Cloning and chromosomal localization of mouse keratocan, a corneal keratan sulfate proteoglycan

The corneal stroma consists of a highly structured extracellularmatrix that is maintained by specialized fibroblasts called kerato-cytes. The transparency of the cornea is dependent on the presenceof highly sulfated proteoglycans (PG) that can bind collagen andcan regulate collagen fibril diameter (Rada et al. 1993). The cor-neal proteoglycans that have been identified contain numerousLeu-rich repeats and all belong to the same gene family, the Leu-rich proteoglycans (LRP). These include a chondroitin/dermatansulfate proteoglycan (CS/DSPG), decorin, and three keratan sul-fate proteoglycans (KSPG), lumican, keratocan, and osteoglycin(Funderburgh et al. 1991, 1993; Blochberger et al. 1992; Corpuz etal. 1996). The corneal KSPGs are glycoproteins exclusively duringearly corneal development before corneal transparency (Cornuet etal. 1994) and in macular corneal dystrophy type I, an inheriteddisease that is characterized by corneal opacity and deposits(Klintworth et al. 1977, 1983; Nakazawa et al. 1984). These pre-vious studies show a direct correlation between corneal transpar-ency and the presence of keratan sulfate. The core protein andpoly-lactosamine side chains alone appear to be insufficient extra-cellular matrix components for sustaining a transparent structure inthe cornea.The primary structure of keratocan, based on the deducedamino acid sequence from bovine and chick cDNA sequences,shows N- and C-terminal globular domains containing four andtwo Cys residues forming two and one disulfide bond, respec-tively, and 11 highly conserved Leu-rich repeats located mainlybetween the two globular domains (Corpuz et al. 1996; Dunlevy etal. 1998). The two-dimensional model of chick keratocan, basedon the X-ray crystallography structure of ribonuclease inhibitor, amolecule comprised entirely of Leu-rich repeats, (Kobe et al. 1993,1995), shows the Leu-rich repeats coiled in a spiral with theseregions in close proximity to each other, forming a horseshoestructure (Dunlevy et al. 1998). This model also shows two out ofthree KS chains extended outward from the outer surface of thehorseshoe with the inner surface more likely to be involved incollagen binding.On the basis of mRNA levels, keratocan is most abundant incornea and sclera, but is also found at substantially lower levels inligament, artery, cartilage, skeletal muscle, and skin (Corpuz et al.1996), and keratocan is more specifically expressed in cornea thanis lumican. Consequentially, we are interested in gaining furtherinsights into its tissue-specific regulation of expression, its role indevelopment and maintenance of corneal transparency. In the pre-sent study, we report the cDNA sequence of mouse keratocan andlocalization of the gene to distal Chromosome (Chr) 10 by segre-gation analysis of restriction fragment length variants (RFLVs) inrecombinant inbred (RI) strains of mice.Total RNA was isolated from mouse whole eyes by use of aguanidinium thiocyanate-phenol-chloroform procedure (Chomc-zynski and Sacchi 1987). An mRNA purification kit (PharmaciaBiotech) was used to further purify the RNA, and ∼10 mgofmRNA was used to prepare a cDNA library (Stratagene CustomLibrary Department) in the Uni-Zap XR vector system. The librarywas screened with a high-specific-activity