mannosylretinylphosphate and Vitamin-A-Deficiency

Male Wistar rats of different vitamin A status (total depletion to moderate deficiency) were administered DDT (5 mg/kg/day) or vehicule (corn oil) i.p. daily for 14 days. Vitamin A-dependent protein mannosylation was measured either by in vivo incorporation of [3H]mannose into liver glycoprotein or by in vitro assay of incorporation of [14C]mannose into mannosylretinyl phosphate. Vitamin A deficiency resulted in a significantly impaired in vivo incorporation of mannose in liver glycoprotein but had no effect on the in vitro transport of mannose via retinyl phosphate. Although DDT induced an increase synthesis of liver proteins in smooth endoplasmic reticulum and caused a diminution of the hepatic vitamin A content, it did not affect vitamin A-dependent protein mannosylation.

Topics: Animals; DDT; Diterpenes; Glycoproteins; Liver; Mannose; Polyisoprenyl Phosphate Monosaccharides; Rats; Reference Values; Subcellular Fractions; Vitamin A; Vitamin A Deficiency

This study was undertaken in order to validate the hypothesis that vitamin A-deficiency alters the structure of thyroglobulin (Tg). For that purpose, four groups of 20 Sprague-Dawley rats were submitted during two months to varying dietary conditions, namely a control diet (C+), a vitamin A-deficient diet (A-), an iodine-deficient diet (I-) and a diet characterized by the association of both deficiencies (A-I-). Both the conventional parameters of thyroid function, the intracellular steps of Tg glycosylation and iodination were analyzed. In the A- and A-I- groups, blood levels of retinol fell to one tenth of the control mean and circulating concentrations of total and free T4 and T3 increased significantly. This biochemical hyperthyroidism contrasted with the maintenance of normal TSH plasma values, suggesting a generalized peripheral refractoriness to thyroid hormones. In both A- and A-I- groups, thyroid cytosol 3H-RPM (retinyl-phosphate-mannose) and 3H-mannose incorporation into the core of the 12S-Tg and 19S-Tg species were reduced by 40-50%. In contrast, cytosolic concentrations of 3H-DPM (dolichyl-phosphate-mannose) rose, suggesting that the N-glycosylation pathways are affected in opposite direction. The sedimentation coefficient in sucrose gradient of the purified dimeric 125I-19S-Tg after guanidine 6M and dithiothreitol denaturation showed that most of the A- Tg molecules were transformed into monomeric 12S species, implying alterations of both noncovalent and covalent bonds. Finally, the radiochromatogram of 125I-iodothyronines recovered after Tg pronase digestion revealed a significant increase in the mono- (MIT) and diiodothyronine (DIT) fractions in contrast with a significant decrease in the T3 and T4 hormonal compounds. These findings are consistent with the view that vitamin A-depletion impairs the endogenous RPM synthesis and, therefore, the normal Tg 0-mannosylation. The growing peptide is characterized by steric hindrance, leading to abnormal closure of disulphide bonds, reduced MIT-DIT coupling reactions and depressed generation of physiologically active thyroid hormones. pure iodine deficit (I-) induces no effects on the above-mentioned glycosylation reactions, but iodine shortage superimposed on preexisting vitamin A-deficit (A-I-) aggravates the Tg dysmaturation.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Animals; Diterpenes; Goiter, Endemic; Iodine; Male; Mannose; Polyisoprenyl Phosphate Monosaccharides; Rats; Rats, Inbred Strains; Subcellular Fractions; Thyroglobulin; Thyroid Gland; Thyroid Hormones; Vitamin A Deficiency

Topics: Animals; Cricetinae; Diterpenes; Dolichol Monophosphate Mannose; Male; Mesocricetus; Polyisoprenyl Phosphate Monosaccharides; Polyisoprenyl Phosphate Sugars; Rats; Vitamin A Deficiency

