342

Biochimica @ Elsevier

et Biophysics Acta, Scientific Publishing

409 (1975) 342-359 Company, Amsterdam

-

Printed

in The Netherlands

BBA 56685

BIOSYNTHETIC STUDIES ON MANNOLIPIDS AND MANNOPROTEINS NORMAL AND VITAMIN A-DEPLETED HAMSTER LIVERS

LUIGI

M. DE LUCA,

CAROL

S. SILVERMAN-JONES

Differentiation Control Section, Bethesda, Md. 20014 (U.S.A.) (Received

May 12th,

Experimental

Pathology

and ROBERT Branch,

OF

M. BARR*

hrational Cancer Institute,

1975)

Summary The incorporation of [l- ’ 4 C] mannose into hamster liver glycolipids and glycoproteins was studied in normal and vitamin A-depleted hamsters. Severely (25% weight loss) and mildly (no weight loss) deficient animals were compared to vitamin A-fed controls. The incorporation of [ ’ 4 C] mannose into glycolipids and glycoproteins decreased in mild and severe vitamin A deficiency by 63-90s compared to vitamin A-fed animals. These results were essentially the same whether expressed per g of wet liver, per DNA or per protein. The size of the pools of mannose, glucose and galactose and their specific radioactivity in liver were determined by gas-liquid chromatography of the boronates of the hexitols (Eisenberg, Jr, F. (1972) Methods Enzymol. XXVIIIB, 168-178) in normal and vitamin A-deficient conditions. It was found that the amount of free hexoses per g of liver was very similar in normal and vitamin A-deficient conditions. The specific radioactivities for mannose and glucose were greater in vitamin A deficiency, thus excluding the possibility that the observed severe decrease in glycopeptide and glycolipid synthesis is a reflection of a similar decrease in the specific radioactivity of the precursor pools. Quantitation of mannose in glycoprotein showed a 79% decrease in vitamin A deficiency. Specific radioactivity of mannose in glycoproteins, 20 min after injection of the label, was 187 dpm/pg of mannose in the normal and 48 dpm/pg of mannose in the vitamin A-deficient livers. It is concluded that vitamin A is necessary for the biosynthesis of liver mannose-containing glycoproteins and glycolipids.

* Present IAA,

address: U.K.

National

Institute

of

Medical

Research,

The

Ridgeway.

Mill

Hill,

London

NW7

343

Introduction The general metabolic role of vitamin A in maintaining growth and epithelial differentiation is still unknown. In vivo and in vitro synthesis of RNA [2-71 and protein [8] is affected by deficiency of the vitamin, but no specific molecular involvement of the vitamin has been found to specifically explain these effects. Epithelial glycoprotein biosynthesis is affected by deficiency [9] and excess [lO,ll] of the vitamin, and the synthesis of a fucose glycoprotein of the epithelial goblet cell was found to depend on vitamin A [ 12,311. Retinol [13,14] and chemically synthesized retinyl phosphate [15,16] are incorporated by mammalian liver membranes into a mannolipid. This compound has column chromatographic properties similar to dolichyl phosphate mannose [17], but it can be partially separated from it by thin-layer chromatography [18]. Retinyl phosphate mannose was found to be more labile to mild acid and base and to hydrogenolysis than dolichyl phosphate mannose [ 141. [ Curbinol- ’ “C] Retinol and [ 3R-4S-43H] mevalonic acid were found to be incorporated, in vivo, in liver and intestinal mannolipids [ 24,251. Helting and Peterson [ 191 have reported that retinol is incorporated into retinyl phosphate galactose and retinyl phosphate mannose by membranes from mouse mastocytoma and rat intestine [42]. They have published preliminary evidence that the galactolipid could function as a donor of galactose to endogenous acceptors. Retinyl pyrophosphate [ 201 and retinyl glycosides [ 211 are synthesized by homogenates of rat thyroid, and retinoyl-glucuronide is synthesized by rat liver [ 22,231. A role of retinyl phosphate monosaccharide in sugar transfer to specific endogenous or exogenous acceptors so far has not been demonstrated. Since we have shown that synthetic retinyl phosphate can function as an acceptor of mannose in a membrane system from liver, it was of interest to establish whether deficiency of the vitamin causes a decrease in the biosynthesis of liver mannolipids and mannoproteins, in vivo. Experimental

Procedures

Preparation of vitamin A deficient Syrian golden hamster: Study on severe vitamin A deficiency. 14 vitamin A-deficient male Syrian golden hamsters were

prepared by placing the mothers on a vitamin A-free diet at birth of the experimental animals [ 261. These were weaned on a vitamin A-deficient diet at 21 days. At 40 days hamsters prepared in this way begin to show signs of vitamin A deficiency such as eye lesions and reduced growth rate. Seven of the fourteen hamsters were kept on the vitamin-free diet and fed ad libitum. The other seven were given 300 pg and 500 pg retinyl acetate in 0.1 ml cotton seed oil by stomach tube on day 39 and 43, respectively. The deficient animals received cotton seed oil only (Fig. 2). The animals were weighed and they were sacrificed at day 44, 20 min after the injection of [l- ’ “C] mannose. A similar nutritional schedule was used to determine uptake of [ ’ 4 C] mannose into liver lipids at different times after intraperitoneal injection of the label, described under legend to Fig. 1. Preparation of labelled glycolipids and glycopeptides. [l- ’ “C] Mannose

