cDNA cloning for a bile canaliculus domain-specific membrane glycoprotein of rat hepatocytes

Abstract

Hepatocytes are polarized cells with distinct sinusoidal, bile canalicular, and basolateral plasma membrane domains. Each domain contains proteins that are specific for it. We have isolated three cDNA clones encoding a rat liver bile canaliculus domain-specific glycoprotein with Mr 110,000 (gp110) by immunologically screening a rat kidney lambda gt11 cDNA library with a rabbit polyclonal antiserum directed against purified gp110. The authenticity of these clones was verified as follows. (i) The antiserum recognizes specifically isopropyl beta-D-thiogalactoside-induced fusion proteins on electrophoretic transfer blots of total lysogen lysates containing these cDNA clones. (ii) Antibodies epitope-selected by these clones are able to interact with gp110 on electrophoretic transfer blots. (iii) The amino acid sequencing derived from the DNA sequence was confirmed by amino acid sequencing of a tryptic peptide of gp110. Rescreening of the same library with the cDNA clones identified a full-length cDNA clone for this glycoprotein. Sequence analysis indicates that the N-linked carbohydrate chains are concentrated on the N-terminal part of this highly glycosylated protein.