The cornea provides transparency, refraction and tensile strength, properties necessary for vision. Most of these properties are due to the unique structure of the extracellular matrix of the stroma. This matrix consists primarily of collagens I, V, & VI and of proteoglycans bearing keratan sulfate (KS) and chondroitin/dermatan sulfate side chains. The arrangement of collagen fibrils in this matrix, their small, uniform diameter and spacing, produces the transparency of the corneal stroma. cDNA cloning has identified lumican as one of the major KS containing proteoglycans of the stroma. The core protein of lumican has been shown to regulate collagen fibril diameter in vitro and the presence of its KS side chains has been shown to correlate with transparency. The long-term goals of this project are to define the interaction of the proteoglycans with collagen and identify the elements that regulate proteoglycan production.
The first aim i s to identify the specific structural elements on lumican that interact with the collagen by producing mutagenized recombinant lumican and testing its ability to inhibit collagen fibril growth.
This aim also proposes to identify the sites on lumican that receive KS by isolating and amino acid sequencing trypsin fragments containing KS.
The second aim i s to search for other lumican- like proteoglycans of the cornea by screening cDNA libraries prepared from embryonic and from adult corneas.
The third aim i s to characterize the expression and routing of the KS sulfotransferases that regulate the maturation of the KS containing proteoglycans by preparing antibodies to the sulfotransferases and using these antibodies, in combination with antibodies to the proteoglycans, to study their rates of synthesis and their routing through the Golgi.
The fourth aim i s to identify the genetic elements that regulate lumican mRNA production by inserting lumican genomic DNA in reporter plasmids and testing for promoter activity by transfection into corneal fibroblasts. The fifth aim is to determine if macular corneal dystrophy type I involves defective sulfotransferases or defective lumican gene by assaying serum for sulfotransferase levels and conducting linkage studies with the lumican gene. This information will serve as a basis for understanding the mechanisms by which corneal transparency is constructed and maintained and will also provide a basis for the eventual development of new therapeutic approaches for the treatment of corneal stromal opacities.
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