DIFFERENTIAL DISTRIBUTION OF LUMICAN AND FIBROMODULIN IN TOOTH CEMENTUM

The objectives of this study were to isolate and characterize the majo r proteoglycans of tooth cementum in relation to the tissue's minerali zation, Cementum was collected from the root apex region of bovine mol ars and pulverized, It was first extracted with 6M guanidine-HCl, pH 7 .4 (G-extract, mineral-unassociated) and then demineralized and extrac ted with 0.5M EDTA (E-extract, mineral-associated). Both extracts were applied to anion exchange and then molecular sieve chromatography to isolate proteoglycans. The fractions collected were assayed for chondr oitin-(CS) and keratan sulfate (KS) containing proteoglycans using the monoclonal antibodies 2-B-6 and 5-D-4, respectively, It was found tha t the KS was the major glycosaminoglycan and was enriched in the G-ext ract fraction, The major KS fraction was then applied to 7.5% SDS-PAGE . The major broad band (69 kDa) was 5-D-4 positive in Western blot ana lysis and separated into two bands (46 kDa and 50 kDa) after treatment with keratanase II and endo-beta-galactosidase. These two proteins we re transfered to PVDF membrane and analyzed for amino acid sequence. T he results showed the major band (46 kDa) to be lumican and the minor (50 kDa) fibromodulin, In addition, based on the immunohistochemical s tudy using a number of mono- and polyclonal antibodies including 5-D-4 , anti-lumican core protein as well as anti-fibromodulin core protein antibodies, the KSPGs were found to be located almost exclusively in n onmineralized portions of cementum such as precementum and the pericem entocyte area, These biochemical as well as immunohistochemical data s uggest that the major KSPGs of cementum, lumican and fibromodulin, hav e a specific tissue distribution and may have regulatory roles in ceme ntum mineralization.