THE JOURNAL OPBIOLOQICAL CHE~~IBTRY Vol. 246, No. 1, Ieaue of January 10, pp. 143-151, 1971

P&ml in U.S.A.

Glycoprotein Biosynthesis

INCORPORATION OF GLYCOSYL GROUPS INTO ENDOGENOUS ACCEPTORS IN A GOLGI APPARATUS- RICH FRACTION OF LIVER*

RONALD R. WAGNER AND MORRIS A. CYNKIN~

From the Tufts University School of Medicine, Department son Avenue, Boston, Massachusetts 02111

(Received for publication, July 7, 1969)

of Biochemistry and Pharmacology, 136 Harri-

SUMMARY

A Golgi apparatus-rich fraction from rat liver was examined for the ability to mediate steps involved in the biosynthesis of glycoproteins. Five enzymatic activities were studied: the transfer of glucosamine, galactose, glucuronic acid, mannose, and N-acetyhreuraminic acid from their sugar nucleotide precursors to endogenous trichloracetic acid-precipitable proteins.

The glucosaminyl transfer was enriched 23-fold in the Golgi membrane fraction, and represented 17% of the total activity of the homogenate. The initial rate of transfer of glucosamine exhibits optima both at pH 6.1 and 7.1. UDP- glucuronic acid stimulates the net transfer of glucosamine at pH 7.1, but not at pH 6.1. GDP-mannose and GTP are also stimulatory at pH 7.1. UDP-glucose, UDP-galactose, UTP, UDP, and UMP are inhibitory.

The Golgi-associated galactosyl transfer was enriched over 24-fold, and accounted for 13 % of the original activity of the homogenate. This transfer activity is stimulated by GDP-mannose. UDP-glucose, UDP-N-acetylglucosamine, and UMP inhibit the transfer of galactose.

The Golgi membrane fraction also transfers glucuronic acid from UDP-glucuronic acid to trichloracetic acid-insolu- ble material. This transfer is stimulated by UDP-N-acetyl- glucosamine. It represents 5 % of the activity of the original homogenate, and is enriched approximately S-fold in the Golgi fraction.

The Golgi-associated transfer of N-acetylneuraminic acid represents 10% of the activity of the homogenate, and is enriched l&fold. This transfer is stimulated by UDP- galactose, and is inhibited by CMP.

Although the transfer of mannose from GDP-mannose to trichloracetic acid-precipitable material is very active in the crude homogenate, little transfer, and rapid destruction of GDP-mannose is observed in the Golgi fraction.

Mild alkaline treatment (0.5 N NaOH, 24”, 90 min) results

* This research was supported by Grant GB 8086 from the Na- tional Science Foundation and Grant 5-ROl-AI03586 from the United States Public Health Service. A preliminary report of this work was presented at the 158th Meeting of the American Chemical Society (1).

$ Holder of a Research Career Development Award, United States Public Health Service.

in the release of 88 to 92% of the radioactivity of the IV- glucuronic acid-labeled product to the trichloracetic acid- soluble fraction. On the other hand, only 32 to 35% of the 14C-glucosamine-labeled product is converted into a tri- chloracetic acid-soluble form by this treatment. When glucosamine transfer is stimulated by the addition of UDP- glucuronic acid, however, approximately 50% of the product is released by alkali. It is suggested that the alkali-stable products might represent glycoproteins, while the more alkali-labile products may be glycosaminoglycans.

Bacterial heparinase degraded 70% of the glucuronic acid- labeled product, and 25% of the glucosamine-labeled prod- uct. Testicular hyaluronidase degraded 35% of the glu- curonic acid-labeled, and 7% of the glucossmine-labeled material.

Pronase digestion converted all of the radioactivity of glucosamine-, glucuronic acid-, or galactose-labeled tri- chloracetic acid-precipitable products into trichloracetic acid- soluble forms. These digestion products sre still of rela- tively high molecular weight, as they are eluted in the void volume of a Sephadex G-50 column.

Acid hydrolysis released all of the radioactivity of the glucosamine-labeled, pronase-digested product as free glu- cosamine, as identified by paper chromatography. The radioactive product of the galactosyl transferase reaction was shown to be 14C-galactose by the same method.

The results of this study are consistent with the hypothesis that the N-acetylglucosamine, galactose, and N-acetyl- neuraminic acid residues of the terminal sialic acid + galac- tose + N-acetylglucosamine sequence of many glycoproteins are added to the nascent glycoprotein within the Golgi ap- paratus, while the sugar residues of the inner core are in- serted at other sites in the endoplasmic reticulum.

The carbohydrate units of glycoproteins are thought to be as- sembled by the stepwise addition of component sugar residues to the previously formed polypeptide. These individual steps are apparently not confined to one subcellular site. Thus, while some glucosamine residues are transferred directly to the ribo-

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144 Glycoprotein Biosynthesis Vol. 246, No. 1

some-bound polypeptide (2-5), additional hexosamines are incorporated after the ribosomal stage of protein synthesis, presumably within the membranes of the rough- and smooth- surfaced endoplasmic reticulum, and it was suggested that si- alit acid is incorporated primarily within the smooth endoplasmic reticulum (3, 6). The deoxycholate-soluble (membranous) frac- tion of liver microsomes catalyzes the transfer of galactose from UDP-galactose to protein acceptors (7), and an enzyme system obtained from rat liver which incorporates N-acetylglucosamine into endogenous glycoprotein acceptors is localized in a fraction consisting primarily of smooth microsomes (8). In addition, the Golgi apparatus is reported to be involved in the glycosylation of proteins (9, 10).