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was supplied as a solution in ethanol/water (9 : 1, v/v). The solvent was removed under Nz and the [ ’ “C] mannose was dissolved in 0.85% NaCl. The solution was injected intraperitoneally (50 @i per hamster), 20 min before killing the animals. These were first ether anesthesized and most of the circulating blood was removed by cardiac bleeding. Livers were weighed, homogenized in two volumes of icecold medium A of Littlefield and Keller [37], unless stated otherwise. The homogenate (1 volume) was extracted with five volumes of chloroform/methanol (2 : 1, v/v), and the lower organic phase was washed once with 0.85% saline. The chloroform extracts were taken to dryness either by evaporation under N2 or by flash evaporation. The lipid extracts to be chromatographed on silicic acid columns were dissolved in a small volume of chloroform/methanol (2 : 1, v/v) which was then adjusted to 8 : 1 ratio by the addition of chloroform. The columns were prepared from a slurry of chloroform/methanol (8 : 1, v/v) and Biosil-A, which had been previously activated by heating at 100°C for 1 h and cooling in a desiccator. After application of the lipid, the columns were eluted with chloroform/methanol (8 : 1, v/v) and, then, in most experiments, a gradient of increasing proportions of methanol in chloroform. DEAE-cellulose chromatography was essentially as previously described [ 251. The lipids, dissolved in a small volume of chloroform/methanol (2 : 1, v/v), were applied to the columns which were first eluted with 99% methanol/l% water and then with a gradient of ammonium acetate in 99% methanol. The lipids eluted by the gradient were freed of the ammonium acetate by adding two volumes of chloroform to the methanol eluate and extracting with 0.2 volumes of water. The chloroform solution was taken to dryness by evaporation under Nz or flash evaporation. Gradients on silicic acid and DEAE-cellulose columns were made using an LKB ultrograd system. Proteolysis. The water phase and interphase material was extracted with one volume of chloroform and lyophilized to dryness. The dry residue was suspended in 20 ml of sterile solution of protease VI, from Sigma, containing 1 mg of enzyme per ml of 0.001 M sodium acetate buffer, pH 7.5. Digestion proceeded for 48 h at 37°C in the presence of toluene. Pronase CB from Calbiochem was added in a sterile solution in acetate buffer to a final concentration of 1 mg per ml. Toluene was added. Digestion continued for 48 additional hours. At this time further addition of protease did not release additional tyrosine, as assayed by Folin-Ciocalteau reagent. Glycopeptides were precipitated by the addition of ethanol as described under Results. Early vitamin A deficiency study, Four groups, a, b, c, and d, of three male hamsters each, were selected at day 34 after birth. These animals had been on the same vitamin A-deficient diet. Groups a and c were selected so that their weight and food consumption was essentially the same. Group c was given 300 pg of retinyl acetate in 0.1 ml of cotton seed oil by stomach tube at days 34 and 37. Their food intake and weight remained the same as group a, which was given only the vehicle cotton seed oil. There was no need for pairfeeding, since group a continued to eat at the same rate as group c. The mean body weight for group a was 52 g and for group c 52.6 g. Group b received the same treatment as c, except that the hamsters were bigger and ate more. Group d received the same treatment as group b except

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that they were given 300 pg of retinoic acid on day 34 and 150 pg of retinoic acid on day 37. The mean body weight for group b was 69 g per animal and for group d 63 g per animal. Animals were injected [l- ’ 4C] mannose as described for the severe deficiency study. They received 20 I.tCi per animal by intraperitoneal injection. Average liver weights were 2.94 g per liver for group a, 3.63 g for group b, 3.2 g for group c and 3.4 g for group d, Table III. 1 ml of homogenate was extracted with 5 ml of chloroform/methanol (2 : 1, v/v). The phases were separated. The organic phase was dried, dissolved in 0.1 ml of 99% methanol and counted. The chloroform/methanol (2 : 1, v/v) powder was collected on filters, washed with at least 100 ml of ice-cold ethanol, dried and combusted in a Packard Oxidizer for determination of radiocarbon. DNA [ 271 and protein [28] were determined in duplicate on each group. Assay of radioactivity. Lipid-bound radioactivity was assayed by liquid scintillation counting, using toluene (1000 ml), 2,5-diphenyloxazone (5 g) and 1,4bis- [ 2]-( 5-phenyloxazolyl)] benzene (50 mg). Efficiency was 66% for ’ 4 C and 34% for 3H. Radioactivity on thin-layer plates was located and assayed by scraping bands of desired width (from 0.1 to 0.5 cm) with an automatic zonal scraper from Analabs, Inc., North Haven, Conn. The scraped thin layer was automatically collected into counting vials and counted. Aqueous phases were counted in Bruno, Christian solution [ 361. Materials. [l-l ‘C] Mannose (150 Cilmol) was obtained from New England Nuclear Co., Boston, Mass. Pregnant Syrian golden hamsters were obtained from A.R. Schmidt, Madison, Wisconsin. Determination of pool sizes and specific radioactivities for mannose, glucose and galactose in normal and vitamin A-deficient livers, 20 min after the injection of ( ’ 4 C] mannose. For determination of differences in specific radioactivities of the precursor pool [ ’ “C] mannose was injected intraperitoneally, 20 min before killing, into one normal and one vitamin A-deficient 44-day-old hamster; their livers were combined with five normal livers and five vitamin A-deficient livers from animals of the same age. The average weight of the normal hamster liver in this experiment was 4.55 g and 3.95 g for the vitamin A-deficient liver. The normal animals were prepared as for the study on glycopeptide synthesis, by feeding the vitamin to partially depleted hamsters. The two liver preparations were homogenized in two volumes of deionized water; the homogenate was extracted with five volumes of chloroform/methanol (2 : 1, v/v). The upper phase was evaporated to dryness in a rotary evaporator below 40°C and resuspended in water. Residual proteins were removed by 5% trichloroacetic acid at 4°C. The precipitate was removed by centrifugation and the supernatants were extracted twice with half the volume of ethyl ether to remove the trichloroacetic acid. The solutions were brought to neutrality with 1 M NaOH. The samples were evaporated to dryness and suspended in water. Boron&ion reaction. This reaction was carried out essentially as described by Eisenberg [ 11. Equal volumes of a solution of’NaBH4 (120 mg/ml) were added to the samples and the reaction proceeded for 15 min. The excess borohydride was removed by addition of 0.5 ml of 4XAG 50 W resin (H’ exchanger). When evolution of hydrogen ceased, 2 mg of arabinose was added

