glycoprotein synthesis in plants - plant · pdf filethe kinetics ofincorporation of 14c, as...

Plant Physiol. (1977) 59, 341-347

Glycoprotein Synthesis in PlantsI. ROLE OF LIPID INTERMEDIATES'

Received for publication July 6, 1976 and in revised form September 27, 1976

MARY C. ERICSON AND DEBORAH P. DELMERMSU/ERDA Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824

ABSTRACT

The enzymic processes involved in glycoprotein synthesis have beenstudied using crude extracts obtained from developing cotyledons ofPhaseolus vulgaris harvested at the time of active deposition of vidin.Radioactivity from GDP-['4CJmannose can be incorporated by crudeextracts into a single chloroform-methanol-soluble product as well asinto insoluble product(s). Mannose is the sole '4C-labeled constituent ofthe lipid. The kinetics of incorporation of 14C, as determined by pulseand pulse-chase experiments using GDP-['4Cjmannose, as weU as directincorporation from added ['4CImannolipid, shows that the mannolipid isan intermediate in the synthesis of the insoluble product(s). The charac-teristics of the mannolipid are consistent with it being a mannosylphosphoryl polyprenol. The mannose is apparently attached to the lipidvia a monophosphate linkage. Of the radioactivity in the insolubleproduct(s), about 20% is pronase-digestible during a "pulse experi-ment." After a chase with unlabeled GDP-mannose, about 40% ispronase-digestible; the other 60% is as yet uncharacterized. A radioac-tive product soluble in a mixture of chloroform-methanol-H20 can beextracted from the insoluble residue obtained during a pulse, but is nolonger present after a chase. This product may be a lipid oligosaccharide,the final intermediate in glycoprotein synthesis. Data are presented onincorporation from UDP-N-[14Cjacetylglucosamine into both chloro-form-methanol-soluble and -insoluble product(s). The results are con-sistent with an involvement of lipid intermediates in the glycosylation ofprotein in this system, and support the concept that the mechanisms ofglycoprotein synthesis in higher plants are similar to those which havebeen reported for mammalian systems.

That lipid-bound sugars act as intermediates in the biosyn-thesis of polysaccharides and glycoproteins has been establishedin bacteria and animals (12, 13). The lipid moieties of theglycolipid intermediates in bacterial systems have been shown tobe polyisoprenoids containing 10 to 12 isoprenoid units (13, 23),whereas the dolichols, a class of polyisoprenoids containing 18 to22 isoprenoid units, are the lipid carriers that have been found inmammalian systems studied (12) and in yeast (2, 10). Thedolichol phosphate sugars are known to act as intermediates inglycoprotein synthesis (12). Although several types of polyiso-prenoid lipids have been found in plant tissues (17, 25), little isknown about lipid intermediates that may be involved either inthe synthesis of polysaccharides or glycoproteins in plants (15).Forsee and Elbein (6, 7) have reported the presence of acidicglycolipids in cotton fibers with properties similar to those foundin bacterial systems. These cotton glycolipids could be involvedin biosynthesis of cell wall polysaccharides. In addition to syn-

I Supported by United States Energy Research and DevelopmentAdministration Contract E(1 1-1 )-1338.

thesis of a mannosyl lipid, Forsee and Elbein (8) reported theincorporation of radioactive mannose, from GDP-['4C]mannose, into lipid-linked oligosaccharides and into pro-tein. These studies represent the best documented case to datefor the involvement of such lipid intermediates in glycoproteinsynthesis in plants; however, a precursor-product relationshipbetween lipid intermediate and the glycoprotein was not conclu-sively demonstrated. Glycoprotein synthesis has also been stud-ied in mung bean shoots where a mannosyl phosphoryl polyiso-prenoid has been implicated as a possible sugar carrier (1, 11).Although either addition of betulaprenol phosphate or dolicholphosphate stimulated incorporation of mannose from GDP-['4C]mannose into mannosyl lipid (1), a precursor-product rela-tionship between the presumed mannosyl phosphoryl polyprenoland the ['4C]mannose-containing glycoprotein was not definitelyshown. Roberts and Pollard (22) have also shown incorporationof radioactivity from UDP-N-acetyl-D-['4C]glucosamine intoboth lipid and protein in mung bean shoots, although, onceagain, no precursor-product relationship was established.In those systems where glycoprotein synthesis has been stud-

