Biomedical subjects

M M Hussain

Publications and source records attributed to M M Hussain.

At least 37 records · Page 2Linked to original sources

PubMed1999

The mammalian low-density lipoprotein receptor family.

The low-density lipoprotein (LDL) receptor (LDL-R) family consists of cell-surface receptors that recognize extracellular ligands and internalize them for degradation by lysosomes. The LDL-R is the prototype of this family, which also contains very-low-density lipoprotein receptors (VLDL-R), apolipoprotein E receptor 2, LRP, and megalin. The family members contain four major structural modules: the cysteine-rich complement-type repeats, epidermal growth factor precursor-like repeats, a transmembrane domain, and a cytoplasmic domain. Each structural module serves distinct and important functions. These receptors bind several structurally dissimilar ligands. It is proposed that instead of a primary sequence, positive electrostatic potential in different ligands constitutes a receptor binding domain. This family of receptors plays crucial roles in various physiologic functions. LDL-R plays an important role in cholesterol homeostasis. Mutations cause familial hypercholesterolemia and premature coronary artery disease. LDL-R-related protein plays an important role in the clearance of plasma-activated alpha 2-macroglobulin and apolipoprotein E-enriched lipoproteins. It is essential for fetal development and has been associated with Alzheimer's disease. Megalin is the major receptor in absorptive epithelial cells of the proximal tubules and an antigenic determinant for Heymann nephritis in rats. Mutations in a chicken homolog of VLDL-R cause female sterility and premature atherosclerosis. This receptor is not expressed in liver tissue; however, transgenic expression of VLDL-R in liver corrects hypercholesterolemia in experiment animals, which suggests that it can be a candidate for gene therapy for various hyperlipidemias. The functional importance of individual receptors may lie in their differential tissue expression. The regulation of expression of these receptors occurs at the transcriptional level. Expression of the LDL-R is regulated by intracellular sterol levels involving novel membrane-bound transcription factors. Other members of the family are not regulated by sterols. All the members are, however, regulated by hormones and growth factors, but the mechanisms of regulation by hormones have not been elucidated. Studies of these receptors have provided important insights into receptor structure-function and mechanisms of ligand removal and catabolism. It is anticipated that increased knowledge about the LDL-R family members will open new avenues for the treatment of many disorders.

Amino Acid Sequence↗

PubMed1998

Amino acids 430-570 in apolipoprotein B are critical for its binding to microsomal triglyceride transfer protein.

Several studies have demonstrated protein-protein interactions between microsomal triglyceride transfer protein (MTP) and apolipoprotein B (apoB). However, the binding sites involved in these interactions have not been elucidated. To identify an MTP binding site in apoB, we have expressed several apoB sequences as fusion proteins with the eight-amino acid FLAG peptide. The chimeras were transiently expressed in COS cells, and conditioned media were used to study the binding of these sequences to either immobilized or soluble MTP. A polypeptide containing amino acids 270-570 (B:270-570), but not 1-300, bound to MTP. AGI-S17, an antagonist of apoB-MTP binding, inhibited the binding of B:270-570 to MTP but not to M2, a monoclonal antibody that recognizes the FLAG peptide. These data indicated that B:270-570 contains an MTP binding site. Next, sequences within 270-570 were subjected to C-terminal truncations at natural proline residues. B:270-509 bound less efficiently than B:270-570, whereas, B:270-430 and other shorter chimeras did not bind to MTP. Furthermore, truncations at amino acids 502 and 509 decreased MTP binding by 73 and 42%, respectively. These data indicate that B:430-570 in the alpha1-globular domain of apoB plays a crucial role in MTP binding and presumably in the initiation and maturation of apoB-containing lipoproteins.

Animals↗

PubMed1998

Lysine and arginine residues in the N-terminal 18% of apolipoprotein B are critical for its binding to microsomal triglyceride transfer protein.

