Exploring the role of glycosylation in vascular biology, immunology,and stem cell biology

Carbohydrates are important modulators in various biological and physiological processes. They are present ubiquitously and coat all cells, forming the glycocalyx of the cell. Glycosylation, a type of post-translational modification (PTM), is an enzymatic process of addition of carbohydrates to lipids and proteins leading to the formation of an abundant and diverse repertoire of glycoconjugates. These glycoconjugates participate in many key biological processes including cell adhesion, molecular trafficking and clearance, receptor activation, signal transduction, and immunomodulation. Carbohydrate-based or -modified therapeutics are becoming increasingly attractive due to their potential roles in angiogenesis and immunomodulation. However, their mechanisms of action in different biological models are not fully explored. In this thesis, three different studies were undertaken to investigate the influence of glycosylation and its importance in different biological cell systems. These studies were aimed at understanding the influence of glycosylation of osteopontin (OPN) in vascular biology, investigating a panel of poly- and oligo-saccharides as potential immunomodulators, and exploring the alteration of extracellular matrix (ECM) components produced in response to variable oxygen concentration in stem cell culture. In Chapter 2, OPN was investigated for its influence on angiogenic functions. OPN is a glycoprotein that upon pre- incubation with endothelial progenitor cells (EPCs) enhances the therapeutic benefit of EPCs after transplantation in a mouse model of hind limb ischemia. It is a pleiotropic molecule with diverse PTMs, including glycosylation and phosphorylation. It binds to a subset of integrin receptors activating intracellular pathways but the roles of PTMs in OPN towards its biological activities are not known. This study investigated the effect of PTMs on OPN for its functional activity using human umbilical vein endothelial cells (HUVECs) as a cell model system. OPN produced in various expression systems, including murine NS0 (rhOPN), bovine milk (bOPN), human breast milk (hOPN) and bacteria (eOPN), were digested with endo- and exo-glycosidases and characterised for their differential PTMs. The digestion studies confirmed the absence of N-linked oligosaccharides on OPN molecules, presence of varied degrees of O-linked oligosaccharides and absence of phosphorylation on all molecules, except bOPN and the absence of PTMs on eOPN. The functional activities of bOPN, rhOPN and their glycoforms along with eOPN were compared to a control, i.e HUVECs incubated without OPN. OPN prolonged the lifetime of tubules, secretion of angioenic factors and increased cell proliferation. It also led to increased activity of p-PI3K- AKT signaling pathway. However, no significant differences were reported between the glycoforms and phosphoforms of OPN, which suggested no significant roles for OPN glycosylation and phosphorylation in these biological functions. In Chapter 3, natural poly- and oligo-saccharides were assessed as potential immunomodulators. Natural polysaccharides offer relatively low toxicity, negligible side effects and ease of accessibility over conventional drugs for immunodulation. These test compounds were characterised for their molecular and physical properties. Upon incubation of human whole blood culture (HWBC) and a monocytic cell line, THP-1, with lipopolysaccharide (LPS) and six oligo- and poly-saccharides (inulin, galacturonan, heteroglycan and fucoidan, mannan and xyloglucan), the supernatant was screened for secreted cytokines and chemokines. TNF-α was measured by flow cytometry in HWBC cell sub-populations. Of the six oligo- and poly-saccharides, inulin, galacturonan, heteroglycan and fucoidan demonstrated pro-inflammatory properties in addition to LPS, while mannan and xyloglucan did not elicit any significant responses. Intracellular TNF-α expression was also increased in the monocytes of HWBC in response to inulin, galacturonan, heteroglycan and fucoidan. This study highlighted inulin and heteroglycan as potential immunomodulatory therapeutics and demonstrated HWBC to have a greater and more varied response in comparison to THP-1 cell, suggesting that HWBC may be a better model than single monocytic cell line to screen test compounds. In Chapter 4, the glycomic and proteomic constituents of mouse stromal cell MS-5 secreted ECM under normoxic and hypoxic conditions were analysed. MS-5 is a feeder cell line that produces ECM that acts as sticky basement for the survival and maintenance of undifferentiated state of hematopoietic stem cells grown above it. When cultured under hypoxic conditions, MS-5 cells improve transcription of pluripotency genes and certain genes involved in mesenchymal cell lineage commitment and their differentiation potential. However, the roles of ECM components in this beneficial effect are not known. To investigate differentially expressed ECM components produced under normoxia and hypoxia, ECM was prepared under normoxic (21% O2) and hypoxic (2% O2) conditions by feeder cells. In silico analyses led to the identification of proteins enriched in each condition and their associated biological functions. Of the identified proteins, 50% of the proteins were found to be ECM-related where they participate in ECM-receptor interactions, focal adhesion and leucocyte transendothelial migration pathway. Based on the abundance of glycosylation related proteins and glycosyltransferases from in silico analysis, a panel of lectins with appropriate specificities were selected for histochemistry of intact ECM in situ. Lectin blotting for the total proteome indicated increased sialylation, fucosylation and mannosylation in hypoxic proteins compared to normoxic. The identified proteins and glycosylation related proteins could be exploited in the development of biomaterials and bioactive scaffolds in the field of tissue engineering. In addition to the above studies, small projects are presented in appendices. In Appendix 1, human OPN was cloned, expressed and purified in bacterial system to obtain OPN devoid of any PTMs. In Appendix 2, the natural oligo- and polysaccharides presented in Chapter 3 were also tested for their angiogenic and antiangiogenic properties using HUVECs and angiogenesis related functional assays were performed. In Appendix 3, a statistical analysis for the lectin microarray profiling data of bovine milk fat globule membrane is presented. With an interdisciplinary approach, diverse roles of carbohydrates were explored using different biological cell models. The studies described in this thesis helped to clarify the roles of PTMs, natural polysaccharides and environmental conditions towards the vascular biology, immunology and stem cell biology. These studies also provided the future directions for further elucidation or study of these molecules towards exploring their therapeutic properties. This thesis hence, provided an opportunity to integrate the field of glycobiology, that was once studied on its own, into the field of therapeutics, immunology and stem biology and utilising the available tools in each field