ied in more detail, the mechanism of lipid transfer of sugar toprotein seems to depend upon the type of carbohydrate-peptidelinkage that the finished glycoprotein will have. In the case of theformation of O-glycosidic linkages between carbohydrate andserine or threonine, such as those studied in yeast (3), the firstsugar is added via a polyprenol monophosphate (in this casedolichol monophosphate) and all subsequent sugars seem to beadded directly to the final acceptor from nucleoside diphosphatesugars. However, in glycoproteins which have the carbohydrateattached by an N-glycosidic linkage between N-acetyl-D-glucosa-mine and asparagine, the mechanism of attachment is morecomplex. The N-acetyl-D-glucosamine residue which will be di-rectly attached to the protein is transferred from UDP-N-acetyl-o-glucosamine to form a polyprenol pyrophosphate derivative.The rest of the sugars that go to make up the carbohydratemoiety are transferred from nucleoside diphosphate sugars topolyprenol monophosphate lipids and from these lipids to thepolyprenol pyrophosphate-N-acetyl-u-glucosamine. In this way,a lipid-bound oligosaccharide is made that is transferred, in itsentirety, to the protein acceptor. In the animal systems wherethis mechanism has been demonstrated, the polyprenol mono-phosphate is dolichol monophosphate and the polyprenol pyro-phosphate is dolichol pyrophosphate (12).

Vicilin, the major storage protein of Phaseolus vulgaris seeds,is a glycoprotein containing mannose and glucosamine (29).Because this glycoprotein is made in large quantity at a specifictime, developing seeds of this plant provide an excellent systemin which to study glycoprotein synthesis. Here, we show thatenzyme preparations from the developing seeds of P. vulgaris(kidney bean) are capable of catalyzing the incorporation ofmannose from GDP-['4C]mannose and of N-acetyl-i-glucosa-mine from UDP-N-acetyl-E-['4C]glucosamine into glycolipidswhich have the properties of sugar phosphoryl polyprenols and

341

www.plantphysiol.orgon May 23, 2018 - Published by Downloaded from Copyright © 1977 American Society of Plant Biologists. All rights reserved.

http://www.plantphysiol.org

ERICSON AND DELMER

of oligosaccharide lipid, as well as into a glycoprotein. From thedata presented below, we conclude that there is a precursor-product relationship between the lipid-bound sugars and theglycoprotein made in seeds of P. vulgaris, and, therefore, thatlipid intermediates can be involved in glycoprotein synthesis inhigher plants in a manner resembling that in mammalian sys-tems.

MATERIALS AND METHODS

MATERIALS

GDP-D-['4C]mannose (68 ,Ci/,umol) and UDP-N-acetyl-D-['4C]glucosamine (53.4 ,tCi/,umol) were obtained from NewEngland Nuclear. GDP, GMP, GDP-mannose, and UDP-N-acetyl-D-glucosamine were obtained from Sigma Chemical Co.Sephadex G-50 and G-200 were purchased from Pharmacia.Silica Gel G plates used in TLC were obtained from BrinkmannInstruments. Pronase (grades B and CB) was purchased fromCalbiochem.

EXTRACTION OF VICILIN FROM DEVELOPING SEEDS

The rate of vicilin accumulation in kidney bean cotyledons wasdetermined. This enabled us to choose the stage of developmentat which glycoprotein synthesis was maximal. Protein was ex-tracted from developing seeds, and vicilin was purified from thisextract as reported by Ericson and Chrispeels (5). Identificationof vicilin was determined by its mobility on sucrose gradients andSDS-acrylamide gels (5). Vicilin constitutes 35 to 50% of thetotal protein in developing cotyledons and the approximateamount of vicilin can be determined on sucrose gradients.

PREPARATION AND ASSAY OF ENZYMES

Bean cotyledons of 14 to 17 mm length and weighing, on theaverage, 0.5 g/cotyledon pair were ground in 50 mm tris-HCI(pH 7.5) containing 1 mm EDTA. One ml of medium was usedper g of tissue. Grinding was done initially with a mortar andpestle and then with a ground glass homogenizer. The homoge-nate was centrifuged at 600g for 10 min and the supernatant wasused as the crude enzyme mixture. The enzyme activities couldbe preserved without appreciable loss upon freezing and thaw-ing. Repeated freezing and thawing (more than three times)resulted in substantial loss of activity.

Incubation mixtures for synthesis of the various products con-tained the following components in a final volume of 0.55 ml:GDP-D-['4C]mannose, 0.3 nmol (35,000 cpm); MnCl2, 5 ,umol;and 0.5 ml of the enzyme preparation. Incubations were allowedto continue for various lengths of time either at room tempera-ture or at 15 C. During pulse-chase experiments, 0.1 ,umol ofunlabeled GDP-D-mannose was added at the time of the chase.

