Biosynthesis of a Fucose-containing Glycopeptide from Rat Small Intestine in Normal and Vitamin A-deficient Conditions*

Abstract The number of goblet cells in the small intestine decreased in the vitamin A-deficient rat. Incorporation of 14C-d-glucosamine into a fucose-containing glycopeptide declined markedly. Ultracentrifugation of this glycopeptide in H2O gave only a single component, with a sedimentation value of 3.6 S, whereas, in 0.1 m phosphate buffer (pH 7.5) two components appeared, with much higher sedimentation constants (6.2 S and 7.2 S). The glycopeptide was found to contain glucosamine, galactose, fucose, and sialic acid in the molar ratios of 3:3:1:0.25, respectively. Synthesis of the fucose-containing glycopeptide in vitro, using uridine-diphosphate-N-acetyl-d-glucosamine-1-14C and 3H-serine, showed a 2- to 3-fold decrease in the incorporation of the labels by rough endoplasmic reticulum prepared from vitamin A-deficient animals as compared with normal animals.

Ultracentrifugation of this glycopeptide in Hz0 gave only a single component, with a sedimentation value of 3.6 S, whereas, in 0.1 M phosphate buffer (pH 7.5) two components appeared, with much higher sedimentation constants (6.2 S and 7.2 S).
Synthesis of the fucose-containing glycopeptide in vifro, using uridine-diphosphate-N-acetyl-D-gh.tcosamine-l-'4C and 3H-serine, showed a 2-to 3-fold decrease in the incorporation of the labels by rough endoplasmic reticulum prepared from vitamin A-deficient animals as compared with normal animals.
Vitnmin h deficiency in rats has been shown to decrease by 50 to 605: the incorporation of 14C'-leucine into proteins in vitro when rough cndoplasmic reticulum is used, but does not appear to affect prot,ein synthesis with free polyribosomes (1). Since vitanlin X deficiency causes a reduction in the number of mucussecreting goblet cells in the small intestine (l), one would expect that glycoproteins, the principal constituents of goblet cells, would also be decreased.
Furthermore, since secreted proteins, such as glycoproteins, are thought to be synthesized on bound rather than free polyribosomes (2,3), such a decrease would be consistent with our findings in V&O. (I).
Much work has been done on the role of vitamin A in the secretion of mucus and biosynthesis of glycosaminoglycans. Some workers found a pronounced effect of the deficiency on this bio-* These st,udies were supported hy National Institutes of Healt,h Grant AM-08732. This paper is Contribution 1628 from the l)epitrtmpnt of Nut,rit.ioll :ind Food Science, Massachusetts Institute of Technology.
synthesis; others could not observe any effect.
Most of the research dealing with the effect of the vitamin on glycosaminoglycnn biosynthesis concerned itself only with incorporation of radioactive precursors into total glycosaminoglycans. Ver) few attempts were made to study a single species of mncromolede (4,5).
'I'he present report describes the biosynthesis of a fucose-containing glycopeptide in z~ivo and in vitro, and its chararterization and behavior in response to I-itamin 4 deficiency.
EXPERIMEIUTAL PROCEDURE Animals and Diets-These were identical to those described in a previous report (1). For the experiment on rccovrr\-from vitamin .4 deficiency, 500 pg of vitamin A acetate in l'wrrn 80 were inject,ed intraperitonenlly according to the method of Levi and Wolf (6) ; concurrently, the same amount was given in oil by mouth, and t'he animals were killed at the times indicated in Table  I. In all experiments, normal control :IJ~ vitamin A-deficient rats were pair-fed. The latter were used after the weight plateau stage, 3 to 5 days after weight loss had begun. Isoktion of 14C-G'lucosamine-labeled Naterial jronl Cell Fractions-W-Glucosamine was injected intraperitonenllg (25 p('i, 200 g, body weight; specific activity, 49 $Zi per pmole) into vit.amin A-normal and -deficient rats. The animals were killed at the time intervals indicated in Fig. 2, and the mucosa was scraped as previously described (I).
The scrapings were homogenized in 6 volumes of ice-cold Medium A of Littlefield and Keller (7) in a Dounce tissue homogenizer wit,h 8 strokes of the loose pestle and 6 strokes of the tight pestle.
An aliquot of the homogenate was diluted twice with a 170 solution of cold glucosamine and precipitated in 5Oh trichloracetic acid. The precipitabe was collected on a Millipore filter (0.45 p pore size), dissolved in Hpamine Hydroxyde 10X and, after addition of 15 ml of Bray's solution (8)) was counted in a liquid scintillation COWter (Nuclear Chicago). Free and membrane-bound polysomes were prepared by the following procedure.
