of structures have been shown in a pancreatic cancer study by Nishimura et al.  which show little or no overlap with 95% confidence intervals for healthy controls over a broad age range. As the majority of methods employ mass spectrometry either as a standalone technique or coupled with chromatography for glycan analysis, improvements in reproducibility and robustness of MS facilitating method transfer between labs are urgently needed.
The use of stable isotope labeled glycans as internal standards and heavy-isotope labeling methods will provide the necessary method precision and robustness acceptable for clinical use.
Internal stable isotope labeled standards for glycan quantification.
The use of internal stable isotope labeled standards for glycan quantification by mass spectrometry has the potential to solve many of the current challenges in MS glycan analyses and to aid in the transfer and acceptance of glycan markers into the clinical practice.
An isotopically enriched and quantified internal standard allows the absolute quantification of individual glycan levels in complex mixtures independent of changes in a glycan profile. A recent roadmap report from the US National Research Council highlighted the need for developing isotopically labeled glycan standards formass spectrometry to overcome “the current practice of relative quantification”.
In analogy to existing clinical methods for quantifying metabolites or drug levels we anticipate that a single or a limited number of glycan disease markers will be most efficiently quantified by isotopic dilution e.g. by employing stable isotope labeled glycans. Glycan synthesis has come a long way and current chemo-enzymatic approaches are now capable of producing well defined pure and strategically labeled glycans for this purpose.
The use of internal standards for absolute glycan quantification accelerates glycan analysis rather than slowing it down as any external calibration is unnecessary. This will be particularly the case for the rapid assessment of individual glycan levels as surrogate metabolites, which reflect impact of hereditary and environmental factors on patient physiology, in a platform independent and robust manner.
|Disease||Aberrant glycosylation||Glycan biomarker||ref|
|Breast cancer||Increased levels of highly sialylated antenna fucosylated glycans.||High G1F, A3F1G1, A4F1G1 and A4F2G2 containing sLex||1|
|Increased levels of fucosylated and sialylated glycans||A2F||2, 3|
|Increased levels of agalactosyl biantennary glycans G0F and glycans containing sLex epitope A3F1G1 and A2F1G1.||Increase A3F1G1 and G1F, d G0F with sLex||4|
|Increased levels of high-mannose type structures.||Man5||5|
|Increased levels of fucosylated glycans. Decreased leveles of sialylated glycans.||G0F, G1F ,G2F, A3G0F||6|
|Gastric cancer||Decreased levels of fucosylated non- and mono-sialylated glycans.||Increased levels of A2||7|
|Decreased levels of biantennary asialo monogalactosylated glycans, and triantennary glycans carrying 2,3-linked sialic acids.
Increased levels of triantennary glycans carrying 2,6-linked sialic acids, and trisialylated triantennary glycans carrying sialyl Lewis X.
|Decrease G1(3), G1(6) Increased A2F on IgG||8|
|Hepatocellular carcinoma||Decreased levels of a tri- and a tetraantennary glycan.||A3G0F, A3G3F, A4||9|
|Increased levels of the fucosylated triantennary glycan.||A3G3F||10|
|Highly increased fucosylation.
Decrease of G2F levels correlates with halting of Hepatoma Cell Migration and Invasion
Increased sialylation and high mannose structures
Increased fucosylation and syalylation of haptoglobin
|AFP-L3, Fucosylated glycoform of AFP||11,12|
|Lung cancer||Increased levels of tri- and tetra-antennary highly sialylated glycans, some with antenna and some with core fucosylation.
Decreased levels of biantennary glycans, mostly with core fucose
|High Tri- and tetra-sialylated glycans in serum||13|
|Ovarian cancer||Increased levels of G0F, and A3G3F||14|
|Increased levels of core fucosylated agalactosyl biantennary glycans and glycans containing sialyl Lewis X||G0F and sLexa3
|Increased levels of tri- and tetra-antennary N-glycans.
