Radka Saldova - Academia.edu (original) (raw)

Papers by Radka Saldova

Analytical Chemistry, Aug 20, 2020

N-glycan alterations in the nervous system can result in different neuropathological symptoms suc... more N-glycan alterations in the nervous system can result in different neuropathological symptoms such as mental retardation, seizures, and epilepsy. Studies have reported the characterization of N-glycans in rodent brains, but there is a lack of spatial resolution as either the tissue samples were homogenized or specific proteins were selected for analysis of glycosylation. We hypothesize that region-specific resolution of N-glycans isolated from the striatum and substantia nigra (SN) can give an insight into the establishment and pathophysiological degeneration of neural circuitry in Parkinson's disease. Specific objectives of the study include isolation of N-glycans from the rat striatum and SN; reproducibility, resolution, and relative quantitation of N-glycome using ultra-performance liquid chromatography (UPLC), weak anion exchange-UPLC, and lectin histochemistry. The total N-glycomes from the striatum and SN were characterized using database mining (GlycoStore), exoglycosidase digestions, and liquid chromatography-mass spectrometry. It revealed significant differences in complex and oligomannose type N-glycans, sialylation (mono-, di-, and tetra-), fucosylation (tri-, core, and outer arm), and galactosylation (di-, tri-, and tetra-) between striatum and SN N-glycans with the detection of phosphorylated N-glycans in SN which were not detected in the striatum. This study presents the most comprehensive comparative analysis of relative abundances of Nglycans in the striatum and SN of rodent brains, serving as a foundation for identifying "brain-type" glycans as biomarkers or therapeutic targets and their modulation in neurodegenerative disorders.

Background: Parkinson's Disease (PD) is a neurodegenerative disorder characterised by the dea... more Background: Parkinson's Disease (PD) is a neurodegenerative disorder characterised by the death of dopaminergic neurons in the substantia nigra. However, the molecular signature of PD is still not fully understood and there is a lack of studies regarding the protein glycosylation patterns in the brain and PD. Little is known on the role of N-glycans in the brain but many of the congenital glycosylation disorders result in neurological abnormalities suggesting their importance[1]. Given that glycans may behave as regulators of protein stability and that PD is associated with the aggregation of aberrant proteins, an understanding of how the glycosylation affects the disease makes it highly attractive to explore in order to comprehend their role in the regulation of PD. Aim: To optimise the isolation of N-glycans from the human striatum and nigra and quantify their relative proportions using ultra performance liquid chromatography (UPLC). Methods: Brain tissue was homogenised using RIPA buffer and the proteins were encapsulated in an acrylamide gel. After reduction and alkylation of the proteins, PNGAseF was used to release the N-glycans and 2-Aminobenzamide to label them. The labelled glycans were purified and analysed on UPLC. Results: Preliminary data shows reproducible chromatographic profiles of 60 peaks, containing one or more glycans, for both striatum and nigra. 13 peaks are significantly different between these regions. Most of the detected glycans in the striatum are neutral (78% both in healthy and PD), however this percentage differs in the nigra (85% in healthy vs 78% in PD). Conclusions: This study describes an UPLC-based method for N-glycan profiling of brain tissue glycoproteins with high reproducibility.

FASEB bioAdvances, Jun 8, 2023

Degeneration of the intervertebral disc is an age‐related condition. It also accompanies the disa... more Degeneration of the intervertebral disc is an age‐related condition. It also accompanies the disappearance of the notochordal cells, which are remnants of the developmental stages of the nucleus pulposus (NP). Molecular changes such as extracellular matrix catabolism, cellular phenotype, and glycosaminoglycan loss in the NP have been extensively studied. However, as one of the most significant co‐ and posttranslational modifications, glycosylation has been overlooked in cells in degeneration. Here, we aim to characterize the N‐glycome of young and mature NP and identify patterns related to aging. Accordingly, we isolated N‐glycans from notochordal cell‐rich NP from porcine discs, characterized them using a combined approach of exoglycosidase digestions and analysis with hydrophilic interaction ultra‐performance liquid chromatography and mass spectrometry. We have assigned over 300 individual N‐glycans for each age group. Moreover, we observed a notable abundance of antennary structures, galactosylation, fucosylation, and sialylation in both age groups. In addition, as indicated from our results, increasing outer arm fucosylation and decreasing α(2,3)‐linked sialylation with aging suggest that these traits are age‐dependent. Lastly, we have focused on an extensive characterization of the N‐glycome of the notochordal cell‐rich NP in aging without inferred degeneration, describing glycosylation changes specific for aging only. Our findings in combination with those of other studies, suggest that the degeneration of the NP does not involve identical processes as aging.