Apolipoprotein B (apoB) and microsomal triglyceride transfer protein (MTP) are essential for the efficient assembly and secretion of triglyceride-rich lipoproteins. We have presented evidence for a high-affinity interaction between these proteins [Hussain, M. M., et al. (1997) Biochemistry 36, 13060-13067]. In this study, we used chemically modified low-density lipoproteins (LDL) and recombinant human apoB18 to identify amino acid residues in apoB that are critical for its interactions with MTP. Acetoacetylation of 74% of lysine residues and cyclohexanedione modification of 54% of arginine residues completely abolished the interactions between LDL and MTP. Regeneration of lysine and arginine residues by hydroxylamine treatment completely restored the binding of modified LDL to MTP. Carboxyethylation of all the histidine residues decreased, but did not abolish, apoB-MTP interactions. In contrast, glycine methyl ester modifications of aspartic and glutamic acid residues, up to 38-44%, had no effect on LDL-MTP interactions. Furthermore, modification of lysine and arginine, but not the aspartic and glutamic acid, residues in apoB18 also completely abolished its interactions with MTP. These studies indicated that lysine and arginine, but not aspartic and glutamic acid, residues are critical for apoB-MTP interactions, whereas histidine residues are not as critical. Since lysine and arginine residues in apoB are known to interact with the LDL receptors and heparin, we studied the effect of different glycosaminoglycans on apoB-MTP interactions. Glycosaminoglycans had no significant inhibitory effect on apoB-MTP interactions, suggesting that the lysine and arginine residues crucial for apoB-MTP interactions are different from those that interact with the LDL receptor and heparin. The lysine and arginine residues in apoB18 may directly interact with negatively charged residues in the MTP molecule, or they may function to maintain the conformation of the recognition site.

Apolipoproteins B↗

PubMed1998

Lysophosphatidylcholine increases apolipoprotein B secretion by enhancing lipid synthesis and decreasing its intracellular degradation in HepG2 cells.

Free fatty acids and lysophosphatidylcholine (lysoPC) are the major lipids bound to human plasma albumin. The effects of fatty acids on the hepatic production of Apolipoprotein B (apo B) have been studied but those of lysoPC have not. In HepG2 cells, lysoPC increased apo B secretion in different experiments by 50-120%, but did not affect the flotation properties of secreted lipoproteins. LysoPC affected neither the cellular protein levels nor apo A-I secretion suggesting that its effect was specific to apo B. Apo B secretion was maximum after incubating cells for 6 h with 0.2 mM lysoPC as equimolar fatty acid free bovine serum albumin (BSA) complexes. LysoPC was metabolized by cells and its fatty acids were used for the synthesis of phosphatidylcholine and triglycerides (TG). Experiments were performed to understand the mechanism of lysoPC action. LysoPC increased the incorporation of 3H-glycerol into newly synthesized cellular (3-fold) and secreted (4-fold) triglycerides, and increased the synthesis (40%) and secretion (4-fold) of phospholipids. LysoPC did not affect apo B synthesis, but inhibited the intracellular degradation of apo B and increased its secretion. Triacsin C (5 microM), an inhibitor of long chain acyl-CoA synthase, completely inhibited the induction of lipid synthesis and abolished the effect of lysoPC on apo B secretion. These studies indicated that lysoPC increased apo B secretion by inducing lipid synthesis; newly synthesized lipids probably protected apo B from intracellular degradation and enhanced secretion. These studies are consistent with the hypothesis that physiologic concentrations of lysoPC can be an important modulator for hepatic apo B secretion.

Apolipoprotein A-I↗

PubMed1997

Apolipoprotein B binding to microsomal triglyceride transfer protein decreases with increases in length and lipidation: implications in lipoprotein biosynthesis.

Microsomal triglyceride transfer protein (MTP), a heterodimer of 97 kDa and protein disulfide isomerase, is required for the assembly of apolipoprotein B (apoB)-containing triglyceride-rich lipoproteins. These proteins have been shown to interact with each other during early stages of lipoprotein biosynthesis. Our studies indicated that binding between apoB and heterodimeric MTP was of high affinity (Kd 10-30 nM) due to ionic interactions. In contrast to MTP, protein disulfide isomerase alone interacted very poorly with lipoproteins, indicating the importance of the heterodimer in these bindings. Preincubation of lipoproteins with detergents enhanced their interaction with MTP. Native VLDL bound poorly to MTP, but its preincubation with Tween-20 resulted in significantly increased binding to MTP. Furthermore, binding of LDL was enhanced by preincubation with taurocholate, indicating that partial delipidation of apoB-containing lipoproteins results in increased binding to MTP. Subsequently, attempts were made to study interactions between C-terminally truncated apoB polypeptides and MTP. Binding of all the polypeptides to MTP was enhanced in the presence of taurocholate. Comparisons revealed that the binding of different apoB polypeptides to MTP was in the order of apoB18 > apoB28 > apoB42 > apoB100. These studies indicated that optimum interactions occur between apoB18 and MTP, and that the increase in apoB length beyond apoB18 has a negative effect on these interactions. Since apoB18 does not assemble triglyceride-rich lipoproteins, these studies suggest that apoB may interact with MTP before its lipidation. It is proposed that steps in lipoprotein biosynthesis may be dictated by the sequential display of different functional domains on the apoB polypeptide.