The mannosyl derivative of phosphorylated vitamin A, mannosylretinylphosphate, is synthesized in vivo and in vitro by most epithelial tissues, including mouse epidermal cells. This compound is but one component of the complex biosynthetic machinery responsible for the assembly of glycoproteins in mammalian membranes; its specific role remains to be understood, even though it has been established that it functions as a carrier of mannose mostly in a direct transfer to protein. The system of five conjugated double bonds appears necessary for this function, since upon saturation the resulting perhydroretinylphosphate is incapable of acting in mannosyl transfer to endogenous microsomal proteins. Using vitamin A - deficient hamsters and rats, retinoic acid was shown to be as active as retinol in restoring the in vivo in corporation of mannose into glycoproteins to normal levels within a short time, in liver and tracheal tissues. Retinoic acid was also active in increasing adhesive properties of spontaneously transformed mouse dermal fibroblasts (3T12 cells) in a reversible manner. The free carboxyl group appeared to be a requirement for this activity inasmuch as the lactonized form of 11-hydroxyretinoic acid was inactive. Even though retinoic acid, as well as retinol, was able to enhance adhesion of 3T12 cells, only the cellular retinoic acid-binding protein was detected, suggesting that the cellular retinol-binding protein is not a requirement for this activity. A metabolite of retinoic acid, compound X, different from retinol, was found incorporated into mannosylretinoidphosphate (MXP) by 3T12 cells. This derivative constitutes up to 40% of the total radioactive pool of derivatives of retinoic acid after 48 hours of labeling. These data suggest that retinol, as mannosylretinylphosphate, and retinoic acid, as mannosylretinoidphosphate, function as mannosyl carriers in biologic membranes.

Topics: Animals; Cell Line; Diterpenes; Dolichol Monophosphate Mannose; Epithelium; Fibroblasts; Hexosephosphates; Mannose; Mannosephosphates; Mice; Mice, Inbred BALB C; Microsomes, Liver; Polyisoprenyl Phosphate Monosaccharides; Polyisoprenyl Phosphate Sugars; Rats; Skin; Tretinoin; Vitamin A; Vitamin A Deficiency

Rat liver microsomal fraction synthesized Ret-P-Man (retinyl phosphate mannose) and Dol-P-Man (dolichyl phosphate mannose) from endogenous Ret-P (retinyl phosphate) and Dol-P (dolichyl phosphate). Ret-P-Man synthesis displayed an absolute requirement for a bivalent cation, and also Dol-P-Man synthesis was stimulated by bivalent metal ions. Mn2+ and Co2+ were the most active, with maximum synthesis of Ret-P-Man occurring at 5-10 mM: Mg2+ was also active, but at higher concentrations. At 5mM-Mn2+ the amount of endogenous Ret-P mannosylated in incubation mixtures containing 5 microM-GDP-mannose in 15 min at 37 degrees C was approx. 3 pmol/mg of protein. In the same assays about 7-10 pmol of endogenous Dol-P was mannosylated. Bivalentcation requirement for Ret-P-Man synthesis from exogenous Ret-P showed maximum synthesis at 2.5 mM-Mn2+ or -Co2+. In addition to Ret-P-Man and Dol-P-Man, a mannolipid co-chromatographing with undecaprenyl phosphate mannose was detected. Triton X-100 (0.5%) abolished Ret-P-Man synthesis from endogenous Ret-P and caused a 99% inhibition of Ret-P-Man synthesis from exogenous Ret-P. The presence of detergent (0.5%) also inhibited Dol-P-Man synthesis from endogenous Dol-P and altered the requirement for Mn2+. Microsomal fraction from Syrian golden hamsters was also active in Ret-P-Man and Dol-P-Man synthesis from endogenous Ret-P and Dol-P. At 5 mM-Mn2+ about 2.5 pmol of endogenous Ret-P and 3.7 pmol of endogenous Dol-P were mannosylated from GDP-mannose per mg of protein in 15 min at 37 degrees C. On the other hand, microsomal fraction from vitamin A-deficient hamsters contained 1.2 pmol of Ret-P and 14.1 pmol of Dol-P available for mannosylation. Since GDP-mannose: Ret-P and GDP-mannose: Dol-P mannosyltransferase activities were not affected, depletion of vitamin A must affect Ret-P and Dol-P pools in opposite ways.