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as a standard. The resulting mixtures were placed on columns containing 0.5 ml of the same resin. The columns were eluted with deionized water, about 4 ml, which was removed by evaporation. The dry residue was washed with methanol twice and the methanol removed by evaporation under N *. Pyridine was added and evaporated to remove methanol. At this stage a viscous residue was obtained. A solution (1 ml) of n-butaneboronic acid (15 mg/ml) was added and the samples were spun to remove the unwanted residue. The supernatants were used for determination of monosaccharides by gas-liquid chromatography. Gas-liguid chromatography. A Hewlett-Packard 5700 A gas chromatograph with flame ionization detector was used. The boronic acid derivatives of the hexitols were analyzed on a 6-ft glass column, internal diameter 2 mm, made by HewlettPackard. The column was packed with OV-17 3%, 100/120 mesh, on Gas Chrom Q, obtained from Supelco. The column temperature was 200°C. The injection temperature was 250“ C, the detection temperature was 300°C and the auxiliary temperature was 300°C. The retention times and other details are given under results. A 10 : 1 splitter permitted the collection of the radioactive materials in 1.75 inches collection capillaries, which were counted directly. A Hewlett-Packard Integrator 3370B was used for collection of peak areas. Counting efficiencies and recovery of radioactive material were determined on known amounts of standards as follows. Glucose, galactose and mannose (2 mg of each) were added to two radioactive mixtures: (1) [ 3H] mannose, [ ’ 4 C] glucose and [ 3H] galactose; (2) [ ’ 4C] mannose, [ 3H] glucose and [ ’ 4 C] galactose. Since the order of elution from the column was first mannose, second glucose and third galactose, the two mixtures were designed to determine the error of collection and whether any spill-over of radioactivity occurred. Areas were collected in capillaries from the peaks of monosaccharides and the intermediate areas. Results are given in the text. Essentially the same proc,edure was used for determination of radioactive monosaccharides incorporated into glycopeptides. These were hydrolyzed as described under Results. Mild alkaline and acid hydrolysis of mannolipids. Lipids eluted from DEAE-cellulose were treated with mild alkali as follows. Eluates were dried under Nz, dissolved in 500 ~1 of chloroform/methanol (1 : 4, v/v), to which 50 ~1 of 1 M NaOH was added, and incubated for 15 min at 37°C. 50 ,ul of 1 M acetic acid was added to neutralize the hydrolyzates, 1600 ,ul of chloroform, 400 ~1 of methanol and 400 (~1 of water were added in sequence and the two phases separated by centrifugation. The two phases were analyzed as specified under Results. For the acid hydrolysis, the dry eluates were dissolved in 400 ~1 of 0.01 M HCl in methanol/water (1 : 1, v/v), Samples were heated at 100°C for 15 min, cooled, neutralized with 400 ~1 of 0.01 M NaOH in methanol. 1000 ~1 of chloroform was added, followed by 200 ~1 of water. The phases were separated by centrifugation and analyzed as specified under Results. Results [l- ’ 4C] Mannose incorporation into normal and vitamin A-deficient hamster liver glycolipids. To study the time course of the incorporation of [l- ’ 4 C] mannose into normal ad libitum fed and severely vitamin A-deficient hamsters a group of

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TIME,h Fig. 1. In viva incorporation of [l- “Cl mannose into lipid extract of hamster liver at different times from the injection of the label. Each hamster was injected (intraperitoneally) with 50 I.rCi of [1-14C1 mannose in saline. Animals were sacrificed in pairs at each of the following time points: 15: 30; 90; 180; 240 min after the injection of the label. Livers were extracted directly into five volumes of chloroform/methanol (2 : 1, v/v) by vigorous homogenization. The final volume of the lipid extract was 15 ml. The lipid phase was extracted with 3 ml of water. twice. One-tenth of the lipid phase was assayed for radioactivity. .-----a, normal; o- - - _ - -0, vitamin A deficient.

twenty 39-day-old hamsters was divided into two groups of 10. One received vitamin A and the other vehicle cotton seed oil as described for preparation of severely deficient and normal hamster under Experimental Procedures. Livers were processed as described. Fig. 1 shows that the highest incorporation of [ ’ “C] mannose in the organic phase occurs at about 20 min from the injection of the label. This time was used for all‘subsequent studies with [ ’ 4 C] mannose. Fig. 1 also shows a very dramatic decrease (90% or more) in the amount of label incorporated into vitamin A-deficient lipids. We examined whether severe vitamin A deficiency affects specific mannolipids or all [l- ’ 4 C] mannose labelled lipids 20 min after the injection of [l- ’ “C] mannose. Two groups of seven normal ad libitum fed and seven severely deficient male hamsters were prepared as described under Fig. 2. Weight curves for each of these animals from age 34 days to day 44, when they were killed, are shown in Fig. 2. Vitamin A-treated animals had not lost weight prior to the administration of the vitamin, which kept them on a normal growth rate. The deficient group had been selected for early signs of vitamin A deficiency, i.e. eye lesions and slower growth rate (Fig. 2b). [l- ’ “C] Mannose-labelled lipids were prepared as described under Experimental Procedures. Normal pooled livers weighed 29.5 g and deficient livers 21.9 g. The organic phases were combined (approx. 500 ml for normal and 386 ml for deficient). The water phase and interphase material as described later was processed for extraction of ’ 4 C-labelled glycopeptides, and under Experimental Procedures. The lipid .phases were dried under Ni?, dissolved in 5 ml of chloroform/methanol (2 : 1, v/v) and chloroform was added to a final concentration of chloroform/methanol (8 : 1, v/v) in preparation for chromatography on silicic acid.

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Fig. 2. Preparation of severely deficient hamsters. A group of 14 hamsters was kept on a vitamin A-deficient diet as described under Experimental Procedures. At age 39 days, they were separated into two groups of seven each. Group A was fed the vitamin, as detailed under Experimental Procedures, and continued to grow normally. Group B only received the vehicle cotton seed oil and lost weight, as evident from the individual weight curves.

Silicic acid chromatography The total lipid extract from normal hamsters (18 . lo6 cpm) and the lipid extract from vitamin A-deficient hamster livers (2.1 . lo6 cpm) were applied to identical columns, which were eluted as indicated under Fig. 3 legend. Approx. 80% of the radioactivity was eluted with chloroform/methanol (8 : 1, v/v),

IO

20

30

50

FRACTION

NUMBER

70

Fig. 3. Chromatography on silicic acid of [ 14C1 mannose-labelled lipid extract. Two columns (2.5 X 30 cm) were prepared as described in the text. The lipid extract from seven vitamin A normal hamster livers, livers, 2.1 . 106 cpm, were applied to their respective 18.0 . 106 cpm, and seven vitamin A-deficient identical columns, as a solution in chloroform/methanol (8 : 1, v/v). The columns were eluted with 216 ml of chloroform/methanol (8 : 1, v/v). fractions l-24. The columns were then eluted with a linear gradient of chloroform/methanol (8 : 1, v/v) to chloroform/methanol (1 : 3. v/v). Out of each fraction of 9 ml, 0.1 ml was used for counting. Only the elution of the vitamin A normal lipid is shown. The deficient lipid had an identical behavior. Recovery of radioactivity from the normal preparation was as follows: a total of 10.2 . 106 cpm was recovered (56.5%). Of the eluted fractions the first peak contained 8.4. 106 cpm. and the second, more polar peak, contained 1.8 . 106 cpm. Recovery of radioactivity from the vitamin A-deficient preparation was as follows: a total of 1.43 106 cpm was recovered (68%). Of the eluted fractions the first peak contained 937 000 cpm and the second peak contained 493 000 cpm. Clearly there was a very dramatic decrease in all fractions from vitamin A-deficient hamsters.