The whole lmrnogcnate was centrifuged at 7,600 x g for 10 min to remove intact cells, cell debris, nuclei, and mitochondria.
The supernatant fraction was then placed on a discontinuous sucrose gradient ( and No11 (9). The material at the interface of the 2.0 and 0.5 M sucrose was removed, diluted I:1 with buffer consisting of Tris (0.05 M), MgClt (5 X 10e3 M), and KC1 (0.025 M) at pH 7.6, and recentrifuged at 105,000 x g for 1 hour. The membrane-bound polysomes were pelleted with all the smooth vesicles (smooth endoplasmic reticulum, Golgi apparatus, and plasma membranes).
After the pellet was redissolved in water, aliquots were precipitated in cold 5% trichloracetic acid, plated on a Millipore filter, dissolved in Hyamine Hydroxyde 10X, and counted in Bray's solution (8). The translucent pellet at the bottom of the discontinuous sucrose gradient represented free polysomes.
Their radioactivity was determined as described above.
Preparation of Glycopeptides-The glycopeptides were prepared from the intestinal scrapings as described by Inoue and Yosizawa (10). The tissue (about 2.5 g/200 g, body weight) was diluted approximately 1: 10 with 80% ethanol and homogenized for 15 min at medium speed in a Virtis homogenizer. After being immersed in a boiling water bath for 15 min, the homogenate was cooled, centrifuged, decanted, and extracted with 80% ethanol, with 100% ethanol, and with ether.
After air drying, the residue (about 200 mg per animal) was resuspended in 10 ml of water to which 2 mg of trypsin and 0.1 ml of toluene were added, and was incubated at 30" for 5 hours. Calcium chloride was then added to give a final concentration of 0.01 M, the pH was adjusted to 8 with sodium hydroxide, and 4 mg of pronase were added together with 0.05 ml of toluene; the suspension was incubated for 42 hours at 37". At the end of the incubation, cold trichloracetic acid was added to give a final concentration of 7%. The suspension was centrifuged and the precipitate discarded.
Two volumes of cold ethanol were added to the supernatant solution and the precipitated glycopeptides were collected by centrifugation, washed with alcohol and ether, dried, and weighed (about 20 mg/200 g, body weight).

DEAE-Xephadex
A-60 Chromatography-The components of the glycopeptide preparation were separated by chromatography on DEAE-Sephadex A-50 columns. The resin was suspended in water and the fine particles were removed by decantation. The resin was then washed several times in 0.5 N HCl and rinsed with water until all chloride ions had disappeared from the washings.
Columns (2 x 100 cm) were packed and the glycopeptides were applied to the columns after being dissolved in 2 ml of water. This material (140 mg) was then placed on a large column of Sepharose 4B (5 x 60 cm) equilibrated with phosphate buffer as described. Three distinct peaks of radioactivity were found in the 20-ml fractions.
Only tubes 18 to 30 were pooled, and the F-glycopeptide was further purified by chromatography on Sepharose 2R (2.5 X 50 cm) equilibrated with the same phosphate buffer as Sepharose 4B.
The eluate showed only one component. This material was used for ultracentrifugal analysis in Hz0 and 0.1 M phosphate buffer at pH 7.5 in the model E Beckman ultrucentrifuge at 22" and 175,000 x g average. Synthesis in vitro of 14C-, 3H-Labeled Fucose-containing Glyco-pep&e-Rough endoplasmic reticulum and pH 5 enzyme fractions were prepared as previously described (1) from tl\-o vit,amin A-normal and two vitamin A-deficient rats 18 hours after ligation of the common bile duct. The cell fractions were incubated for 45 min at 37" in the presence of 1.25 PCi of uridine diphosphate-A-acetylglucosamine-1-W (40 mCi per mmole) and 2.5 PCi of L-serine-3H (5 Ci per mmole) ; 8.2 mg of rough endol)lnsmic reticulum protein and 5 mg of pH 5 enzyme protein were used in each incubation.
All other components of the incubation were identical with those used previously (1). At the end of the incubation, 2 mg of cold F-glycopeptide were added as carrier to both preparations.
The preparations were digested as described for F-glycopeptide, and the glycopeptides were placed successively on DERE-Sephadex (1 x 12 cm) and Sepharose 2B (1 X 12 cm) columns.
