M.R. Lakshman

Alcohol and molecular regulation of protein glycosylation and function

Chronic alcohol exposure leads to the appearance of carbohydrate-deficient transferrin (CDT), a N-glycosylated protein and sialic acid-deficient apolipoprotein E (apoE), an O-glycosylated protein. We show that chronic ethanol treatment destabilizes sialyltransferase (ST) mRNA resulting in a concomitant decreased steady-state level of ST mRNA. As a result, alcohol markedly decreases the hepatic synthetic rate of ST. This leads to impaired sialylation of transferrin and apoE. Consequently, apoE content in plasma high-density lipoproteins (HDL) is decreased. ApoE plays a significant role in the delivery of HDL cholesterol to the liver via apo B/E receptor, a process called reverse cholesterol transport (RCT). Desialylation of apoE results in its decreased association with HDL. Thus, the dissociation constant of HDL for binding to sialo-apoE is 90 +/- 35 nM, whereas that for desialo-apoE is 1010 +/- 250 nM. More importantly, the uptake of labeled cholesterol by human HepG2 cells is decreased by 30-40% from reconstituted HDL particles (rHDL)-containing desialo-apoE compared to rHDL with sialo-apoE. We conclude that chronic alcohol exposure down-regulates the expression of sialyltransferase genes resulting in impaired sialylation of apoE. This leads to its decreased binding to plasma HDL and thereby, impairs the RCT function of HDL.

Long-term ethanol exposure impairs glycosylation of both N- and O-glycosylated proteins in rat liver

Carbohydrate residues of glycoproteins play important roles in their functions. We have previously shown that long-term ethanol treatment in rats alters the normal glycosylation pattern of plasma transferrin and apolipoprotein (apo) E. Glycosylation of proteins is a posttranslational process that is regulated by both glycosyltransferases and glycosidases, the resident enzymes of hepatic subcellular organelles. In this investigation using rat transferrin and apo E as model N- and O-glycosylated proteins, respectively, we have explored the effects of long-term ethanol treatment on the (1) incorporation of various labeled sugar precursors into these specific glycoproteins, (2) activities of mannosyltransferase, galactosyltransferase, and sialytransferases, and (3) hepatic synthetic rate of N-acetyl glucosamine (GlcNAc) alpha 2,6-sialyltransferase (2,6-ST). The relative ratio of labeled sugar to leucine incorporation (glycosylation index) showed a 43% (P < .01) decrease for relative mannosylation of transferrin molecule at both the microsomal and Golgi level in the ethanol group (AN) versus the control group (CN). For apo E, relative mannosylation was reduced by 48.9% (P < .01) and 46.9% (P < .01), respectively, at the microsomal and Golgi level in the AN versus CN. More importantly, relative sialation of transferrin was reduced by 86% (P < .001) in AN as compared with CN. Relative sialation of apo E was reduced by 35% (P < .01) in AN as compared with CN.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and Partial Characterization of a Cellular Carotenoid-binding Protein from Ferret Liver

A cellular carotenoid-binding protein was purified to homogeneity from β-carotene-fed ferret liver utilizing the following steps: ammonium sulfate precipitation, ion exchange, gel filtration, and affinity chromatography. The final purification was 607-fold. [14C]β-Carotene co-purified with the binding protein throughout the purification procedures. SDS-PAGE of the purified protein showed a single band with an apparent molecular mass of 67 kDa. Scatchard analysis of the specific binding of the purified protein to β-carotene showed two classes of binding sites, a high affinity site with an apparent K d of 56 × 10−9 m and a low affinity site with aK d of 32 × 10−6 m. The B max for β-carotene binding to the high affinity site was 1 mol/mol, while that for the low affinity site was 145 mol/mol. The absorption spectrum of the complex showed a 32-nm bathochromic shift in λmax with minor peaks at 460 and 516 nm. Except for α-carotene and cryptoxanthin, none of the model carotenoids or retinol competed with β-carotene binding to the protein. Thus, a specific carotenoid-binding protein of 67 kDa has been characterized in mammalian liver with a high degree of specificity for binding only carotenoids with at least one unsubstituted β-ionone ring.

