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O-Glycosylation changes in misfolded proteins are of particular interest in understanding neurodegenerative conditions such as Parkinson's disease (PD) and incidental Lewy body disease (ILBD). This work outlines optimizations of a microwave-assisted nonreductive release to limit glycan degradation and employs this methodology to analyze O-glycosylation on the human striatum and substantia nigra tissue in PD, ILBD, and healthy controls, working alongside well-established reductive release approaches. A total of 70 O-glycans were identified, with ILBD presenting significantly decreased levels of mannose-core (p = 0.017) and glucuronylated structures (p = 0.039) in the striatum and PD presenting an increase in sialylation (p < 0.001) and a decrease in sulfation (p = 0.001). Significant increases in sialylation (p = 0.038) in PD were also observed in the substantia nigra. This is the first study to profile the whole nigrostriatal O-glycome in healthy, PD, and ILBD tissues, outlining disease biomarkers alongside benefits of employing orthogonal techniques for O-glycan analysis.

Traumatic spinal cord injury (SCI) results in disruption of tissue integrity and loss of function. We hypothesize that glycosylation has a role in determining the occurrence of regeneration and that biomaterial treatment can influence this glycosylation response. We investigated the glycosylation response to spinal cord transection in

Glycosylation is crucial in cellular metabolism and survival. Of interest is the role of N-linked and O-linked glycans in disease states. Robust analytical methods must be defined to identify suita...

Glycosylation is crucial in cellular metabolism and survival. Of interest is the role of

Afamin is an 87 kDa glycoprotein with five predicted N-glycosylation sites. Afamin's glycan abundance contributes to conformational and chemical inhomogeneity presenting great challenges for molecular structure determination. For the purpose of studying the structure of afamin, various forms of recombinantly expressed human afamin (rhAFM) with different glycosylation patterns were thus created. Wild-type rhAFM and various hypoglycosylated forms were expressed in CHO, CHO-Lec1, and HEK293T cells. Fully nonglycosylated rhAFM was obtained by transfection of point-mutated cDNA to delete all N-glycosylation sites of afamin. Wild-type and hypo/nonglycosylated rhAFM were purified from cell culture supernatants by immobilized metal ion affinity and size exclusion chromatography. Glycan analysis of purified proteins demonstrated differences in micro- and macro-heterogeneity of glycosylation enabling the comparison between hypoglycosylated, wild- type rhAFM, and native plasma afamin. Because antibody fragments can work as artificial chaperones by stabilizing the structure of proteins and consequently enhance the chance for successful crystallization, we incubated a Fab fragment of the monoclonal anti-afamin antibody N14 with human afamin and obtained a stoichiometric complex. Subsequent results showed sufficient expression of various partially or nonglycosylated forms of rhAFM in HEK293T and CHO cells and revealed that glycosylation is not necessary for expression and secretion

Acute phase proteins (APPs) are a group of circulating plasma proteins which undergo changes quantitatively or qualitatively at the time of inflammation. Many of these APPs are glycosylated, and it has been shown that alterations in glycosylation may occur in inflammatory and malignant conditions. Changes in glycosylation have been studied as potential biomarkers in cancer and also in chronic inflammatory conditions and have been shown to correlate with disease severity in certain conditions. Serine protease inhibitors (serpins), many of which are also APPs, are proteins involved in the control of proteases in numerous pathways. Alpha-1 Antitrypsin (AAT) is the most abundant serpin within the circulation and is an APP which has been shown to increase in response to inflammation. The primary role of AAT is maintaining the protease/antiprotease balance in the lung, but it also possesses important anti-inflammatory and immune-modulating properties. Several glycoforms of AAT exist, and they possess differing properties in regard to plasma half-life and stability. Glycosylation may also be important in determining the immune modulatory properties of AAT. The review will focus on the role and importance of glycosylation in acute phase proteins with particular attention to AAT and its use as a biomarker of disease. The review describes the processes involved in glycosylation, how glycosylation changes in differing disease states, and the alterations that occur to glycans of APPs with disease and inflammation. Finally, the review explores the importance of changes in glycosylation of AAT at times of inflammation and in malignant conditions and how this may impact upon the functions of AAT.