Animal Reproduction Science, Dec 1, 2022

The files are comma separated and contain measurements for 46 N-glycan peaks on 1581 serum sample... more The files are comma separated and contain measurements for 46 N-glycan peaks on 1581 serum samples (corresponding to 1565 unique participants) from SDRNT1BIO and from a pool of healthy controls. N-glycan peaks are expressed as absolute levels in ng/µL in volume of serum and as percentage areas (in this case, result files also contain a Man3 column). File 'sample_script.R' contains code to read these files, recompute percentage areas after exclusion of Man3, and derivation of 18 summary measures that aggregate the sums of peaks having features related to antennary structure, galactosylation, fucosylation, or sialylation.

Glycosylation, one of the most fundamental post-translational modifications, is altered in many c... more Glycosylation, one of the most fundamental post-translational modifications, is altered in many cancers. These alterations have been proven to impact on the progression of the tumor cell and promote survival. In literature published over the last half century, it is obvious there is a clear link between (a) chemo-resistance and hypoxia (b) hypoxia and epigenetics and more recently (c) glycosylation and epigenetics. We aim to bring these paradigms together with the remit of offering a more complete story and opening up new avenues of approach for the detection, diagnosis and treatment of breast and ovarian cancer. Ovarian and breast cancer cells were exposed to differential hypoxic conditions (0.5%, 1.0%, 2.0%) and normoxia. Firstly, we compared the methylation status of hypoxia exposed cells to the normoxic controls. A combination of hydrophilic interaction liquid chromatography (HILIC) and statistical analyses allowed comparisons of the secreted N-glycans from cells exposed to differential hypoxic conditions. Western blots assessed apoptosis, senescence, autophagy and epithelial to mesenchymal transition. The OrisTM migration assay assessed the migration of the cells pre- and post-differential hypoxic exposure. RT-qPCR was used to measure gene expression of appropriate glycosyltransferases and possible transcription factors (TFs) in these samples to determine any associations with changes seen in N-glycosylation. There were non-significant trends observed between the percentage oxygen that the cells were exposed to and resultant changes seen in branching and sialylation on secreted N-glycans from the breast and ovarian cancer cell lines. While only some of these changes could be explained by RT-qPCR data, GATA2/3 transcripts, identified in-silico as possible TFs, have been shown to significantly correlate with ST3GAL4 and MGAT5 glycosyltransferases, respectively. Hypoxia exposed cells also displayed increased migration with a greater effect seen in the 0.5% hypoxia exposed samples. The GATA2 and GATA3 transcription factors are gaining popularity in recent years, with links to cancer stage, increased invasiveness and as possible therapeutic targets. Our recent data shows a strong correlation between GATA2 and GATA3 and the levels of glycosyltransferases involved in branching and sialyation. These glycan changes are known to be strongly involved in cancer cell survival and metastases. Here for the first time we may have a possible mechanism of action linking GATA2 and 3 and invasiveness of breast and ovarian cancer cells. Citation Format: Gordon Greville, Esther Llop, Rosa Peracaula Miró, Amanda McCann, Pauline M. Rudd, Radka Saldova. Hypoxia regulates tumor cell invasiveness through altered glycosylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2423.