Topics: Animals; Cations, Divalent; Chromatography, Thin Layer; Cricetinae; Detergents; Diterpenes; Dolichol Monophosphate Mannose; Dolichol Phosphates; In Vitro Techniques; Kinetics; Male; Mesocricetus; Microsomes, Liver; Octoxynol; Polyethylene Glycols; Polyisoprenyl Phosphate Monosaccharides; Polyisoprenyl Phosphate Sugars; Polyisoprenyl Phosphates; Rats; Serum Albumin, Bovine; Vitamin A; Vitamin A Deficiency

A study was conducted to determine whether mannosyl retinyl phosphate occurred in rat liver and intestine in vivo, and, if so, to partially purify it and investigate its properties. After injection of [(3)H]retinol and [(14)C]mannose, a chloroform-methanol 2:1 extract of rat liver and small intestinal mucosa yielded two (3)H/(14)C-labeled peaks on DEAE-cellulose column chromatography: peak I eluted with 10 mM and peak II eluted with 29 mM ammonium acetate. Peak II, subjected to silicic acid column chromatography, gave principally two (3)H/(14)C-labeled fractions, one eluted with chloroform-methanol 2:1 and the other with chloroform-methanol 1:1. The latter showed, on thin-layer chromatography in a chloroform-methanol-water 60:25:4 system, an R(f) of 0.25 (with coincidence of the (3)H and (14)C radioactivity), which is identical to the R(f) of authentic mannosyl retinyl phosphate. The chloroform-methanol 1:1 peak, on mild acid hydrolysis, yielded [(3)H]retinol (identified by two thin-layer chromatography systems), [(14)C]mannose, and [(14)C]-mannose phosphate (identified by paper chromatography). On mild alkali hydrolysis, the peak yielded [(3)H]retinol and [(14)C]mannose phosphate. The substance eluted in the chloroform-methanol 1:1 peak from silicic acid was therefore concluded to be mannosyl retinyl phosphate. When chromatographed on silicic acid, peak I from the DEAE-cellulose column primarily showed a fraction eluted with chloroform-methanol 2:1. When chromatographed on thin-layer plates in the above solvent, this fraction showed an R(f) of 0.3, with coincidence of (3)H and (14)C radioactivity; it was resistant to mild acid hydrolysis, mild and strong alkali hydrolysis, and glucuronidase action. Mannosyl retinyl phosphate occurs, therefore, in vivo in liver and intestinal mucosa, and it is accompanied by a closely similar, though slightly less polar, compound that remains unidentified.

Topics: Animals; Chromatography, DEAE-Cellulose; Diterpenes; Intestinal Mucosa; Liver; Mannose; Polyisoprenyl Phosphate Monosaccharides; Polyisoprenyl Phosphate Sugars; Rats; Vitamin A; Vitamin A Deficiency

Upon incubation of [14C]mannose-labeled mannosyl retinyl phosphate with a membrane fraction from rat liver, mannose was transferred to an endogenous acceptor precipitable withchloroform/methanol to the extent of about 7%. The reaction proceeded linearly with time for 120 min at a pH optimum of about 7.0. The acceptor thus labeled with mannose could be solubilized by sodium dodecyl sulfate/mercaptoethanol. More than half of this acceptor appeared in the void volume of a Sephadex G-100 column. When it was digested with Pronase, a substantial proportion of it appeared between the void and bed volumes of a Sephadex G-100 column, thus indicating that it was a glycopeptide. In high-voltage paper electrophoresis, this glycopeptide moved to the cathode at low pH and to the anode at highpH. When digested with highly purified jack bean alpha-mannosidase, the glycopeptide released almost 50% of its radioactivity as mannose. That this transfer of mannose to glycoprotein from mannosyl retinyl phosphate does not take place via dolichyl mannosyl phosphate was shown by the fact that it is Mn2+ and Mg2+ independent, it is not inhibited by the presence of a 10-fold molar excess of nonradioactive GDP-mannose, and neither 14C-labeled dolichyl mannosyl phosphate nor 14-C labeled lipid pyrophosphoryl oligosaccharide could be detected during the incubation.

Topics: Animals; Carrier Proteins; Cell Membrane; Diterpenes; Kinetics; Liver; Mannose; Polyisoprenyl Phosphate Monosaccharides; Polyisoprenyl Phosphate Sugars; Rats; Vitamin A; Vitamin A Deficiency