Non-Glycanated Biglycan and LTBP4: Leveraging the extracellular matrix for Duchenne Muscular Dystrophy therapeutics

Abstract

The extracellular matrix (ECM) plays key roles in normal and diseased skeletal and cardiac muscle. In healthy muscle the ECM is essential for transmitting contractile force, maintaining myofiber integrity and orchestrating cellular signaling. Duchenne Muscular Dystrophy (DMD) is caused by loss of dystrophin, a cytosolic protein that anchors a transmembrane complex and serves as a vital link between the actin cytoskeleton and the basal lamina. Loss of dystrophin leads to membrane fragility and impaired signaling, resulting in myofiber death and cycles of inflammation and regeneration. Fibrosis is also a cardinal feature of DMD. In this review, we will focus on two cases where understanding the normal function and regulation of ECM in muscle has led to the discovery of candidate therapeutics for DMD. Biglycan is a small leucine rich repeat ECM protein present as two glycoforms in muscle that have dramatically different functions. One widely expressed form is biglycan proteoglycan (PG) that bears two chondroitin sulfate GAG chains (typically chondroitin sulfate) and two N-linked carbohydrates. The second glycoform, referred to as 'NG' (non-glycanated) biglycan, lacks the GAG side chains. NG, but not PG biglycan recruits utrophin, an autosomal paralog of dystrophin, and an NOS-containing signaling complex to the muscle cell membrane. Recombinant NG biglycan can be systemically delivered to dystrophic mice where it upregulates utrophin at the membrane and improves muscle health and function. An optimized version of NG biglycan, 'TVN-102', is under development as a candidate therapeutic for DMD. A second matrix-embedded protein being evaluated for therapeutic potential is latent TGFβ binding protein 4 (LTBP4). Identified in a genomic screen for modifiers of muscular dystrophy, LTBP4 binds both TGFβ and myostatin. Genetic studies identified the hinge region of LTBP4 as linked to TGFβ release and contributing to the "hyper-TGFβ" signaling state that promotes fibrosis in muscular dystrophy. This hinge region can be stabilized by antibodies directed towards this domain. Stabilizing the hinge region of LTBP4 is expected to reduce latent TGFβ release and thus reduce fibrosis.

Figure 1

NG biglycan and the dystrophin/utrophin-associated protein complex (D/UPC) in skeletal muscle. The complex is anchored by the actin binding proteins utrophin or dystrophin (not shown) that in turn associate with three membrane-associated subcomplexes: the dystroglycans, sarcoglycan-sarcospan, and the signaling nNOS/α-dystrobrevin/syntrophin (NODS) [2]. The α-dystroglycan binds laminin and agrin via a complex carbohydrate that includes matriglycan [–89]. NG Biglycan binds to α-sarcoglycan and γ-sarcoglycan via distinct domains and also collagen VI, thus providing a second linkage of the D/UPC to the ECM [34, 89]. Proteins indicated by red stars are down-regulated in biglycan mutant mice and upregulated by systemic delivery of recombinant NG biglycan in mdx and/or biglycan mutant mice [18, 37]. Abbreviations: Cav3, caveolin 3; nNOS, neuronal nitric oxide synthase; SP, sarcospan. See text for details.

Figure 2

Distinct functions of biglycan glycoforms. Endogenous biglycan is expressed in forms bearing (PG) or lacking (NG) GAG side chains. Only NG-biglycan counters dystrophic pathology and stabilizes synaptic specializations. NG-biglycan is the basis for a recombinant protein, TVN-102, that is currently being developed as a therapeutic for DMD and other neuromuscular disorders. PG biglycan is a proinflammatory danger signal that requires GAG side chains for this activity [38, 39, 41]. Note that both forms have two N-linked carbohydrates. See text for details.

Figure 3

Schematic of LTBP4 action in sequestering TGF-β and myostatin. LTBP4 is an ECM-associated protein that binds the latent forms of TGF-β (orange) and myostatin (blue) [59]. LTBP4 was identified as a modifier of muscular dystrophy in mice and humans with DMD [, –68]. TGF-β family members like TGF-β 1, 2, and 3 and myostatin are produced as latent forms containing a prodomain linked to the active C-terminal domain (blue and orange ovals represent the active domains of myostatin and TGF-β, respectively). These latent forms are held inactive in the matrix until released, which then undergo further processing to release the active domains that subsequently engage cell surface receptors (purple). The TGF-β receptor and Alk4/5 are the primary receptors, and once engaged by their ligands, these receptors promote SMAD activation regulating a cascade of gene expression changes (reviewed in [90]). The hinge region in LTBP4 undergoes proteolytic cleavage associated with release of latent TGF-β [47, 58]. Antibodies directed at LTBP4’s hinge region are being developed by Solid Biosciences as a biological agent to treat DMD with the goal of limiting active TGF-β and myostatin.