Animals↗

PubMed1997

Nickel is a specific antagonist for the catabolism of activated alpha 2-macroglobulin.

The multifunctional low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP) binds and degrades several ligands involved in protease and lipoprotein metabolism. We previously reported that nickel (Ni2+) specifically inhibits the binding of activated alpha 2-macroglobulin (alpha 2 M*) at 4 degrees C to LRP and had no effect on the binding of other ligands to the receptor (Hussain et al. (1995) Biochem. 34, 16074-16081). In the current investigation, we have examined the effect of Ni2+ on the catabolism of 125 I-labeled alpha 2M*, receptor-associated protein (RAP) and lactoferrin at physiologic temperatures by fibroblasts. Nickel completely inhibited the degradation of alpha 2M* over a wide range of concentrations (0.3-2.4 nM); 50% inhibition for the degradation of 1.2 nM alpha 2M* was observed at 0.5 mM Ni2+. Furthermore, nickel inhibited the binding, internalization and degradation of 125I-alpha 2M* in a dose- and time-dependent manner. In contrast, the degradation of several concentrations of 125I-RAP by fibroblasts was not affected by different amounts of Ni2+ for various times. Similarly, Ni2+ did not inhibit the degradation of lactoferrin either before or after treating the cells with heparitinase to remove cell-surface proteoglycans. The degradation of lactoferrin was, however, inhibited by the RAP indicating that lactoferrin degradation was mediated by the LRP. These data suggest that Ni2+ is a specific inhibitor for the degradation of alpha 2M*.

Animals↗

PubMed1997

Measurement of apolipoprotein B in various cell lines: correlation between intracellular levels and rates of secretion.

We have standardized simple but sensitive enzyme-linked immunoassays to understand a relationship between intracellular levels and secretion rates of apoB. The assays were based on commercially available antibodies and were specific to human apoB. A monoclonal antibody, 1D1, was immobilized on microtiter wells and incubated with different amounts of low density lipoproteins to obtain a standard curve. Conditioned media were added to other wells in parallel, and the amount of apoB was quantitated from a linear regression curve. To standardize conditions for the measurement of intracellular apoB, cells were homogenized and solubilized with different concentrations of taurocholate. We found that 0.5% taurocholate was sufficient to solubilize all the apoB in HepG2, Caco-2, and McA-RH7777 cells. Next, a standard curve was prepared in the presence of taurocholate and used to determine intracellular levels of apoB in different cell lines. The intracellular levels (pmol/mg cell protein) and the rates of secretion (pmol/mg/h) of apoB100 were positively correlated (r2 = 0.81, P = 0.0009) in HepG2 cells. Furthermore, a positive correlation (r2 = 0.88, P < 0.0001) was found between intracellular and secreted apoB42 in stably transfected McA-RH7777 cells. In contrast, no correlation was observed for human apoB28 and apoB18 in stably transfected cells that were secreted either partially associated or completely unassociated with lipoproteins. These studies indicated that the rate of secretion of lipid-associated apoB, but not the lipid-free apoB, was tightly controlled.

Apolipoproteins B↗

PubMed1997

Assembly and secretion of VLDL in nondifferentiated Caco-2 cells stably transfected with human recombinant ApoB48 cDNA.