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from both columns and the rest as a broad peak at approximately chloroform/methanol (1 : 1, v/v). The material eluted at chloroform/methanol (8 : 1, v/v) was analyzed by thin-layer chromatography on silica gel plates in chloroform/methanol/water (60 : 25 : 4, v/v). The RF for both normal and vitamin A-deficient preparation was 0.8. The product was not characterized further. The main peaks of radioactivity eluted off silicic acid with chloroform/ methanol (1 : 1, v/v) were collected in tubes 44 + 70 for normal and 39 --f 65 for vitamin A-deficient preparations. They were extracted with 0.2 volumes of water. About one-third of the radioactivity became water soluble. An aliquot (5000 dpm) of the remaining lipid phases were chromatographed on thin-layer plates in chloroform/methanol/water (60 : 25 : 4, v/v). 0.5 cm bands were counted. Counting efficiency was 40-45%. Three peaks of radioactivity were found: One at RF 0.21 (similar to the retinyl phosphate mannose synthesized in vitro by rat and hamster liver membranes [15,16] from retinyl phosphate and guanosine diphosphate mannose); a second at RF 0.29 and a third at R, 0.5. All three peaks were drastically affected by vitamin A deficiency, with at least 75% decrease in the amount of label incorporated in each peak. Subsequent studies on the purification of the mannolipids were conducted only on the normal hamster liver preparation. DEAE-cellulose chromatography Further purification of the ’ 4 C-labelled glycolipid from silicic acid was obtained by chromatography on DEAE-cellulose [25] . The column was prepared as detailed under Experimental Procedures. An aliquot (about 270 000

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Fig. 4. DEAE-cellulose chromatography of normal [ L4 Cl mannose-labelled lipid eluted from silicic acid by the chloroform/methanol 8 : 1 to 1 : 3 (v/v) gradient. DEAE-cellulose (1.2 X 12 cm) was prepared as lipid eluted at about chloroform/ previously reported [251. An aliquot of 270 000 dpm of 14C-labelled methanol (1 : 1. v/v), Fig. 2, was partitioned between one volume of water and five volumes of chloroform/methanol (2 : 1, v/v). The lipid phase applied to DEAE contained about 180 000 dpm of 14C-labelled lipid. The non-phosphorylated lipids were eluted with 99% methanol (about 60% of original radioactivity) in fractions 3-7. A gradient of WO.l M ammonium acetate was aDplied, and for 8 h 5ml fractions were collected. 0.5 ml was assayed for radioactivitu. after removing the solvent. Lipids 1. 2 and 3 were pooled as indicated by the solid bars, dried and stored under liquid nitrogen.

350

cpm) of the silicic acid lipid (tubes 44-70) was dried under N2 and repartitioned between 5 ml of chloroform/methanol (2 : 1, v/v) and 1 ml of water. The lipid phase (about 180 000 cpm) was applied to a column of DEAE-cellulose (Fig. 4) (120 X 12 mm), equilibrated with 99% methanol. 99% methanol eluted about 60% of the radioactivity (tubes 3-7). This lipid was labelled Lipid I. This lipid chromatographed at RF 0.5 on silica gel plates. It did not give any water-soluble product when heated with diluted acid (0.1 M) at 100°C for 20 min, conditions which are known to hydrolyze prenyl phosphate mannose compounds. The gradient eluted a major peak of radioactivity with 0.025 M ammonium acetate and a small peak at 0.050 M. These were designated Lipid 2 (tubes 19-24) and Lipid 3 (tubes 27-35), respectively. Lipids 2 and 3 were dried, the salt removed by partition between water and chloroform/methanol (2 : 1, v/v). A total of 150 000 cpm out of the original 180 000 cpm were recovered in the lipid phase. Upon thin-layer chromatography Lipid 2 gave a broad peak of radioactivity between RF 0.2 and 0.32 and a smaller peak at RF 0.37. Lipid 3 gave a compound at RF 0.24. Ultraviolet absorption on plates consistantly coincided with the compound with low mobility. This internal ultraviolet absorbing indicator was useful because variation in mobility of the labelled lipids was observed among different batches of plates. When [ ’ ‘C] niannolipids are synthesized in vitro from guanosine diphosphate [ ’ “C] mannose and are chromatographed by DEAE-cellulose and silicic acid their behavior is similar to that of Lipid 2. Thus this compound should contain both retinyl phosphate mannose and dolichyl phosphate mannose, if they exist in vivo. This was confirmed by using as specific precursors [carbinol’ “C] retinol and [ 3R, 4S-4 ‘H] mevalonic acid. The results of the labelling experiment are reported elsewhere [25]. Lipids 2 and 3 were tested for their stability to mild alkali and acid. Hydrolyses

33 000 cpm of Lipid 2 was treated with mild alkali, as specified under Experimental Procedures. Under these conditions retinyl phosphate mannose releases mannose and mannose l-phosphate in the aqueous phase [ 14,251, but dolichyl phosphate mannose is stable [ 171. Lipid 2 released about 30% (11 500 cpm) of its radioactivity into the water phase. Mannose l-phosphate and mannose accounted for about 60% of the radioactive product. In a separate experiment [ 251 [carbinol- ’ “C] retinol and [ 3R, 4S-4 3H] mevalonic acid were both incorporated in Lipid 2 from hamster liver. Mild alkali treatment hydrolyzed the [carbinol’ “C] retinol lipid but did not hydrolyze the [ 3H] dolichyl phosphate mannose. This was hydrolyzed with mild acid [ 251. The same procedure was followed for the alkali-resistant lipid. After alkaline hydrolysis, the organic phase was chromatographed on a short column of silicic acid (0.9 X 6 cm). 34% (6400 cpm) of the lipid was eluted with chloroform/methanol (8 : 1, v/v), possibly a breakdown product. 66% (15 100 cpm) was eluted with chloroform/methanol (1 : 1, v/v), and behaved as synthetic dolichyl phosphate mannose in chloroform/methanol/water (60 : 25 : 4, v/v). Upon mild acid hydrolysis 46% of the radioactivity became water soluble. The aqueous phase was chromatographed on Whatman 3MM paper, using butanol/pyridine/water (9 : 5

