A theoretical estimate for nucleotide sugar demand towards Chinese Hamster Ovary cellular glycosylation

Glycosylation greatly influences the safety and efficacy of many of the highest-selling recombinant therapeutic proteins (rTPs). In order to define optimal cell culture feeding strategies that control rTP glycosylation, it is necessary to know how nucleotide sugars (NSs) are consumed towards host cell and rTP glycosylation. Here, we present a theoretical framework that integrates the reported glycoproteome of CHO cells, the number of N-linked and O-GalNAc glycosylation sites on individual host cell proteins (HCPs), and the carbohydrate content of CHO glycosphingolipids to estimate the demand of NSs towards CHO cell glycosylation. We have identified the most abundant N-linked and O-GalNAc CHO glycoproteins, obtained the weighted frequency of N-linked and O-GalNAc glycosites across the CHO cell proteome, and have derived stoichiometric coefficients for NS consumption towards CHO cell glycosylation. By combining the obtained stoichiometric coefficients with previously reported data for specific growth and productivity of CHO cells, we observe that the demand of NSs towards glycosylation is significant and, thus, is required to better understand the burden of glycosylation on cellular metabolism. The estimated demand of NSs towards CHO cell glycosylation can be used to rationally design feeding strategies that ensure optimal and consistent rTP glycosylation

Analysis of N-Glycosylation Sites in HIV glycoprotein 160

HIV infection is a condition caused by the human immunodeficiency virus. The condition gradually destroys the immune system, which makes it harder for the body to fight infections. HIV presents a complex knot for scientists to unravel. An envelope protein of the human HIV that is encoded by the env gene contains numerous glycosylation sites. It serves as a precursor for both the GP120 and the GP41. Here statistical investigation was done to study the sequential aspects of amino acids around the N-glycosylated protein from HIV virus. Sequences containing N-glycosylated asparagine were selected from the uniprot database of N-glycosylated proteins. The frequency of occurrence of amino acid residues around the glycosylated asparagine showed that there are increased numbers of isoleucine and threonine residues around the N-glycosylation sites in comparison with the nonglycosylated asparagine residues. Preferential occurrence
of amino acid residues around the glycosylation site shows that T has the maximum preference around the N-glycosylation site. T at 3 and/or -3 positions strongly favors glycosylation irrespective of other glycosylation sites. The data presented in the present work clearly indicate that there is a pronounced positional preference for the hydrophobic and neutral amino acids at various positions around the N-glycosylation site. In the future it will be of much interest to investigate further the possible structural and conformational implications of some
of these suggested positional preferences of the various amino acids around the site of glycosylation. This is a potentially important study, and such analyses will surely contribute an important part of our knowledge base in the future on HIV research. These results will be of interest to molecular biologists and protein engineers to identify N-glycosylation sites important in molecular recognition processes in HIV virus

Griffithsin Retains Anti-HIV-1 Potency with Changes in gp120 Glycosylation and Complements Broadly Neutralizing Antibodies PGT121 and PGT126.

Griffithsin (Grft) is an antiviral lectin that has been shown to potently inhibit HIV-1 by binding high-mannose N-linked glycosylation sites on HIV-1 gp120. A key factor for Grft potency is glycosylation at N295 of gp120, which is directly adjacent to N332, a target glycan for an entire class of broadly neutralizing antibodies (bNAbs). Here, we unify previous work on the importance of other glycans to Grft potency against HIV-1 and Grft's role in mediating the conformational change of gp120 by mutating nearly every glycosylation site in gp120. In addition to a significant loss of Grft activity by the removal of glycosylation at N295, glycan absence at N332 or N448 was found to have moderate effects on Grft potency. Interestingly, in the absence of N295, Grft effectiveness could be improved by a mutation that results in the glycan at N448 shifting to N446, indicating that the importance of individual glycans may be related to their effect on glycosylation density. Grft's ability to alter the structure of gp120, exposing the CD4 binding site, correlated with the presence of glycosylation at N295 only in clade B strains, not clade C strains. We further demonstrate that Grft can rescue the activity of the bNAbs PGT121 and PGT126 in the event of a loss or a shift of glycosylation at N332, where the bNAbs suffer a drastic loss of potency. Despite targeting the same region, Grft in combination with PGT121 and PGT126 produced additive effects. This indicates that Grft could be an important combinational therapeutic

