Activation of dolichol-phosphate mannosyltransferase by dibutryl cyclic AMP in rat liver.

Radiolabeled mannose incorporation into secretory glycoproteins and immunoprecipitable fibronectin in the incubation media significantly increased (105 and 32 percent respectively) with a corresponding increase in the levels of dolichol-phosphate mannose, dolichol-diphosphate oligosaccharides and dolichol-phosphate mannosyltransferase activity in the rat liver slices when incubated with dibutryl cAMP and ATP. Dibutryl cAMP activated maximally this enzyme in the presence of ATP in the incubation medium. The activation of the enzyme resulted in a two fold increase in Vmax with no apparent change in the Km for GDP mannose. Phosphorylation the rat liver microsomes with catalytic subunit of cAMP dependent protein kinase, resulted in the activation of dolichol-phosphate mannosyltransferase. These results suggest that cAMP modulates protein glycosylation by activating dolicholphosphate mannosyltransferase activity. The activation of this enzyme could be through phosphorylation/dephosphorylation mechanism involving a cAMP dependent protein kinase.

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.

Stimulation by dolichol phosphate-mannose of N-acetylglucosaminyl-lipid biosynthesis by membranes from class E Thy-1-negative mutant mouse lymphoma cells which are defective in dolichol phosphate-mannose biosynthesis.

Dolichol phosphate-mannose has been shown previously to stimulate the biosynthesis of N-acetylglucosaminyl-diphosphate-dolichol (E. L. Kean (1985) J. Biol. Chem. 260, 12561-12571). Although the class E Thy-1-negative mutant mouse lymphoma cells are unable to synthesize dolichol phosphate-mannose, the addition of this compound exogenously to membranes from the mutant cells brought about a stimulation of N-acetylglucosaminyl-lipid synthesis similar to that obtained with membranes from wild type cells. The retention of this activity by the mutant cells supports the suggestion of a regulatory role for dolichol phosphate-mannose as an intrinsic property of the glucosaminyltransferase which catalyzes the initial reaction of the dolichol pathway.

Mannosylation of endogenous and exogenous phosphatidic acid by liver microsomal membranes. Formation of phosphatidylmannose.

Hamster liver post-nuclear membranes catalyze the transfer of mannose from GDP-mannose to endogenous dolichyl phosphate and to a second major endogenous acidic lipid. This mannolipid was believed to be synthesized from endogenous retinyl phosphate and was tentatively identified as retinyl phosphate mannose (Ret-P-Man) (De Luca, L. M., Brugh, M. R. Silverman-Jones, C. S. and Shidoji, Y. (1982) Biochem. J. 208, 159-170). To characterize this endogenous mannolipid in more detail, we isolated and purified the mannolipid from incubations containing hamster liver membranes and GDP-[14C]mannose and compared its properties to those of authentic Ret-P-Man. We found that the endogenous mannolipid was separable from authentic Ret-P-Man on a Mono Q anion exchange column, did not exhibit the absorbance spectrum characteristic of a retinol moiety, and was stable to mild acid under conditions which cleave authentic Ret-P-Man. The endogenous mannolipid was sensitive to mild base hydrolysis and mannose was released from the mannolipid by snake venom phosphodiesterase digestion. These properties were consistent with the endogenous acceptor being phosphatidic acid. Addition of exogenous phosphatidic acid, but not phospholipids with a head group blocking the phosphate moiety, to incubations containing hamster liver membranes and GDP-[14C]mannose resulted in the synthesis of a mannolipid with chromatographic and physical properties identical to the endogenous mannolipid. A double-labeled mannolipid was synthesized in incubations containing hamster liver membranes, GDP-[14C]mannose, and [3H]phosphatidic acid. Mannosyl transfer to exogenous phosphatidic acid was saturable with increasing concentrations of phosphatidic acid and GDP-mannose and specific for glycosyl transfer from GDP-mannose. Class E Thy-1-negative mutant mouse lymphoma cell membranes, which are defective in dolichyl phosphate mannose synthesis, also fail to transfer mannose from GDP-mannose to exogenous phosphatidic acid or retinyl phosphate. Amphomycin, an inhibitor of dolichyl phosphate mannose synthesis, blocked mannosyl transfer to the endogenous lipid, and to exogenous retinyl phosphate and phosphatidic acid. We conclude that the same mannosyltransferase responsible for dolichyl phosphate mannose synthesis can also utilize in vitro exogenous retinyl phosphate and phosphatidic acid as well as endogenous phosphatidic acid as mannosyl acceptors.

Effect of herpes simplex virus type 1 on cellular pools of oligosaccharide-lipid.

