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D.R. Ledee, M.E. Fini; Discovering Matrix Metalloproteinase–9 Interacting Proteins in the Cornea . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4880.
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© ARVO (1962-2015); The Authors (2016-present)
Matrix metalloproteinases (MMPs) are a family of zinc metalloproteinases that mediate tissue remodeling. One member, MMP–9 or gelatinase B, is of particular relevance to ocular surface biology, as it plays a role in normal corneal re–epithelialization, failure to heal, and the pathology of dry eye disease. In order to understand more about how MMP–9 takes part in these processes, we sought to identify interacting proteins.
An MMP–9 cDNA encoding the catalytic domain including the Zn–binding domain (amino acids 109 – 730) was used as bait to screen a mouse cornea cDNA library using the yeast two– hybrid system (Clontech). The N–terminal propeptide domain functions to keep MMP–9 in an inactive state, necessitating its exclusion. Positive clones were identified by the ability to grow on quadruple drop–out media plates and the expression of α–galactosidase, presenting blue colonies. Positive library cDNAs were sequenced and identities determined by BLAST searches of GenBank. The GST–pulldown assay was then applied as a first step in verifying true versus non–specific interactions.
Screening with MMP–9 bait yielded 151 positive clones, 49 encoding in–frame proteins. The encoded proteins could be categorized as extracellular matrix, transmembrane, membrane–associated, or signaling proteins. Presently, 12 of the 49 clones have been examined using GST–pulldown assays confirming that at least 9 of these clones interact with MMP–9 in vitro. 3 of the 9 (clusterin, envoplakin and cathepsin D) have published data supporting roles for each in ocular surface disorders. Envoplakin, a component of desmosomes, is a target in the autoimmune disease, paraneoplastic pemphigus, exhibiting mucosal and cutaneous lesions with ∼66–72% eye involvement. Clusterin, a secreted glycoprotein, is down–regulated in ocular surface diseases like Stevens–Johnson syndrome and ocular cicatricial pemphigoid; and cathepsin D, a lysosomal protein, is up–regulated during bacterial infections or chemical injury to the ocular surface. The others have little or no published data on their role in the cornea.
Screening of a corneal cDNA library with MMP–9 has yielded expected and novel potential MMP–9 interactions. Functional characterization of these interacting proteins should provide insight into the biological functions and signaling mechanisms of MMP–9.
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