A previous report (11) has described the transfer in vitro of glucosamine from UDP-N-acetylglucosamine to protein acceptors within a Golgi apparatus-rich fraction isolated from rat liver. Evidence is presented here suggesting that both glycoproteins and mucopolysaccharides are products of this transfer. In addi- tion, this fraction catalyzes the transfer of galactose from UDP- galactose, N-acetylneuraminic acid from CMP-N-acetylneur- aminic acid, and glucuronic acid from UDP-glucuronic acid to endogenous macromolecular acceptors, which have the properties of glycoproteins and mucopolysaccharides, respectively.

EXPERIMENTAL PROCEDURES

Materiuls-UDP-N-acetylglucosamine-l-14C (specific activity 12.3 PCi per pmole) was prepared as previously described (12). UDP-14C-glucuronic acid (uniformly labeled, 233 PCi per pmole), UDP-14C-galactose (uniformly labeled, 280 $Zi per pmole), and GDP-**C-mannose (uniformly labeled, 152 PCi per pmole) were purchased from New England Nuclear. CMPXXV-acetyl- neuraminic acid (2.4 MCi per pmole) was the generous gift of Dr. J. Hickman. All nucleotides and nonradioactive sugar nucleo- tides were obtained from Sigma. Pronase was purchased from Calbiochem. Hyaluronidase (from bovine testes, type VI) and ar-amylase (from hog pancreas) were from Sigma. Heparinase (from Flavobacterium heparinum) was the kind gift of Dr. J. E. Silbert.

Isolation of Golgi Apparatus-rich Fraction-Golgi membranes were prepared by a modification of the procedure of Morrh et al. (13). Fasted male Wistar rats weighing 150 to 200 g were killed by decapitation. Livers were immediately removed, chilled on ice, and finely minced with a razor blade. Minced tissue (5 g) was homogenized with a few strokes of a motor-driven glass- teflon homogenizer in 12.5 ml of a medium consisting of 0.5 M

Tris-maleate buffer, pH 6.5, and the homogenate was centrifuged for 20 min at 2,000 x g in a Spinco SW-39L rotor. The super- natant was removed, and the upper third to one-half of the pellet was suspended in 1 to 1.5 ml of homogenization medium by swirling. This suspension was layered over 3.5 ml of homogeni- zation medium containing 1.25 M sucrose, and centrifuged for 30 min at 71,000 x g. The material at the interphase of the two sucrose solutions was removed, diluted with 0.5 M sucrose ho- mogenization medium, and centrifuged for 30 min at 4,500 X g to obtain the Golgi membranes as a pellet. Pellets from 5 g of liver, original wet weight, were suspended in 0.3 to 0.6 ml of buffer for use, as described below.

Assay of Enzymatic Activity-Transfer of 14C-glycosyl groups to protein was measured as radioactivity precipitated with 5% trichloracetic acid. The standard incubation mixture contained, in a total volume of 100 ~1: 20 to 80 ~1 of suspended Golgi mem-

branes (0.2 to 0.5 mg of protein), 0.5 pmoles of MgC12, 0.1 pmole of EDTA, 4.0 pmoles of Tris-maleate (pH 6.1) or Tris (pH 7.1) buffer, and as indicated, 0.8 nmole (15,000 cpm) of UDP-N- acetylglucosamine-14C, 1.0 nmole (22,000 cpm) of UDP-glucu- ronic acid-14C, 1.0 nmole (22,000 cpm) of UDP-galactose-14C, 1.0 nmole (27,000 cpm) of GDP-mannose-14C, or 5 nmoles (20,000 cpm) of CMP-N-acetylneuraminic acid-14C. Incubations were carried out at 37”, and reactions were terminated by the addition of 3 ml of cold 5% trichloracetic acid. Trichloracetic acid pre- cipitates were washed twice with 3 ml of 5% trichloracetic acid, once with 2 ml of absolute ethanol, 2 ml of ethanol-ether (l:l, v/v), and 2 ml of ether. Dried precipitates were suspended in 1 ml of hydroxide of hyamine, and heated at 65” until dissolved. The digested samples were added to 10 ml of Liquauor, and radioactivity was measured by liquid scintillation spectrometry. Efficiency for counting l4C under these conditions was approxi- mately 60%.

Proteolytic Digestion-Washed trichloracetic acid precipitates from large scale incubations (3 to 5 mg, dry weight) were sus- pended by sonic disruption in 3.0 ml 0.05 M Tris, pH 8.1, con- taining 0.01 M CaCls and 5% ethanol. Pronase (3 mg) was added, and the mixtures were incubated at 37” for approximately 30 hours. Samples were then adjusted to 5 y0 trichloracetic acid concentration, and the insoluble residues were removed by cen- trifugation. The supernatant solutions were extracted with ether to remove trichloracetic acid, concentrated in a vacuum, and applied to a Sephadex G-50 column, 2 x 60 cm, equilibrated with water. Elution was carried out with water, and 3-ml frac- tions were collected. The radioactivity of an aliquot of each fraction was measured by liquid scintillation spectrometry with Bray’s mixture (14).