The act'ivity of 3H and 14C was measured in Bray's medium with a Beckman LS-150 liquid scintillation counter. Analytical Techniques-Uranic acid was determined by the method of Bitter and Muir (11). Hexosamines were measured by the method of Rondle and Morgan (12) and by chromatography on the short column of the Beckman amino acid analyzer at pH 5.28 after hydrolysis in 4 N HCl for 5 hours in a sealed ampoule under nitrogen at, 100" (13). Total hexoses were de-IThe abbreviation used is F-glycopeptide, fucose-containitlg glycopeptide.
termined by the anthrone method of Trevelyan and Harrison (14) and gnlactose by the galactostat reagent method (after hydrolysis in 1 N HCl for 1 hour at 80").
Paper chromatography of the sugars was done according to Trevelyan, Procter, and Harrison (15) after hydrolysis in 0.1 N HCl for 1 hour at 80". N-Acetylneuraminic acid was determined by the method of Warren (16) after hydrolysis of the glycopeptide by 0.1 N H&SO4 for 1 hour at 90" in a closed ampoule under nitrogen, fucose according to Aminoff and Morgan (17), aldopentoses by Dische and Borenfreund's method (18), SO,' by the method of Antonopoulos (19), and protein by the method of Lowry et al. (20). The amino acid composition of F-glycopeptide was determined on the Beckman amino acid analyzer after hydrolysis of a sample at 105" in 6 N HCl for 12 hours.
Materials-n-glucosamine hydrochloride A-deficient rats. The de"ficient rats lost about 5% of their maximum weight and the normal animals were pair-fed. The animals were killed at the indicated time intervals after injection of the label, and the small intestines were processed as described under "Experimental Procedure." Wet weight of the mucosa from each animal was about 2.5 g.
into free and membrane-bound polyribosomes was determined at different time intervals; although the level of incorporation into membrane-bound polyribosomes was about 10 times that into the free ones, no effect of the vitamin deficiency was detectable. It is noteworthy that maximum incorporation for both deficient and normal mucosa is attained 2 hours after intraperitoneal injection of the label (Fig. 1). All subsequent experiments were performed at this time interval after injection of i4C-glucosamine.
Glycopeptides from eight normal and eight vitamin A-deficient rats were prepared by the method described in "Experimental Procedure." The total amount of material obtained for the two groups was almost identical (122 mg of glycopeptide material from the normal and 120 mg from the deficient), as was the total radioactivity (about 1,200,OOO dpm for both).
Therefore, no difference could be det,ected when we considered the mixture of all glycopeptides. Since our aim was to ascertain whether or not the vitamin was necessary for synthesizing a specific glycopeptide, the normal and deficient preparations were placed on two identical DEAE-Sephadex A-50 columns. A Two peaks of radioactivity were eluted by 0.2 M LiCI; in both preparations these two peaks contained the same amount of radioactivity (about 210,000 dpm). The 0.4 M LiCl eluted a peak that we henceforth term "Peak III" and that was greatly affected by vitamin A deficiency. The peak from deficient rats contained only about 407, the radioactivity of the normal (Fig. 3). By increasing the concentration of LiCl to 0. The rats were injected with 'Gglucosamine 2 hours prior to killing, and the glycopeptides were prepared according to the method of Inoue and Yosizawa (10). This experiment used eight normal and eight vitamin A-deficient rats.
In all, 122 mg of the normal glycopeptide mixture and 120 mg of the deficient were obtained. The glycopeptides were dissolved separately in 27 ml of water, and 22 ml were applied to two identical columns (2 X 100 cm) of DEAE-Sephadex; 12.5-ml fractions were collected with am automatic fraction collector, and the radioactivit,y was assayed on 0.5 ml. a Per group of six rats. Normal F-glycopeptide was prepared from tubes 6 to 12 (Fig. 4A); deficient F-glycopeptide was prepared from tubes 10 to 25 (Fig. 4B) B, elution pattern of F-glycopeptide prepared as follows. Tubes 18 to 30 from Sepharose 4B chromatography were combined and, after extensive dialysis against twice-distilled water and concentration to 30 ml, F-glycopeptide was precipitated by adding 60 ml of cold ethanol.
The precipitate was placed on a column (2.5 X 50 cm; void volume, 50 ml) of Sepharose 2B equilibrated in the same way as for the Sepharose 4B. Fractions of 25 ml were collected and radioactivity assayed on 0.1 ml. F-glycopeptide was then precipitated after dialysis, washed with ethanol and ethyl ether, and used for composition analysis.