An improved separation of the human serum N-glycome using hydrophilic interaction chromatography technology with UPLC is described, where more than 140 N-glycans were assigned. Using this technique, serum samples from 107 healthy controls and 62 newly diagnosed breast cancer patients were profiled. The most statistically significant alterations were observed in cancer patients compared with healthy controls: an increase in sialylation, branching, and outer-arm fucosylation and a decrease in high-mannosylated and biantennary core-fucosylated glycans. In the controls and cases combined systemic features were analyzed; serum estradiol was associated with increase in digalactosylated glycans, and higher mammographic density was associated with increase in biantennary digalactosylated glycans and with decrease in trisialylated and in outer-arm fucosylated glycans. Furthermore, particular glycans were altered in some features of the breast carcinomas; bisected biantennary nonfucosylated glycans were decreased in patients with progesterone receptor positive tumors, and core-fucosylated biantennary bisected monogalactosylated glycans were decreased in patients with the TP53 mutation. Systemic features show more significant associations with the serum N-glycome than do the features of the breast carcinomas. In conclusion, the UPLC-based glycan analysis technique described here reveals highly significant differences between healthy women and breast cancer patients. Significant associations with breast carcinoma and systemic features are described.

Tissue inhibitor of metalloproteinases-1 (TIMP-1) inhibits matrix metalloproteinases (MMPs) by binding at a 1:1 stoichiometry. Here we have shown the involvement of N-glycosylation in the MMP inhibitory ability of TIMP-1. TIMP-1, purified from HEK 293 cells overexpressing TIMP-1 (293 TIMP-1), showed less binding and inhibitory abilities to MMPs than TIMP-1 purified from fibroblasts or SF9 insect cells infected with TIMP-1 baculovirus. Following deglycosylation of TIMP-1, all forms of TIMP-1 showed similar levels of MMP binding and inhibition, suggesting that glycosylation is involved in the regulation of these TIMP-1 activities. Analysis of the N-glycan structures showed that SF9 TIMP-1 has the simplest N-glycan structures, followed by fibroblast TIMP-1 and 293 TIMP-1, in order of increasing complexity in their N-glycan structures. Further analyses showed that cleavage of outer arm fucose residues from the N-glycans of 293 TIMP-1 or knockdown of both FUT4 and FUT7 (which encode for fucosyltransferases that add outer arm fucose residues to N-glycans) enhanced the MMP-binding and catalytic abilities of 293 TIMP-1, bringing them up to the levels of the other TIMP-1. These results demonstrate that the ability of TIMP-1 to inhibit MMPs is at least in part regulated by outer arm fucosylation of its N-glycans.

Lung cancer has a poor prognosis and a 5-year survival rate of 15%. Therefore, early detection is vital. Diagnostic testing of serum for cancer-associated biomarkers is a noninvasive detection method. Glycosylation is the most frequent post-translational modification of proteins and it has been shown to be altered in cancer. In this paper, high-throughput HILIC technology was applied to serum samples from 100 lung cancer patients, alongside 84 age-matched controls and significant alterations in N-linked glycosylation were identified. Increases were detected in glycans containing Sialyl Lewis X, monoantennary glycans, highly sialylated glycans and decreases were observed in core-fucosylated biantennary glycans, with some being detectable as early as in Stage I. The N-linked glycan profile of haptoglobin demonstrated similar alterations to those elucidated in the total serum glycome. The most significantly altered HILIC peak in lung cancer samples includes predominantly disialylated and tri- and tetra-antennary glycans. This potential disease marker is significantly increased across all disease groups compared to controls and a strong disease effect is visible even after the effect of smoking is accounted for. The combination of all glyco-biomarkers had the highest sensitivity and specificity. This study identifies candidates for further study as potential biomarkers for the disease.

