Wlodzimierz Sasak

RECENT DEVELOPMENTS IN STUDIES ON BIOLOGICAL FUNCTIONS OF VITAMIN A IN NORMAL AND TRANSFORMED TISSUES

Abstract A biochemical pathway of phosphorylation and glycosylation of vitamin A has recently been found in hepatic, intestinal and epidermal tissues. More recent work suggests that mannosylretinylphosphate functions as a donor of mannose to membrane glycoconjugates. These reactions might ultimately explain the effects of vitamin A deficiency and some of the effects of excess vitamin A on biological systems. Studies of the effect of retinoids on cellular in vitro systems showed an increase in the adhesive properties of spontaneously-transformed mouse fibroblasts in culture (Balb/c 3T12–3 cells). These cells are usually detached from the culture dish surface in an EDTA adhesion assay. After culturing in presence of 3.3 x 10-6 to 3.3 x 10-5M retinol or retinoic acid the cells are no longer lifted from the plate and their morphology and adhesion resemble those of normal fibroblasts. This phenomenon of increased adhesion is observed as early as two days after exposure to the retinoid and it is readily reversible upon culturing in medium without exogenous retinoid. A variety of retinoids was tested in the adhesion assay. The most active compounds were retinol, retinyl-phosphate, retinoic acid, 5,6-epoxyretinoic acid and the TMMP and DACP derivatives of retinoic acid. All these compounds possess biological activity in other systems. Anhydroretinol, perhydromonoeneretinol, the phenyl derivative of retinoic acid, which do not have biological activity in other systems, did not increase adhesion of 3T12 cells. Other polyprenoid compounds without vitamin A activity were also tested in this assay. Dolichol, dolichylphosphate juvenile hormone, abscisic acid, β-ionone, dibutyryl cyclic adenosine monophosphate and sodium butyrate did not induce adhesion. The mechanism by which retinol and retinoic acid increase the adhesive properties of 3T12 cells was investigated. Cyclic adenosine monophosphate and guanosine monophosphate levels were not significantly altered by retinoid treatment at least at 6, 24, 48 and 72 hours after treatment with 3.3 x 10-5M retinoic acid, when most of the cells remain attached. Retinoic acid stimulated the incorporation of (2-3H) mannose into glycoproteins of 3T12 cells. (11, 123H and carboxyl-14C)Retinoic acid was incorporated into a compound (Metabolite I) which had chromatographic properties of a glycosylretinylphosphate. The synthesis of this compound was time-dependent and was not carried out by formalin -fixed 3T12 cells. Mild alkaline conditions which release anhydroretinol from retinylphosphate, also cleaved Metabolite I to yield a product with the polarity of a hydrocarbon, but slightly more polar than anhydroretinol. It is suggested that retinoic acid can be reduced to an alcohol, probably after metabolic modification. It is further suggested that such “retinol-like” compound would follow the same route of phosphorylation and glycosylation as shown for retinol in other systems. Microsomes from 3T12 cells were active as the intact cells in synthesizing mannosylretinylphosphate and dolichyl mannosylphosphate. Exogenous retinylphosphate specifically stimulated the synthesis of mannosylretinylphosphate. Thus it appears that vitamin A is involved in glycosyl transfer reactions in the 3T12 system, as well as in normal membranes. It remains to be established whether the observed increased adhesion is the result of such involvement. A novel reaction for retinol was found in 3T12 cells. Up to 55% of exogenously supplied retinol was converted to the hydrocarbon anhydroretinol in 48 hours. The same reaction was also carried out by microsomes from 3T12 cells, which converted 7% of retinol to anhydroretinol in 30 minutes at 37°C. This reaction may well represent a detoxification mechanism for the transformed cell.

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.

Mannosyl transfer from mannosylretinylphosphate to glycoconjugates of rat liver membranes

The metabolic mode of action of vitamin A in ma~t~ning growth and normal phenotypic expression is still unknown, although its molecular function in the visual cycle has been elucidated [ 1,2]. Some insight into a possible metabolic function of the ‘oldest’ of the vitamins was gained, when it was observed that its deficiency caused a drastic reduction in the incorporation of labelled monosaccharides into glycoproteins of a variety of tissues {3--S]. The greatest effect ~90%~ was noted on the incorporation of [ I4 Clmannose into liver glycoproteins [6]. Conversely, excessive doses of this essential nutrient caused an increase in the amount of [ 14C] mannose incorporated into rat liver glycoconjugates [7]up to 600% over controls. On the basis of these and other data, it was proposed that retinol may be phosphorylated to retinylphosphate and that the phospho~lated vitamin may function as a carrier of glycosyl residues for the biosynthesis of some glycoproteins [8] in a manner akin to the mode of action of bacterial and mammalian polyprenols [9,10]. ~ospho~l [l I-131 and glycosylphosphoryl [ 14-181 derivates of vitamin A have been characterized from several systems in vivo and in vitro, However, the function of MRP as donor of mannose to glycoproteins is not clear at present. Here we demonstrate that mannosylretinyl-

Separation of mannosylretinylphosphate from dolichylmannosylphosphate by chromatography on columns of DEAE-Sephacel

Abstract A one-step column chromatographic procedure on DEAE-Sephacel allows the separation of mannosylretinylphosphate from dolichylmannosylphosphate with minimal breakdown of the mannosylretinylphosphate. Using this procedure, subcellular fractions of rat liver were shown to be active in synthesizing both mannolipids from GDP-[ 14 C]mannose in the absence or presence of exogenous retinylphosphate.

Mannosyl Retinyl Phosphate: Its Role as a Donor of Mannose to Glycoconjugates in Rat Liver Membranes

Publisher Summary The chapter describes the role of vitamin A (retinol) in the visual cycle. Phosphate and glycosyl phosphate derivatives of retinol are synthesized in vitro and in the whole animal. These compounds are labile to yield anhydroretinol, which is a compound with a characteristic absorption spectrum with maxima at 350, 370, and 390 nm. Monophosphate and glycosyl monophosphate derivatives of dolichol are stable under such conditions. In vivo, rat liver cells synthesize mannosyl retinylphosphate (MRP) and dolichyl mannosyl phosphate (DMP) in the ratio of approximately 1:1, whereas in vitro mostly DMP is obtained from guanosine diphosphate mannose. Addition of retinyl phosphate (RP) to the liver microsomalsystem specifically enhances MRP synthesis. MRP transfers mannosyl residues to endogenous glycoconjugates of rat liver microsomes.