A novel regulatory mechanism of the BMP signaling pathway Jose Maria Amich Manero under the direction of Prof. Vicki Rosen Dr. Jonathan Lowery Harvard School of Dental Medicine Research Science Institute July 30, 2013 Abstract The BMP signaling pathway is a pivotal morphogenetic signal involved in a wide spectrum of cellular processes. The fact that the number of ligands far exceeds the number of receptors, and how a limited canonical pathway can accomplish pleiotropic effects demonstrate that the regulation of this pathway is, at present, poorly understood. In this study, we propose N-linked glycosylation as a specific regulatory mechanism of the BMP type 2 receptors (ACVR2A, ACVR2B and BMPR2). Computational screening for glycosylated asparagine residues in BMPR2 reveals three putative sites, which we show to be glycosylated by means of site-directed mutagenesis. Furthermore, we demonstrate that BMPR2 glycosylation is essential for ligand binding but that glycosylation of ACVR2A prevents binding. Collectively, our findings provide the first mechanistic insight into the regulation of the BMP signaling pathway through glycosylation of BMP type 2 receptors. Summary Numerous organismic processes such as embryonic development and bone growth are controlled by a cell regulatory mechanism known as the bone morphogenetic protein (BMP) pathway. This pathway is activated when a signaling protein binds to the membrane receptor, transmitting in turn an order to the nucleus. In an attempt to shed light on BMP pathway activation, we focused on receptor-bound sugar chains in view of their protein-specific signa- ture. To study the role of individual sugar chains, we systematically blocked their function until we were able to pinpoint three key sites. We also assessed if these sugars affected the receptor's ligand-binding ability, and found that they promoted binding to the receptor in some cases and prevented binding in others. Thus, our findings provide the first mechanistic explanation for the BMP pathway regulation at a receptor-specific level. 1 Introduction 1.1 The TGF-β superfamily The bone morphogenetic protein (BMP) signaling pathway plays a key role in the regulation of many cellular processes, such as embryo and cell growth, differentiation and apoptosis [1]. The BMP pathway has been observed to be necessary for the maintenance of vascular and bone homeostasis, leading to severe disease when mutations occur in members of the pathway [2]. The BMP ligands belong to the TGF-β superfamily, featuring many other proteins such as growth and differentiation factors (GDFs) that regulate cartilage and skeletal development [3], Activins (Acts) and inhibins (Inhs) that regulate pituitary hormone secretion [4], the M¨ullerianinhibiting substance (MIS) that determines sex during embryonic development [5] and bone morphogenetic proteins (BMPs) that regulate vertebrate development [6]. Receptor extracellular ligand binding is necessary for the activation of the BMP signaling pathway. Mechanistically, the initial activation of the pathway is achieved via the binding of transforming growth factor β-like (TGF-β-like) ligands to the Activin/TGF-β and BMP receptors, leading to cellular response through the regulation of target genes. 1.2 Structure and function of BMPs BMPs have recently been under extensive study due to their implications in human disease. BMPs were originally described by Urist in 1965 [7], who observed that extracts of this protein had the ability to induce osteogenesis after intramuscular implantation. Since then, BMPs have been reported to contribute to a wide range of processes, such as cartilage development, osteogenesis and oocyte development [8]. BMPs bind to the extracellular domain (ECD) of the type II receptor (BMPR2, ACVR2A and ACVR2B) [9], which dimerizes with another receptor of its type. The dimeric type II 1 Figure 1: The activation of the BMP signaling pathway occurs when a BMP ligand triggers dimerization of type 1 (ALK2, 3, 6) and type 2 (BMPR2, ACVR2A, ACVR2B) receptors, leading to phosphorylation of SMAD1/5/8 and translocating to the nucleus together with SMAD4 for transcription of genes containing the SMAD binding elements. receptors will phosphorylate the GS box of the type I receptors (ALK2, ALK3 and ALK6) [9], inducing the type I receptor to phosphorylate the C-terminus of several receptor-activated (RA) SMAD proteins (SMAD1, SMAD5 and SMAD8) [10]. The RA-SMAD complex will then bind to SMAD4 and translocate as a RA-SMAD/SMAD4 complex to the nucleus, facilitating the transcription of genes by binding to SMAD binding elements (SBEs) (Figure 1) [11]. The number of characterized BMP ligands outweighs the number of known receptors, giving rise to a competitive and synergistic receptor activation network. Competition for the bone morphogenetic protein 2 (BMP2) has been identified as a potent regulator of the canon- ical SMAD pathway (SMAD1/5/8) [12]. Several studies have reported that affinity of BMP2 to type 2 receptors ACVR2B and ACVR2A is higher than to BMPR2 [13, 14]. As ACVR2A and ACVR2B also bind to Activin, eliciting the receptor-activated SMAD2/3 canonical 2 pathway, which is often opposite in effect to SMAD1/5/8, it is possible that BMPR2 has a regulatory role in SMAD signaling based on BMP and Activin competition. However, many of these studies have been performed using bacterially-expressed proteins, which might lead to artifactual differences based on altered posttranslational modifications. One such possible modification is N-linked glycosylation, which does not occur in bacteria. 