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Isolation and characterization of a Mr = 110,000 glycoprotein localized to the hepatocyte bile canaliculus
J K Petell1, M Diamond, W J Hong
1Department of Biological Sciences, State University of New York at Buffalo 14260.
Abstract:
A Mr = 110,000 glycoprotein, GP 110, was partially purified using wheat germ agglutinin-Sepharose affinity chromatography from a bile canalicular-enriched membrane fraction denoted N2u of rat liver. This fraction was subjected to preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the Mr = 110,000 polypeptide was excised and used as an immunogen in rabbits. The antisera were found to specifically recognize a Mr = 110,000 polypeptide, named GP 110, in the N2u membrane fraction. In isolated hepatocytes, GP 110 was readily accessible to cell surface iodination catalyzed by lactoperoxidase at 4 degrees C and was judged by immunoprecipitation studies to contain about 2% of total radioactivity incorporated into externally oriented proteins of the cell. Immunoprecipitated GP 110 was shown by two-dimensional polyacrylamide gel electrophoresis to migrate with an approximate pI of 4.9. Indirect immunofluorescence on frozen liver sections demonstrated that GP 110 was primarily localized in the bile canaliculus. In corroborative studies employing subcellular fractionation, it was found that GP 110 was enriched nearly 19-fold in P2, a plasma membrane fraction primarily derived from the sinusoidal domain, and 44-fold in N2u. In contrast, only low levels of GP 110 were present in endoplasmic reticulum, mitochondrial, cytosolic, and nuclear-enriched fractions of liver. The physiological function of GP 110 is as yet unknown; antisera to it did not immunoprecipitate other known bile canalicular proteins of similar molecular weights. GP 110 was found to be extensively glycosylated relative to other known membrane proteins; approximately 33% of the apparent molecular weight appear to be carbohydrate. In agreement, limited removal of N-linked carbohydrate chains indicated that there are approximately eight chains/GP 110 polypeptide. Neuraminidase treatment of GP 110 resulted in a desialylated Mr = 85,000 polypeptide suggesting that the majority of carbohydrate chains on GP 110 are of the complex type.
Insights
Researchers purified and characterized GP 110, a novel glycoprotein found in rat liver plasma membranes. This study details its specific localization to bile canaliculi and its extensive glycosylation, paving the way for future functional studies.
Area of Science:
- Hepatology and Cell Biology
- Membrane Protein Research
- Glycoprotein Characterization
Background:
- Bile canaliculi are crucial for bile excretion in the liver.
- Understanding membrane proteins in hepatocytes is key to liver function.
- GP 110 is a glycoprotein identified in rat liver membrane fractions.
Purpose of the Study:
- To partially purify and characterize a Mr = 110,000 glycoprotein (GP 110) from rat liver.
- To determine the cellular localization and accessibility of GP 110.
- To investigate the glycosylation status of GP 110.
Main Methods:
- Wheat germ agglutinin-Sepharose affinity chromatography for partial purification.
- Preparative SDS-PAGE for polypeptide isolation and antibody generation.
- Lactoperoxidase-catalyzed iodination and immunoprecipitation for cell surface labeling.
- Two-dimensional polyacrylamide gel electrophoresis for pI determination.
- Indirect immunofluorescence and subcellular fractionation for localization studies.
- Neuraminidase treatment to assess carbohydrate chains.
Main Results:
- GP 110 was purified from a bile canalicular-enriched membrane fraction (N2u).
- Antisera specifically recognized GP 110, which is externally oriented on isolated hepatocytes.
- GP 110 is primarily localized to the bile canaliculus and enriched in plasma membrane fractions.
- GP 110 is extensively glycosylated, with approximately 33% of its mass being carbohydrate, including complex-type N-linked chains.
Conclusions:
- GP 110 is a novel, extensively glycosylated glycoprotein localized to the bile canalicular domain of rat hepatocytes.
- Its physiological function remains unknown, but its specific localization suggests a role in bile transport or related processes.
- Further research is needed to elucidate the functional significance of GP 110.