351

: 4, v/v) as solvent, together with standard D-glucose, D-galactose and D-mannose and their glycosyl phosphates. 60% of the radioactivity cochromatographed with D-mannose, 30% with methyl-P-mannoside. Stronger acid hydrolyses (0.1 M HCl) gave higher yields of water-soluble products. Lipid 3 did not behave as either retinyl phosphate mannose or dolichyl phosphate mannose, when hydrolyzed under the same conditions as Lipid 2. In a separate experiment neither [carbinol- ’ “C] retinol nor [3R, 4S-4 3H]mevalonate were incorporated into this lipid [ 251. In conclusion, only Lipid 2 of the in vivo labelled lipids, 20 min after the injection of [ ’ “C] mannose, had properties similar to in vitro synthesized mannolipids. This lipid represents only 5-10% of the total lipid-bound ’ 4C radioactivity; in contrast W---90% of the lipid-soluble [ ’ “C] mannose, after incubation with liver membranes and guanosine diphosphate [ ’ “C] mannose, is accounted for by the mannolipids. As in the case of the in vitro synthesized compound, the in vivo synthesized mannolipid (Lipid 2) appears to contain two compounds which are partially resolved by thin-layer chromatographys in chloroform/methanol/water (60 : 25 : 4, v/v). The compound with low mobility is labile to mild alkali, giving mannose l-phosphate and mannose. [l- ’ 4C] Mannose incorporation into glycopeptides of normal and severely deficient hamster livers The digested glycopeptides from normal and vitamin A-deficient hamster livers, obtained as described under Experimental Procedures, were precipitated in the presence of 5 mg of hyaluronic acid per ml of the glycopeptide solution, total volume 20 ml, by addition of 60 ml of ice-cold ethanol. The precipitate was allowed to sediment at -20°C for 24 h, was collected by centrifugation, washed with 99% ethanol and ethyl ether, and dried. An aliquot was counted. No further precipitation occurred after 3 days at -20°C. The supernatant contained only dialyzable material. The normal glycopeptide preparation had 11 725 200 dpm dissolved in 23 ml, the deficient one had 2 982 500 dpm dissolved in 20 ml; 5.75 ml (2 931 300 dpm) for normal and 5 ml (745 600 dpm) for deficient was applied to columns of DEAE-Sephadex A-50 (Figs 5A and 5B). The columns were eluted with a gradient of 0.5 --f 1 M LiCl, in a similar manner for the normal and vitamin A-deficient preparations. Three main peaks of radioactivity were collected for normal glycopeptides as shown in Fig. 5. These areas were designated I, II, III prefixed by N for normal. Peak NI comprised tubes 6-17 (2 584 300 dpm); Peak NII tubes 25-32 (211 300 dpm); Peak NIII tubes 38-47 (234 200 dpm). Three main peaks of radioactivity were eluted for vitamin A-deficient glycopeptides. Peak DI comprised tubes 7-17 (506 700 dpm); Peak DII comprised tubes 19-25 (169 000 dpm); Peak DIII comprised tubes 45-51 (38 600 dpm). The elution was different for normal and deficient peaks and identical roman numerals are not intended to indicate identity of glycopeptides. Peaks NI (2 584 300 dpm) and DI (506 700 dpm) obtained from DEAE-Sephadex A50 were lyophilized to dryness, dissolved in 5 ml of water and chromatographed on columns of Sephadex G-100 (2.5 X 40 cm) equilibrated with 0.02 M LiCl. Radioactive peaks were eluted later (150-225 ml) than the void volume (69 ml); in the area of elution of standard [ ’ “C] mannose, which was

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Fig. 5. DEAE-Sephadex chromatography of [ 14CImannose-labelled glycopeptides from vitamin A normal and deficient hamster liver. The glycopeptides were prepared by proteolysis. as described. Two columns of DEAE-Sephadex A50 (2.5 X 31 cm) were equilibrated with 0.05 M LiCl. One-fourth of the total glycopeptides from normal animals and 745 600 dpm glycopeptides, i.e. 2 931 300 dpm of “C-labelled of 14C-labelled glycopeptides from vitamin A-deficient hamsters, dissolved in 0.05 M LiCl. were placed the columns. A convex gradient of 0.05-l M LiCl was applied by an automatic LKB gradient maker. The gradient was collected in 48 h. Fractions of 12 ml were collected for the vitamin A-deficient and 19 ml for the vitamin A normal preparations. No radioactivity was eluted above 0.3 M LiCl. Recovery of radioactivity was 102% for normal and 96% for deficient 14C-labelled glycopeptides. Three main radioactive peaks were pooled separately for normal (Peaks NI, NII and NIII) and deficient (Peaks DI, DII. DIII) glycopeptides. Peak NI comprised tubes 6-17 (2 584 300 dpm); Peak NII comprised tubes 25-32 (211 300 dpm); Peak NH1 comprised tubes 38-47 (234 200 dpm). Peak DI comprised tubes 7-17 (506 700 dpm); Peak DII comprised tubes 19-25 (169 000 dpm): Peak DIII comprised tubes 45--51 (38 600 dpm).

run separately. Hyaluronic acid, used as carrier in the precipitation of glycopeptides, is usually eluted at the void volume. Recovery of radioactivity was, for Peak NI 2 521 900 dpm and for Peak DI 426 000 dpm. These were pooled, lyophilized, dissolved in 5 ml of water and applied to columns of Sephadex G 25 superfine. Sephadex

G-25 superfine

A 2.5 X 40 cm column was prepared and equilibrated with 0.02 M LiCl, V,, was 92.8 ml, [ ’ “C] mannose was eluted between 170 and 175 ml. Peaks NI and DI were pooled and lyophilized. They were dissolved in 5 ml of water and applied to the column. Identical chromatographies were performed with Peaks NII, NIII, DII and DIII. NIA was eluted between V, (92.8 ml) and 120 ml, similar to Peak DIA. Peak NIA had 2 431 500 dpm and Peak DIA 108 400 dpm, a reduction in incorporation of [l- ’ “C] mannose of approx. 95%. Peak NIA represent 80% of the total [ ’ 4 C] mannose incorporated into normal liver glycopeptides. Peak NIB was eluted in the area of mannose, similar to Peak DIB. Peak NIB had 306 500 dpm and Peak DIB 242 000 dpm. Peaks NII, NIII, DII and DIII were lyophilized and chromatographed directly on Sephadex G-25 superfine. Each peak gave at least two components; (A) of higher molecular weight; the other, (B) eluted in the area of [ ’ “C] mannose. Table I summarizes the results.

