Differential regulation of the synthesis of mannosylphosphoryl derivatives of dolichol and retinol in HeLa cells.

We have shown earlier that in HeLa S3G cells, glucocorticoids stimulate the synthesis of dolichyl phosphorylmannose (Dol-P-Man) with a concomitant increase in the glycosylation of proteins (Ramachandran, C.K., Gray, S.L. and Melnykovych G. (1982) Biochem. J. 208, 47-52). Although controversial, there have been several lines of evidence suggesting that the synthesis of retinyl phosphorylmannose (Ret-P-Man) and Dol-P-Man may be carried out by the same enzyme. We examined this possibility and conclude that in HeLa S3G cells the syntheses of Dol-P-Man and Ret-P-Man are catalyzed by two different enzymes located in the same microenvironment. Our conclusion is based on the following observations: exogenously added dolichyl phosphate and retinyl phosphate did not compete with each other; when the cells were grown in the presence of 1 microM dexamethasone, the microsomal synthesis of Dol-P-Man was stimulated, without affecting the Ret-P-Man synthesis; Arrhenius plots on Ret-P-Man and Dol-P-Man synthesis showed breaks at 22 and 37.7 degrees C.

Effect of inhibiting dolichol phosphate-mannose formation on the biosynthesis of the N-acetylglucosaminylpyrophosphoryl polyprenols by the retina.

Previously (Kean, E.L. (1982) J. Biol. Chem. 257, 7952-7954), it was shown that dolichol phosphate-mannose stimulated the formation of the N-acetylglucosamine-containing mono-, di- and trisaccharide intermediates of the dolichol pathway. Consistent with this activating role, the present report demonstrates that inhibition of the formation of dolichol phosphate-mannose by the antibiotics, showdomycin and diumycin, also blocked the stimulatory phenomenon.

Glycoprotein biosynthesis in intestinal epithelial cells during differentiation. Incorporation of [14C]mannose from GDP-[14C]mannose into dolichol derivatives.

Epithelial cells of the rat small intestine were collected as a gradient of villus to crypt cells. Homogenates of these cells incubated with GDP-D-[14C]mannose in the presence of MnCl2 incorporated radioactivity into dolichyl mannosyl phosphate and a mixutre of dolichyl pyrophosphate oligosaccharides varying in the size of their oligosaccharide moiety. The labeled oligosaccharides formed in villus cell homogenates appeared shorter than those formed in crypt cell homogenates. The addition of dolichyl phosphate greatly stimulated the synthesis of dolichyl mannosyl phosphate. The initial rate of synthesis of dolichyl mannosyl phosphate from GDP-D-[14C]mannose and exogenous dolichyl phosphate was highest in an intermediate cell fraction having a low specific activity of sucrase and alkaline phosphatase and an intermediate specific activity of thymidine kinase. To compare the rates of dolichyl mannosyl phosphate synthesis in the different cell fractions, it was essential to control degradation of GDP-D-[14]mannose by the addition of AMP to the incubation, since villus cells degraded GDP-D-[14C]mannose much faster than crypt cells.

Effect of progesterone, estrogen withdrawal and secondary estrogen treatment of mannosylphosphoryldolichol synthesis in chick oviduct membranes.

Oviduct membranes from chicks treated with diethylstilbestrol have a fully induced level of an enzyme that transfers mannose from GDP-Man to form mannosylphosphoryldolichol (Lucas, J.J. and Levin, E. (1977) J. Biol. Chem.252, 4330--4336). Withdrawal of diethylstilbestrol for 5 days causes a decrease in oviduct weight, lysozyme, and 60% of the mannosyltransferase activity. Chicks withdrawn from treatment for 10 days followed by secondary stimulation with diethylstilbestrol exhibit a more rapid increase in the mannosyltransferase activity than chicks that have not been previously treated with diethylstilbestrol. Further experiments indicate that the decrease in mannosylphosphoryldolichol synthesis after hormonal withdrawal may be the result of decreased levels of endogenous dolichyphosphate in the membrane preparations.

Inflammation-induced increases in dolichol synthesis and hydroxymethylglutaryl-coenzyme A reductase activity in mouse liver are prevented by a high-cholesterol diet but not by fasting.

The inflammatory response in mammals is characterized by the synthesis in the liver of several N-linked serum glycoproteins called acute-phase reactants. In C57BL/6J mice, turpentine-induced inflammation was accompanied by increases in 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity, dolichol synthesis, and dolichyl phosphoryl mannose synthesis. Cholesterol feeding, but not fasting, prevented these inflammation-induced increases in reductase activity and dolichol synthesis. However, the rate of incorporation of [3H]mannose into total serum glycoproteins was not affected by the high-cholesterol diet, and this rate increased during acute inflammation in control and cholesterol-fed mice.

Study of nuclear mannosyl-transferase: lipids intermediates.

Mannosyl-transferase of rat liver nuclei catalyzed the transfer of mannose, from GDP-mannose, to endogeneous lipids and proteins. Two solubility-different glycolipids were characterized: a mannosyl-phosphoryl-dolichol and an oligosaccharide-lipid. The labeling of the two lipids was consistent with a role in mannose transfer to nuclear glycoproteins, but not according to the Lennarz' scheme.