Serum N-glycans are promising biomarkers for systemic disease states. Better understanding of the serum N-glycome of patients with resectable periampullary adenocarcinoma may identify novel prognostic markers for this disease. Serum N-glycans in 70 patients with resectable periampullary adenocarcinoma, 15 patients with benign periampullary tumor, and 129 healthy individuals were quantified using ultra performance liquid chromatography. High-sensitivity C-reactive protein (hsCRP) was analyzed for all samples using an immunoturbidimetric method. The N-glycome was compared to clinical and histopathological data, and to the acute phase response as measured by hsCRP. Whole-genome tumor tissue mRNA expression data were used for correlation and enrichment analysis to investigate underlying biological processes giving rise to changes in the serum N-glycome. Significant changes were found in the serum N-glycome of patients with periampullary adenocarcinoma (n = 70) compared to healthy individuals (n = 129). No significant differences were found between patients with benign (n = 15) and malignant periampullary tumors (n = 70). Many alterations in the N-glycome correlated with systemic acute phase response as measured by hsCRP. Enrichment analysis indicated that immunologic pathways of the cancer microenvironment correlate with specific features of the serum N-glycome. Certain glycans were associated with poor overall and disease free survival in patients with pancreatobiliary type of periampullary adenocarcinoma. Our study supports the hypothesis that certain factors secreted by the tumor affect liver and plasma cells to orchestrate the changes in the serum N-glycome observed. The serum N-glycome could potentially reflect modified phenotypes of the host and/or tumor microenvironment. The prognostic impact of the serum N-glycome should be evaluated in larger, prospective studies.

Galactose intoxication and over-restriction in galactosemia may affect glycosylation pathways and cause multisystem effects. In this study, we describe an applied hydrophilic interaction chromatography ultra-performance liquid chromatography high-throughput method to analyze whole serum and extracted IgG N-glycans with measurement of agalactosylated (G0), monogalactosylated (G1), and digalactosylated (G2) structures as a quantitative measure of galactose incorporation. This was applied to nine children with severe galactosemia (genotype Q188R/Q188R) and one child with a milder variant (genotype S135L/S135L). The profiles were also compared with those obtained from three age-matched children with PMM2-CDG (congenital disorder of glycosylation type la) and nine pediatric control samples. We have observed that severe N-glycan assembly defects correct in the neonate following dietary restriction of galactose. However, treated adult galactosemia patients continue to exhibit ongoing N-glycan processing defects. We have now applied informative galactose incorporation ratios as a method of studying the presence of N-glycan processing defects in children with galactosemia. We identified N-glycan processing defects present in galactosemia children from an early age. For G0/G1, G0/G2, and (G0/G1)/G2 ratios, the difference noted between galactosemia patients and controls was found to be statistically significant (p = 0.002, 0.01, and 0.006, respectively).

Alpha-1 antitrypsin (AAT) is the major physiological inhibitor of a range of serine proteases, and in the lung, it maintains a protease-antiprotease balance. AAT deficiency (AATD) is an autosomal co-dominant condition with the Z mutation being the most common cause. Individuals homozygous for Z (PiZZ) have low levels of circulating mutant Z-AAT protein leading to premature emphysematous lung disease. Extensive glycoanalysis has been performed on normal AAT (M-AAT) from healthy individuals and the importance of glycosylation in affecting the immune modulatory roles of AAT is documented. However, no glycoanalysis has been carried out on Z-AAT from deficient individuals to date. In this study, we investigate whether the glycans present on Z-AAT differ to those found on M-AAT from healthy controls. Plasma AAT was purified from 10 individuals: 5 AATD donors with the PiZZ phenotype and 5 PiMM healthy controls. Glycoanalysis was performed employing N-glycan release, exoglycosidase digestion and UPLC analysis. No difference in branched glycans was identified between AATD and healthy controls. However, a significant increase in both outer arm (α1-3) (p = 0.04) and core (α1-6) fucosylated glycans (p < 0.0001) was found on Z-AAT compared to M-AAT. This study has identified increased fucosylation on N-glycans of Z-AAT indicative of ongoing inflammation in AATD individuals with implications for early therapeutic intervention.