Experiments involving incubation with UDP-N-acetylglucosa-mine were performed as above except 4.31 nmol (400,000 cpm)UDP-N-acetyl- [4C]glucosamine was substituted for the labeledGDP-mannose and 0.1 ,umol unlabeled GDP-mannose wasadded to each reaction mixture. During pulse-chase experi-ments, 0.1 ,tmol of unlabeled UDP-N-acetyl-D-glucosamine wasadded at the time of the chase.

ANALYSIS OF PRODUCTS

Reaction mixtures described above were terminated by theaddition of 1.5 ml of chloroform-methanol, 1:2, with thoroughmixing. The mixtures were incubated at 37 C for 20 min to

ensure complete extraction of the lipids. After incubation, 0.5ml of CHCl3 and 0.5 ml 0.15 M NaCl in 0.01 M HCI were addedto each mixture. Phases were separated by centrifugation andthe lower organic phase was removed and counted. The upper

(aqueous) layer and the interface were recentrifuged, the upperlayer discarded, and the pellet or interface product was washedthree times with water and once with methanol. The washeswere discarded and the pellet was counted.

In some experiments, pellets were further extracted threetimes with 2 ml of chloroform-methanol-water, 1: 1:0.3, and theextracts were pooled. The material soluble in chloroform-metha-nol-water, 1:1:0.3, was evaporated with a stream of N2, redis-solved in a small volume of the same solution, and counted.

Radioactivity was determined using a Packard Tri-Carb liquidscintillation spectrometer. Quench curves and counting efficien-cies were determined for various counting conditions, and alldata converted to dpm. The radioactivities of all fractions weremeasured in a scintillation fluid containing I part Bio-Solve(obtained from Beckman Instruments) to 10 parts standard,toluene-based scintillation fluid. A summary of the extractionprocedure is illustrated in Figure 1.

THIN LAYER CHROMATOGRAPHY

Thin-layer chromatography of the lipid products was done onsilica gel plates in three solvent systems: solvent system A,chloroform-methanol-water, 65:25:4, v/v/v; solvent system B,chloroform-methanol-acetic acid-water, 25:15:4:2, v/v/v; andsolvent system C, chloroform-methanol-concentrated ammo-nium hydroxide, 75:25:4, v/v/v. The plates were activated at100 C for 30 min before use. Samples were evaporated in astream of N2 and redissolved in 50 ,ul of either chloroform-methanol (1:1; for organic phase lipids; see above) or chloro-form-methanol-water (1:1:0.3; for oligosaccharide lipids) andwere applied to a silica gel plate. After chromatography, thestrips were dried and cut into 1-cm sections which were counteddirectly in toluene scintillation fluid.

MILD ACID TREATMENT OF LIPIDS

The procedure for mild acid hydrolysis was similar to that usedby Richards and Hemming (21). Samples of dried radioactivelipid were mixed with 1 ml of methanol-water, 1:1, containing0.01 N HCI and heated at 100 C for 10 min. The mixture wasthen neutralized by adding 1 ml NaOH (0.01 N in methanol),and the lipids were extracted by adding 2 ml chloroform, 0.5 mlmethanol, and 1.5 ml H2O. After thorough mixing, the mixturewas separated into two layers by low speed centrifugation. Aportion of the chloroform layer was assayed for radioactivity byliquid scintillation counting. The aqueous phase was dried andchromatographed together with standard sugars on WhatmanNo. 4 chromatography paper in a solvent system containingpyridine-ethyl acetate-water, 2:8:1. Lipid samples untreatedwith acid were dissolved in 50% methanol and heated along withthe acid-treated samples. The lipids were extracted and assayedfor radioactivity just as were the acid-treated samples.

ALKALI TREATMENT OF LIPIDS

Samples for alkaline saponification were suspended in chloro-form-methanol, 1:4, v/v, containing 0.1 N NaOH and incubatedat 37 C for 30 min. The samples were neutralized with 0.1 ml 1 NHCI, and chloroform and water were added to bring the chloro-form-methanol-water ratio to 1:1:1. Control samples were incu-bated at 37 C for 30 min in chloroform-methanol, 1:4. Radioac-tivity in the organic phases was then determined.

