Biosynthesis of Proteoglycans and Hyaluronic Acid by Rat Oral Epithelial Cells (Keratinocytes) in Vitro

Biosynthesis of complex carbohydrates, including sulfated glycoproteins, hyaluronic acid (HA), and proteoglycans (PGs), synthesized by rat oral epithelial cells (keratinocytes) in culture were studied by metabolic labeling protocols using [35S]sulfate and [3H]glucosamine in combination with differential enzymatic digestion and analytical gel filtration. The epithelial cells synthesized a major sulfated glycoprotein species with an apparent molecular size ∼50 kDa, which accounted for approximately 46% of the total 35S incorporation. HA was a relatively minor component of 3H-labeled macromolecules (∼4% of the total 3H incorporation), and almost all of it was secreted into the medium. PGs accounted for approximately half of the 35S incorporation, of which about 30% was secreted into the medium and the remainder associated with the cell layer. The majority of PGs (75% of the secreted and 97% of the cell-associated) contained heparan sulfate (HS) and had an apparent molecular weight of ∼150,000. Cell-associated HSPGs had a core protein of ∼70 kDa with HS chains of ∼64 kDa, while HSPG in the medium had a core protein of ∼50 kDa with HS chains of the same average size as those of the cell-associated HSPG. Of the total cell-associated HSPGs, glycosylphosphatidyl inositol-anchored forms, plasma membrane intercalated forms and those associated with basolateral pericellular matrix accounted for ∼3%, 56% and ∼4% of the total, respectively. Approximately one third of the cell-associated HSPGs were intracellular components most likely generated through intracellular degradation processes following endocytosis. Cell surface HSPGs synthesized by keratinocytes may be involved in some biological roles such as the regulation of normal epithelial turnover and defense mechanisms involving interactions with various oral pathogens.