Atypical antipsychotic drugs, such as olanzapine, have been shown to alleviate the positive, negative and, to a lesser degree, the cognitive symptoms of schizophrenia in many patients. However, the detailed mechanisms of action of these drugs have yet to be elucidated. We have carried out the first investigation aimed at evaluating the effects of olanzapine treatment on the glycosylation of serum proteins in schizophrenia patients. Olanzapine treatment resulted in increased levels of a disialylated biantennary glycan and reduced levels of a number of disialylated bi- and triantennary glycans on whole serum glycoproteins. These changes were not observed on a low-abundance serum protein fraction. alpha 1 acid glycoprotein was identified as a carrier of some of the detected altered oligosaccharides. In addition, glycan analysis of haptoglobin, transferrin, and alpha 1 antitrypsin reported similar findings, although these changes did not reach significance. Exoglycosidase digestion analysis showed that olanzapine treatment increased galactosylation and sialylation of whole serum proteins, suggesting increased activity of specific galactosyltransferases and increased availability of galactose residues for sialylation. Taken together, these findings indicate that olanzapine treatment results in altered glycosylation of serum proteins.

Glycosylation is highly variable depending on many environmental factors. Using our fully quantitative high-throughput normal phase hydrophilic interaction liquid chromatography platform we have identified glycosylation changes associated with medication in the plasma N-glycome from three different population cohorts: ORCADES from the Orkney Islands in Scotland and CROATIA-Vis and CROATIA-Korcula from the Croatian islands of Vis and Korcula. Associations between glycosylation and the use of hormones (oral contraceptives, hormone replacement therapy), nonsteroidal anti-inflammatory drugs (aspirin and other NSAIDs), oral steroids (prednisolone) and steroid inhalers (beclomethasone) were investigated. Significant differences associated with usage of oral contraceptives were found with increased core-fucosylated biantennary glycans. Decreases in core-fucosylated biantennary glycans, core-fucosylated triantennary glycans with outer-arm fucose, and high mannosylated glycans were associated with the use of anti-inflammatory drugs. All of the changes in glycosylation were independent of blood group status. In conclusion, hormones and anti-inflammatory medication were associated with changes in glycosylation, possibly as a result of the modulatory effect of these drugs on the inflammatory response. In general, cancer is associated with inflammation, and many glycoproteins in the plasma are acute phase related to the host response. These preliminary data indicate the importance of correcting the levels of glycans used as biomarkers for the effects of medication.

Schizophrenia is a major neuropsychiatric disorder that affects 2% of the population worldwide. No biochemical diagnostic tests are available, and patients must undergo lengthy clinical evaluation periods before an accurate diagnosis can be given. Blood and cerebrospinal fluid are candidates for the identification of potential biomarkers for this disease. We have identified several N-glycans that distinguish first onset, unmedicated schizophrenia patients from healthy individuals. This is the first report of the N-glycome from low abundance serum proteins and cerebrospinal fluid. The tetraantennary tetrasialylated glycan with a polylactosamine extension, A4G4LacS4, from low abundance serum proteins showed a 2-fold increase in serum from male schizophrenia patients. Gender specificity was also demonstrated as the triantennary trisialylated glycan containing the SLex epitope was increased significantly in male schizophrenia patients on both high and low abundance serum proteins. Levels of bisecting and sialylated glycans in the cerebrospinal fluid showed a general down-regulation in schizophrenia patients and a 95% positive predictive power for distinguishing patients from controls. These changes are consistent with the reported down-regulation of beta-1,4-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyltransferase III and beta-galactoside alpha-2,3/6-sialyltransferases in the prefrontal cortex from schizophrenia patients. These alterations in the N-glycosylation signature could be used potentially for early diagnosis and monitoring of patients after treatment.