CHARACTERIZATION OF THE INTERFACE PRODUCT

Proteolytic Digestion. The washed pellets (Fig. 1) from sam-

ples which had been incubated with GDP-['4C]mannose, with or

without a subsequent chase with unlabeled GDP-mannose, were

suspended in 1 ml 0.05 M sodium phosphate buffer (pH 7.3).

342 Plant Physiol. Vol. 59, 1977



GLYCOPROTEIN SYNTHESIS IN PLANTS

INCUBATION MIXTURE

STEP 1 EXTRACTION WITHCHLOROFORM/METHANOL (1:2)CENTRIFUGATION

LOWER LAYER(chloroform:methanolsoluble products)

UPPER LAYERAND INTERFACE

STEP 2 CENTRIFUGATION

PELLET

STEP 3

PELET(Insoluble product usedin most experiments)

STEP 4(used for experiments

indicated in the text)

PELLET

AQUEOUS PHASE(discard)

WASH WITH WATER (3 X)AND METHANOL (1 X)

WASH(discard)

EXTRACT WITH CHLOROFORM:METHANOL:WATER(1:1:0.3)

CHLOROFORM:METHANOL:WATER(1:1:0.3) soluble lipids

FIG. 1. Protocol for isolation of reaction products.

Two mg of pronase and drop of toluene (to retard bacterialgrowth) were added to each sample. The samples were thenincubated at 37 C for 72 hr. After the first 24 hr, an additional 2mg of pronase and drop of toluene were added to each incuba-tion mixture. The reactions were terminated by placing thesamples in a boiling water bath for 10 min. Insoluble materialwas then removed by centrifugation.

Elimination. In order to determine the nature of the carbo-hydrate -* peptide linkage of the protein portion of the interfaceproduct, samples of this product were subjected to mild alkalinehydrolysis. Samples of insoluble product from standard incuba-tions were suspended in 3 ml of 0.1 M NaOH for 24 hr, at roomtemperature. At the end of this incubation, 3 ml of 15% trichlo-roacetic acid were added to the mixtures, which were thencentrifuged. The radioactivity of pellets and portions of thesupernatant fluids was then assayed.

INCUBATION OF [14CIMANNOLIPID WITH ENZYME PREPARATION

I'4ClMannolipid was recovered in the organic phase of thechloroform-methanol, 1:2, extract from several standard incuba-tions. This I'4Clmannolipid was dried in a stream of N, andresolubilized in 0.1% Triton X-100. Incubation mixtures forsynthesis of insoluble product, using I'4Clmannolipid as thesugar donor, contained the following components in a final

volume of 0.625 ml: 100 1ld of ['4C]mannolipid in 0.1% TritonX-100 (3,000 cpm); MnC12, 5 /.mol; and 0.5 ml of the enzymepreparation. This mixture was incubated for 20 min at roomtemperature. The reaction was terminated by the addition of 1.5ml chloroform-methanol, 1:2. Lipid and interface products werethen prepared as described above. Control samples containing100 ,ul 0.1% Triton X-100 but using GDP-['4C]mannose as thesugar donor were also analyzed for radioactive products.

RESULTS

Vicilin Content of Kidney Bean Cotyledons. The in vivoaccumulation of the storage protein vicilin in kidney bean cotyle-dons during seed development is illustrated in Figure 2. Cotyle-dons grow from 8 to 20 mm in length in approximately 10 or 12days. As can be seen, vicilin is continuously accumulated overthis period. Synthesis of the glycoprotein vicilin increases rapidlyin cotyledons 16 to 18 mm in length. We used cotyledons of thissize as a source of crude enzyme for our in vitro studies.Enzymic Transfer of Mannose from GDP-['4C]Mannose to

Endogenous Acceptors. When the enzyme preparation fromcotyledons of P. vulgaris was incubated with GDP-['4C]man-nose, radioactivity was incorporated into a chloroform-metha-nol-soluble product and insoluble interface product. The kineticsof incorporation into these products is shown in Figure 3. The

343Plant Physiol. Vol. 59, 1977



ERICSON AND DELMER

incorporation of labeled mannose into an insoluble interfaceproduct continued over the course of the entire 20-min incuba-tion period. The incorporation into chloroform-methanol-solu-ble material was very rapid during the first few minutes ofincubation, but reached a plateau after 6 to 7 min.To determine whether there was a precursor-product relation-

ship between the chloroform-methanol-soluble product and theinsoluble interface product, a pulse-chase experiment was per-formed. An excess of unlabeled GDP-mannose was added after6 min of incubation with GDP-['4C]mannose. Preliminary ex-periments at room temperature showed that radioactivity waschased from the chloroform-methanol-soluble fraction within 4to 5 min, and continued to rise in the insoluble interface product(Fig. 4A) until radioactivity was depleted from the chloroform-methanol-soluble product. Subsequent experiments were run at15 C to slow the reactions to permit more detailed observation ofkinetics (Fig. 4B). The initial rate of increase in radioactivity ofthe insoluble interface product can be accounted for by the

25r

20[

ca0

co

.2_

IC

concurrent loss of radioactivity from the chloroform-methanol-soluble product.