Intestinal cells secrete apoB48-containing very low density lipoproteins (VLDLs) and chylomicrons for the transport of biliary and dietary lipids. The molecular mechanisms regulating the assembly of intestinal lipoproteins are not known due to a lack of reliable and specific cell culture models. Caco-2 (a human colon carcinoma) cells have been used to study intestinal lipid metabolism. These cells have been shown to secrete both apoB100- and apoB48-containing triglyceride (TG)-rich lipoproteins only after differentiation into enterocyte-like cells. To study lipoprotein assembly in nondifferentiated Caco-2 cells, we stably expressed human recombinant apoB48 cDNA under the control of a constitutive cytomegalovirus promoter. Pulse-chase analysis revealed that the majority (> 50%) of apoB48 synthesized was degraded intracellularly in the presence or absence of oleic acid. Transfected nondifferentiated cells secreted lipoproteins with flotation densities similar to those of plasma HDL or LDL when cultured in serum-free or serum-containing media, respectively. Incubation of cells with media containing serum and oleic acid resulted in the secretion of VLDL-like particles. Secretion of VLDL was inhibited (> 80%) by triacsin C due to > 60% inhibition of oleate-induced TG synthesis. However, inhibition of cholesteryl ester synthesis by 70% with an acyl coenzyme A:cholesterol acyltransferase inhibitor did not affect VLDL secretion. Efficient assembly of lipoproteins usually requires the microsomal TG transfer protein (MTP). The presence of MTP in transfected Caco-2 cells was investigated by measuring TG transfer activity in microsomal fractions. Microsomal fractions had 0.2% TG transfer activity per hour per microgram of protein, which corresponds to 30% to 60% of the MTP activity present in liver-derived cells. To determine whether MTP activity was required for lipoprotein assembly, transfected cells were incubated in the presence of the MTP inhibitor CP-10,447. This compound completely abolished the secretion of apoB. These data show that the transfected cell lines secrete lipoproteins of different densities under different culture conditions and that the assembly of larger VLDL particles requires active TG synthesis and MTP activity. Thus, in nondifferentiated Caco-2 cells, the amount of apoB secreted and not the MTP activity is the limiting factor for lipoprotein assembly.

Apolipoprotein B-48↗

PubMed1997

Uptake of chylomicrons by the liver, but not by the bone marrow, is modulated by lipoprotein lipase activity.

We have shown that chylomicrons are catabolized by the liver and bone marrow in rabbits and marmosets. In the present investigation, we studied the role of various apolipoproteins and lipoprotein lipase in the clearance of these particles by the liver and bone marrow in rabbits. Incubation of chylomicrons with purified apolipoprotein (apo) E or C-II resulted in more rapid clearance of these particles from the plasma, whereas incubation of chylomicrons with apoA-I, apoC-I, apoC-III1, or apoC-III2, did not affect their clearance rates. Analysis of tissue uptake revealed that the increased plasma clearance rate of chylomicrons enriched with apoE or apoC-II was primarily due to enhanced uptake by the liver. The uptake of chylomicrons by the bone marrow increased after their enrichment with apoA-I but decreased after their enrichment with apoC-II. Because apoC-II is a cofactor for lipoprotein lipase, we hypothesized that the increased clearance rates were due to faster hydrolysis of chylomicrons and rapid generation of chylomicron remnants. To test this hypothesis, lipoprotein lipase activity was inhibited by injection of an antilipoprotein lipase monoclonal antibody. Inhibition of lipoprotein lipase retarded clearance of chylomicrons from the plasma and decreased their uptake by the liver but did not affect their uptake by the bone marrow. These studies suggest that bone marrow can take up chylomicrons in the absence of lipoprotein lipase activity and provide an explanation for the presence of foam cells in the bone marrow of type I hyperlipoproteinemic patients.

Animals↗

PubMed1996

Up-regulation of the low density lipoprotein receptor-related protein by dexamethasone in HepG2 cells.

Dexamethasone has been shown to decrease the expression of the low density lipoprotein (LDL) receptor, but its effect on other members of the LDL receptor family is not known. We studied the effect of dexamethasone in HepG2 cells on the expression of the LDL receptor family members using radiolabeled receptor associated protein (RAP) which binds to all the members of the family. Treatment of HepG2 cells with increasing concentrations of dexamethasone resulted in a 2-fold increase in the binding and degradation of RAP. To identify the receptor responsible for the increased binding and degradation of RAP, we used specific ligands. For LDL receptor, we used LDL itself. For the LDL receptor-related protein/alpha 2-macroglobulin receptor, we used activated alpha 2-macroglobulin. The binding of LDL to HepG2 cells was decreased, whereas binding and degradation of activated alpha 2-macroglobulin was increased by 2-fold suggesting that dexamethasone increased LRP expression. Increased LRP expression was positively correlated with the increase in the steady-state levels and transcript numbers of the LRP mRNA; no changes in RAP or gamma-actin mRNA levels were observed. Increased mRNA levels were not due to an increased rate of transcription of the gene as assessed by nuclear run-on experiments. These studies indicate that dexamethasone increases cell-surface LRP activity in HepG2 cells by increasing the steady state mRNA levels and suggest that post-transcriptional mechanisms play a role in controlling LRP mRNA levels.

