ABSTRACT
Matrix metalloproteinase 9 (MMP9), a protease implicated in multiple diseases, is secreted as an inactive zymogen and requires proteolytic removal of the pro-domain for activation. The relative levels and functionality of the pro- and active-MMP9 isoforms in tissues are not characterized. We generated a specific antibody that distinguishes an active form of MMP9, F107-MMP9, from the inactive pro-MMP9 isoform. Using multiple in vitro assays and specimen types, we show that F107-MMP9 expression is localized and disease-specific compared with its more abundant parental pro-form. It is detected around sites of active tissue remodeling, including fistulae of inflammatory bowel and dermal fissures in hidradenitis suppurativa, and is expressed by myeloid cells, including macrophages and neutrophils. Together, our findings provide insights into the distribution and potential role of MMP9 in inflammatory diseases.
ABSTRACT
Matrix metalloproteinase 9 (MMP9) is a member of a large family of proteases that are secreted as inactive zymogens. It is a key regulator of the extracellular matrix, involved in the degradation of various extracellular matrix proteins. MMP9 plays a pathological role in a variety of inflammatory and oncology disorders and has long been considered an attractive therapeutic target. GS-5745, a potent, highly selective humanized monoclonal antibody inhibitor of MMP9, has shown promise in treating ulcerative colitis and gastric cancer. Here we describe the crystal structure of GS-5745·MMP9 complex and biochemical studies to elucidate the mechanism of inhibition of MMP9 by GS-5745. GS-5745 binds MMP9 distal to the active site, near the junction between the prodomain and catalytic domain, and inhibits MMP9 by two mechanisms. Binding to pro-MMP9 prevents MMP9 activation, whereas binding to active MMP9 allosterically inhibits activity.
Subject(s)
Antibodies, Monoclonal, Humanized/chemistry , Colitis, Ulcerative/drug therapy , Matrix Metalloproteinase 9/chemistry , Matrix Metalloproteinase Inhibitors/chemistry , Stomach Neoplasms/drug therapy , Allosteric Site , Antibodies/chemistry , Catalytic Domain , Crystallography, X-Ray , Drug Design , Drug Evaluation, Preclinical , Gelatin/chemistry , Gene Deletion , HEK293 Cells , Humans , Inhibitory Concentration 50 , Protein Binding , Recombinant Proteins/chemistry , Surface Plasmon ResonanceABSTRACT
Guava is one of the most widely grown plants in the tropics; however, it is affected by many fruit rot diseases. Fruit diseases decrease the marketability of fresh fruit and fruit for processing. A survey of scab disease was conducted at the USDA/ARS Tropical Plant Genetic Resource Management Unit in Hilo, HI, where more than 50 accessions of guava are grown. Symptoms observed were gray/light brown lesions surrounded by dark brown borders on leaves and brown, raised, corky, necrotic lesions on the exocarp of fruit which progressed as the fruits matured. Seventeen isolates from infected fruit, six isolates from lesions on leaves, and nine isolates from additional crops surrounding the guava trees were collected. The main fungi consistently isolated from symptomatic leaves and fruit were Pestalotiopsis spp. Morphology, colony characteristics, and pathogenicity of the isolates were examined and potential sources of host resistance were identified for germplasm characterization studies. Molecular methods were used to identify four Pestalotiopsis taxa (P. clavispora, P. microspora, P. sp. GJ-1, and P. disseminata) on guava in Hawaii. To our knowledge, this is the first report of traditional and molecular methods of identification and characterization being used for fungal pathogens of guava in Hawaii.
