Expression of the extracellular domain of endoglin in mammalian cells and bacteria

Class I histocompatibility molecules, consisting of a heavy chain, a light chain (ß2-microglobulin) and peptide, are assembled in the endoplasmic reticulum before being exported to the Golgi apparatus and the cell surface where they are surveyed by cytotoxic T cells. Assembly of the heavy/light chain heterodimer with peptide occurs in the peptide loading complex, which consists of the transporter associated with antigen processing, tapasin, ERp57, calnexin and calreticulin. In the first part of this thesis, I have used a mutational approach to investigate the physical organization of the peptide loading complex with a particular emphasis on tapasin. Several residues were identified to be involved in the interaction between H-2 D d and tapasin and mutations at these positions resulted in phenotypes of low surface expression, altered endoplasmic reticulum to Golgi transport and mildly affected peptide loading. These complex observations, analyzed in the context of similar studies performed with HLA-A2 and H-2 Ld suggest that the organization of the peptide loading complex can vary depending on the specific class I molecule examined. In the second part of this thesis, I investigated later events in the biogenesis of class I molecules, namely their export from the endoplasmic reticulum to the Golgi apparatus. I found that the putative cargo receptor Bap31 binds to two allotypes of mouse class I molecules with the interaction initiated at the time of heavy chain association with ß2-microglobulin and maintained until the class I molecule has left the endoplasmic reticulm. Consistent with an important role in recruiting class I molecules to transport vesicles, I showed that in the absence of Bap31 and its binding partner Bap29, there is a loss of class I colocalization with p137, a component of mammalian COPII coats. This observation is also associated with a delay in class I traffic from the endoplasmic reticulum to the Golgi. These observations are consistent with the view that class I molecules are largely recruited to endoplasmic reticulum exit sites by Bap29/31 and that Bap29/31 is a cargo receptor for major histocompatibility complex class I molecules., Endoglin is a homodimeric membrane glycoprotein predominantly expressed on endothelial cells. Cloning and sequencing of betaglycan, the transforming growth factor (TGF)-$\beta$ receptor III, had revealed homology with endoglin, particularly in the cytoplasmic domain. These observations led to the identification of endoglin as component of the TGF-$\beta$ receptor complex. Although betaglycan can be released from the plasma membrane by enzymatic cleavage, no soluble form of endoglin has been identified. The purpose of the current study was to produce a recombinant soluble form of endoglin to be used in functional and structural studies. We engineered several constructs of endoglin, including natural variants, and expressed them in COS-1 cells. Although all fragments were detected intracellularly, only $\Delta$586, corresponding to the complete extracellular domain was secreted. Our results demonstrate that a recombinant soluble form of endoglin can be secreted in a stable and dimeric form by COS-1 cells. (Abstract shortened by UMI.)