Cell Line↗

PubMed1996

Chylomicron assembly and catabolism: role of apolipoproteins and receptors.

Chylomicrons are lipoproteins synthesized exclusively by the intestine to transport dietary fat and fat-soluble vitamins. Synthesis of apoB48, a translational product of the apob gene, is required for the assembly of chylomicrons. The apob gene transcription in the intestine results in 14 and 7 kb mRNAs. These mRNAs are post-transcriptionally edited creating a stop codon. The edited mRNAs chylomicrons from the shorter apoB48 peptide remains to be elucidated. In addition, the roles of proteins involved in the assembly pathway, e.g. apobec-1, MTP and apoA-IV, needs to be studied. Cloning of enzymes involved in the intestinal biosynthesis of triglycerides will be crucial to fully appreciate the assembly of chylomicrons. There is a need for cell culture and transgenic animal models that can be used for intestinal lipoprotein assembly. The catabolism of chylomicrons is far more complex and efficient than the catabolism of VLDL. Even though the major steps involved in the catabolism of chylomicrons are now known, the determinants for apolipoprotein exchange, processing of remnants in the space of Disse, as well as the mechanism of uptake of these particles by extra-hepatic tissue needs further exploration.

Animals↗

PubMed1996

In situ hybridization studies of alpha 2-macroglobulin receptor and receptor-associated protein in human prostate carcinoma.

We have utilized in situ hybridization and immunocytochemistry techniques to examine the expression of both alpha 2-macroglobulin (alpha 2-MR) and the 39 kDa receptor-associated protein (RAP) in 8 benign (BPH) and 34 malignant human prostate tissues, including 4 metastases. The levels of alpha 2-MR mRNA expression (but not RAP) increased significantly in high Gleason score carcinomas ( > 8) and in metastatic lesions, suggesting that alpha 2-MR expression is associated with advanced cancer. Semi-quantitative analysis with computer-assisted system analysis (CASA) confirmed this interpretation. This is the first report of alpha 2-MR expression being associated with advanced prostate cancer.

Base Sequence↗

PubMed1996

Corynebacterium pseudotuberculosis infection and lymphadenitis (taloa or mala) in the camel.

Pure cultures of Corynebacterium pseudotuberculosis were obtained from 11 cases of lymphadenitis (known locally as taloa or mala) in camels. Camel isolates produced typical taloa in camels experimentally inoculated subcutaneously at the base of the external ear with 10(10) colony forming units. A sheep strain of C. pseudotuberculosis inoculated into camels produced a local abscess at the site of inoculation but did not produce taloa. Re-infection of camels recovered from experimental inoculation did not produce taloa suggesting the possibility of the development of a vaccine against lymphadenitis in camels.

Abscess↗

PubMed1995

Nickel is a specific inhibitor for the binding of activated alpha 2-macroglobulin to the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor.

The low density receptor-related protein/alpha 2-macroglobulin receptor (LRP/alpha 2-MR) binds to several ligands involved in lipoprotein and protease clearance. The receptor-associated protein (RAP) inhibits the binding of all known ligands. We studied the inhibition by Ni2+ of the binding of different ligands to cells and to the purified LRP/alpha 2-MR. Ni2+ inhibited all of the specific binding of radiolabeled methylamine-activated alpha 2-macroglobulin (125I-alpha 2-M*) to rabbit aortic smooth muscle cells (SMC), rat hepatoma Fu5AH, and mouse fibroblast L cells. Ni2+ also inhibited the binding of trypsin-activated alpha 2-macroglobulin to SMC but did not affect the binding of RAP, Pseudomonas exotoxin A, or low-density lipoproteins. The inhibition of alpha 2-M* binding by Ni2+ was not due to its interaction with alpha 2-M*. Preincubation of SMC with Ni2+ followed by ligand binding suggested that Ni2+ binds to cell-surface molecules and inhibits the binding of alpha 2-M* but does not affect RAP binding. Most of the binding of alpha 2-M* to SMC was due to its binding to the LRP/alpha 2-MR, as opposed to the recently described signaling receptor, as demonstrated by the inhibition of this binding by the RAP. Moreover, the inhibition of alpha 2-M* binding to the LRP/alpha 2-MR by Ni2+ was demonstrated using purified receptor immobilized on microtiter plates. Two to three molecules of 63Ni2+ bound to the immobilized receptor with equal affinity but not to alpha 2-M*. The specific binding of alpha 2-M* to the immobilized receptor was inhibited in the presence of nickel.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Chylomicron metabolism in normal, cholesterol-fed, and Watanabe heritable hyperlipidemic rabbits. Saturation of the sequestration step of the remnant clearance pathway.