Incorporation of [3H]mannose into cellular pools of mannosylphosphoryl dolichol (Man-P-Dol), oligosaccharide-lipid, and glycoprotein was measured and compared in herpes simplex virus type 1 (HSV-1)-infected cells and -uninfected cells. While mannose incorporation into the monosaccharide-dolichol fraction was similar in infected or uninfected Vero cells, incorporation into the oligosaccharide-lipid fraction was markedly reduced in HSV-1-infected cells (64% of control levels). In contrast, mannose incorporation into glycoprotein was significantly increased in virus-infected cells versus uninfected cells (194% of control levels). The kinetics of incorporation into the various fractions was examined and it was determined that there was minimal increase in mannose incorporation into oligosaccharide-lipid after 8 hr postinfection in virus-infected cells. This corresponded to the time at which nonglycosylated precursors of the HSV-1 glycoproteins were first detected in association with the nuclear fraction. These data suggest that there is an accelerated turnover of oligosaccharide-lipid in virus-infected Vero cells which is most likely due to extensive glycoprotein synthesis.

Glycoprotein biosynthesis in Chlamydomonas. A mannolipid intermediate with the properties of a short-chain alpha-saturated polyprenyl monophosphate.

A crude membrane preparation of the unicellular green alga Chlamydomonas reinhardii was found to catalyse the incorporation of D-[14C]mannose from GDP-D-[14C]-mannose into a chloroform/methanol-soluble compound and into a trichloroacetic acid-insoluble polymer fraction. The labelled lipid revealed the chemical and chromatographic properties of a short-chain (about C55-C65) alpha-saturated polyprenyl mannosyl monophosphate. In the presence of detergent both long-chain (C85-C105) dolichol phosphate and alpha-unsaturated undecaprenyl phosphate (C55) were found to be effective as exogenous acceptors of D-mannose from GDP-D-[14C]mannose to yield their corresponding labelled polyprenyl mannosyl phosphates. Exogenous dolichyl phosphate stimulated the incorporation of mannose from GDP-D-[14C]mannose into the polymer fraction 5-7-fold, whereas the mannose moiety from undecaprenyl mannosyl phosphate was not further transferred. Authentic dolichyl phosphate [3H]mannose and partially purified mannolipid formed from GDP-[14C]mannose and exogenous dolichyl phosphate were found to function as direct mannosyl donors for the synthesis of labelled mannoproteins. These results clearly indicate the existence of dolichol-type glycolipids and their role as intermediates in transglycosylation reactions of this algal system. Both the saturation of the alpha-isoprene unit and the length of the polyprenyl chain may be regarded as evolutionary markers.

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.

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.

The effect of tsushimycin on the synthesis of lipid-linked saccharides in aorta.

The antibiotic, tsushimycin, inhibits the formation of dolichyl phosphate mannose, dolichyl phosphate glucose and dolichyl pyrophosphate N-acetylglucosamine in the particulate enzyme preparation from pig aorta. Although this antibiotic also inhibits the incorporation of mannose and glucose into lipid-linked oligosaccharides, these reactions are less sensitive to antibiotic than those involved in the synthesis of lipid-linked monosaccharides. In the presence of tsushimycin, most of the mannose incorporated into lipid-linked oligosaccharides is into one oligosaccharide that has the properties of the heptasaccharide Man5GlcNAc2, whereas in the absence of antibiotic most of the mannose is in larger-sized oligosaccharides. On the other hand, the glucose-labelled lipid-linked oligosaccharides appear to be similar in size in the presence or absence of antibiotic. Tsushimycin also inhibits the formation of lipid-linked monosaccharides by the solubilized enzyme preparation of aorta. Various concentrations of dolichyl phosphate or the detergent, Nonidet P40, had no effect on antibiotic inhibition. Some evidence indicates that tsushimycin binds to the particulate enzyme.

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.

[Mannosylation of lipid and protein acceptors in sheep lung microsomes]. / Mannosylation des accepteurs lipidiques et protéiniques des microsomes de poumons de mouton.

In Sheep lungs, we have shown that a mannosyl-transferase activity is involved at microsomal level. This enzymatic system is able to catalyze the incorporation of mannose from GDP-mannose into two different endogenous acceptors: lipids and proteins. The mannolipid has the properties of a mannosyl-phosphoryl-polyprenol. The kinetic studies of product biosynthesis demonstrate that no precursor-product relationship exists in this system; this is an original behaviour of the lung in glycosylation phenomenon.

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.

[Demonstration and characterization of mannosyl phosphoryl dolichol synthesized in the outer membrane of liver mitochondria]. / Mise en évidence et caractérisation d'un mannosyl-phosphoryl-dolichol synthétisé dans la membrane externe des mitochondries de foie.

There is in mitochondrial outer membrane a mannosyl-transferase which is able to catalyse the mannose transfer on a polyprenic acceptor, to give a mannosyl-phosphoryl-dolichol. This enzyme is stimulated by Mn2+, Mg2+ and dithiothreitol. It is inhibited by GDP, mersalyl and EGTA.