Analytical methods-Protein was estimated by the method of Lowry et al. (15) with bovine serum albumin as standard. Sugars were detected on paper chromatograms with a triphenyl tetrazolium chloride reagent (16). Labeled sugars were located by scanning paper chromatograms for radioactivity (Nuclear Chicago Actigraph III).

RESULTS

Fig. 1 shows the appearance of Golgi membranes isolated by the procedure of Morrb et al. (13), as viewed with the electron microscope. On the basis of morphological, chemical, and enzy- matic analyses, it was concluded (17, 18) that 70 to 90% of the fractions prepared by this procedure consist of Golgi apparatus- derived material. Golgi-rich fractions containing 2 to 3 mg of protein were obtained from 5 g of liver in a typical preparation.

The Golgi apparatus-rich fraction actively transfers several l*C-monosaccharides from their sugar nucleotide precursors to trichloracetic acid-precipitable endogenous acceptors, as shown in Table I. No conclusions can be drawn from the apparent en- richment factors and recoveries expressed for the mannosyl and glucuronyl transfer activities, since considerable destruction of GDP-mannose and UDP-glucuronic acid was observed when in- cubation mixtures were examined by paper electrophoresis under conditions which separated the sugar nucleotides from the corres- ponding sugar l-phosphates and free sugars. Thus, in the ex- periment illustrated in Table I, all of the UDP-glucuronic acid in the homogenate incubation mixtures, and all of the GDP- mannose in the Golgi fraction incubation mixtures had been de- graded after 15 min, or less, of incubation. In this experiment, glycosyl transfer was assayed after a lo-min incubation period.

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FIG. 1. Golgi apparatus-rich fraction, prepared as described under “Experimental Procedures.” Golgi pellets were suspended in water, and aliquots mixed with an equal volume of 1.2% phosphotungstic acid (pH 6.8) on formvar-coated grids. taken with a Philips 200 electron microscope; a, X 71,000, b, enclosed area in a at X 185,000.

Photograph was

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146 Glycoprotein Biosynthesis Vol. 246, No. 1

TABLE I Glycosyl transfer activities in homogenate and Golgi fraction

Incubation mixtures contained, in a total volume of 100 ~1: 80 ~1 of homogenate or Golgi suspension, 0.5 pmole of MgC12, 0.1 pmole of EDTA, 40 pmoles of sucrose, 0.8 mg of dextran, 4.0 pmoles of Tris-maleate, pH 6.5, and r4C-sugar nucleotides as described in the text. Incubation time was 10 min.

W-Sugar nucleotide Specilic activity Total activity

UDP-GlcNAc. .......... UDP-GlcUA. ........... UDP-galactose ......... CMP-NANAb. ......... GDP-mannose. .........

Transfer of “C-sugar

pmdes/mg protein pm&s/g liver

8.9 205 1 414 71 3.7 27.7 175 8.2 7.0 171 375 49.3

91.5 1686 4945 485 38.0 9.91 1837 2.8

--

%

23.0 17 7.5 5

24.4 13 18.4 10 0.3 0.2

a Refers to the ratio: specific activityin Golgi fraction to specific

b NANA, N-acetylneuraminic acid. activity in homogenate.

I / I %e%---30 40 50 6i

MINUTES

FIG. 2. Effect of UDP-glucuronic acid on glucosamine (Cl&) transfer at pH 7.1. Incubation conditions were as described in the text, with the addition of 4 X W4 M UDP-glucuronic acid (0 ) ; no additions (0 ) .

On the other hand, from 40 to 90% of the added UDP-N-acetyl- glucosamine, UDP-galactose, and CMP-N-acetylneuraminic acid remained after 15 min of incubation with either the homogenate or the Golgi fraction.

Transfer of Glucosamine-Fig. 2 shows the transfer of W- glucosamine (presumably as N-acetylglucosamine) from UDP- N-acetyl-14C-glucosamine to trichloracetic acid-insoluble material as a function of time.. The initial rate of transfer exhibits optima both at pH 6.1 and 7.1 (Fig. 3). Transfer of glucosamine at pH 7.1 is stimulated (up to 50%) by the addition of unlabeled UDP- glucuronic acid (optimal concentration, 4 x 10h4 M) to the stand- ard incubation mixture (Fig. 2). In contrast, glucosamine trans- fer at pH 6.1 is not stimulated by UDP-glucuronic acid (Table II). GDP-mannose stimulates transfer at pH 7.1, but this ap- pears to be a nonspecific effect, since GTP is also stimulatory, and since the Golgi-associated transfer of mannose from GDP- mannose to trichloracetic acid-precipitable material does not occur to an extent which could account for the observed stimula-

j 1, , , , , , , , ( , 5.7 6.1 6.5 6.9 7.3

P”

FIG. 3. Effect of pH on initial rate of glucosamine (GZcN) trans- fer. Conditions of incubation were as described in the text. In- cubation time was 4 min. 0, average values from the results of experiments with two different Golgi preparations; 0, average values from the results of experiments with three different prepa- rations of Golgi membranes.