Role of N-glycosylation in oral cancer

Oral squamous cell carcinoma represents more than 90% head and neck cancers with high incidence rate and morbidity. To date, little is known about the molecular mechanisms responsible for OSCC initiation and progression to advanced disease. Thus, identifying key pathways involved in OSCC pathobiology is likely to lead to the identification of new druggable targets and future anti-cancer therapies. Work from our laboratory has linked the metabolic pathway of protein N-glycosylation with OSCC biology. Specifically, overexpression of the first N-glycosylation gene, DPAGT1, in human OSCC tumor specimens was shown to be associated with aberrant activation of canonical Wnt signaling and inhibition of mature E-cadherin junctions. The purpose of this study was to examine how increased N-glycosylation was associated with OSCC growth and metastatic properties in cellular and murine models. We show that high level of DPAGT1 expression correlates with increased cell surface modification of malignant OSCC HSC-3 cells with complex N-glycans. Further, HSC-3 cells are hypersenitive to the N-glycosylation inhibitor, tunicamycin, suggesting that aggressive properties of OSCC cells depend, in part, on the N-glycosylation pathway. Lastly, we show that orthotopic HSC-3 cell-derived tumor xenografts are inhibited by tunicamycin both in overall growth and metastases, indicating that targeting DPAGT1 and N-glycosylation may represent a new strategy for the treatment of OSCC in human patients

Characterization of Cell Glycocalyx with Mass Spectrometry Methods.

The cell membrane plays an important role in protecting the cell from its extracellular environment. As such, extensive work has been devoted to studying its structure and function. Crucial intercellular processes, such as signal transduction and immune protection, are mediated by cell surface glycosylation, which is comprised of large biomolecules, including glycoproteins and glycosphingolipids. Because perturbations in glycosylation could result in dysfunction of cells and are related to diseases, the analysis of surface glycosylation is critical for understanding pathogenic mechanisms and can further lead to biomarker discovery. Different mass spectrometry-based techniques have been developed for glycan analysis, ranging from highly specific, targeted approaches to more comprehensive profiling studies. In this review, we summarized the work conducted for extensive analysis of cell membrane glycosylation, particularly those employing liquid chromatography with mass spectrometry (LC-MS) in combination with various sample preparation techniques

Diagnostic accuracy of urinary prostate protein glycosylation profiling in prostatitis diagnosis

Introduction: Although prostatitis is a common male urinary tract infection, clinical diagnosis of prostatitis is difficult. The developmental mechanism of prostatitis is not yet unraveled which led to the elaboration of various biomarkers. As changes in asparagine-linked-(N-)-glycosylation were observed between healthy volunteers (HV), patients with benign prostate hyperplasia and prostate cancer patients, a difference could exist in biochemical parameters and urinary N-glycosylation between HV and prostatitis patients. We therefore investigated if prostatic protein glycosylation could improve the diagnosis of prostatitis. Materials and methods: Differences in serum and urine biochemical markers and in total urine N-glycosylation profile of prostatic proteins were determined between HV (N = 66) and prostatitis patients (N = 36). Additionally, diagnostic accuracy of significant biochemical markers and changes in N-glycosylation was assessed. Results: Urinary white blood cell (WBC) count enabled discrimination of HV from prostatitis patients (P < 0.001). Urinary bacteria count allowed for discriminating prostatitis patients from HV (P < 0.001). Total amount of biantennary structures (urinary 2A/MA marker) was significantly lower in prostatitis patients compared to HV (P < 0.001). Combining the urinary 2A/MA marker and urinary WBC count resulted in an AUC of 0.79, 95% confidence interval (CI) = (0.70-0.89) which was significantly better than urinary WBC count (AUC = 0.70, 95% CI = [0.59-0.82], P = 0.042) as isolated test. Conclusions: We have demonstrated the diagnostic value of urinary N-glycosylation profiling, which shows great potential as biomarker for prostatitis. Further research is required to unravel the developmental course of prostatic inflammation

Glycosylation of Candida albicans cell wall proteins is critical for induction of innate immune responses and apoptosis of epithelial cells.