Radioactive chloroform-methanol-soluble product (1,400dpm) resuspended in 0.1% Triton X-100 was also successfully

E*0

/

0

0 4 8 12 16 20

Cotyledon Length (mm)

FIG. 2. In vivo synthesis of vicilin during development of kidneybean seeds. The graph represents the amount of vicilin per bean as afunction of length of the cotyledons.

p

insoluble product .-f/

I

20Incubation Time (min)

FIG. 3. Formation of chloroform-methanol (c:m)-soluble and insolu-ble products as a function of time of incubation.

A

chase

E EJ

2000 - 2000 -

chsechose

1000-Dbods 1000lj Att000 ~~~c:m-soluble product c:m-soluble product

0 0o 10 20 0 10 20

Incubation Time (min) 0 RT Incubation Time (min) @ 15°C

FIG. 4. Pulse-chase experiments. Samples were incubated as described under "Materials and Methods" with GDP-('4C]mannose. A: Sampleswere incubated at room temperature (RT) for 6 min, an excess of unlabeled GDP-mannose was then added to each mixture. B: Samples wereincubated at 15 C for 10 min, an excess of unlabeled GDP-mannose was then added to each mixture; ( ): pulsed products; (---): chased products.


15F



GLYCOPROTEIN SYNTHESIS IN PLANTS

used as the only donor of radioactive sugar. Control samplescontaining the same amounts of Triton X-100 but using GDP-['4C]mannose as the sugar donor indicated that the presence ofTriton X-100 did not interfere with the normal course of thereaction. During the course of the incubation, 600 dpm disap-peared from the chloroform-methanol-soluble substrate, andwas accompanied by the appearance of 400 dpm in the insolubleinterface product. It is possible that an inability to solubilize thelipid substrate completely, or an inaccessibility of the enzyme ornonglycosylated protein precursor was primarily responsible forthe failure to transfer all of the radioactive sugar from thesubstrate to the interface product.The formation of mannosyl phosphoryl polyprenol from GDP-

mannose is a reversible reaction. If mannose is attached to thelipid by a monophosphate linkage, then the reaction should bereversible by GDP. If the mannose is attached to the lipid by apyrophosphate linkage, then the reaction should be reversible byGMP. The accumulation or synthesis of the mannolipid wasfound to be reversed by the addition of GDP but not GMP(Table I). First, the crude extract was incubated with GDP-['4C]mannose to synthesize the mannolipid. After an initial pe-riod (2 or 5 min), incubation was continued with no additions orin the presence of added GMP or GDP for a total of 15 min. Atthe end of the incubation time, the radioactivity in the lipidfraction was the same in those samples treated with GMP as inthose to which no additions were made. However, in thosesamples to which GDP was added, radioactivity disappearedfrom the lipid. Thin layer chromatography of the aqueous phaseof these samples on cellulose plates in 95% ethanol-I M ammo-nium acetate-7:3 (pH 3.8) showed that all of the radioactivitywas contained in GDP-mannose. These results suggest that themannose is attached to the lipid by a monophosphate linkage. Insamples treated with GDP, there was a decrease in the radioac-tivity transferred to insoluble product, as would be expected.There was also a small decrease in transfer of radioactivity ininsoluble product in the samples treated with GMP.Thin Layer Chromatography of the Product Soluble in Chlo-

roform-Methanol. The product soluble in chloroform-methanolwas subjected to TLC in three different solvent systems. Onlyone peak of radioactivity was detected on chromatograms run ineach of these three solvent systems. The RF value of the productwas 0.44 in solvent system A, 0.90 in solvent system B, and 0.19in solvent system C. As expected for a glycolipid of the poly-prenol phosphate type, the radioactivity showed relative to thesolvent front, rapid migration in the acidic solvent, slow migra-tion in the basic solvent, and intermediate migration in theneutral solvent (4).Treatment of Chlorofonn-Methanol-soluble Product with

Mild Acid and Alkali. Glycolipids of the polyprenol phosphatetype are stable to mild alkali but are extremely labile to mild acidtreatment (9). On the other hand, glycolipids such as dimannosyldiglycerides and steryl mannosides are labile to alkali and re-quire vigorous acid treatment before they are hydrolyzed (14).

