Objective
The overall goal of this project is the identification of proteins selectively expressed on the surface (surfaceome) of β-cell and non-β-cell populations of the islet (including, but not limited to, α-cells, δ-cells, islet endothelial cells, and passenger leukocytes). This information will be essential for the development of affinity reagents that are design to for the purification of primary human b-cells for functional studies. The development of tools for targeted drug delivery to the b-cells. Development of ligands or multivalent ligands designed to target and enhance b-cell function and viability.
Background Rationale
Insulin-dependent or type 1 diabetes (T1D) is an autoimmune disease characterized by the selective destruction of insulin producing β-cells that occurs during an inflammatory reaction in and around pancreatic islets. The progression to T1D is characterized by an asymptomatic period where functional β-cell mass is lost in the absence of phenotypic expression of the disease. While inflammatory mediators released during islet inflammation likely contribute to disease development by impairing the function and reducing the viability of β-cells, the mechanisms contributing to the loss of β-cells during this asymptomatic period are poorly defined. The realization that cell-cell interactions are necessary for proper β-cell function underscores the importance of knowing the identity of cell surface glycoproteins and the glycan structures on these surface proteins may participate in autoimmunity. Currently a comprehensive view of the cell surface glycoproteome and glycome of human islet cell types at a depth necessary to inform molecular mechanisms of disease is lacking; however, we believe that understanding this landscape will identify novel targets for therapeutic payload delivery as well as biomarkers of disease. The overall goal of this study is to define the cell surface glycoproteome and cellular glycome of human β-cells and non-β-cells.
Description of Project
Insulin-dependent or type 1 diabetes (T1D) is an autoimmune disease characterized by the selective destruction of insulin producing β-cells that occurs during an inflammatory reaction in and around pancreatic islets. The progression to T1D is characterized by an asymptomatic period where functional β-cell mass is lost in the absence of phenotypic expression of the disease. While inflammatory mediators released during islet inflammation likely contribute to disease development by impairing the function and reducing the viability of β-cells, the mechanisms contributing to the loss of β-cells during this asymptomatic period are poorly defined. The realization that cell-cell interactions are necessary for proper β-cell function underscores the importance of knowing the identity of cell surface glycoproteins and the glycan structures on these surface proteins may participate in autoimmunity. Currently a comprehensive view of the cell surface glycoproteome and glycome of human islet cell types at a depth necessary to inform molecular mechanisms of disease is lacking; however, we believe that understanding this landscape will identify novel targets for therapeutic payload delivery as well as biomarkers of disease. The overall goal of this study is to define the cell surface glycoproteome and cellular glycome of human β-cells and non-β-cells.
Using innovative mass spectrometry (MS) approaches developed in our laboratory, we have recently generated the first views of the cell surface glycoproteome and glycome of islet cells. Using Microscale Cell Surface Capture (μCSC) technology, we have experimentally defined the cell surface glycoproteome of primary rat β-cells and non-β-cells. This catalog includes 378 proteins, many of which have not been previously described in β-cells. Using glyPAQ technology, we have performed the first-ever structural glycomics analysis of human islets, revealing 496 distinct glycan structures. The glycomics data provide the first clues to the key glycosylation machinery in the islet and complements the cell surface glycoproteome data to inform which surface accessible molecules may be involved in immune-mediated destruction or could be exploited to develop remote sensing biomarkers of disease and therapeutic alpha cell payload delivery targets. Our proposed studies will apply these technologies to experimentally identify and quantify accessible epitopes on the cell surface and provide detailed characterization glycan structures. Both approaches are proven applicable to very small numbers of primary human cells.
Anticipated Outcome
Define the cell surface glycoproteome on human β-cells and non β-cells
Define the extracellular domains of proteins found on the cell surface can be shed during disease development and could potentially serve as valuable biomarkers for active β-cell killing during the asymptomatic period of disease progression.
Development of reagents that allow for a positive purification of naïve human β-cells without genetic manipulation.
Define the glycome in the islet and islet cell types can be exploited to fully define cell surface glycoproteins (which structures are on which proteins)
Develop a comprehensive surfaceome maps of primary human cells will facilitate benchmarking of transdifferentiation-derived and stem cell-derived islet subtypes
Relevance to T1D
Insulin-dependent or type 1 diabetes (T1D) is an autoimmune disease characterized by the selective destruction of insulin producing β-cells that occurs during an inflammatory reaction in and around pancreatic islets. The progression to T1D is characterized by an asymptomatic period where functional β-cell mass is lost in the absence of phenotypic expression of the disease. While inflammatory mediators released during islet inflammation likely contribute to disease development by impairing the function and reducing the viability of β-cells, the mechanisms contributing to the loss of β-cells during this asymptomatic period are poorly defined. The realization that cell-cell interactions are necessary for proper β-cell function underscores the importance of knowing the identity of cell surface glycoproteins and the glycan structures on these surface proteins may participate in autoimmunity. Currently a comprehensive view of the cell surface glycoproteome and glycome of human islet cell types at a depth necessary to inform molecular mechanisms of disease is lacking; however, we believe that understanding this landscape will identify novel targets for therapeutic payload delivery as well as biomarkers of disease. The overall goal of this study is to define the cell surface glycoproteome and cellular glycome of human β-cells and non-β-cells.