High-yield purification and characterization of human asialoglycoprotein receptor.
The human asialoglycoprotein receptor (ASGPR) represents a major component of the hepatocellular membrane. To study its native composition, approximately 30% of receptor activity from liver specimens was recovered in highly purified ASGPR preparations. Discontinuous, denaturing SDS-gel electrophoresis based on Tris-Tricine buffer indicated the presence of a multimeric ASGPR corresponding to H1 and H2 polypeptides as confirmed by peptide-specific immunoblotting. FPLC-gel filtration of ASGPR preparations revealed a molecular mass for receptor complexes at 150 and 95 kDa, suggesting functional heterotrimers and dimers of H1/H2 subunits. Gel filtration of SDS-denatured protein indicated a single peak at 50 kDa apparently corresponding to dissociated subunits H1 and H2. beta-Mercaptoethanol treatment followed by affinity chromatography separated functionally active and inactive receptors. The H2 subunit was strikingly enriched in the inactive fraction of receptors. Both active and inactive ASGPR preparations consistently showed peaks at 150 and 95 kDa by gel filtration. Receptor activity retained in such heteromers was linked to a lower glycosylation state of ASGPR. These results suggest that native human ASGPR consists of sulfide- and non-sulfide-linked heterotrimers and -dimers from H1 and H2 subunits with a functional restriction to their glycosylation states.
Treichel
U
U
Department of Internal Medicine, Johannes Gutenberg University of Mainz, Federal Republic of Germany.
Schreiter
T
T
Meyer zum Büschenfelde
K H
KH
Stockert
R J
RJ
D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins / Q000378:metabolism*; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015151:Immunoblotting; D008099:Liver / Q000737:chemistry*; D008565:Membrane Proteins / Q000302:isolation & purification; D011487:Protein Conformation; D011956:Receptors, Cell Surface / Q000302:isolation & purification*
D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins; D008565:Membrane Proteins; D011956:Receptors, Cell Surface