Effect of dietary omega-3 fatty acids and chronic ethanol consumption on reverse cholesterol transport in rats.

We previously showed that chronic ethanol feeding leads to a decrease of apolipoprotein E (apoE) in high-density lipoprotein (HDL), whereas supplementing this diet with 2.8% of total dietary calories as omega3-fatty acids (omega3FAs) restores HDL-apoE to the control values. Since HDL containing apoE plays a major role in reverse cholesterol transport (RCT), we measured the effects chronic ethanol intake and omega3-FAs on RCT in the present study. Four groups of rats, control normal fat (CN), alcohol-normal fat (AN), control omega3FA fat (CF), and alcohol-omega3FA fat (AF), were fed their respective diets for 8 weeks, after which hepatocytes and HDLs from each group were evaluated for RCT capacity (cholesterol efflux from macrophages and uptake by liver cells). Compared with the control diet (CN), chronic ethanol (AN) feeding inhibited the cholesterol efflux capacity of HDL by 21% (P < .01), whereas omega3FA feeding (2.8% of total dietary calories) stimulated this capacity by 79% (P < .01) and 25% (P < .01) in CF and AF rats, respectively. With respect to cholesterol uptake by the liver, there were no significant 3-way or 4-way interactions between the 4 factors, HDL-alcohol, HDL-fish oil, hepatocyte-alcohol, and hepatocyte-fish oil. The main effects for HDL-alcohol, HDL-fish oil, and hepatocyte-alcohol were all highly significant (P = .0001, .0001, and .007, respectively). There was a significant HDL-alcohol and HDL-fish oil interaction (P = .0001). Hepatocyte-alcohol was not a factor in any 2-way interactions. Our study indicates no evidence of an interaction between the effects of omega3FAs and the effects of alcohol on hepatocytes in terms of RCT function. Thus, feeding as little as 2.8% of the total dietary calories as omega3FA not only restored the impaired RCT function of HDL caused by chronic ethanol intake, but also enhanced by severalfold the ability of HDL to promote RCT even in normal animals.

The effects of dietary taurocholate, fat, protein, and carbohydrate on the distribution and fate of dietary β‐carotene in ferrets

Dietary beta-carotene has been shown to have cancer chemopreventive action on the basis of epidemiologic evidence and studies in animals. Because the anticarcinogenic property of beta-carotene may be exerted per se, it is desirable to achieve the maximum absorption and accumulation of intact beta-carotene in various parts of the body. Therefore the effects of dietary taurocholate, fat, protein, and carbohydrate on the absorption, accumulation, and fate of dietary beta-carotene (3.730 nmol/g diet) in selected tissues of ferrets were explored. Taurocholate (0.2-1.0% wt/wt) and fat (6-23% wt/wt) caused two- to threefold (p < 0.05) increases in the absorption and accumulation of beta-carotene in the liver, lungs, and adipose tissue in a dose-dependent manner. In contrast, neither dietary protein (10-40% wt/wt) nor carbohydrate (25-55% wt/wt) affected the absorption and accumulation of beta-carotene in various tissues. Significantly, taurocholate, 23% fat, or 40% protein also markedly increased the amounts of hepatic retinol and retinyl esters derived from dietary beta-carotene. These results indicate that dietary taurocholate, fat, and high protein have a marked influence on the exposure of beta-carotene to intestinal carotene cleavage enzyme or its activity. Thus an ideal combination of dietary components (wt/wt) in ferrets for the maximal absorption and accumulation of beta-carotene in different tissues is 0.5% taurocholate and 13.4% fat, whereas 1% taurocholate, 23% fat, or 40% protein stimulates its conversion to vitamin A.