Phenyl phosphate as a water-soluble analog of dolichyl phosphate in microsomal mannosyltransferase systems.

Incubation of rat liver microsomes with GDP-[14C]mannose and phenyl phosphate for 30 min, followed by paper-chromatographic analysis, revealed the presence of a new radioactive compound that was isolated and characterized as phenyl beta-D-mannosyl phosphate. The enzyme catalyzing this reaction was very closely related to a dolichyl beta-D-mannosyl phosphate-synthesizing activity found in the same microsomes. The two activities were affected in a parallel fashion by mild heating and were found in the same proportions in whole microsomes, in rough-surfaced microsomes, and in smooth-surfaced mirosomes. Increasing the concentration of phenyl phosphate from 0 to 15 mM resulted in a progressive reduction in the rate of mannose incorporation into endogenous dolichyl phosphate. It is concluded that the two reactions are catalyzed by a single enzyme. The labeled product from phenyl phosphate was rapidly excluded from the microsomes, while the product from dolichyl phosphate was retained in the microsomes, suggesting that phenyl phosphate may act as a mannose-trapping agent, thereby inhibiting mannosyltransferase reactions involving dolichyl phosphate as a glycosyl carrier.

The biosynthesis of a mannolipid containing a metabolite of retinoic acid by 3T12 mouse fibroblasts.

Retinol and retinoic acid (RA) increase the adhesive properties of spontaneously transformed mouse fibroblasts (BALB/c 3T12-3 cells) and the incorporation of [2-3H]mannose into cellular glycoconjugates. Therefore we searched for a mannolipid of retinoic acid similar to mannosylretinylphosphate (MRP) in these cells. Radioactively labeled RA was incorporated into a compound of chromatographic characteristics similar to those of standard MRP. This metabolite contained the same 3H:14C ratio as the precursor [11, 12-3H, 15-14C]retinoic acid, demonstrating that no decarboxylation had occurred. A doubly labeled mannolipid was obtained from cells incubated with [2-3H]mannose and [15-14C]retinoic acid. This mannolipid was readily cleaved by mild acid, yielding [3H]mannosephosphate and a compound that migrated as standard anhydroretinol on thin layer of silica gel in toluene:chloroform:methanol (4:1:1) at Rf 0.93. Standard all trans-MRP yields all-trans-anhydroretinol under these conditions. An ion pair reverse phase HPLC system was developed to further characterize the mannolipids obtained from retinol and retinoic acid in 3T12 cells. [15-3H]Retinol and [15-14C]retinoic acid were incorporated into mannolipids that cochromatographed upon HPLC with standard MRP. The mixture of the [15-3H]retinol and [15-14C]retinoic acid derived mannolipids was subjected to mild acid hydrolysis, after purification by HPLC. Nearly 100% of the compounds was hydrolyzed, yielding all-trans-[3H]anhydroretinol and a 14C]labeled product which was eluted from HPLC as a slightly more polar compound than all-trans-anhydroretinol. The retinoic acid-derived mannolipid (MXP) represented approximately 4% of the total radioactivity in the methanolic extract of 3T12 cells incubated for 20 hours in the presence of labeled retinoic acid. However, if the cells were incubated for an additional 20 hours in the absence of the radioactive precursor, MXP represented 40% of the total extracted radioactivity. These results demonstrate that 3T12 cells synthesize mannosylretinylphosphate from retinol and a mannosylretinoidphosphate (MXP) from retinoic acid. These results exclude the possibility of a reduction of retinoic acid to retinol, but suggest that a closely related compound is formed from RA and that this retinol-like compound (X) is incorporated into MXP.

Recent studies on the involvement of retinyl phosphate as a carrier of mannose in biological membranes.

Rat liver microsomes synthesized [14C]mannosylretinylphosphate and dolichyl [14C]mannosylphosphate from guanosinedisphosphate [14C]mannose, retinylphosphate and dolichylphosphate. Two distinct enzyme activities were shown to be responsible for the biosynthesis of the two mannolipids. A higher affinity mannosyl transferase (EA I), responsible for dolichylmannosylphosphate synthesis, displayed a Km for GDP-mannose of 1.7 microM; while a lower affinity enzyme (EA II), responsible for mannosylretinylphosphate synthesis, displayed a Km for GDP-mannose of 12.5 microM. These Km values were unaffected by the addition of either dolichylphosphate for EA II, or retinylphosphate for EA I. The same Km values were found before and after solubilization of the enzyme activity with 1% Triton X-100. Differential solubilization of EA I and EA II was demonstrated, utilizing different concentrations of Triton X-100. Triple-labeled mannosylretinylphosphate was prepared from [3H]retinylphosphate, retinyl[32P]phosphate and GDP-[14C]mannose from incubations containing rat liver microsomes. This compound was shown to donate [14C]mannose to endogenous acceptors of rat liver microsomes.

Effects of exogenous retinol and retinoic acid on the biosynthesis of 14C-mannose labelled glycolipids and glycoproteins in rat liver.