Decreased levels of glycans containing a bisecting GlcNAc.
|bA2F,A3G0F, A3G3F, A4||16|
|Increased levels of sialylated glycans and a small group of truncated glycans.
Decreased levels of several neutral glycans, including high-mannose-type glycans
|MGn(3)F, MGn(6), A2||17|
|MUC1 O-glycans in serum||Sialylated core 1type O-glycans||18|
|Prostate cancer||Increased core fucosylation and increase a2,3-sialic acid compared with BPH patients serum
G0 significantly increased and G1 and α2-6 syalilation decreased in the IgG heavy chains of patients with prostate cancer
|Decreased A3G3, A4FS4, Increased A4S4||19, 38|
|Decreased sialylation of PSA subforms in seminal plasma||F3 PSA subform with low S1 and S2
F3 PSA subform with high a2,3 sial and low core fucose
F4 PSA subform with high S1
|Poor prognosis is associated with decreased levels of fucosylated glycans and increased levels of sialylated compounds||22|
|Pancreatic cancer||Increase of core fucosylation and Lex in tri-antennary glycans||Fucosylated haptoglobin||23, 24, 25|
|Colon cancer||Levels of FUT3-7 and FUT9, responsible for a1,3-4 fucosylation at haptoglobin Asn241||a1,3-4 fucosylation at haptoglobin Asn241||26–28|
|Disease||Aberrant glycosylation||Glycan biomarker||ref|
Rheumatoid arthritis, Systemic lupus erythrematosus, Sjogren’s syndrome, Juvenile-onset chronic arthritis, Crohn´s syndrome, Tuberculosis
|Elevated proportion of IgG G0 glycoforms
Reduced degree of antennae sialylation and galactosylation for IgG N glycans
|Follicular lymphoma||IgG and IgM variable region glycans containing one or more oligomannose sugars. Significant increase in potential glycosylation sites in the variable region.||Man5-Man9||32|
|IgA nephropathy||Aberrant glycosylation has been linked to diseases in which there is reduced clearance of IgA. Aberrant O-glycosylation of serum IgA1 in patients is ascribed to a decrease in terminal galactosylation and sialylation.||Man5, G0F
|Primary Sjogren’s syndrome||Levels of serum IgA1 and IgA2 are elevated as a result of increased sialylation of the glycans.|
|Congenital Disorders of Glycosylation||Glycan analysis of serum proteins, such as the Igs that collectively account for up to 50% of the serum glycoproteins, provides diagnostic information that can pinpoint the faulty step in the pathway.||mono–antennary fucosylated and α2,3 sialylated|
|Schistosomiasis||Unusual O-glycan, glycan epitopes of soluble egg antigens of Schistosoma mansoni||Fuc1-2Fuc1-3GalNAc1- 4(Fuc1-2Fuc1-3)|
|Maturity-onset diabetes of the young (MODY)||Lewis a in tri- biantennary N-glycans in serum and plasma||Lewis a in tri- biantennary N-glycans in serum and plasma||33|
|Diabetes||Severity and duration of glucose dysregulation in individuals can be estimated by monitoring the levels of O-GlcNAc simultaneously at specific sites on several key proteins in erythrocytes. Increase in antenna fucosylation of AGP in plasma from patients with Type 1 Diabetes Mellitus||O-GlcNAc||34|
|Type 2 Diabetes Mellitus||Increased levels of glycans carrying α1-6 linked fucose||G0F, G1F ,G2F,|
|Aging||Individuals of ages above 50 had increased levels of non-galactosylated glycans, while the levels of galactosylated structures decreased with increasing age. Bisecting N-acetylglucosamine showed an age-dependency: bisecting GlcNAc is generally increasing with age and seems to reach a plateau at 50 years of age.||bA2F||35|
|Aging, smoke and lipid profiles||Very large biological variability in glycosylation. Increased ratio of bisecting GlcNAc in IgG of smokers||bA2F||36|
|Alzheimer’s disease||The glycosylation pattern of Reelin decrease in fucose content|
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