Differentiation of Intrahepatic Membrane-Bound and Secretory Apolipoprotein B by Monoclonal Antibodies: Membrane-Bound Apolipoprotein B Is More Glycosylated

Wong, Laurence; Torbati, Aliza

doi:10.1021/bi00173a040

Cited by 9 publications

(3 citation statements)

References 26 publications

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“…6) suggested strongly that the secretion-competent VLDL particles utilize membrane-associated apoB100 during assembly. The observed difference in Endo H sensitivity between medium (Endo H-resistant) and membrane-associated apoB100 (Endo H-sensitive) is reminiscent of a previous report that membrane-bound apoB in rat hepatocytes had oligosaccharide moieties distinct from that of apoB in the plasma (31). Although evidence abounds, the significance of apoB association with membranes during VLDL assembly is unknown, nor is the physical nature of apoB-membrane interactions clear.…”

Section: Discussionsupporting

confidence: 49%

Intracellular Assembly of Very Low Density Lipoproteins Containing Apolipoprotein B100 in Rat Hepatoma McA-RH7777 Cells

Tran¹,

Thorne-Tjomsland²,

DeLong³

et al. 2002

Journal of Biological Chemistry

109

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Previous studies with McA-RH7777 cells showed a 15-20-min temporal delay in the oleate treatmentinduced assembly of very low density lipoproteins (VLDL) after apolipoprotein (apo) B100 translation, suggesting a post-translational process. Here, we determined whether the post-translational assembly of apoB100-VLDL occurred within the endoplasmic reticulum (ER) or in post-ER compartments using biochemical and microscopic techniques. At steady state, apoB100 distributed throughout ER and Golgi, which were fractionated by Nycodenz gradient centrifugation. Pulsechase experiments showed that it took about 20 min for newly synthesized apoB100 to exit the ER and to accumulate in the cis/medial Golgi. At the end of a subsequent 20-min chase, a small fraction of apoB100 accumulated in the distal Golgi, and a large amount of apoB100 was secreted into the medium as VLDL. VLDL was not detected either in the lumen of ER or in that of cis/ medial Golgi where apoB100 was membrane-associated and sensitive to endoglycosidase H treatment. In contrast, VLDL particles were found in the lumen of the distal Golgi where apoB100 was resistant to endoglycosidase H. Formation of lumenal VLDL almost coincided with the appearance of VLDL in the medium, suggesting that the site of VLDL assembly is proximal to the site of secretion. When microsomal triglyceride transfer protein activity was inactivated after apoB had exited the ER, VLDL formation in the distal Golgi and its subsequent secretion was unaffected. Lipid analysis by tandem mass spectrometry showed that oleate treatment increased the masses of membrane phosphatidylcholine (by 68%) and phosphatidylethanolamine (by 27%) and altered the membrane phospholipid profiles of ER and Golgi. Taken together, these results suggest that VLDL assembly in McA-RH7777 cells takes place in compartments at the distal end of the secretory pathway.

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Section: Discussionsupporting

confidence: 49%

Intracellular Assembly of Very Low Density Lipoproteins Containing Apolipoprotein B100 in Rat Hepatoma McA-RH7777 Cells

Tran¹,

Thorne-Tjomsland²,

DeLong³

et al. 2002

Journal of Biological Chemistry

109

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“…Hence, apoE appears segregated from albumin in the hepatic Golgi apparatus. In a less extensive study, apoB antigenicity (using anti-rat apoB characterized as in Wong and Pino (1987) and Wong and Torbati (1994)) was restricted to the saccular distensions of the Golgi (Fig. 6, c and d).…”

Section: Heterogeneous Distribution Of Apoe But Not Albumin In Golgi Apparatusmentioning

confidence: 96%

Concentration of intracellular hepatic apolipoprotein E in Golgi apparatus saccular distensions and endosomes.