Scientific Reports, Jun 28, 2023

Endometriosis is a chronic inflammatory gynaecological disease characterized by the growth of end... more Endometriosis is a chronic inflammatory gynaecological disease characterized by the growth of endometrial tissue outside the uterine cavity. There are currently no definitive non-invasive diagnostic tools. Glycosylation is the most common posttranslational modification of proteins and altered glycosylation has been found in many diseases, including chronic inflammatory conditions and cancer. Sialylation and galactosylation on serum IgG have previously been found to be altered in endometriosis and serum sialylation changed after Zoladex (Goserelin Acetate) therapy. Using IgG and whole serum glycoproteins, we investigated N-glycosylation in two clinical cohorts of women with and without endometriosis. PNGase F-digested serum samples were fluorescently labelled and N-glycans were profiled by ultra-performance liquid chromatography. Clinical data was collected to link glycomic findings with metabolic and hormonal profiles. Total serum glycoprotein and IgG glycosylation differed in patients with endometriosis compared to control cases. The most significantly altered was glycan peak 3 from IgG, containing bisected biantennary glycans, which was decreased in the endometriosis cohorts (p = 0.0000005-0.018). In conclusion, this is the first pilot study to identify changes in N-glycans from whole serum glycoproteins associated with endometriosis. A larger validation study is now warranted and such studies should include the follow-up of surgically and pharmacologically treated patients. Endometriosis (EMS) is a chronic inflammatory gynaecological disease of unknown aetiology characterized by the growth of endometrial tissue outside the uterine cavity 1-5. Affecting 6-10% of women of reproductive age, EMS is a leading cause of female infertility as well as causing significant dysmenorrhoea and pelvic pain 1,4. It has also been associated with increased risks of cancer and autoimmune diseases 1,2,4. The aetiology of EMS is multifactorial and implicated factors include genetic predisposition, prenatal exposure to endocrine-disrupting chemicals and alterations in the microbiome, the immune system and sex hormones 4,6. Laparoscopy is the gold standard for definitive EMS diagnosis 1. There are no comprehensive non-invasive diagnostic tools currently available 7 , although ovarian endometriomas and deep nodular forms of the disease can be detected by ultrasonography and MRI 3. Treatment depends on surgical intervention and hormone therapy and recurrence rates are high 1. Early recognition and treatment of EMS improves patient fertility, dysmenorrhoea and pelvic pain, thereby improving quality of life 1. Furthermore, EMS represents a significant public health problem due to its effect on the quality of life of women 5. This in turn causes a substantial economic burden with EMS-related

bioRxiv (Cold Spring Harbor Laboratory), May 20, 2022

Parkinson's Disease (PD) associated state of neuroinflammation due to the aggregation of aberrant... more Parkinson's Disease (PD) associated state of neuroinflammation due to the aggregation of aberrant proteins is widely reported. One type of post-translational modification involved in protein stability is glycosylation. Here, we aimed to spatially characterise the human Parkinsonian nigro-striatal Nglycome, and related transcriptome/proteome, and its correlation with endoplasmic reticulum stress and unfolded protein response (UPR), providing a comprehensive characterisation of the PD molecular signature. Significant changes were seen upon PD: 3% increase in sialylation and 5% increase in fucosylation in both regions, and 2% increase in oligomannosylated N-glycans in the substantia nigra. In the latter, a decrease in the mRNA expression of sialidases and an upregulation in the UPR pathway were also seen. This complete characterisation of the human nigro-striatal N-glycome provides an insight into the glycomic profile of PD through a transversal approach while combining the other PD "omics" pieces, which can potentially assist in the development of glyco-focused therapeutics.

Journal of Proteome Research, Jun 30, 2021

O-Glycosylation changes in misfolded proteins are of particular interest in understanding neurode... more 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.

Biology of Reproduction, Apr 26, 2022

Sialic acid occupies terminal positions on O-glycans of cervical mucins, where they contribute to... more Sialic acid occupies terminal positions on O-glycans of cervical mucins, where they contribute to the increased viscosity of mucin thereby regulating sperm transport. This study characterized the sialylated cervical mucins from follicular phase mucus of six European ewe breeds with known differences in pregnancy rates following cervical artificial insemination (AI) using frozen-thawed semen at both synchronized and natural estrus cycles. These were Suffolk (low fertility) and Belclare (medium fertility) in Ireland, Ile de France and Romanov (both with medium fertility) in France, and Norwegian White Sheep (NWS) and Fur (both with high fertility) in Norway. Expression of mucin and sialic acid related genes was quantified using RNA-sequencing in cervical tissue from Suffolk, Belclare, Fur, and NWS only. Cervical tissue was also assessed for the percentage of cervical epithelial populated by mucin secreting goblet cells in the same four ewe breeds. Biochemical analysis showed that there was an effect of ewe breed on sialic acid species, which was represented by Suffolk having higher levels of Neu5,9Ac 2 compared with NWS (P < 0.05). Suffolk ewes had a lower percentage of goblet cells than Fur and NWS (P < 0.05). Gene expression analysis identified higher expression of MUC5AC, MUC5B, ST6GAL1, and ST6GAL2 and lower expression of ST3GAL3, ST3GAL4, and SIGLEC10 in Suffolk compared with high fertility ewe breeds (P < 0.05). Our results indicate that specific alterations in sialylated mucin composition may be related to impaired cervical sperm transport.