E$ects of nucleotides and sugar nucleotides on transfer of

TABLE II

glucosamineJ4C Glucosamine transfer was assayed as described in the text. In-

cubation time was 30 min. All additions were at 4 X W4 M con- centration. Experiments at pH 6.1 and 7.1 were performed with different preparations of Golgi membranes.

Additions

None ...................... UTP ...................... UDP ...................... UMP. .................... GTP ...................... GDP-mannose ............. UDP-glucose. .............. UDP-galactose ............ UDP-GlcUA. ............. UDP-GlcNAc. .............

- I

t .-

i

-

pH 7.1

GlUCOSa- mine-WI ransferred

bmoles/mg protein

366 288 257 230 422 443 273 288 521 34

- !

ercentagl f change

%

-21 -30 -37 -kl5 -l-21 -25 -21 -t42 -91

_

e 1

__

-

pH 6.1

Glucosa- mine-W

xansferred

yy,“g

425 244 181 138 424 420 299 279 388

-

ercentage ,f change

%

-43 -57 -68

-0.2 -1.2

-30 -34 -9

tion on the basis of newly synthesized acceptor sites alone. Thus, these substances may spare UDP-iV-acetylglucosamine from enzymatic hydrolysis by a relatively nonspecific pyrophos- phatase. UDP-glucose, UDP-galactose, UTP, UDP, and UMP are inhibitory at either pH. At 4 x 10e4 M, UMP is the most inhibitory of these substances, all of which are capable of being readily converted to UMP enzymatically. UTPase and UDPase activities are reported to be present in the Golgi fraction, and to be considerably enriched over the activities of the original ho- mogenate (17).

The stimulation of glucosamine transfer by UDP-glucuronic acid may be the result of the synthesis of glucosamine acceptor sites, protection from enzymatic hydrolysis, or both, since only 33% of the original UDP-N-acetylglucosamine is found intact after 30 min of incubation.

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Issue of January 10, 1971 R. R. Wagner and M. A. Cynlcin 147

400 -

300 -

YE 2 P n F 200 - 5;

2

0.008

0.006

lIi?zl

0.004

0.002

III!“““’ (6 10 20 30 40

UDP-GLcNAc (J.IM)

FIG. 4. Effect of UDP-N-acetylglucosamine concentration on transfer of glucosamine at pH 6.1 (a) and pH 7.1 (b). Assay con- ditions were as described for the standard incubation mixture. Incubation time was 4 min. The inset shows the data plotted by the method of Lineweaver and Burk.

I- -. I I ,

10 20 30 40 50 60

MINUTES

FIG. 5. Glucuronic acid (GUA) transfer at pH 7.1, with the addition of 2 X 10-4 M UDP-N-acetylglucosamine (0); no addi- tions ( l ). Incubation conditions were as described for the trans- fer of glucuronic acid.

The effect of UDP-N-acetylglucosamine concentration on glucosamine transfer at pH 6.1 and 7.1 is illustrated in Fig. 4. From these experiments, apparent Michaelis constants for UDP- N-acetylglucosamine of 10.1 X 10e6 M for transfer at pH 6.1, and 7.95 x 10-G M for transfer at pH 7.1, were calculated. The

140

0.02

. ..m 1

. ” 0.01

0.1 a2

, I I I I I , I

10 20 30 41

UDP-GUA t&t)

FIG. 6. Effect of UDP-glucuronic acid (UDP-GUA) on transfer of glucuronic acid at pH 6.1. Assay conditions were as described. Incubation time was 4 min. The inset shows the Lineweaver- Burk plot of the data.

I I I I 1 \

10 20 30 40 50 60

MINUTES

FIG. 7. Transfer of galactose from UDP-galactose to trichlor- acetic acid-insoluble material at pH 7.1. Incubation conditions as described in the text.

Golgi-associated transfer of glucosamine to endogenous acceptor accounts for 17% of the total activity of the original homogenate (Table I), and is enriched 23-fold. In preliminary experiments, plasma membrane and 105,000 x g supernatant fractions were essentially devoid of this activity.l

Transfer of Glucuronic Acid-The transfer of glucuronic acid- 14C from UDP-glucuronic acid-14C to trichloracetic acid-pre- cipitable material is stimulated by UDP-N-acetylglucosamine (optimal concentration, 2 X 10m4 M), as illustrated in Fig. 5. This transfer activity is enriched approximately S-fold in the Golgi fraction, and accounts for 5% of the activity of the total homogenate (Table I). The pH optimum of this transfer is approximately 6.1 in Tris-maleate buffer. The apparent K, for UDP-glucuronic acid is 1.29 X 10m5 M (Fig. 6).