C. albicans is one of the most common fungal pathogen of humans, causing local and superficial mucosal infections in immunocompromised individuals. Given that the key structure mediating host-C. albicans interactions is the fungal cell wall, we aimed to identify features of the cell wall inducing epithelial responses and be associated with fungal pathogenesis. We demonstrate here the importance of cell wall protein glycosylation in epithelial immune activation with a predominant role for the highly branched N-glycosylation residues. Moreover, these glycan moieties induce growth arrest and apoptosis of epithelial cells. Using an in vitro model of oral candidosis we demonstrate, that apoptosis induction by C. albicans wild-type occurs in early stage of infection and strongly depends on intact cell wall protein glycosylation. These novel findings demonstrate that glycosylation of the C. albicans cell wall proteins appears essential for modulation of epithelial immunity and apoptosis induction, both of which may promote fungal pathogenesis in vivo

Mapping the complete glycoproteome of virion-derived HIV-1 gp120 provides insights into broadly neutralizing antibody binding

The surface envelope glycoprotein (SU) of Human immunodeficiency virus type 1 (HIV-1), gp120SU plays an essential role in virus binding to target CD4+ T-cells and is a major vaccine target. Gp120 has remarkably high levels of N-linked glycosylation and there is considerable evidence that this “glycan shield” can help protect the virus from antibody-mediated neutralization. In recent years, however, it has become clear that gp120 glycosylation can also be included in the targets of recognition by some of the most potent broadly neutralizing antibodies. Knowing the site-specific glycosylation of gp120 can facilitate the rational design of glycopeptide antigens for HIV vaccine development. While most prior studies have focused on glycan analysis of recombinant forms of gp120, here we report the first systematic glycosylation site analysis of gp120 derived from virions produced by infected T lymphoid cells and show that a single site is exclusively substituted with complex glycans. These results should help guide the design of vaccine immunogens

Structure analysis of biologically important prokaryotic glycopolymers

Of the many post-translational modifications organisms can undertake, glycosylation is the most prevalent and the most diverse. The research in this thesis focuses on the structural characterisation of glycosylation in two classes of glycopolymer (lipopolysaccharide (LPS) and glycoprotein) in two domains of life (bacteria and archaea). The common theme linking these subprojects is the development and application of high sensitivity analytical techniques, primarily mass spectrometry (MS), for studying prokaryotic glycosylation. Many prokaryotes produce glycan arrangements with extraordinary variety in composition and structure. A further challenge is posed by additional functionalities such as lipids whose characterisation is not always straightforward. Glycosylation in prokaryotes has a variety of different biological functions, including their important roles in the mediation of interactions between pathogens and hosts. Thus enhanced knowledge of bacterial glycosylation may be of therapeutic value, whilst a better understanding of archaeal protein glycosylation will provide further targets for industrial applications, as well as insight into this post- translational modification across evolution and protein processing under extreme conditions. The first sub-project focused on the S-layer glycoprotein of the halophilic archeaon Haloferax volcanii, which has been reported to be modified by both glycans and lipids. Glycoproteomic and associated MS technologies were employed to characterise the N- and O-linked glycosylation and to explore putative lipid modifications. Approximately 90% of the S-layer was mapped and N-glycans were identified at all the mapped consensus sites, decorated with a pentasaccharide consisting of two hexoses, two hexuronic acids and a methylated hexuronic acid. The O-glycans are homogeneously identified as a disaccharide consisting of galactose and glucose. Unexpectedly it was found that membrane-derived lipids were present in the S- layer samples despite extensive purification, calling into question the predicted presence of covalently linked lipid. The H. volcanii N-glycosylation is mediated by the products of the agl gene cluster and the functional characterisation of members of the agl gene cluster was investigated by MS analysis of agl-mutant strains of the S-layer. Burkholderia pseudomallei is the causative agent of melioidosis, a serious and often fatal disease in humans which is endemic in South-East Asia and other equatorial regions. Its LPS is vital for serum resistance and the O-antigen repeat structures are of interest as vaccine targets. B. pseudomallei is reported to produce several polysaccharides, amongst which the already characterised ‘typical’ O-antigen of K96243 represents 97% of the strains. The serologically distinct ‘atypical’ strain 576 produces a different LPS, whose characterisation is the subject of this research project. MS strategies coupled with various hydrolytic and chemical derivatisation methodologies were employed to define the composition and potential sequences of the O-antigen repeat unit. These MS strategies were complemented by a novel NMR technique involving embedding of the LPS into micelles. Taken together the MS and NMR data have revealed a highly unusual O-antigen structure for atypical LPS which is remarkably different from the typical O-antigen. The development of structural analysis tools in MS and NMR applicable to the illustrated types of glycosylation in these prokaryotes will give a more consistent approach to sugar characterisation and their modifications thus providing more informative results for pathogenicity and immunological studies as well as pathway comparisons.Open Acces