The plasma clearance of radiolabeled chylomicrons was compared in normal, cholesterol-fed, and Watanabe heritable hyperlipidemic (WHHL) rabbits. Chylomicron clearance was rapid in normal rabbits but was significantly retarded in cholesterol-fed and WHHL rabbits. At 40 min after the injection of chylomicrons, 14-17% of the injected dose remained in the plasma of normal rabbits, whereas approximately 40-50% of the injected dose remained in the plasma of cholesterol-fed and WHHL rabbits. The differences were reflected in the reduced plasma clearance by the liver and bone marrow of the cholesterol-fed and WHHL rabbits. The hyperlipidemic rabbits expressed normal levels of low density lipoprotein (LDL) receptor-related protein/alpha 2-macroglobulin receptor in the liver. In contrast, the hepatic levels of LDL receptors were lower in hyperlipidemic rabbits; as expected, they were significantly lower in WHHL rabbits compared with normal and cholesterol-fed rabbits. Furthermore, it was demonstrated that lipoproteins accumulating in the plasma of the hyperlipidemic rabbits competed for and retarded the clearance of chylomicrons from the plasma. Competition was demonstrated by cross-circulation of normal and cholesterol-fed or normal and WHHL rabbits, in which the rapid influx of plasma containing the accumulated plasma lipoproteins from cholesterol-fed or WHHL rabbits was shown to impair the uptake of chylomicrons by the liver and bone marrow of normal rabbits. These observations were extended by infusing isolated lipoproteins into normal rabbits. The rabbit d < 1.02 g/ml (remnant) fraction and the canine cholesterol-rich high density lipoproteins (HDL) with apolipoprotein E (HDLc) inhibited chylomicron clearance, whereas human LDL and HDL from humans and rabbits did not. We conclude that the low LDL receptor activity in the cholesterol-fed and WHHL rabbits may contribute, at least in part, to the impaired clearance by decreasing remnant uptake and causing the accumulation of chylomicron and/or very low density lipoprotein remnants. The accumulated remnant lipoproteins then compete for and saturate the mechanism responsible for the initial rapid clearance of chylomicrons from the plasma. We speculate that saturation of the initial rapid clearance may occur at the sequestration step, which involves the binding of remnants to heparan sulfate proteoglycans in the space of Disse.

Animals↗

PubMed1995

Characterization of recombinant human apoB-48-containing lipoproteins in rat hepatoma McA-RH7777 cells transfected with apoB-48 cDNA. Overexpression of apoB-48 decreases synthesis of endogenous apoB-100.

We studied the effect of overexpression of apolipoprotein (apo) B-48 on the synthesis and secretion of endogenous apoB-100 in rat hepatoma McA-RH7777 cell lines stably transfected with human apoB-48 cDNA under the control of the cytomegalovirus promoter. Three cell lines that secrete 40 to 60 ng human apoB.mg cell protein-1.h-1 were used. The recombinant human apoB-48 exhibited physicochemical characteristics (buoyant density, 1.06 to 1.21 g/mL; beta-electrophoretic mobility and diameters, 16 to 20 nm) indistinguishable from those of endogenous rat apoB-48. Overexpression of the recombinant human apoB-48 resulted in a 50% decrease in the secretion of endogenous apoB-100 but did not affect the secretion of apoE or apoA-I. Several possible mechanisms for the decreased secretion of apoB-100 were evaluated. First, recruitment of lipids into lipoproteins was shown to be unaffected since no major changes in the physicochemical properties of apoB-100-containing lipoproteins were observed. Second, the intracellular degradation of apoB-100 was not altered as the intracellular retention half-time and secretion efficiency remained unaffected by apoB-48 overexpression. Third, the posttranslational regulatory mechanisms for apoB-100 remained normal, as demonstrated by a twofold increase in apoB-100 secretion after supplementation with oleic acid. Unexpectedly, a 35% to 50% decrease in the steady-state synthesis of endogenous apoB-100 was observed in apoB-48-transfected cells compared with control cells. These data suggested that decreased secretion of apoB-100 was secondary to decreased synthesis. The decreased apoB-100 synthesis was not due to decreased steady-state levels of rat apoB-100 mRNA. These results suggest that overexpression of recombinant human apoB-48 may interfere with posttranscriptional events, possibly at the translation-translocation level, and decrease translational yield of apoB-100. These posttranscriptional events prior to the complete synthesis of the apoB-100 polypeptide can be important in the control of apoB-100 secretion.