353

TABLE

I

SEPHADEX

G-25

BELLED Each

of

(2.5 ml)

the

X 40 of

the

all eluted pooled

SUPERFINE

GLYCOPEPTIDES six

cm)

14C-labelled

columns

column just

after

separately.

equilibrated and paper

was

with

of

glycopeptides Sephadex

superfine

with

the

void

volume

was

removed

dextran

and

water.

by The

N stands

DPM

obtained

G-25

determined

deionized

OF

HAMSTER

LIVER

[14Cl

MANNOSE-LA-

DEAESEPHADEX

LiCl

chromatography.

14C

CHROMATOGRAPHY FROM

peaks

as described equilibrated

blue B and

a second

desalted

the

C in the

area

peaks

Results

0.02 of on

A

was

M LiCl.

of mannose

V,

chromatography radioactive

for normal,

free

was

to

void

volume

170

ml.

[3Hlmannose.

an identical

were

applied

The

used

(92.8

Peaks

A were

Peaks

A were

column

for strong

identical

of

Sephadex

acid hydrolysis

D for deficient. C

B

A

and

under with

A+B+C

Recovery __.__

(%‘o)

Normal 2 621900

2 431

500

NII

NI

211300

190

200

NIII

234

200

DI

426

000

400

242

DII

169

000

24 300

100

38 600

3 100

33 000

306

2 738

000

108

190

200

90

142

200

60

000

350

400

82

400

124

700

74

30 800

79

500

0 33 300

75900

Deficient

DIII

The small molecular by paper chromatography gas-liquid chromatography Analysis of radioactivity

108

13 200

14 500

weight component was shown to be [l- ’ 4 C] mannose in butanol/pyridine/water (9 : 5 : 4, v/v) and by of the boronic acid derivatives of the hexitols. in A and Bpeaks

off Sephadex

G-25 superfine

Peaks were desalted on Sephadex G-25, lyophilized, hydrolyzed by 2 M HCl for 3 h at 100°C in vials sealed under N *. Vials contained 5000 dpm of each glycopeptide, except for Peak DIII, 500 dpm. HCl was removed by flash evaporation and the dry residue was resuspended in a few drops of deionized water and dried. The dry residue was dissolved in 30 ~1 of water and applied to Whatman No. 1 paper for chromatography in butanol/pyridine/water (9 : 5 : 4, v/v) along with standard mannose, glucose and galactose. Monosaccharides were localized by reduction of a AgN03 solution. Radioactivity was found to coincide with mannose. The identification of [ ’ “C] mannose in Peaks NIA and DIA was also determined by gas-liquid chromatography of the boronic acid derivatives of the hexitols, after strong acid hydrolysis. About 58 000 ’ 4C dpm of Peak NIA and 14 600 dpm of Peak DIA was used for this analysis. Table II has the results. For Peak NIA 93% of the radioactivity was recovered in the area of mannose, for Peak DIA 80%. An important finding emerged on the amount of mannose present in the two biological conditions. 1 g of wet normal liver contained 0.94 mg of mannose in Peak NIA. 1 g of wet vitamin A-deficient liver contained 0.19 mg of mannose in Peak DIA, a reduction of 79%, when wet weight of livers had dropped only 26%. Interestingly the specific radioactivities of [ ’ 4 C] mannose in Peak NIA was 187 dpm/pg of mannose and in Peak DIA 48 dpm/pg of mannose, Table II.

354

TABLE

II

HYDROLYSIS GAS-LIQUID

AND

DETERMINATION

CHROMATOGRAPHY

OF OF

THE

SPECIFIC BORONIC

RADIOACTIVITY ACID

OF

DERIVATIVES

[‘4C1MANNOSE OF

THE

BY

HEXITOLS

An aliquot from Peak NIA (58 000 dpm) and Peak DIA (14 600 dpm) obtained from DEAE-Sephadex, Sephadex G-100 and two consecutive passages on Sephadex G-25 superfine. was hydrolyzed for 3 h at 100°C in 2 M HCI in a N2 atmosphere. The dry residue after removal of HCI was treated as specified under Experimental Procedures to reduce the hexoses to hexitols and to obtain the boronic acid derivaindicates the area collected tives 111. Collection of monosaccharides was as for previous work: “Before” before the mannose peak. Conditions were standardized as described. Efficiency of recovery and counting in capillaries of radioactive monosaccharides was 27%. Results are given in cpm as determined off the machine and calculated per g of liver for comparative purposes, since relatively more of the Peak DIA sample was used. The boronic acid derivatives were dissolved in 0.5 ml of pyridine. 50h for Peak NIA and 1OOh for Peak DIA was evaporated to dryness and redissolved in 5h suitable for injection. Condensates were collected corresponding to total volume eluted before mannose and the volumes of mannose, glucose and galactose and counted in 20 ml of toluene, PPO, POPOP.

Volumes

Before Mannosr Glucose Galactow

CPM Peak NIA

146 1465 212 6 ,ug of Hexose

Mannose Glucose Galactose

Mannosr Glucose Galactose

CPM Peak DIA

Total dpm 14C/g liver in: ._~_ ____~._~ ~.