Transfer of Galactose-The incorporation of galactose into tri- chloracetic acid-insoluble material is shown in Fig. 7. Thirteen

1 R. R. Wagner, D. J. Morre, and M. A. Cynkin, unpublished observations.

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148 Glycoprotein Biosynthesis Vol. 246, No. 1

TABLE III Effects of nucleotides and sugar nucleotides on transfer of

galactose-14C

Conditions of incubation were as described in the text. Incu- bation time was 30 min. All additions were at 4 X W4 M con- centration.

Additions Galactose-‘4C transferred

Percentage of change

None ............................. UDP-glucose ..................... UDP-GlcNAc. .................... UDP-GlcUA ...................... GDP-mannose. ................... UMP .............................

pmoles/mg protein

61 42 43 67 75 34

%

-31 -30 +10 +23 -44

I$ 600

B h

E” > 2

iti _a 400 - 0.006

D

z . 5

- ;

1 0.004 v

B 200 - 2 0.002

i 0.1 0.2

zv (3)

10 20

UDP-GAL (&I)

30 A0

FIG. 8. Effect of UDP-galactose concentration on transfer of galactose at pH 7.1. Assay conditions as described. Incubation time was 4 min. Inset, Lineweaver-Burk plot of the data.

per cent of the total activity of the crude homogenate is re- covered in the isolated Golgi fraction, at over 24 times the specific activity (Table I). Table III shows the effects of various addi- tions on this transfer activity. As was observed with glucos- amine, the transfer of galactose is strongly inhibited by UMP (44%) and by substances such as UDP-N-acetylglucosamine and UDP-glucose, which can readily give rise to UMP, with the ex- ception of UDP-glucuronic acid, which is slightly stimulatory. This activity is also stimulated by GDP-mannose, which again probably results from a nonspecific sparing effect on UDP- galactose. The apparent K, for UDP-galactose is 2.62 X 1O-5 M at pH 7.1 (Fig. 8).

Transfer of N-Acetylneuraminic Acid-Fig. 9 shows the effect of UDP-galactose on the transfer of 14C-N-acetylneuraminic acid from CMP-W-N-acetylneuraminic acid to trichloracetic acid- insoluble material. N-Acetylneuraminic acid is found linked to galactose in the oligosaccharide chains of many glycoproteins, and since galactose is actively incorporated within the Golgi fraction, it is possible that the attachment of N-acetylneuraminic acid to newly inserted galactose residues is the basis for the ob- served stimulation. Furthermore, if this were a nonspecific ef- fect, such as protection from enzymatic hydrolysis, UDP-glucose a.nd UDP-N-acetylglucosamine might also be expected to stimu-

Y I I I I I I 10 20 30 40 50 60

MINUTES

FIG. 9. Effect of UDP-galactose on transfer of N-acetylneura- minic acid (NANA) at pH 7.1. Incubation conditions as de- scribed in the text, with the addition of 4 X lO+ M UDP-galactose (0); no additions (0).

late, and they do not (Table IV). GDP-mannose does not stim- ulate this transfer. This is consistent with the observation that very little destruction of CMP-N-acetylneuraminic acid (39% after 30.min incubation) is seen in the Golgi fraction. It is interesting to note that CMP inhibits the transfer of N-acetyl- neuraminic acid from CMP-N-acetylneuraminic acid, just as UMP inhibits the transfer of glucosamine and galactose from their UDP derivatives. UMP does not inhibit N-acetylneu- raminic acid transfer.

The Golgi-associated transfer of N-acetylneuraminic acid ac- counts for 10% of the original activity of the homogenate, and is enriched approximately l&fold (Table I).

Transfer of Mannose-The transfer of mannose from GDP- mannose to trichloracetic acid-precipitable endogenous acceptors is very active in the original homogenate (Table I). However, only a trace amount (0.2y0) is recovered in the Golgi apparatus fraction, with a specific activity lower than that of the homoge- nate. This result may arise from an enrichment of GDP-man- nose pyrophosphatase in the Golgi fraction, since GDP-mannose is destroyed very rapidly within this fraction, in contrast to the other sugar nucleotides studied. Thus, for the experiment illus- trated in Table I, the GDP-mannose is completely absent at 15 min of incubation, or less, while over half of the original amount of this material remains in the homogenate at the end of the same period. Hence, the actual extent of mannose transfer within the Golgi fraction has not been determined.

Characterization of Products-Over 97% of the radioactive products of the glucosaminyl, glucuronyl, galactosyl, and N- acetylneuraminyl transfer reactions remains associated with Golgi membranes sedimented from 0.05 M Tris, at 105,000 x g for 1 hour. Treatment of labeled Golgi membranes with ultra- sonic vibrations (Bronwill Biosonik II) for 1 min at one-half maximum intensity released from 32 to 54 To of the incorporated radioactivity to the 105,000 x g supernatant, from which it could be precipitated with 5% trichloracetic acid.