Glycobiology, Nov 27, 2020

We investigated associations of quantitative levels of N-glycans with hemoglobin A 1c (HbA 1c), r... more We investigated associations of quantitative levels of N-glycans with hemoglobin A 1c (HbA 1c), renal function and renal function decline in type 1 diabetes. We measured 46 total N-glycan peaks (GPs) on 1565 serum samples from the Scottish Diabetes Research Network Type 1 Bioresource Study (SDRNT1BIO) and a pool of healthy donors. Quantitation of absolute abundance of each GP used 2AB-labeled mannose-3 as a standard. We studied cross-sectional associations of GPs and derived measures with HbA 1c , albumin/creatinine ratio (ACR) and estimated glomerular filtration rate (eGFR), and prospective associations with incident albuminuria and final eGFR. All GPs were 1.4 to 3.2 times more abundant in SDRTN1BIO than in the healthy samples. Absolute levels of all GPs were slightly higher with higher HbA 1c , with strongest associations for triantennary trigalactosylated disialylated, triantennary trigalactosylated trisialylated structures with core or outer arm fucose, and tetraantennary tetragalactosylated trisialylated glycans. Most GPs showed increased abundance with worsening ACR. Lower eGFR was associated with higher absolute GP levels, most significantly with biantennary digalactosylated disialylated glycans with and without bisect, triantennary trigalactosylated trisialylated glycans with and without outer arm fucose, and core fucosylated biantennary monogalactosylated monosialylated glycans. Although several GPs

Carbohydrate chemistry, 2011

Considering that glycans are ubiquitously present on all cell surfaces, the fact that several hum... more Considering that glycans are ubiquitously present on all cell surfaces, the fact that several human diseases involve disturbed glycan processing pathways comes as no surprise to the emerging field of Glycobiology. Alterations in cellular signalling pathways (either caused by disease specific mutatio...

Springer eBooks, 2021

Therapeutic monoclonal antibodies (mAbs) are mostly of the IgG class and constitute highly effica... more Therapeutic monoclonal antibodies (mAbs) are mostly of the IgG class and constitute highly efficacious biopharmaceuticals for a wide range of clinical indications. Full-length IgG mAbs are large proteins that are subject to multiple posttranslational modifications (PTMs) during biosynthesis, purification, or storage, resulting in micro-heterogeneity. The production of recombinant mAbs in nonhuman cell lines may result in loss of structural fidelity and the generation of variants having altered stability, biological activities, and/or immunogenic potential. Additionally, even fully human therapeutic mAbs are of unique specificity, by design, and, consequently, of unique structure; therefore, structural elements may be recognized as non-self by individuals within an outbred human population to provoke an anti-therapeutic/anti-drug antibody (ATA/ADA) response. Consequently, regulatory authorities require that the structure of a potential mAb drug product is comprehensively characterized employing state-of-the-art orthogonal analytical technologies; the PTM profile may define a set of critical quality attributes (CQAs) for the drug product that must be maintained, employing quality by design parameters, throughout the lifetime of the drug. Glycosylation of IgG-Fc, at Asn297 on each heavy chain, is an established CQA since its presence and fine structure can have a profound impact on efficacy and safety. The glycoform profile of serum-derived IgG is highly heterogeneous while mAbs produced in mammalian cells in vitro is less heterogeneous and can be "orchestrated" depending on the cell line employed and the culture conditions adopted. Thus, the gross structure and PTM profile of a given mAb, established for the drug substance gaining regulatory approval, have to be maintained for the lifespan of the drug. This review outlines our current understanding of common PTMs detected in mAbs and endogenous IgG and the relationship between a variant's structural attribute and its impact on clinical performance.