134 632 87 23 in Peak NIA/g Liver

Peak NIA

Peak DIA

17 500 176 600 25 563 716

1990 9 400 1 290 341

pg Hexosr

in Peak DIAig Liver

942 13 342 211

197 328 47

Specific Radioactivity in dpm 14C/pg Hexose in Peak NIA

Specific Radioactivity in dpm 14C/pg Hexosc in Peak DIA

187 1.9 3.4

48 3.9 7.1

Study of early vitamin A deficiency

Four groups, a, b, c, d, of three male hamsters each were prepared as described under Experimental Procedures. The animals were sacrificed at day 38 when the weight for the deficient hamsters, group a, was at the plateau or preplateau stage. It is important to state here that precautions were taken to avoid infection: The hamsters were kept in cages with filter tops and their hardwood bedding was changed every other day. Labelling and processing of liver lipids and protein by [l-’ “C] mannose was as described under Experimental Procedures. The results are given in Table III. The lipid phase showed a 63% drop in the incorporation of [ ’ 4 C] mannose into the vitamin A-deficient group a (26 000 cpm/g liver) compared to group c (70 900 cpm/g liver), which had identical weight as a. Group b had 99 000 cpm/g liver and the retinoic acid-fed animals 57 400 cpm/g liver. These results do not change significantly if expressed as cpmimg of liver protein or cpm/mg of DNA. Both protein and DNA values are given in Table III. [ ’ 4 C] Mannose incorporation into glycoproteins was 67 300 cpm/g liver for group a, 184 000 for group b, 186 600 for group c,

355

TABLE [14Cl

III MANNOSE

INCORPORATION

INTO

GLYCOLIPIDS

AND

GLYCOPROTEINS

OF

HAMSTER

LIVERS Groups group,

a, b. c, d were homogenized

associated animals

with is the

lipid

average

Group -

prepared

in and

two

proteins

weight

Body

as described

volumes

of

Experimental A.

was determined

per animal.

Weight .___

under

medium Body

Liver _____

Protein

as described

weight

Weight

DNA

under

and liver

(g)

Procedures.

and

weight

Livers

were

Experimental is given

mg Protein/g

were

combined

in triplicate.

Procedures.

Liver

mg

a

52

2.94

173.2

1.69

69

3.63

204.1

1.17

C

52.6

3.2

175.4

1.41

d

63

3.4

198

1.03

14C-Labeled

cpm

Protein/

cpm

mg Protein

a

365

b

901

C

CPM-Lipid/g

Liver

g Wet

of

Liver

Protein/

Liver

63 300

156

300

184

000

132

000

186

600

184

800

191300

CPM-Lipid/mg

DNA/g

14C-Labeled

cpm

39 600

966

Group

Protein/

mg DNA

1063

d

14C-Labeled

Weight

in g.

b

Group

per

Radioactivity

Protein

CPM-Lipid/mg

a

26 000

150

15312

b

99 000

485

84 112

C

70 900

405

50 176

d

57 400

289

55 458

DNA

and 191 300 for group d. Clearly [ ’ 4 C] mannose incorporation into liver glycoproteins and glycolipids depends on the vitamin A status of the animal and its drastic drop becomes evident before loss of appetite and weight occurs. Pool

studies

It was desirable to determine whether the observed decrease in the incorporation of [ i “C] mannose into mannolipids and mannoproteins, in vitamin A deficiency, was simply a reflection of a decreased specific radioactivity of the pool of [ ’ “C] mannose and whether a change in this pool was due to decreased or increased conversion of this monosaccharide to glucose and galactose. For this purpose one normal and one vitamin A-deficient 44-day-old hamster was injected with [ i 4 C] mannose as described under Experimental Procedures. The animals were bled from the heart, killed 20 min later, and processed together with five normal and five vitamin A-deficient 44-day-old hamster livers, for the purpose of measuring pool sizes and specific radioactivities of mannose, glucose and galactose. The efficiency of collection of radioactivity was determined by using about 1000 cpm of each of the following compounds, in two different experiments: [ ’ 4 C] mannose, [ 3H] glucose and [ ’ 4 C] galactose, and [ 3H] mannose, [ ’ 4 C] glucose, and [ 3H] galactose. The peaks were collected as well as the intermediate areas. The results demonstrated that no cross contamination occurred and that each radioactive standard was collected in its own area only. The efficiency of collection was from 65 to 71% of the injected material.

356 TABLE

IV

DETERMINATION OF POOL SIZES OF MANNOSE, GLUCOSE SPECIFIC RADIOACTIVITIES, IN LIVER, 20 MIN AFTER THE INTO NORMAL AND VITAMIN A-DEFICIENT HAMSTERS

AND GALACTOSE AND THEIR INJECTION OF [‘4C1MANNOSE

Boronate derivatives of hexitols were prepared from normal and vitamin A-deficient hamster livers as described under Experimental Procedures. A 10 : 1 splitter was used for determination of radioactivity. No spillover of radioactivity was found between different monosaccharide areas, as determined by two [3Hlglucose experiments with [ 3Hlmannose, [ 14Cl glucose and [ 3H] @lactose and with [ *4Clmannose, and [ 14C] gala&we, as detailed in Results. The samples were collected in 1.75 inches tapered capillary tubes, for counting. The nitrogen flow rate was 45 ml per min and the retention times for arabinose (used as an internal standard) was 1.43 min, mannose 5.1 min, glucose 6.13 min and galactose 7.4 min. Recovery of radioactivity in the collection tubes was determined with known standards and found to be between 65 and 71’%. All measurements of radioactivity were done to an accuracy of at least 2.5% of standard deviation and the amounts of radioactivity per vial were in alI cases greater than 500 cpm, at an efficiency of about 70%. This experiment was repeated with very similar results. Normal

Pool siLe in nMolfg liver

Total radioactivity in dpmig liver

Specific radioactivity in dpm/nMol

Mannosc Glucose Galactosr

699 15870 32

1.6 0.95 0.04

228 5.98 125

560 13 700 79

4.9 105 I .9 * 105 0.10~10~ _..-

Vitamin

10” 105

I.05

A

Deficient Mannosc Glucow Galactose

~__~__~_

875 13.5 126

The results of the pool size and specific radioactivity studies are shown in Table IV. Clearly, the amounts of free mannose and gmcose are very similar for normal and deficient livers; the size of the pool for galactose is very low and great accuracy cannot be expected for galactose. In contrast to the identity of the size of the pools, for normal and deficient preparations, the total amount of radioactive mannose and glucose is greater in vitamin A-deficient livers by $-fold for mannose and 2-fold for glucose. Thus the specific radioactivity for normal glucose and mannose is also lower than for vitamin A deficiency by the same factor (Table IV). It is concluded that the decrease in incorporation of [ 1‘C] mannose into deficient glycopeptides and glycolipids is not a reflection of a decrease in the specific radioactivity of the precursor pools. In fact, these are much greater in vitamin A deficiency than in normal livers. Another interesting fact emerged from these studies. The extent of metabolism of mannose to glucose after 20 min from the injection of Iabelled mannose appears to differ in the two biological conditions: 42% of the total recovered radioactivity is present as glucose in the normal preparation and only 27% in the vitamin A-deficient preparation. This may be a reflection of a decreased utilization of radioactive mannose in vitamin A deficiency for biosynthesis of glycoproteins or glycolipids. It is also of interest to note that although 42% of the total radioactivity, 20 min after the injection of [ i 4 C] mannose, is associated with [ ’ 4 C] glucose, only small amounts of radioactive glucose are found incorporated into glycopeptides. This indicates a separation of pools of free monosaccharides from