Table V shows the effect of mild alkali on the glucuronic acid- l*C- and glucosamineJ4C-labeled products. It has been shown (19) that the 0-glycosidic linkages to serine and threonine which occur in glycosaminoglycans and some glycoproteins are cleaved by treatment with dilute alkali under mild conditions, in contrast

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TABLE IV TOLE V Effects of nucleotides and sugar nucleotides on transfer of Alkali lability of transfer products

N-acetylneuraminic acid-W Duplicate reaction mixtures were incubated for 30 min. Non- Incubation conditions as described in the text. Incubation radioactive UDP-N-acetylglucosamine, when present, was at

time was 30 min. All additions were at 4 X 1OV M concentration. 2 X 1OV M, UDP-glucuronic acid at 4 X lo+ M concentration. Different preparations of Golgi fraction were used for experi-

UC-N- Additions Acetylneuraminic Percentage

of change ments at pH 6.1 and 7.1. Trichloracetic acid-insoluble pellets

acid transferred were washed as usual, and the radioactivity of one set of pellets

pmozes/mg protein % was measured as described. The remaining pellets were sus- peaded in 0.5 ml of 0.5 N NaOH, and incubated at 24”, for 90 min.

None........................... 2608 The mixtures were then neutralized, adjusted to 5% trichloracetic CMP 1740 -33 acid concentration, and centrifuged. Precipitates were washed UMP 2600 -0.3 with organic solvents, digested with Hyamine hydroxide, and UDP-glucose 2348 -10 radioactivity measured as described. Radioactivity released to UDP-galactose 3190 +22 the trichloracetic acid supernatant fraction was then calculated UDP-GlcNAc. . 2428 -7 hv diffowncc?. GDP-mannose. . 2623

I +0.6

to the more stable aspartamido-type linkages found in most

plasma glycoproteins. Treatment with 0.5 N NaOH (24”, 90 min) converted 88 to 92yo of the glucuronic acid-W-labeled

material into a form soluble in 5’$.. trichloracetic acid. Treat- ment of glucosamine-14C-labeled product in this manner con- verted from 32 to 51 y0 into a trichloracetic acid-soluble form, the extent of conversion being dependent upon stimulation of gluco- samine transfer by UDP-glucuronic acid. Alkali released 32% of the radioactivity of the product labeled with glucosamine at pH 7.1. When transfer of glucosamine at this pH was stimu- lated by UDP-glucuronic acid (38% stimulation), 51y0 of the product was rendered trichloracetic acid-soluble. Product which had been labeled with glucosamine at pH 6.1 behaved similarly on treatment with alkali (35”“/, release) to the material

labeled at pH 7.1. When UDP-glucuronic acid was added to the assay mixture at pH 6.1, no stimulation of glucosamine transfer was observed (6% inhibition) although 50% of this product was now degraded by alkali. Thus it seems that while the net trans- fer of glucosamine is not increased, the addition of UDP-glucu- ronic acid does stimulate the incorporation of glucosamine into acceptors of the alkali-labile type, at the expense of its incorpora- tion into the more alkali-stable acceptors. It is possible that the inhibitory effect is through the action of UMP (Table II) which might be formed from hydrolysis of UDP-glucuronic acid, al- though this phenomenon remains to be investigated in greater detail.

For purposes of comparison, it is interesting to note that treat- ment of material labeled with galactose-14C under these condi- tions releases only 16% of the incorporated radioactivity to the trichloracetic acid-soluble fraction.

Testicular hyaluronidase converts 35%, and heparinase 70% of the glucuronic acidXXabeled trichloracetic acid-insoluble product into a form soluble in 5 y0 trichloracetic acid (Table VI). On the other hand, hyalmonidase degrades only 7%, and hep- arinase 25% of the glucosamine-labeled material. Pancreatic cY-amylase failed to release any radioactivity from the galactose- ‘%-labeled product.

Incubation with pronase resulted in the conversion of 98 to 100% of the radioactive products into trichloracetic acid-soluble forms, regardless of whether UDP-N-acetylglucosamine, UDP- glucuronic acid, or UDP-galactose was the 14C-glycosyl donor. In every case, the radioactivity of the pronase-digested material was eluted with the void volume on chromatography with Sephadex G-50. Radioactive fractions were pooled, concen-

Reaction system

WI-Sugar nucleotide pH Additions

UDP-GlcNAc 7.1 None

7.1 UDP-GlcUA 6.1 None 6.1 UDP-GlcUA

UDP-GlcUA 7.1 None

7.1 UDP-GlcNAc 6.1 None

6.1 UDP-GlcNAc

‘“C-Sugar transfer

pmozes

91

125 93 88 24

28 15

19

-.

T

I

Release by alkali

pneozes y& 29 32 64 51

33 35 44 50

21 88 25 89 14 92 17 92

TABLE VI

Action of mucopolysaccharidases on transfer products

Incubations were carried out in triplicate at pH 6.1 for 30 min. The radioactivity of one set of trichloracetic acid pellets was measured as described. One set of pellets was subjected to diges-

tion with hyaluronidase (0.5 mg, at 37”, for 16 hours in 1.0 ml of 0.1 M sodium acetate-O.15 M sodium chloride, pH 5.0) and the re- maining set to digestion with heparinase (0.5 mg, 24”, 17 hours in 1.0 ml of 0.1 M sodium acetate, pH 6.0). Reaction mixtures were adjusted to 5% trichloracetic acid concentration, and precipitates were treated as described under “Experimental Procedures.” The amount of radioactivity rendered trichloracetic acid-soluble

by this treatment was obtained by difference. Experiments with UDP-N-acetylglucosamine and with UDP-glucuronic acid were conducted with different preparations of Golgi fraction.

