apical surface forms the bile canaliculi and is specialized for We have identified B10, a plasma membrane protein bile secretion. A variety of apical and basolateral domainpreviously defined by a monoclonal antibody, as an alkaspecific proteins have been identified, which carry out the line phosphodiesterase I (APDE) expressed in the unique functions of each plasma membrane domain. 1 B10, an plasma membrane of rat hepatocytes and enterocytes, apical hepatocyte plasma membrane protein, was originally with a restricted apical distribution. B10 complemenidentified by monoclonal antibodies generated to rat hepatotary DNA (cDNA) was cloned from a rat intestinal library cytes. 2 Its exclusive apical location was shown to be altered screened with a polyclonal antibody directed to the hein the fetus and the young rat, 3 during intrahepatic and expatic protein. Two distinct B10 clones with an open readtrahepatic cholestasis, 4 and after microtubule disruption. 4,5 ing frame of 2,625 bp were obtained that differed onlyChanges in B10 expression were observed in chemically inby 12 bases in the coding region. One B10 clone had a duced liver carcinomas, 6,7 and in hepatoma cell lines. 8 Howsingle base difference with gp130 RB13-6 cDNA, which was ever, it was not possible to evaluate the consequences of such recently cloned in rat fetal brain. B10/gp130 RB13-6 had 50% disorders because B10 differed from proteins already identiidentity at the amino acid level with the plasma cell antified at the bile canalicular surface, and its function remained gen PC-1, an APDE cloned in the mouse and in human.unknown. Anti-B10 antibodies immunoprecipitated 34% of theIn this report, we describe the identity of B10 with APDE activity in liver plasma membranes and over 95% gp130 , a recently cloned fetal brain protein that is also of the APDE activity in intestinal cells. Most of the reexpressed by brain tumor cells but is absent from normal maining activity in hepatocytes (44%) could be immunoadult brain cells. 9 We show that B10 is a member of the precipitated by antibodies directed to PC-1. APDE activfamily of alkaline phosphodiesterase I (APDE) enzymes, the ity immunoprecipitated with anti-B10 antibodies was first member of which was the plasma cell antigen PC-1, a found in the apical rat liver plasma membrane fractions membrane glycoprotein cloned in the mouse 10 and in huon a sucrose gradient whereas most of the remaining man. 11 PC-1 was originally characterized as an antigen that APDE activity was associated with the basolateral fracis selectively expressed on the surface of B lymphocytes when tions, which contained PC-1. By immunofluorescence, they reach their final stage of differentiation into antibody-B10 was localized to the apical surfaces of hepatocytes secreting cells. 12 We show that B10 is the major APDE of and enterocytes whereas PC-1 was present on the basointestinal epithelial cells, whereas in hepatocytes, there are lateral surfaces of hepatocytes. B10/gp130 RB13-6 and rat at least two main APDE enzymes, B10 and PC-1. T...
Alkaline phosphodiesterase (APDE) is associated with the cellular plasma membrane of many organs. Several isoforms are also detected in normal human serum and their respective amounts vary in liver diseases but their significance is unknown. The aims of this study were: 1) to identify a serum form of B10, an APDE exclusively localized at the apical pole of the plasma membrane of rat hepatocytes and biliary cells; 2) to gain insight into its origin; and 3) to investigate its behavior, in two liver diseases in which an abnormal membrane expression of B10 has been reported, namely cholestasis and cholangiocarcinoma. A soluble form of B10 was immunoprecipitated from normal rat serum, which amounted to 13% of total serum APDE activity. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the size of the serum enzyme was 125 kd, which is slightly lower than that found in the plasma membrane (130 kd). In bile, a 120-kd and a 130-kd form was found. A sixfold and fivefold increase of B10 APDE activity was observed in the serum of bile duct-ligated rats and in the Long-Evans Cinnamon (LEC) rats which spontaneously develop cholangiocarcinoma. The molecular size of the form present in serum was unchanged. A threefold increase was also observed in LEC rats which had not yet developed a cholangiocarcinoma. In conclusion, we identified a soluble form of B10 in normal rat serum. The increase in serum B10 in the experimental and pathological conditions investigated does not seem to result from passage of the biliary form to the serum but seems to be caused by increased cleavage of the membrane form. Its rise early during the onset of cholangiocarcinoma suggests that B10 in the serum might be a marker of carcinogenesis and/or be involved in the development of cholangiocarcinoma.
Glycosylation was considered the major signal candidate for apical targeting of transmembrane proteins in polarized epithelial cells. However, direct demonstration of the role of glycosylation has proved difficult because non-glycosylated apical transmembrane proteins usually do not reach the cell surface. Here we were able to follow the targeting of the apical transmembrane glycoprotein NPP3 both when glycosylated and non-glycosylated. Transfected in polarized MDCK and Caco-2 cells, NPP3 was exclusively expressed at the apical membrane. The transport kinetics of the protein to the cell surface were studied after metabolic (35)S-labeling and surface immunoprecipitation. The newly synthesized protein was mainly targeted directly to the apical surface in MDCK cells, whereas 50% transited through the basolateral surface in Caco-2 cells. In both cell types, the basolaterally targeted pool was effectively transcytosed to the apical surface. In the presence of tunicamycin, NPP3 was not N-glycosylated. The non-glycosylated protein was partially retained intracellularly but the fraction that reached the cell surface was nevertheless predominantly targeted apically. However, transcytosis of the non-glycosylated protein was partially impaired in MDCK cells. These results provide direct evidence that glycosylation cannot be considered an apical targeting signal for NPP3, although glycosylation is necessary for correct trafficking of the protein to the cell surface.
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