Dahan

Ahluwalia

Wong

et al. 1994

The Journal of Cell Biology

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Abstract. The intrahepatic distribution of apolipoprotein E has been assessed by immunogold labeling of cryosections as well as by Western blotting of organelles isolated from liver homogenates. Both techniques supported the prior analytical fractionation studies of Wong (1989) who concluded that intrahepatic apoE was largely endosomal. All endosomal components decorated by gold particles indicative of apoE antigenicity in cryosections appeared filled with lipoprotein-like particles thereby accounting for this prominent morphological feature of isolated liver endosomes. The distribution of gold particles about the hepatic Golgi apparatus revealed a high content of apoE in closely apposed endosomes, ca. 400 nm in diameter, double labeled for apoE and internalized HRP. Remarkably, apoE (but not internalized HRP) was also observed within saccular distensions of all saccules of stacked Golgi cisternae but absent from the flattened saccular components as was also observed for apoB. This contrasted with albumin, the major secretory protein, which was uniformly distributed throughout the hepatic Golgi apparatus. These observations support a growing body of evidence for intra-Golgi sorting of secretory material in hepatic Golgi apparatus. The lack of any immunoreactive apoE or albumin in small 70-90 nm vesicles about the Golgi cisternae suggests limits to current models of vesicle-mediated intra-Golgi transport.

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“…In recent studies, a majority of endogenous apoB was protected from exogenous protease in membrane vesicles isolated from HepG2 cells (Ingram and Shelness, 1996;Leiper et al, 1996). Also, the extent of apoB glycosylation suggested that apoB was fully translocated into the lumen of the ER (Wong and Torbati, 1994;Leiper et al, 1996).…”

mentioning

confidence: 99%

Identification of Two Regions in Apolipoprotein B100 that are Exposed on the Cytosolic Side of the Endoplasmic Reticulum Membrane

Stoops

Mertz

et al. 1998

The Journal of Cell Biology

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Protease protection assays of apolipoprotein B100 (apoB) in digitonin-permeabilized HepG2 cells indicated that multiple domains of apoB are exposed to the cytosol through an extensive portion of the secretory pathway. The intracellular orientation of apoB in the secretory pathway was confirmed by immunocytochemistry using antibodies recognizing specific domains of apoB in streptolysin-O (STP-O)– and saponin-permeabilized HepG2 cells. Lumenal epitopes on marker proteins in secretory pathway compartments (p63, p53, and galactosyltransferase) were not stained by antibodies in STP-O–treated cells, but were brightly stained in saponin-treated cells, confirming that internal membranes were not perforated in STP-O–treated cells. An anti-apoB peptide antibody (B4) recognizing amino acids 3221–3240 caused intense staining in close proximity to the nuclear membrane, and less intensely throughout the secretory pathway in STP-O–permeabilized cells. Staining with this antibody was similar in STP-O– and saponin-treated cells, indicating that this epitope in apoB is exposed to the cytosol at the site of apoB synthesis and throughout most of the remaining secretory pathway. Similar results indicating a cytosolic orientation were obtained with monoclonal antibody CC3.4, which recognizes amino acids 690–797 (79–91 kD) in apoB. Two polyclonal antibodies made to human LDL and two monoclonal antibodies recognizing amino acids 1878–2148 (D7.2) and 3214–3506 (B1B6) in apoB did not produce a strong reticular signal for apoB in STP-O–treated cells. The anti-LDL and B1B6 antibodies produced almost identical punctate patterns in STP-O–treated cells that overlapped with LAMP-1, a membrane marker for lysosomes. These observations suggest that the B1B6 epitope of apoB is exposed on the surface of the lysosome. The results identify two specific regions in apoB that are exposed to the cytosol in the secretory pathway.

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Differentiation of Intrahepatic Membrane-Bound and Secretory Apolipoprotein B by Monoclonal Antibodies: Membrane-Bound Apolipoprotein B Is More Glycosylated

Cited by 9 publications

References 26 publications

Intracellular Assembly of Very Low Density Lipoproteins Containing Apolipoprotein B100 in Rat Hepatoma McA-RH7777 Cells

Intracellular Assembly of Very Low Density Lipoproteins Containing Apolipoprotein B100 in Rat Hepatoma McA-RH7777 Cells

Concentration of intracellular hepatic apolipoprotein E in Golgi apparatus saccular distensions and endosomes.

Identification of Two Regions in Apolipoprotein B100 that are Exposed on the Cytosolic Side of the Endoplasmic Reticulum Membrane

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