Nature Communications, May 25, 2023

The establishment of the gut microbiota in the perinatal period is a key event in early life. The... more The establishment of the gut microbiota in the perinatal period is a key event in early life. The microbes that colonise the infant's digestive tract are, among other functions, essential for educating the immune system 1-3. Perturbations in the gut microbiota composition in early life, for example as a result of antibiotic treatment, are also associated with gastrointestinal infection, atopic disease and obesity 4,5. Concurrent with birth, the neonatal gut becomes colonised by socalled founder species, many of which are representatives of the genera Escherichia, Enterococcus, and Lactobacillus (more recently reassigned as several distinct genera). This facilitates subsequent colonisation by strict anaerobes such as members of the Bifidobacterium and Bacteroides genera, which frequently dominate a healthy infant gut within 1-4 weeks 6-10. Vertical transmission of bacteria, i.e., from mother to infant, is assumed to be key in the establishment of the infant gut microbiome 11,12. Shotgun metagenomic analyses can infer strain transmission, either by aligning species-specific marker genes 7,8,13,14 or via alignment of metagenomic reads to reference genomes 15. These methods have indicated that 50-80% of all species in the infant gut at 4-months are shared with the maternal microbiome. However, strain transmission rates vary between species 7,8 , with

Journal of Proteome Research

Traumatic spinal cord injury (SCI) results in disruption of tissue integrity and loss of function... more 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 Xenopus laevis and rat. Transected rats received an aligned collagen hydrogel. The response compared regenerative success, regenerative failure, and treatment in an established nonregenerative mammalian system. In a healthy rat spinal cord, ultraperformance liquid chromatography (UPLC) Nglycoprofiling identified complex, hybrid, and oligomannose N-glycans. Following rat SCI, complex and outer-arm fucosylated glycans decreased while oligomannose and hybrid structures increased. Sialic acid was associated with microglia/macrophages following SCI. Treatment with aligned collagen hydrogel had a minimal effect on the glycosylation response. In Xenopus, lectin histochemistry revealed increased levels of N-acetyl-glucosamine (GlcNAc) in premetamorphic animals. The addition of GlcNAc is required for processing complex-type glycans and is a necessary foundation for additional branching. A large increase in sialic acid was observed in nonregenerative animals. This work suggests that glycosylation may influence regenerative success. In particular, loss of complex glycans in rat spinal cord may contribute to regeneration failure. Targeting the glycosylation response may be a promising strategy for future therapies.

Diabetes Research and Clinical Practice

Annals of Nutrition and Metabolism, 2022

Introduction: Metabolic or inflammatory markers may predict adverse outcomes in women with obesit... more Introduction: Metabolic or inflammatory markers may predict adverse outcomes in women with obesity. We sought to describe metabolic-obesity phenotypes of women using novel staging tools and investigate relationships with inflammation. Methods: In a cross-sectional study, we collected fasting blood samples from sixty-four females with body mass index (BMI) ≥28 kg/m2. Participants were classified as metabolically healthy or metabolically unhealthy obesity (MUO) using the cardiometabolic disease staging system (CMDS) and Edmonton obesity staging system (EOSS). Data were analyzed using independent sample t tests, Pearson’s correlations, and multiple logistic regression. Results: Mean (SD) age was 40.2 (9.3) years with median (IQR) BMI 31.8 (30.3–35.7) kg/m2. The prevalence of MUO was 46.9% and 81.3% using CMDS and EOSS criteria, respectively. Women with raised CMDS scores had higher C3 (1.34 [0.20] vs. 1.18 [0.15], p = 0.001) and C-reactive protein (CRP) (2.89 [1.31–7.61] vs. 1.39 [0.74...

As more therapeutic options become available there is an increasing need for clinical markers tha... more As more therapeutic options become available there is an increasing need for clinical markers that will provide more sensitive and specific early detection of disease. At the same time, improved technologies for monitoring disease progression and response to therapy are required. In many cases, single assays of existing biomarkers are neither sensitive nor specific enough for use as sole screening methods and in general a combination of markers is required. Many systemic diseases, particularly cancer, have been linked with many systems, from genomics to glycomics. Therefore we have developed an automated 96-well plate based strategy for identifying, quantifying and screening potential glycans released from proteins in body fluids as clinical markers. We have constructed a data base of the serum glycome of healthy controls to compare with that of clinical controls and of patients with various diseases including schizophrenia, rheumatoid arthritis, breast, ovarian, lung, stomach, pros...