Studies were conducted to investigate the in vivo and in vitro effects of retinol and retinoic acid on the synthesis of mannolipids and mannopeptides in rat liver. The incorporation of 14C-mannose into glycolipids and glycoproteins showed a decrease in vitamin A-depleted rats as compared with vitamin A-fed rats. By means of DEAE-cellulose, silicic acid and thin-layer chromatography, the mannose-containing lipids were separated into mannosyl retinyl phosphate (MRP, Rf 0.2) and dolichyl mannosyl phosphate (DMP, Rf 0.4), respectively. A rapid increase in the synthesis of labelled MRP was observed, exhibiting a peak between 25 and 60 min after intraperitoneal administration of retinol to vitamin A-depleted rats. Similarly, administration of retinoic acid brought about elevation of 14C-mannolipid (Rf 0.2) synthesis with a peak at 60 min after injection. On the other hand, the incorporation of 14C-mannose into DMP (Rf 0.4) remained unchanged by such treatment. In vitro addition of retinyl phosphate, but not retinoyl phosphate, markedly stimulated the synthesis of 14C-mannolipid (Rf 0.2), using crude membrane of rat liver and GDP-14C-mannose as the donor. These findings strongly suggest that not only retinol but also retinoic acid plays an important biological role in mannosyl transfer reaction in rat liver. However, the molecular participation of a metabolite of retinoic acid in the formation of a mannolipid and the structure of such a metabolite remain to be established.

Effect of protein malnutrition on glycoprotein synthesis by testes of 20-day-old rats.

The effects of protein malnutrition during the nursing period on glycoprotein biosynthesis by testes of 20-day-old rats was studied. Pregnant Wistar rats were housed individually. On the day of delivery they were divided into two groups: one was fed a control diet (25% casein) and the other a low-protein diet (8% casein) for a period of 20 days. Body and testis weights of pups suckled by the malnourished mothers were significantly lower than those of the pups suckled by normally-fed mothers. The seminiferous tubules o malnourished rats showed a significant decrease in diameter and in the stage of development of spermatogenesis. Whole testes of normally-fed 20-day-old rats showed significantly greater [2-3H]mannose incorporation into glycoproteins than did the testes of malnourished rats of the same age. The microsomes of normally-fed rats showed significantly higher GDP: mannose polyprenyl mannosyl transferase activity than did microsomes from malnourished rats, and this difference increased when exogenous dolichyl-phosphate was added to the incubation medium. These results indicate that protein malnutrition decreases GDP: mannose polyprenyl mannosyl transferase activity in the microsomes of testes from 20-day-old rats.

Two mutants of Dictyostelium discoideum that lack a sulfated carbohydrate antigenic determinant synthesize a truncated lipid-linked precursor of N-linked oligosaccharides.

Dictyostelium discoideum glycoproteins contain mannose-6-SO4 in highly immunogenic N-linked oligosaccharides. To more precisely define the structural requirements of the antigenic determinant, we have analyzed the oligosaccharides synthesized by two mutant strains (HL241 and HL243) that lack it. Both mutant strains synthesize N-linked oligosaccharides which are very similar to each other but are smaller and less charged than those derived from the wild-type. Both mutants contain substantial amounts of Man-6-SO4, and only a single residue of Man-6-P-OCH3 per chain, in contrast to the wild-type which may have 1 or 2 such residues. Neutral species are similar to the wild-type in that they can still be modified by the addition of residues of fucose and N-acetylglucosamine. Both mutant strains synthesize a truncated lipid-linked oligosaccharide, Man6GlcNAc2, with the most probable structure being: (sequence; see text) based on Jack bean alpha-mannosidase, alpha-1,2-specific mannosidase digestions and methylation analysis. The presence of this small oligosaccharide appears to result from the loss of the mannosyltransferase(s) needed to synthesize structures larger than Man6GlcNAc2 and not from the absence of dolichol phosphate or dolichol-P-mannose synthetase. These data along with the analysis of another mutant strain suggest that the expression of the antigenic determinant requires a specific arrangement of Man-6-SO4 on the alpha-1,6 branch of the oligosaccharide linked to the beta-mannose.

Novel mannose carrier in the trypanosomatid Crithidia fasciculata behaving as a short alpha-saturated polyprenyl phosphate.

Crithidia fasciculata cells incubated with [14C]glucose or membranes derived from the same protozoan incubated with GDP-[14C]mannose were found to synthesize a lipid monophosphate mannose. No glucosylated mild acid-labile compound was formed in vivo or in vitro when UDP-[14C]glucose was used instead of GDP-[14C]mannose. The lipid moiety of the mannosyl derivative formed behaved as a polyprenol having 11 isoprene residues as judged by t.l.c. and be gel filtration in sodium deoxycholate-containing buffers. The mannolipid was not broken on treatment with hot phenol, suggesting the existence of an alpha-saturated isoprene unit. This is the first case reported in which a mannosyl phospholipid involved in sugar transfer in a eukaryotic cell behaves as if it was similar to that of bacterial polyprenols, although having its putative alpha-isoprene unit saturated to the same extent as dolichols from higher organisms.