[23] Enzymatic conversion of all-trans-β-carotene to retinal

Enzymatic conversion of all-trans-beta-carotene to retinal by a partially purified enzyme from rabbit, rat, and human neonatal intestinal mucosa has been demonstrated. The enzymatic product was characterized based on the following evidence. First, the product gave rise to its O-ethyl oxime by treatment with O-ethylhydroxylamine with an absorption maximum at 363 nm in ethanol characteristic of authentic retinal (O-ethyl) oxime. High-performance liquid chromatography of this derivative yielded a sharp peak with a retention time of 7.99 min, corresponding to the authentic compound. The enzyme blank and boiled enzyme blank failed to show any significant HPLC peaks corresponding to retinal (O-ethyl) oxime or retinal or retinol. Second, the mass spectrum of the O-ethyl oxime of the enzymatic product was identical to that of authentic retinal (O-ethyl) oxime (m/z 327, 45%; m+ and m/z 282, 100%, methoxy). Third, the 14C radioactivity persisted to constant specific activity even after repeated crystallization of the retinal (O-ethyl) oxime isolated from the enzyme reaction with purified beta-[14C]carotene. Fourth, the enzymatic product exhibited an absorption maximum at 370 nm in light petroleum characteristic of authentic retinal. Furthermore, it was reduced by horse liver alcohol dehydrogenase to retinol with an absorption maximum at 326 nm in light petroleum. This retinol was enzymatically esterified to retinyl palmitate by rat pancreatic esterase with a retention time of 10 min on HPLC, corresponding to authentic retinyl palmitate. Thus, the enzymatic product of beta-carotene cleavage by the partially purified intestinal enzyme has been unequivocally confirmed to be retinal. Similarly, enzymatic conversion of all-trans-beta-carotene to retinal by an intestinal mucosal enzyme from autopsy samples of human neonates has also been demonstrated. Based on the observed activities among intestinal samples from 12 premature infants, the BCC enzyme activity ranged from 3.3 to 1210 pmol/mg mucosal protein/hr. However, the observed activities in the human autopsy samples may be markedly underestimated, presumably because of marked loss of enzyme activity from the time of death to the time of assay. Therefore, the true activity of the enzyme can be assessed only after the extent of the loss of its activity on storage of the human samples can be accurately measured. Nonetheless, the demonstration of BCC enzyme activity in human neonates shows that beta-carotene may be an important source of vitamin A nutrition during gestation.

Purification and characterization of cellular carotenoid-binding protein from mammalian liver.

A cellular carotenoid-binding protein was purified to homogeneity from beta-carotene-fed ferret liver utilizing the following steps: ammonium sulfate precipitation, ion exchange, gel filtration, and affinity chromatography. The final purification was 607-fold. beta-[14C]Carotene copurified with the binding protein throughout the purification procedures. SDS-PAGE of the purified protein showed a single band with an apparent molecular mass of 67 kDa. Scatchard analysis of the specific binding of the purified protein to beta-carotene showed two classes of binding sites; a high-affinity site with an apparent Kd of 56 x 10(-9) M and a low-affinity site with a Kd of 32 x 10(-6) M. The Bmax for beta-carotene binding to the high-affinity site was 1 mol/mol whereas that for the low-affinity site was 145 mol/mol. The absorption spectrum of the complex showed a 32-nm bathochromic shift in lambda max with minor peaks at 460 and 516 nm. Except for alpha-carotene and cryptoxanthin, none of the model carotenoids or retinol competed with beta-carotene binding to the protein. Thus, a specific carotenoid-binding protein of 67 kDa size has been characterized in mammalian liver with a high degree of specificity for binding only carotenoids with at least one unsubstituted beta-ionone ring.