M, another broad peak of radioactivity could be detected; the peak from deficient animals had less radioactivity than the normal. The effect of the vitamin A deficiency, however, was not as pronounced as in Peak III. The 1 M LiCl eluted a final peak of radioactivity that showed an increased incorporation of *'Cglucosamine in the deficient preparation. From these results it is clear that if total glycopeptides were analyzed, no difference between the vitamin A-normal and -deficient preparations could be detected. The ratio of Peak III to Peak V (Fig. 3) for the vitamin Anormal preparation is 0.5; the same ratio in vitamin A deficiency is 0.1 due to decreased incorporation of '"C-glucosamine into Peak III (from 145,000 dpm for the normal to 60,000 for the deficient) and increased incorporation into glycopeptide V (from  Vol. 245,No. 17 deficient). This experiment was repeated three times with identical results.
We next wanted to determine whether vitamin A administration to deficient rats initiated Peak III biosynthesis and restored to normal the ratio of Peak III to Peak V. Table I provides the answer. Four hours after vitamin A injection the ratio was unchanged. After 18 hours the ratio increased to 0.56, and after 26 hours the value was higher than normal (3.86).  We found previously (1) that vitamin A deficiency caused the number of goblet cells of the small intestine to decrease from 17.3 3tr 1.3 cells per crypt in the vitamin A-normal rat to 11.0 i 1.9 cells in the vitamin A-deficient rat. If the goblet cells produce Peak III, its increase after vitamin A injection could be due to the appearance of new goblet cells. For this reason the goblet cells were counted at the various time intervals after vitamin A administration. Table II gives the results. Even 4 hours after injection of vitamin A there is a statistically significant increase in the number of goblet cells per crypt of Lieberkiihn (p < 0.01). The greatest increase occurs between 10 and 18 hours after injection.
Further attempts to fractionate Peak III on Sephadex G-200 were fruitless because of the large size of the glycopeptides. On Sepharose 4B we were able to obtain two major radioactive peaks and a shoulder of radioactivity after the second peak. The first eluted peak is the only one to contain fucose, as determined both by the fucose assay (17) and by paper chromatography (15). This latter technique also showed galactose to be the only hexose present in the first eluted peak. From Fig. 4 it is evident that the only peak to be affected by vitamin A deficiency is the fucosecontaining glycopeptide of larger molecular weight. From Table III we can see that, although the total amount of Fglycopeptide seems to be higher in vitamin A deficiency, the specific radioactivity of 14C-glucosamine is decreased 4-fold. This indicates that there is an accumulation of F-glycopeptide (perhaps due to lack of secretion) in vitamin A deficiency and at the same time a decrease in synthesis. This is also found in the F-glycopeptide synthesized in vitro. Figs. 5 and 6 show a 2-to a-fold decrease in the incorporation of UDP-N-acetylglucosa-mineJ4C and serine-3H in F-glycopeptide synthesized on vitamin A-deficient rough endoplasmic reticulum.
To characterize F-glycopeptide better, we prepared it in large quantities from 40 rats 2 hours after the injection of 14C-glucosamine. After DEAE-Sephadex chromatography, Peak III, eluted with 0.4 M LiCl, was placed on Sepharose 4B. Fig 7A  clearly shows the presence of three radioactive components. To avoid any contamination from the second peak, F-glycopeptide was prepared from tubes 18 to 30 by the usual ethanol precipitation method.
It was then rechromatographed on Sepharose 2B (Fig. 7B), which gave a single peak just after the void volume. As can be seen in Table IV, F-glycopeptide contains glucosamine, galactose, fucose, and sialic acid in the molar ratios 3 : 3 : 1: 6.25.
No sulfate and aldopentoses were found. The uranic acid present is probably due to contamination from the adjacent peak.
Typically for glycopeptides, threonine is the most abundant amino acid and the amino acid content represents 15% of the total F-glycopeptide.
When ultracentrifugal patterns at three different concentrations of F-glycopeptide in Hz0 were run, only one sharp peak could be detected, with a theoretical sedimentation value of 3.6 R. However, wheu the same analysis was run in 0.1 M phosphate buffer at pH 7.5, two distinct peaks appeared with sedimentation values of 6.2 and 7.2 (Fig. 8). The possibility exists that the two components are two subunits of the same glycoprotein and that they radically change t,heir configuration from linear to spherical in an alkaline environment.. Further studies are in progress to clarify this point.

DISCUSSION
In an attempt to find a metabolic function for vitamin A at the molecular level, we showed in previous experiments (1) that in intestinal mucosa, a target organ of the vitamin, protein synthesis on membrane-bound but not on free polyribosomes is partially dependent on the vitamin. Having observed that the number of mucus-secreting goblet cells in the intestine of vitamin A-deficient rats is decreased (l), and since secreted proteins are thought to be synthesized on membrane-bound polyribosomes, we considered it pertinent to determine whether the synthesis of any intestinal glycopeptide in vivo and in vitro would be affected by the vitamin. If we could relate the biosynthesis of a particular and defined macromolecule in vitro to the presence of the vitamin, the mechanism whereby the latter influences the former might become apparent.
