Intracellular traffic of the ecto-nucleotide pyrophosphatase/phosphodiesterase NPP3 to the apical plasma membrane of MDCK and Caco-2 cells: apical targeting occurs in the absence of N-glycosylation.

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.

Identification of B10, an alkaline phosphodiesterase of the apical plasma membrane of hepatocytes and biliary cells, in rat serum: increased levels following bile duct ligation and during the development of cholangiocarcinoma.

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.

Biochemical and molecular identification of distinct forms of alkaline phosphodiesterase I expressed on the apical and basolateral plasma membrane surfaces of rat hepatocytes.

We have identified B10, a plasma membrane protein previously defined by a monoclonal antibody, as an alkaline phosphodiesterase I (APDE) expressed in the plasma membrane of rat hepatocytes and enterocytes, with a restricted apical distribution. B10 complementary DNA (cDNA) was cloned from a rat intestinal library screened with a polyclonal antibody directed to the hepatic protein. Two distinct B10 clones with an open reading frame of 2,625 bp were obtained that differed only by 12 bases in the coding region. One B10 clone had a single base difference with gp130RB13-6 cDNA, which was recently cloned in rat fetal brain. B10/gp130RB13-6 had 50% identity at the amino acid level with the plasma cell antigen PC-1, an APDE cloned in the mouse and in human. Anti-B10 antibodies immunoprecipitated 34% of the APDE activity in liver plasma membranes and over 95% of the APDE activity in intestinal cells. Most of the remaining activity in hepatocytes (44%) could be immunoprecipitated by antibodies directed to PC-1. APDE activity immunoprecipitated with anti-B10 antibodies was found in the apical rat liver plasma membrane fractions on a sucrose gradient whereas most of the remaining APDE activity was associated with the basolateral fractions, which contained PC-1. By immunofluorescence, B10 was localized to the apical surfaces of hepatocytes and enterocytes whereas PC-1 was present on the basolateral surfaces of hepatocytes. B10/gp130RB13-6 and rat PC-1 are a unique example of distinct molecules having similar enzymatic activity but different apical/basolateral location, and possibly different functions.