RAPID COMMUNICATIONS Heparan Sulfate Proteoglycans Are Concentrated on the Sinusoidal Plasmalemmal Domain and in Intracellular Organelles of Hepatocytes

The distribution of cell surface heparan sulfate proteoglycans (HSPGs) was determined in rat liver by immunocytochemistry. A polyclonal antibody was raised against HSPGs purified from rat liver microsomes which specifically immunoprecipitated liver membrane HSPGs. It was shown to recognize both the heparin-releasable and membrane-intercalated form of membrane HSPGs and to recognize determinants on the core protein of these HSPGs. By immunocytochemistry membrane HSPGs were localized to hepatocytes. The distribution of HSPGs at the cell surface of the hepatocyte was restricted to the sinusoidal domain of the plasmalemma; there was little or no staining of the lateral or bile canalicular domains. Intracellularly, HSPGs were occasionally detected in cisternae of the rough endoplasmic reticulum and were regularly found in Golgi cisternae--usually distributed across the entire Golgi stack. HSPGs were also localized in some endosomes, lysosomes, and cytoplasmic vesicles of hepatocytes. We conclude that the HSPGs recognized by this antibody have a restricted distribution in rat liver: they are largely confined to the sinusoidal plasmalemmal domain and to biosynthetic and endocytic compartments of hepatocytes. Heparan sulfate proteoglycans (HSPGs) ~ are widely distributed in mammalian tissues and cell cultures. They have been found in association with cell surfaces (1-5), intracellular organdies (6, 7), and extracellular matrices (especially basement membranes) (8-11), and have been proposed to participate in a variety of functions such as cell-cell interactions (12), cell migration (13), and cell adhesion (14, 15). HSPGs isolated from different sources have distinct structures in terms of their overall size (70-400 kD) and the size of their glycosaminoglycan chains (14-70 kD). One of the best characterized populations of HSPGs are those isolated from rat liver membranes (3, 15). These HSPGs are relatively small (75 kD) and consist of a core protein with about four attached heparan sulfate (HS) side chains (14 kD). Three forms of HSPG have been purified from rat liver membranes (3, 16) and from primary hepatocyte cultures (15, 17)--one that behaves as a peripheral membrane protein in that it can be displaced from cells by exogenous heparin, another that behaves as an integral membrane protein in that Abbreviations used in this paper." ER, endoplasmic reticulum; HS, heparan sulfate; HSPGs, heparan sulfate proteoglycans; PMs, p lasma m e m b r a n e fractions. its core protein is intercalated into the lipid bilayer, and a third form, recently characterized, which consists of HS oligosaccharides and is believed to be associated with lysosomes (Kj611en, L., H. Pertofi, A. Oldberg, M. H66k, submitted for publication). The heparin-displacable and membrane-intercalated forms are known to be present in rat liver microsomal and plasma membrane fractions, but their precise localization in rat liver in situ is unknown. In this study, an antibody was generated against rat liver membrane HSPGs and used to determine the distribution of these HSPGs in rat liver. MATERIALS AND METHODS Materials: Molecular weight standards for SDS PAGE and Sepharose 4B-Protein A beads were obtained from Pharmacia Fine Chemicals (Uppsala, Sweden). lodobeads were obtained from Pierce Chemical Co. (Rockford, IL), and Na[t2~I] was obtained from Amersham Corp. (Arlington Heights, IL). Fab fragments of sheep anti-rabbit IgG conjugated to horseradish peroxidase were obtained from the Institute Pasteur Productions (Marnes La Coquette, France). 3,3 diaminobenzidine and pepstatin A were obtained from Sigma Chemical Co. (St. Louis, MO). Fluorescein isothiocyanate-conjugated rabbit anti-goat IgG and goat anti-rabbit F(ab')2 were obtained from Cappel Laboratories, Inc. (Cochranville, PA), and l0 nm colloidal gold coupled to sheep anti-rabbit IgG was obtained from Janssen Life Science Products (Piscataway, N J). Plateletderived heparitinasc was prepared as previously described (3). THe JOURNAL OF CELL BIOLOGY . VOLUME 100 MARCH 1985 975-980 © The Rockefeller University Press • 0021-9525/85J0310975/06 $1.00 975 on O cber 1, 2015 jcb.rress.org D ow nladed fom Published March 1, 1985