A unique heparan sulfate in the nuclei of hepatocytes: Structural changes with the growth state of the cells

Growing and confluent cultures of a rat hepatocyte cell line were labeled with ³⁵SO₄^2- and the heparan sulfate in the culture medium, the pericellular matrix, the nucleus, the nuclear outer membrane, and the remaining cytoplasmic pool was purified by DEAE-cellulose chromatography. The heparan sulfate in all pools from the confluent cells was bound more strongly on the DEAE-cellulose column than the corresponding pools from the growing cells. Gel filtration of each pool before and after β-elimination showed that the heparan sulfate from the nuclear and nuclear membrane pools was composed of primarily free chains, whereas the heparan sulfate in all of the other pools was mixture of proteoglycans and free chains. The heparan sulfate in each pool was cleaved with nitrous acid to obtain mixtures of di- and tetrasaccharides. Analysis of these mixtures showed that the structural features of the heparan sulfates in each pool were different and were altered significantly when the growing cells became confluent. The nuclear-plus-nuclear membrane pools represented 6.5% and 5.4% of the total cell-associated heparan sulfate in the growing cells and confluent cells, respectively. The structural features of the heparan sulfate in the two nuclear pools were very similar to each other, but were markedly different from each of the heparan sulfate from the other pools or from any previously described heparan sulfate or heparin. The most unusual aspect of these structures was the high content of β-D-glucuronosyl(2-SO₄)→D-glucosamine-N,O-(SO₄)₂ disaccharide units in these sequences. The mode of biosynthesis and delivery of these unusual sequences to the nucleus and the potential significance of these observations are discussed.