Chronic ethanol consumption leads to destabilization of rat liver β-galactoside α2,6-sialyltransferase mRNA

Chronic ethanol consumption in rats is accompanied by decreased levels of Galβ1,4GlcNAc α2,6-sialyltransferase (2,6-ST) activity in the liver. Our previous studies have shown that there is a concomitant decrease in the levels of 2,6-ST mRNA. In this study, the alteration in the regulation of 2,6-ST expression by chronic ethanol consumption was assessed by Northern hybridization, nuclear run-on experiments, and 2,6-ST mRNA stability studies. 2,6-ST downregulation was found at 4 weeks of feeding an ethanol diet (36% of calories from ethanol) and remained up to 8 weeks. The decrease in 2,6-ST mRNA levels was found to be dose-dependent, with lower dose of ethanol (12% and 24% of total dietary calories from ethanol) being ineffective and the effects being manifested only when 36% of the dietary calories were from ethanol. The effects of chronic ethanol feeding could be completely reversed within 1 week after ethanol consumption was stopped, when 2,6-ST mRNA levels were restored to normal. The downregulation was not sensitive to actinomycin D, indicating that the regulation was not affected at the transcriptional level but at the posttranscriptional level. This was confirmed by nuclear run-on experiments showing that the rate of 2,6-ST mRNA transcription was unaffected by ethanol. Finally, mRNA stability experiments showed that the half-life of 2,6-ST mRNA was reduced 50% in ethanol-fed rat livers compared with control rat livers. Taken together, the results show that 2,6-ST mRNA is regulated at the posttranscriptional level and chronic ethanol intake downregulates 2,6-ST expression by destabilizing its mRNA.

Carotenoid-protein complexes.

This chapter provides an updated report of carotenoprotein-related research. Very few carotenoproteins have been purified; however, their presence in aqueous extracts may be indicated by spectroscopic evidence. Carotenoproteins have been isolated, purified, and characterized from the ectoderm, exoskeleton, eggs, and ovaries of marine invertebrates, especially crustaceans. Water-soluble and detergent-soluble carotenoid-protein complexes have also been isolated from the cytoplasmic membrane of some cyanobacteria, Mangifera indica, and carrots. Recently, we have been able to partially purify a beta-carotene-protein complex from fresh livers of rats fed beta-carotene. Studies are currently in progress to purify and characterize the protein. This is the first successful isolation of a vertebrate carotenoprotein. The isolation of carotenoproteins is generally by standard techniques of protein chemistry. Purification, crystallization of the complex, and reconstitution of apoprotein and carotenoid components have been achieved for some crustacean carotenoproteins. The complex is very sensitive to bright light and to temperatures above that of refrigeration. However, it is best preserved in solutions of high ionic strength or as a precipitate in strong ammonium sulfate solutions.

Conversion of all trans β-carotene to retinal by an enzyme from the intestinal mucosa of human neonates☆

Abstract The enzymatic conversion of all trans β-carotene to retinal by an intestinal mucosal enzyme (β-carotene cleavage enzyme, BCC) from autopsy samples of human neonates was demonstrated for the first time. The enzymatic product was characterized as its O -ethyl oxime, which, on high pressure liquid chromatography (HPLC), yielded a sharp peak corresponding to an authentic retinal ( O -ethyl) oxime. The enzyme blank and boiled enzyme blank failed to show any significant HPLC peaks corresponding to retinal ( O -ethyl) oxime, retinal, or retinol. Based on the observed activities among intestinal samples from 14 premature infants, the BCC enzyme activity ranged from 3.3–1210 pmoles per mg mucosal protein per hr. Studies on the stability of the enzyme using the rat as the experimental animal revealed that as much as 80% of the original activity of the fresh intestine is lost in storage of the dead animal for 8 hr at 25° C followed by storage at 4° C for 16 hr. More importantly, 70% of the fresh enzyme activity is lost after storage of the animals at 4° C for only 8 hr. Thus, the observed activities in the human autopsy samples appear to be markedly underestimated because of the marked loss of enzyme activity from the time of death to the time of assay. Therefore, the true activity of the enzyme can be assessed only after the extent of loss of activity on storage of the human samples can be accurately measured. In spite of repeated attempts, no detectable BCC activity was found in the placentas of pre-term or term infants. Nonetheless, the demonstration of BCC enzyme activity in the intestinal mucosa of human neonates shows that β-carotene can serve as an important source of vitamin A in newborn infants.