1.3 The role of glycosylation in BMP binding kinetics Glycosylation is a protein posttranslational modification based on the addition of polysac- charides to key residues, playing a role in protein structure and function [15]. The importance of glycosylation in determining a protein's properties has been shown to be critical in a num- ber of cases, such as hormones and cytokines [15]. In the case of secreted signaling proteins, glycosylation can alter their conformation and binding kinetics, and thus control pathway activation [15]. N-linked glycolysation occurs when a monosaccharide, N-acetylglucosamine, attaches to a nitrogen molecule in an asparagine residue. In eukaryotes, glycosylation takes place in the endoplasmic reticulum (ER) lumen, for a further cleavage to the cellular membrane [16]. While N-linked glycosylation has been well characterized in a number of proteins, its effect on the type 2 BMP receptor is still to be clarified. Kang et al [17] showed that a non- N-glycosylated pro-VEGF region significantly reduced VEGF secretion in Saccharomyces cerevisiae, demonstrating that N-linked glycosylation has a bearing in protein stability. In another study, Zheng et al [18] found that thermal stability and susceptibility to degradation by papain were dependent on glycosylation status, although protein secondary and tertiary structure were unchanged. 3 1.4 Research approach In this study, we aim to explore the structural and functional repercussions of aberrant or null BMP type 2 receptor glycosylation in BMP2 binding. Using several BMPR2 models containing single, double and triple substitutions in glycosylated asparagine residues, we assess BMP2 binding to BMPR2, as well as to the other type II receptors (ACVR2A and ACVR2B) to further understand the specific role of glycosylation at the individual receptor level. We hope our findings will shed light on BMP type 2 receptor function, enabling a clearer understanding of their complex and highly specific regulatory mechanism. 2 Methods 2.1 hBMPR2 plasmid constructions Prior to the start of RSI, several plasmids were generated in order to study the effects of site- directed mutagenesis on hBMPR2 function. A shuttle plasmid encoding the human BMPR2 was subcloned into the carbenicillin-resistant pcDNA3.1/V5-His-TOPO expression vector (Invitrogen), which appends C-terminal V5 and His epitope tags in-frame, via standard PCR using GoTag HotStart DNA Polymerase (Promega). The obtained plasmids were hBMPR2, hBMPR2- N55Q, hBMPR2 -N110Q, hBMPR2- N126Q, hBMPR2- N55Q/ N110Q, hBMPR2- N55Q/ N110Q, hBMPR2- N55Q/ N126Q, hBMPR2- N110Q/ N126Q and hBMPR2- N55Q/ N110Q/ N126Q. The primers used for plasmid sequencing plasmids are shown in the ap- pendix (Appendix A.1). 2.2 W20 transfections Mutant species of hBMPR2 were obtained via the transfection of W20 cells with plasmids containing the single, double and triple mutant hBMPR2 sequences. Before transfecting 4 the cells, Escherichia coli bacteria were transformed in order to propagate the plasmids. To effectuate the transformation, we firstly transfered 50µl of bacteria and added 1µg of plasmid DNA. To permeabilize the bacterial membrane, the culture was heated up to 42oC during 20 seconds and selection was performed in carbenicillin-containing media. The culture was further incubated at 32oC with shaking, and 100µl of the bacteria were then transferred into a new agar plate for further growth overnight. After incubation, the cultures were pelleted at 13000rpm and the plasmids isolated following the QIAprep Spin MiniPrep kit (QIAGEN) instructions. The transfection was performed using the X-tremeGENE 9 DNA Transfection Reagent (Roche) on W20 cells, previously passaged in 10% DMEM. To perform the transfection, the X-tremeGENE 9 DNA Transfection Reagent was vortexed and 3µl of the reagent was added to 100µl of serum-free medium (to a concentration of 3:1 reagent per DNA ratio). At this point, 1µg of DNA was added to the 100µl of diluted reagent, and the solution was incubated for 15 minutes at room temperature. As the W20 cells were grown in a 25cm2 dish, 300µl of the diluted reagent was further added to the cell culture vessels in a dropwise manner without removal of the media. Lysis of the cells was performed using RIPA buffer 72 hours after transfection, where the proteins were retrieved in the supernatant phase and subsequently prepared for western blotting. 2.3 Immunoblotting Western blots were performed on lysates from transfected W20 cells and BMPR2-ECD/Fc chimeras. Proteins were resolved on Novex Tris Glycine gels (Invitrogen) and transferred to Amersham Hybond ECL nitrocellulose membranes (GE Healthcare).