Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal dominant genetic disorder that results from a mutation of one of two key signaling receptors for the transforming growth factor beta (TGFβ) superfamily: endoglin and activin receptor-like kinase 1 (ALK1). These mutations result in development of HHT Type 1 and HHT Type 2, respectively. Patients suffering from HHT experience spontaneous blood vessel growth that can lead to telangiectasia, arteriovenous malformation (AVM) development, and other related health problems.
ALK1 is a serine/threonine kinase receptor found on the cell membrane of endothelial cells. ALK1 and its co-receptor endoglin, are activated by binding to the circulating BMP9 ligand. The ALK1-endoglin-BMP9 complex will then regulate endothelial proliferation by activating the SMAD pathway by phosphorylation. Mutations in the ACVRL1 gene can form a modified ALK1 protein that has a high potential to inhibit this function, causing the hyperproliferation of endothelial cells and the development of AVMs, and ultimately HHT Type 2.
It is believed, however unproven, that ALK1 is heavily glycosylated in the extracellular domain. My thesis research was aimed at studying the glycosylation of ALK1 and at exploring the relevance of this glycosylation to the development of HHT. The glycosylation of ALK1 was investigated by using: (i) a computational prediction approach (NetNGlyc 1.0 bioinformatics server), (ii) a glycosylation inhibiting drug (tunicamycin), (iii) an in vitro enzymatic approach of glycosylation breakdown, and (iv) site-directed mutagenesis to identify the ASP residue glycosylated on ALK1.
The bioinformatics software NetNGlyc predicted a N-linked glycosylation site on an asparagine (ASN) residue located at position 98 in the extracellular domain of ALK1. I further found that, based on western blot analysis, ALK1 proteins shifted to a lighter molecular weight (5-8 kDa) when treated with tunicamycin, as well as endo H and PNGase F enzymes, which represent two glycosidases able to remove N-linked oligosaccharides on proteins. Western blot analysis also revealed an identical shift in protein size (5-8 kDa) when comparing wild type ALK1 to an asparagine98-to-alanine (N98A) mutant ALK1 construct.
The 5-8 kDa shift observed in the drug and enzymatic experiments indicate the removal of a bulky oligosaccharide from the wild type ALK1 protein. This 5-8 kDa shift observed in the mutagenesis experiment indicated that the same oligosaccharide addition could not occur on ALK1 when ASP98 was missing. Thus proving that the asparagine at the 98th position of ALK1 is involved in N-linked glycosylation. These important findings on ALK1 modification offer a greater understanding of the mechanisms behind ALK1 regulation and function, especially its role in controlling angiogenesis. Furthermore, this data provides grounds for further research into the importance of ALK1 glycosylation in the pathogenesis of HHT, as well as the investigation into new treatment regiments.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/17018 |
| Date | 18 June 2016 |
| Creators | Gadaleta, Erick Michael |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
Page generated in 0.002 seconds