RESUMO
High-molecular-weight (HMW) hyaluronic acid (HA) is a highly abundant natural polysaccharide and a fundamental component of the extracellular matrix (ECM). Its size and concentration regulate tissues' macro- and microenvironments, and its upregulation is a hallmark feature of certain tumors. Yet, the conformational dynamics of HMW-HA and how it engages with the components of the ECM microenvironment remain poorly understood at the molecular level. Probing the molecular structure and dynamics of HMW polysaccharides in a hydrated, physiological-like environment is crucial and also technically challenging. Here, we deploy advanced magic-angle spinning (MAS) solid-state NMR spectroscopy in combination with isotopic enrichment to enable an in-depth study of HMW-HA to address this challenge. This approach resolves multiple coexisting HA conformations and dynamics as a function of environmental conditions. By combining 13C-labeled HA with unlabeled ECM components, we detect by MAS NMR HA-specific changes in global and local conformational dynamics as a consequence of hydration and ECM interactions. These measurements reveal atom-specific variations in the dynamics and structure of the N-acetylglucosamine moiety of HA. We discuss possible implications for interactions that stabilize the structure of HMW-HA and facilitate its recognition by HA-binding proteins. The described methods apply similarly to the studies of the molecular structure and dynamics of HA in tumor contexts and in other biological tissues as well as HMW-HA hydrogels and nanoparticles used for biomedical and/or pharmaceutical applications.
Assuntos
Ácido Hialurônico , Espectroscopia de Ressonância Magnética , Peso Molecular , Ácido Hialurônico/química , Humanos , Matriz Extracelular/química , Matriz Extracelular/metabolismoRESUMO
Hyaluronic acid (HA) is a naturally occurring polysaccharide that is abundant in the extracellular matrix (ECM) of all vertebrate cells. HA-based hydrogels have attracted great interest for biomedical applications due to their high viscoelasticity and biocompatibility. In both ECM and hydrogel applications, high molecular weight (HMW)-HA can absorb a large amount of water to yield matrices with a high level of structural integrity. To understand the molecular underpinnings of structural and functional properties of HA-containing hydrogels, few techniques are available. Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for such studies, e.g. 13C NMR measurements can reveal the structural and dynamical features of (HMW) HA. However, a major obstacle to 13C NMR is the low natural abundance of 13C, necessitating the generation of HMW-HA that is enriched with 13C isotopes. Here we present a convenient method to obtain 13C- and 15N-enriched HMW-HA in good yield from Streptococcus equi subsp. zooepidemicus. The labeled HMW-HA has been characterized by solution and magic angle spinning (MAS) solid-state NMR spectroscopy, as well as other methods. These results will open new ways to study the structure and dynamics of HMW-HA-based hydrogels, and interactions of HMW-HA with proteins and other ECM components, using advanced NMR techniques.
Assuntos
Ácido Hialurônico , Proteínas , Ácido Hialurônico/química , Peso Molecular , Proteínas/química , Espectroscopia de Ressonância Magnética , Hidrogéis/químicaRESUMO
Barth syndrome (BTHS) is a life-threatening genetic disorder with unknown pathogenicity caused by mutations in TAFAZZIN (TAZ) that affect remodeling of mitochondrial cardiolipin (CL). TAZ deficiency leads to accumulation of mono-lyso-CL (MLCL), which forms a peroxidase complex with cytochrome c (cyt c) capable of oxidizing polyunsaturated fatty acid-containing lipids. We hypothesized that accumulation of MLCL facilitates formation of anomalous MLCL-cyt c peroxidase complexes and peroxidation of polyunsaturated fatty acid phospholipids as the primary BTHS pathogenic mechanism. Using genetic, biochemical/biophysical, redox lipidomic and computational approaches, we reveal mechanisms of peroxidase-competent MLCL-cyt c complexation and increased phospholipid peroxidation in different TAZ-deficient cells and animal models and in pre-transplant biopsies from hearts of patients with BTHS. A specific mitochondria-targeted anti-peroxidase agent inhibited MLCL-cyt c peroxidase activity, prevented phospholipid peroxidation, improved mitochondrial respiration of TAZ-deficient C2C12 myoblasts and restored exercise endurance in a BTHS Drosophila model. Targeting MLCL-cyt c peroxidase offers therapeutic approaches to BTHS treatment.