‘“C-Sugar nucleotide

Hyalumnidase Heparinase

pm&s pm&s % pmo1es %

UDP-GlcUA. 27 10 I 35 19 70 UDP-GlcNAc 79 5 7 20

I

25

trated in a vacuum, and aliquots were subjected to acid hy- drolysis for release and identification of radioactive sugars.

Recovery of 14C-Xugars from Radioactive Products-Hydrolyzed, pronase-digested samples (2 N HCI, loo”, 4 hours for release of galactose; 4 N HCl, loo”, 6 hours for release of glucosamine) were evaporated in a vacuum at 50” to remove HCl, and aliquots were chromatographed on Whatman 3MM paper together with ap- propriate standard sugars in a solvent system consisting of ethyl acetate-pyridine-water (12 : 5 :4, v/v/v). Under these condi- tions, all of the radioactivity from the galactose-14C-labeled

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150 Glycoprotein Biosynthesis Vol. 246, No. I.

product cochromatographed with authentic galactose, while all of the radioactivity from the glucosamine-14C-labeled material migrated with standard glucosamine.

DISCUSSION

The results of several investigations suggest that the carbo- hydrate units of glycoproteins are assembled by the stepwise transfer of monosaccharides to the completed polypeptide. Evi- dence indicates that the first sugar attached, glucosamine, may be transferred directly to the ribosome-bound polypeptide (2-5)) while additional sugar residues are transferred subsequent to the ribosomal stage of protein synthesis, as the nascent glycoprotein travels through the channels of the rough and smooth endoplas- mic reticulum, en route to excretion from the cell.

The role of the Golgi complex in the concentration and packag- ing of secretions has been described (2&22). More recently, however, radioautographic methods of analysis have prompted the suggestion that this organelle may actually be a site of bio- synthesis of complex polysaccharides. Thus a concentration of radioactive label may be observed in the Golgi region of histo- logical sections, after administration of radioisotopic glucose (9, IO), and other precursors (23-25), to intact animals. These results are not definitive, however, and may reflect transport and assembly, rather than actual biosynthesis. In order to test this hypothesis further, we have examined an isolated Golgi appa- ratus-rich fraction for the ability to catalyze steps involved in the biosynthesis of glycoproteins in vitro. The above report de- scribes some characteristics of this system, which actively trans- fers glucosamine, galactose, and N-acetylneuraminic acid from their sugar nucleotide precursors, to endogenous macromolecular acceptors which have the properties of proteins. Seventeen per cent of the glucosaminyl transferase and 13% of the galactosyl transferase activities of the original homogenate are recovered in the Golgi fraction, at 23 and 24 times the specific activity, re- spectively. In contrast, only 0.2’% of the transfer of mannose from GDP-mannose to endogenous acceptors appears to be localized in the Golgi fraction, at a specific activity lower than that of the crude homogenate. While no reliable conclusions can be drawn from this data, because of the rapid destruction of GDP-mannose, as mentioned earlier, it is nevertheless still pos- sible that the mannosyl transferase is not associated with the Golgi apparatus. This would be consistent with the results of radioautographic studies, which indicate that galactose, but not mannose is taken up by the Golgi complex of thyroid cells (26). A large number of glycoproteins, including thyroglobulin (27) and fibrinogen (28) contain oligosaccharide chains with the ter- minal sequence: sialic acid --f galactose --f N-acetylglucosamine. This trisaccharide is usually attached to an inner core region con- taining alternating mannose and N-acetylglucosamine residues (29). I f the view is correct that newly synthesized protein is glycosylated sequentially as it migrates from rough to smooth endoplasmic reticulum, and thence to the Golgi complex (3, 30), it would be reasonable to expect that the terminal sugars would be attached near the final stages of the process, just prior to secre- tion, possibly within the Golgi apparatus, or the plasma mem- brane. It is thus possible that the N-acetylglucosamine, galac- tose, and N-acetylneuraminic acid residues of the terminal trisaccharide are transferred to the glycoprotein within the Golgi apparatus, after the core sugars have been inserted at other sites within the endoplasmic reticulum. The findings that gluco- samine, galactose, and N-acetylneuraminic acid transfer are

highly concentrated in the Golgi fraction are consistent with this hypothesis.

The Golgi region has also been shown to take up 35S04, which has led to the claim that this organelle mediates a step or steps in the biosynthesis of the sulfated glycosaminoglycans (24, 25). This hypothesis is supported by our findings that glucuronic acid is transferred to endogenous acceptors within the isolated Golgi fraction (5% of the activity of the total homogenate, at 73 times the specific activity). This transfer of glucuronic acid is stimu- lated by UDP-N-acetylglucosamine. The products formed are very alkali-labile, and are degraded to a significant extent by acid mucopolysaccharidases (35 y0 by hyaluronidase, 70 70 by hepari- nase). Conversely, it has been shown that the transfer of gluco- samine is stimulated by UDP-glucuronic acid. This observa- tion, coupled with the findings that glucosamine is transferred to (at least) two distinct species of acceptors, which differ in sus- ceptibility to alkaline degradation, and to the action of muco- polysaccharidases, strongly suggest that glucosamine is trans- ferred to both endogenous glycoprotein and glycosaminoglycan acceptors within this Golgi fraction. The occurrence of two pH optima for the initial rate of transfer of glucosamine also suggests the presence of two distinct transferase enzymes. With alkali lability as a basis for estimation, it would appear that glucosa- mine is incorporated into acceptors of the glycoprotein type to the extent of 65 to 70% of the total transfer, and that when this transfer is stimulated by UDP-glucuronic acid, approximately equal quantities of both species of product are formed.