357

those destined branes.

for the biosynthesis

of glycoproteins

and glycolipids

in mem-

Discussion Studies with liver, intestinal membranes and fibroblasts have shown that retinol [13,14,19,40] and chemically synthesized retinyl phosphate [ 15,161 are incorporated into retinyl phosphate saccharide compounds. In vivo studies have tended to confirm the in vitro findings [24,25]. However, the in vivo synthesized compounds have not been fully characterized, mostly because the available techniques have not allowed good resolution of retinyl phosphate saccharide compounds between themselves and from dolichyl phosphate saccharide compounds [ 24,251. Because of this, a direct demonstration of whether or not retinyl phosphate saccharides transfer their saccharidic components to specific acceptors has been difficult. A second approach, in our laboratory, has been to use vitamin A-deficient animals in the study of glycoprotein synthesis. This approach is not without difficulties, due mostly to the fact that severe vitamin A deficiency leads to inappetence, weight loss and inanition. In the past, to avoid nutritional differences, animals have been fed the same amount of food as vitamin A-deficient animals [ 121 or vitamin A-deficient animals have been force-fed a high protein diet [34,35]. Either of these approaches may be criticized: A normal animal eating as little as a severely deficient animal is not a proper control, but a stressed animal. Conversely a vitamin A-deficient force-fed animal is equally stressed and will not digest properly. If a biochemical difference is a direct consequence of vitamin A deficiency, it should become apparent prior to loss of appetite, which has been listed as the earliest symptom of vitamin A deficiency. The stimulus to consume food is obviously a result of nervous, digestive and other functions which are impaired in the deficient animal, in which taste preferences are also altered [39]. Our measurements were made at different stages of vitamin A deficiency, with severely deficient hamsters which weighed 25% less than their controls, and with hamsters at a stage of deficiency when weight and appetite were the same as vitamin A-treated controls. We have previously demonstrated that deficiency of the vitamin causes a decrease in the synthesis of epithelial glycoproteins [9,12,29,30]. The glycoprotein from rat intestinal goblet cell has been isolated and characterized as dependent on the vitamin for its synthesis [9,12,31]. However, in all the preceding studies on glycoprotein synthesis, mannose, a component of membrane glycoproteins, serum glycoproteins, hormonal glycoproteins and immunoglobulins, was never used as a labelled precursor. In this work we have demonstrated for the first time that the incorporation of [ ’ “C] mannose into liver glycolipids and glycoproteins depends on vitamin A, even at a time in the progression of deficiency, when the weight of the animals and their food consumption were normal. The marked decrease in incorporation of [ ’ “C] mannose into both classes of compounds did not change, whether results were expressed per DNA, per protein or per wet weight. Determination of the size of the pools for mannose, glucose and galactose

358

and their specific radioactivity showed accumulation of labelled monosaccharides in vitamin A deficiency. It also showed 27% (for deficient) and 42% (for normal) conversion to [ ’ 4C] glucose, 20 min after intraperitoneal injection of [ ’ 4 C] mannose. However, hydrolyses of labelled glycopeptides showed very little covalently bound [ ’ 4 C] glucose. A most important finding emerging from these studies is that the amount of covalently bound mannose in glycopeptides decreased by 79% in vitamin A deficiency. Moreover, specific radioactivity of [ ’ “C] mannose in glycopeptides was still much smaller in deficient livers. To our knowledge differences as big as those described in this paper for the biosynthesis of mannolipids and mannoproteins have not been reported for other biosynthetic activities at an early or late stage of vitamin A deficiency. At least ten enzyme activities have been described as dependent on vitamin A (for a review, see refs 32 and 33). Controlled nutritional studies were done by Rogers [33] on at least three of these enzymes: ATP sulfurylase, A5-3-fl-hydroxysteroid dehydrogenase and Lgulonolactone oxidase. The conclusions were that the effects of vitamin A deficiency reported for these enzymes were due to inanition and stress and that the different effects obtained with pair-fed controls were due to the particular feeding protocol used in different laboratories. Biosynthesis of RNA was also found to be dependent on vitamin A [2-6]. However, these observations were made at a point in deficiency when the animals had lost weight. Moreover, the reported effects were small and no actual decrease in weight of RNA except for tRNA [ 3 ] was ever reported even in advanced deficiency. The reported results allow us to conclude that vitamin A deficiency leads to severe impairment of the biosynthesis of mannose-containing glycolipids and glycoproteins. Whether this is a direct result of a molecular involvement of retinol or a metabolite in glycosyl transfer reactions remains to be established. It must be emphasized that although in vivo labelled compounds have been isolated with properties similar to in vitro synthesized retinyl phosphate mannose, the possibility still exists that the two compounds are different. Since vitamin A is present in very small amounts and intermediates may be present in smaller concentrations, a full elucidation of the structure of this compound(s) has to await isolation of enough material for chemical analysis. The biological activity of the retinoic acid deserves some comment. This compound has partial vitamin A activity: it replaces vitamin A in the growth function but not in vision and reproduction [41]. Table III shows that retinoic acid promotes [ ’ 4 C] mannose incorporation into glycopeptides to the same extent as retinol, but the restoration of incorporation into the lipid is only partial. We do not know the significance of this observation. If the formation of retinyl phosphate sugar has physiological significance, the problem of how and whether retinoic acid can fulfill the same function as retinol in tissues other than the testes must also be solved. In conclusion, depletion of retinol severely impairs the biosynthesis of mannose containing glycolipids and glycoproteins of the liver, at a very early stage of deficiency. In the light of these results, we propose that the transfer of mannose from hydrophilic to hydrophobic environments occurs via more than one lipid intermediate and that retinyl phosphate, which we have recently isolated and characterized (Frot-Coutaz, J.P. and De Luca, L.M., unpublished),

359

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