Amino Acid Sequence↗

PubMed1994

Decreased expression of the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor in invasive cell clones derived from human prostate and breast tumor cells.

The importance of receptors involved in the clearance of proteases and protease/inhibitor complexes in tumor invasion is unknown. We studied the expression of the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP/alpha 2-MR), the major receptor involved in the clearance of protease/inhibitor complexes, in invasive and noninvasive subclones derived from tumor cells. The receptor activity was 2- to 3-fold lower in invasive subclones compared to noninvasive subclones derived from human prostate PC-3 and DU 145 and melanoma A2058 cells. The receptor activity was decreased in breast cancer (MCF-10A) cells transfected with mutated Ha-ras compared to nontransfected cells. Furthermore, invasive cells derived from ras-transfected MCF-10A cells expressed lower levels of receptor compared to their non-invasive counterparts. These studies indicate a correlation between invasive phenotype and low receptor expression in different tumor cells. Experiments were performed to understand two possible mechanisms (decreased transcription of the receptor and increased transcription of an inhibitory protein) for the decreased cell-surface expression of the receptor in invasive cells. The decreased expression in invasive subclones was due to 2- to 3-fold lower levels of LRP/alpha 2-MR mRNA in all cells. In invasive PC-3 subclones, but not in DU 145, A2058, and MCF-10A subclones, 2-fold higher levels of the 39 kDa receptor-associated protein (an inhibitor of the receptor) mRNA were observed. These studies showed that the decreased expression of LRP/alpha 2-MR activity in invasive subclones was generally correlated with the decreased steady-state mRNA levels of the receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗

PubMed1993

Role of heparan sulfate proteoglycans in the binding and uptake of apolipoprotein E-enriched remnant lipoproteins by cultured cells.

Addition of apolipoprotein (apo) E to rabbit beta-very low density lipoproteins (beta-VLDL) has been shown to result in a marked enhancement of their binding and uptake by various cell types. Apolipoprotein E binds to lipoprotein receptors and proteoglycans. To distinguish between apoE binding to these sites, cells were treated with heparinase. Heparinase treatment of receptor-negative familial hypercholesterolemic (FH) fibroblasts and human hepatoma cells (HepG2) released 30-40% of newly synthesized cell surface 35S-labeled proteoglycans and decreased the binding of beta-VLDL+apoE to FH and normal fibroblasts and HepG2 cells by more than 80%. Furthermore, heparinase treatment significantly decreased the uptake of fluorescently labeled beta-VLDL+apoE by HepG2 cells and decreased cholesteryl ester synthesis in FH fibroblasts by 75%. Likewise, canine chylomicron remnants enriched in apoE demonstrated enhanced binding that was 80% inhibited by heparinase treatment of HepG2 cells. Heparinase treatment did not affect beta-VLDL (without added apoE) or low density lipoprotein (LDL) binding to these cells or the binding activity of beta-VLDL+apoE to the LDL receptor-related protein (LRP) or to the LDL receptor on ligand blots. Chinese hamster ovary (CHO) mutant cells lacking the synthesis of either heparan sulfate (pgsD-677) or all proteoglycans (pgsA-745) did not display any enhanced binding of the beta-VLDL+apoE. By comparison, wild-type CHO cells demonstrated enhanced binding of beta-VLDL+apoE that could be abolished by treatment with heparinase. These mutant cells and wild-type CHO cells possessed a similar amount of LRP, as determined by ligand blot analyses and by alpha 2-macroglobulin binding, and possessed a similar amount of LDL receptor activity, as determined by LDL binding. Therefore, we would interpret these data as showing that heparan sulfate proteoglycan may be involved in the initial binding of the apoE-enriched remnants with the subsequent involvement of the LRP in the uptake of these lipoproteins. It remains to be determined whether the heparan sulfate proteoglycan can function by itself in both the binding and internalization of the apoE-enriched remnants or whether the proteoglycan is part of a complex with LRP that mediates a two-step process, i.e. binding and subsequent internalization by the receptor.

Animals↗