Volume 126, Issue 7, Pages 1819-1827 (June 2004) Molecular characterization of hepatocystin, the protein that is defective in autosomal dominant polycystic liver disease  Joost P.H. Drenth, Jose A. Martina, Rene H.M. Te Morsche, Jan B.M.J. Jansen, Juan S. Bonifacino  Gastroenterology  Volume 126, Issue 7, Pages 1819-1827 (June 2004) DOI: 10.1053/j.gastro.2004.02.023

Figure 1 Schematic representation of hepatocystin, showing its different domains or motifs, and the positions of mutations found in PCLD patients. Notice that these mutations are dispersed throughout the protein. SP, signal peptide; LDLa, low-density lipoprotein receptor domain A; EF, EF-hand calcium-binding domains; Glu-rich, glutamic acid-rich region; MPR, mannose 6-phosphate receptor domain; HDEL, endoplasmic reticulum targeting sequence. The scale indicates the number of amino acids. Gastroenterology 2004 126, 1819-1827DOI: (10.1053/j.gastro.2004.02.023)

Figure 2 Immunoblot analysis of normal hepatocystin and analyses of glycosylation of normal and mutant hepatocystin. (A ) Extracts from the human organs indicated in the figure were subjected to SDS-PAGE on 4%–20% acrylamide gels, and immunoblot analysis was performed with an antibody to hepatocystin. The positions of molecular mass markers (in kilodaltons) are shown on the right. Notice that the antibody detected variable amounts of an approximately 90-kilodalton species in all tissue samples. (B and C ) HA-tagged full-length or truncated hepatocystin was expressed by transfection in HeLa cells. Hepatocystin was immunoprecipitated with a specific anti-HA monoclonal antibody. The immunoprecipitates were subsequently incubated in the presence (+) or absence (−) of PNGase F (B) or Endo H (C ). Carboxypeptidase Y, a highly glycosylated protein, was included as a positive control for deglycosylation. Gastroenterology 2004 126, 1819-1827DOI: (10.1053/j.gastro.2004.02.023)

Figure 3 Immunoblot analysis of normal and mutant hepatocystin and assembly of hepatocystin. (A ) Equivalent amounts (20 μg of protein) of liver from a healthy individual (control) and from a patient bearing the 1338–2A→G mutation in hepatocystin, as well as liver cyst wall tissue for the same patient, were resolved and analyzed by immunoblotting with antibodies to the α subunit of glucosidase II, hepatocystin and calreticulin (control). (B) Varying amounts of lysate from Epstein-Barr virus (EBV)-transformed B lymphoblasts (numbers represent millions of cells) derived from a healthy individual (control; left) and from the 1338–2A→G patient (right) were analyzed as in (B). Tubulin was included as a control. In both (B) and (C ), notice the lower levels of hepatocystin and α-glucosidase in the patient’s tissues. (C ) HeLa cells were transfected with HA-tagged full-length or truncated hepatocystin and blotted with a monoclonal anti-HA antibody (left 2 lanes) or a polyclonal antibody directed to the central part of hepatocystin (middle 2 lanes). Liver samples from a healthy individual and from the 1338–2A→G patient were similarly analyzed by using this anti-hepatocystin antibody (right 2 lanes). Notice that the anti-hepatocystin antibody recognizes the overexpressed truncated protein but that this is absent in the patient’s liver. (D) HA-tagged full-length or truncated hepatocystin was expressed by transfection in HeLa cells, and hepatocystin was immunoprecipitated with anti-HA monoclonal antibody (left 2 lanes). Western blot analysis demonstrates that native α-glucosidase (αG arrow) is coimmunoprecipitated with full-length but not with truncated hepatocystin. The right 2 lanes represent experiments in which HeLa cells were co-transfected with an α-glucosidase construct and with full-length or truncated hepatocystin. Here, too, α-glucosidase is coimmunoprecipitated with full length but not with truncated hepatocystin. Gastroenterology 2004 126, 1819-1827DOI: (10.1053/j.gastro.2004.02.023)

Figure 4 Immunofluorescence microscopy localization of normal and mutant hepatocystin (HC). HeLa cells were transiently transfected with constructs encoding HA-tagged normal (A—C) and mutant (D—F ) hepatocystin. Cells were fixed, permeabilized, and stained with antibodies to the HA epitope (A and D) or to endogenous calreticulin (B and E ), followed by appropriate secondary antibodies; (C ) and (F ) show merged images. Notice that both normal and truncated hepatocystin colocalize with calreticulin in the ER (bars = 10 μm). Gastroenterology 2004 126, 1819-1827DOI: (10.1053/j.gastro.2004.02.023)

Figure 5 Fate of normal and truncated hepatocystin. (A and B) HeLa cells were transfected with either truncated or full-length HA-tagged hepatocystin, metabolically labeled with [35S]methionine/cysteine for 20 minutes, and chased for the indicated time periods. Cell lysates (A) and supernatants (B) were immunoprecipitated by using anti-HA monoclonal antibody and analyzed by SDS-PAGE and fluorography (C and D). HeLa cells were transfected with either truncated or full-length HA-tagged hepatocystin, and protein synthesis was inhibited by incubation with cycloheximide (10 μg/mL). After 3, 6, and 9 hours, cells and supernatants were collected and immunoprecipitated by using anti-HA antibody and analyzed by SDS-PAGE, followed by immunoblot analysis with the same antibody to HA. Notice that truncated hepatocystin is secreted into the medium, whereas the full-length hepatocystin is not secreted in either assay. The positions of molecular mass markers (in kilodaltons) are shown on the right. Gastroenterology 2004 126, 1819-1827DOI: (10.1053/j.gastro.2004.02.023)