An electron microscope autoradiographic study of the carbohydrate recognition systems in rat liver. I. Distribution of 125I-ligands among the liver cell types.

Abstract

Electron microscope autoradiography was used to study the cellular localization of seven glycoproteins rapidly cleared from the circulating plasma of rats and taken up by the liver. I and 15 min after intravenous administration of the 125I-glycoproteins, livers were fixed in situ by perfusion and processed for autoradiography. Autoradiographic grains in the developed sections were found to represent the intact 125 I-ligand. A quantitative analysis of the distribution and concentration (density) of autoradiographic grains over the three major cell types of the liver was then performed. Three molecules, asialo-fetuin, asialo-orosomucoid, and lactosaminated RNase A dimer, the oligosaccharide chains of which terminate in galactose residues, were bound and internalized almost exclusively (>900 /0) by hepatocytes. Conversely, four molecules, the oligosaccharide chains of which terminate in either N-acetyl-glucosamine (agalacto-orosomucoid) or mannose (ahexosamino-orosomucoid, preputial,8-glucuronidase, and mannobiosaminated RNase A dimer), were specifically bound and internalized by cells lining the blood sinusoids-that is, by Kupffer cells and endothelial cells. Endothelial cells were two to six times more active (on a cell volume basis) than were Kupffer cells in the internalization of these four 125 I-ligands. Mannose and N-acetylglucosamine-terminated glycoproteins competed with each other for uptake into either endothelial cells or Kupffer cells, indicating that a single system recognized mannose or Nacetyl-glucosamine residues. Finally, agalacto-orosomucoid and ahexosaminoorosomucoid were also associated with hepatocytes, but competition experiments utilizing excess asialo-orosomucoid demonstrated that residual galactosyl residues were responsible for this association.J. CELL BIOLOGY

Quantitative Analysis of the Targeting of Mannose‐Terminal Glucocerebrosidase

“…Both Kupffer cells and endothelial liver cells are known to express mannose-specific receptors [8,91. To identify the cell type(s) responsible for the hepatic uptake, rats were injected with Alglucerase; 10 min later, parenchymal, Kupffer and endothelial cells were isolated from the liver and assayed for Alglucerase enzyme activity.…”

“…However, endothelial liver cells also express functional mannose receptors, which are immunologically indistinghuisable from the receptors on Kupffer cells and other macrophages [8 -101. Moreover, endothelial liver cells have been found to be more active in uptake of mannose-terminal ligands than Kupffer cells [8,91. The earlier studies on targeting of Alglucerase only distinguished between uptake by parenchymal and sinusoidal liver cells.…”

Receptor-mediated endocytosis of asialoglycoproteins by rat hepatocytes: receptor-positive and receptor-negative endosomes.

“…Pellets from S. aureus immunoadsorptions were fixed overnight at 4°C in 0.5% glutaraldehyde, 1.5% formaldehyde, 0.1 M Na caeodylate, pH 7.4, 2.0 mM CaCI2, 2.0 mM MgC12 and processed as described previously (23). For electron microscopic visualization of ASOR, ~zSI-ASOR-HRP was prepared as described (53) and mixed with ~I-ASOR.…”

Isolation and partial characterization of the sialoglycoprotein fraction of murine erythrocyte ghosts.

“…Since the preparation of large amounts of murine erythrocyte ghosts for the isolation of milligram amounts of sialoglycoproteins is a tedious and expensive operation, we used a radiochemical approach to determine the best gel system and to define the monomeric units and their pattern of aggregation. We labeled the sialoglycoproteins with K12'I with the LPO-GO methods (16), resolved individual components by SDS PAGE, eluted each band electrophoretically and analyzed the eluates by SDS PAGE in both Steck-Yu and Laemmli gels . When the [125 1]sialoglycoproteins were analyzed by SDS PAGE on the Laemmli system (Fig .…”

“…The composition and the buffer system of each acrylamide gel are indicated in the legend of each figure . As molecular weight markers we used : ,B-galactosidase from Escherichia coli (130,000), rabbit muscle phosphorylase B (94,000), bovine serum albumin (68,000), ovalbumin (43,000), bovine chymotrypsinogen (25,700), soybean trypsin inhibitor (21,500), and human globin (16,500 Recovery of Ghost Sialyl Residues during the Isolation of the Sialoglycoprotein Fraction by LIS Sialyl residues were released by 0.025 M H Z S04 for 1 h at 80°C and assayed according to Warren (44) using readings at 562 and 549 nm and 2-deoxyribose and NeuNAc as standards, as previously detailed (32) . All recovery figures are calculated on the basis of ghosts .…”