RESUMEN
Chondroitin sulfate proteoglycans (CSPGs) are up-regulated following spinal cord injury and are partly responsible for failed regeneration. Experimental paradigms in vivo that degrade chondroitin sulfate glycosaminoglycan chains with the bacterial enzyme, chondroitinase, greatly enhance the ability of axons to regenerate through the glial scar. Unfortunately, enthusiasm for this treatment paradigm is diminished by the lack of a minimally invasive and sustained delivery method. To address these deficits, we have engineered a Tet-On adenoviral vector encoding chondroitinase AC and have characterized its enzymatic function in vitro. U373 human astrocytoma cells were transduced with adenovirus and subsequently induced with doxycycline to secrete enzymatically active chondroitinase as detected by western blot and kinetic analyses. Enzymatic activity demonstrated biological relevance in studies where neurite outgrowth into and across CSPG-adsorbed regions pre-treated with conditioned media from chondroitinase secreting astrocytes was significantly increased compared with untreated controls (p < 0.0001). We also measured important parameters of enzyme activity including: pH, temperature, and enzyme stability that are fundamental to harnessing the true therapeutic potential of this approach. The use of resident cells for continuous secretion of CSPG-degrading enzymes at the site of the glial scar promises to be of greater clinical relevance than contemporary methods.
Asunto(s)
Axones/fisiología , Condroitín Liasas/fisiología , Sulfatos de Condroitina/antagonistas & inhibidores , Sulfatos de Condroitina/farmacología , Proteoglicanos/antagonistas & inhibidores , Proteoglicanos/farmacología , Adenoviridae/genética , Animales , Western Blotting , Línea Celular Tumoral , Células Cultivadas , Pollos , Condroitín Liasas/química , Condroitín Liasas/genética , Clonación Molecular , Doxiciclina/farmacología , Ganglios Espinales/citología , Ganglios Espinales/efectos de los fármacos , Humanos , Concentración de Iones de Hidrógeno , Inmunoprecipitación , Regeneración Nerviosa/efectos de los fármacos , Neuronas Aferentes/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , TemperaturaAsunto(s)
Polisacáridos/metabolismo , Animales , Coagulación Sanguínea , Cartílago/fisiología , Condroitín Liasas/fisiología , Sulfatos de Condroitina/metabolismo , Dermatán Sulfato/metabolismo , Humanos , Neoplasias/metabolismo , Osteoartritis/etiología , Polisacáridos/química , Transducción de SeñalRESUMEN
Polysaccharide lyases (or eliminases) are a class of enzymes (EC 4.2.2.-) that act to cleave certain activated glycosidic linkages present in acidic polysaccharides. These enzymes act through an eliminase mechanism, rather than through hydrolysis, resulting in unsaturated oligosaccharide products. Acidic polysaccharides are ubiquitous and so are the lyases that degrade them. This review article examines lyases that act on acidic polysaccharides of plant, animal, and microbial origin. These lyases are predominantly of microbial origin and come from a wide variety of both pathogenic and nonpathogenic bacteria and fungi. The lyases discussed include alginate lyase (EC 4.2.2.3), pectin lyase (EC 4.2.2.10), pectate lyase (EC 4.2.2.2), oligogalacturonide lyase (EC 4.2.2.6), exopolygalacturonate lyase (EC 4.2.2.9), chondroitin lyases (EC 4.2.2.4 and EC 4.2.2.5), hyaluronate lyase (EC 4.2.2.1), heparin lyase (EC 4.2.2.7), heparan lyase (EC 4.2.2.8), and other unclassified lyases. This review examines the sources, regulation, purification, and properties of these polysaccharide lyases.