The effect of vitamin A on the synthesis of mucopolysaccharides and glycoproteins in a variety of tissues has been investigated by many workers with varying results. Colon mucopolysaccharide was reported to be depressed (22), whereas there was no change in mast cell heparin (5). Bone mucopolysaccharide was actually found to increase upon deficiency (23). The difficulty in interpreting many of these reports has been the lack of separation of the isolated material into definite chemical entities.
We have focused our attention on the biosynthesis of total glycopeptide as well as that of specific components. We found no difference in the l-14C-glucosamine incorporation into total glycoprotein in intestinal mucosa from vitamin A-deficient and pair-fed normal control rats (Fig. 1). Similarly, vitamin A deficiency did not affect the synthesis of bulk glycoproteins in viva on membrane-bound polyribosomes or on free polyribosomes at various times after a pulse of labeled glucosamine (Fig. 2). Kinetic data of incorporation were also very similar for deficient and normal mucosa.
It should be noted that the incorporated radioactivity was about 10 times greater in rough endoplasmic reticulum than in free polyribosomes, consistent with recent reports (24) that glycoproteins are synthesized mainly on rough endoplasmic reticulum. When total intestinal mucosal glycopeptides were prepared 2 hours after the intraperitoneal injection of labeled glucosamine, no difference was noticed in total amounts and total radioactivity between normal and deficient preparations, a result that would indicate that the vitamin has no effect on glycopeptide biosynthesis.
However, upon separation on DEAE-Sephadex of bulk glycopeptide into its constituents (Fig. 3)) it was obvious that '4C-glycosamine incorporation into the glycopept,ide fraction eluted with 0.4 M LiCl was strongly depressed in vitamin A deficiency (Peak III, Fig. 3). Simultaneously, it, became apparent that another glycopeptide (Peak V) increased in vitamin A deficiency.
These results suggested a precursor-product relationship between glycopeptides III and V, with accumulation of the precursor when the product synthesis is interrupted. They also show that the effect of the vitamin could not be detected by measuring bulk glycopeptide synthesis because the effect was obscured by a variety of unaffected glycopeptides, in addition to a glycopeptide that increased during deficiency. This explains the extreme variability of the results obt,ained by other researchers.
Recovery studies (Table I)  The specific radioactivity of glucosamine in F-glycopeptide was 4-fold lower than in normal F-glycopeptide (Table III and Fig. 4). Table III shows that the actual amount of F-glycopeptide present in vitamin A-deficient mucosa is increased, although the synthesis is impaired, possibly because of lack of secretion. Because the decreased biosynthesis of F-glycopeptide parallels its accumulation in vitamin ,4 deficiency, one can hypothesize that the rate of degradation of F-glycopeptide is decreased to a higher extent than the rate of synthesis, resulting in accumulation The chemical composition of F-glycopeptide is very similar to that of the blood group substances of which fucose, glucosamine, galactose, and sialic acid are common constituents (25). Moreover, the amino acid composition of the different human blood group-specific substances isolated from ovarian cyst fluids (25) shows thrconine as the most abundant amino acid; this is also the most abundant amino acid in F-glycopeptide. The ultracentrifugal behavior of F-glycopeptide in Hz0 and phosphate buffer indicates that, below pH 6, F-glycopeptide has an elongated, rod-shaped configuration, whereas, at pH 7.5, it has a globular or hydrated structure that allows faster sedimentation and the separation into two components (Fig. 8). The study of the biosynthesis in vitro of the fucose-containing glycopeptide in the presence of rough endoplasmic reticulum and pH 5 enzyme fractions clearly indicates that the biosynthesis of both the polysaccharide and the peptide moieties is affected by vitamin A deficiency. Moreover, this experiment excludes any effect of the vitamin prior to the formation of UDP-N-acetylglucosamine.
We have recently reported  Vol. 245,No. 17 presence of a microsomal preparation from rat liver. The compound has the same properties as the mannosyl-l-phosphoryl undecaprenol isolated from Micrococcus lysodeikticus (27)) which has been shown to participate in the biosynthesis of a membrane-associated mannan. Since vitamin A is a Tetrametric derivative of isoprene, it might be performing in mammals the same function as the undecaprenol performs in microorganisms: that of carrying mono-or oligosaccharides for the biosynthesis of glycoproteins.