It is interesting to note that the incorporation of N-acetyl- neuraminic acid from CMP-N-acetylneuraminic acid is inhibited by CMP (Table IV), a product of the transfer reaction. Simi- larly, transfer of glucosamine and galactose from their UDP- derivatives is strongly inhibited by UMP (Tables II and III). Conceivably, inhibition of glycosyl transfer by these nucleoside monophosphates could serve to regulate the rate of glycoprotein biosynthesis in &JO. If UDP, and not UMP, is the product of the glucosamine and galactose transfer reactions, UDPase would be a necessary complement of such a regulatory system. UDP- ase activity is reported to be enriched in the Golgi apparatus frac- tion (17). Alternatively, however, if a lipid intermediate of the type described for bacterial cell wall (31) and lipopolysaccharide (32) biosynthesis should be involved in the transfer of glycosyl groups to proteins in mammalian liver, as has recently been sug- gested (33, 34), nucleoside monophosphates might inhibit by a different mechanism. In this case, UMP would be a direct product of sugar-lipid intermediate formation (32), and its in- hibitory effect would be caused by the reversibility of the re- action.

The inhibition of glucosamine transfer by UDP-glucose and UDP-galactose, and of galactose transfer by UDP-glucose and UDP-N-acetylglucosamine, might reflect the presence of sugar nucleotide pyrophosphatase activity in the Golgi fraction. This would result in the conversion of the uridine diphospho sugars to the corresponding sugar l-phosphates and UMP, and the latter would inhibit the transfer of galactose and glucosamine. The observation that GDP-mannose stimulates glucosamine and galactose transfer (Tables II and III) may indicate that a rela- tively nonspecific pyrophosphatase hydrolyzes both the GDP- and UDP-monosaccharides. High concentrations of GDP- mannose would then spare the UDP-sugars from hydrolysis. CMP-N-acetylneuraminic acid would not be hydrolyzed by such a pyrophosphatase, and hence GDP-mannose would not be ex- pected to stimulate N-acetylneuraminic acid transfer (Table IV).

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Issue of January 10, 1971 R. R. Wagner and M. A. Cynkin 151

The failure to detect stimulation of glucosamine transfer by UDP-glucuronic acid at pH 6.1 might also be the result of the action of a pyrophosphatase. If this enzyme were more active near pH 6.1 than at pH 7.1, the rate of formation of UMP from UDP-glucuronic acid would then be increased. The greater in- hibition by UMP could mask the stimulation of glucosamine transfer by UDP-glucuronic acid.

The stimulation of N-acetylneuraminic acid transfer by UDP- galactose may result from the attachment of N-acetylneuraminic acid to newly incorporated galactose residues. This effect is specific for UDP-galactose, since UDP-glucose and UDP-N- acetylglucosamine fail to stimulate. The finding that galactose is actively incorporated within the Golgi fraction, and the ob- servation that N-acetylneuraminic acid is often found linked to galactose residues of plasma glycoproteins also lend support to this hypothesis.

I f galactose is transferred to glucosamine residues, as sug- gested, UDP-N-acetylglucosamine might be expected to stimu- late the incorporation of galactose. Again, however, UMP formed from UDP-N-acetylglucosamine may mask this stimula- tion.

A recent report (35) describes the isolation of a Golgi ap- paratus-rich fraction from bovine liver by zonal ultracentrifuga- tion. This fraction catalyzes the transfer of galactose from UDP-galactose to free N-acetylglucosamine. This activity is enriched SO-fold in the Golgi fraction, and is inferred to be involved in, or related to glycoprotein biosynthesis. Some support is lent this assumption by the finding (36) that a purified preparation from calf thyroid transfers galactose from UDP- galactose to thyroglobulin glycopeptides from which both sialic acid and galactose have been removed, as well as to free N-acetylglucosamine.

It must be borne in mind that the transfer activities described in this report reflect the presence of both transferase enzymes and the appropriate endogenous acceptors within a particular frac- tion, and do not necessarily represent the total transferase activity of the cell or its subfractions.

Acknowledgments-We are indebted to Dr. W. D. Belt for electron micrographs, and to Dr. D. J. Morrb for making available the details of the Golgi membrane isolation procedure prior to publication.

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Ronald R. Wagner and Morris A. CynkinOF LIVER

ENDOGENOUS ACCEPTORS IN A GOLGI APPARATUS-RICH FRACTION Glycoprotein Biosynthesis: INCORPORATION OF GLYCOSYL GROUPS INTO

1971, 246:143-151.J. Biol. Chem. 

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