Table 1. Effects of Adding an Excess of Nucleotides to Reaction MixturesContaining GDP-( C]-Mannose and Extract of Cotyledons

Reactions were allowed to proceed for 2 or 5 min to allow synthesis ofradioactive lipid to occur normally, after which nucleotides were addedto give a concentration of 1.0 mM. All reactions were termina,ed at 15min with the addition of chloroform:methanol (1:2).

Nucleotide Added Recovery of 14CCompound Time of nucleotide Lipid Insoluble

addition Productmin dpm x 10 3

None - 9.5 26.0

GMP 2 9.2 19.8

GDP 2 1.4 16.6

GMP 5 9.6 19.6

GDP 5 1.7 16.9

When samples of labeled chloroform-methanol-soluble lipid(15,500 dpm/sample) were treated with mild acid at 100 C for10 min, all of the radioactivity was released as water-solublematerial. This radioactive material was shown by means ofchromatography to be exclusively mannose. The radioactivity ofcontrol samples of lipid heated in 50% methanol (without HCl)at 100 C for the same length of time remained in the organicphase. When samples of ['4C]mannolipid (20,500 dpm) weretreated with mild alkali, all radioactivity was retained as lipid-bound material.

Characterization of the Insoluble Interface Product. About20% of the insoluble radioactivity from the interface productwas digested by pronase (Table IIA), indicating that 20% of theinterface product, at the end of a standard incubation, is glyco-protein in nature. Since, at this time, the pellets had not beenextracted with chloroform-methanol-water, 1:1:0.3, to removeany possible oligosaccharide lipid (18), the remaining 80% ofthe interface product could be oligosaccharide lipid, other prod-ucts such as mannan, or a mixture of both.

Pulse-chase experiments were then performed and the pelletswere extracted with the 1:1:0.3 solvent. A radioactive, chasea-ble lipid product was found (Fig. 5). Radioactivity was trans-ferred from this lipid product at a much slower rate than fromthe chloroform-methanol-soluble lipid during the chase. Whensubjected to TLC in solvent system A, the radioactivity of thischaseable product remained at the origin, as would be expectedfor an oligosaccharide lipid.

Pellets of insoluble interface product from samples which hadbeen incubated with GDP-['4C]mannose for 20 min and thenchased with excess unlabeled GDP-mannose for 10 min weresubjected to pronase digestion. The results (Table IIB) showthat in contrast to the results in Table IIA, in radioactive sampleswhich have been treated with unlabeled GDP-mannose forenough time to ensure complete transfer of radioactivity throughall potential lipid intermediates to protein, 42% of the interfaceproduct is pronase-digestible.

In order to determine the nature of the carbohydrate-peptidelinkage of the glycoprotein present, samples of insoluble product(33,000 dpm/sample) were treated with mild alkali. No radioac-

Table II. A. Solubilization of the Radioactive, Interface Product byDigestion with Pronase

Interface product was obtained from samples incubated for 20 minutes withGDP-[14C]-mannose. Details of the digestion procedure are given inMaterials and Methods.

Sample Radioactivity % solubilized(dpm)

Interface product(before proteolytic 11,380digestion)

Interface product(after proteolytic 9,550digestion)

Counts solubilizedby proteolytic 1,020 19.3digestion

Table II. B. SoZubilization of the Radioactive, "Chased", Intel-faceProduct by Digestion with Pronase

Interface product was obtained from samples incubated for 5 minutes withGDP-[14CJ-mannose and for an additional 15 minutes with unlabeled GDP-mannose. Details of the digestion procedure are given in Materials andMethods.

Sample Radioactivity Xsolubilized(dpm)

Interface product(before proteolytic 4700digestion)

Interfare product(after proteolytic 2720digestion)

Counts solubilizedby proteolytic 1980 42.0digestion

345Plant Physiol. Vol. 59, 1977



ERICSON AND DELMER

6001

400-

E

1500-

0

-~~~0

o0

2001

0O 10 20

insoluble product

1000p

E

5001

0 10 20

Incubation Time (min)

FIG. 5. Formation, as a function of time of incubation at roomtemperature, of radioactive product soluble in chloroform-methanol-water, 1:1:0.3. After 7 min of incubation with GDP-['4C]mannose, anexcess of unlabeled GDP-mannose was added to some of the samples;

): pulsed product; (----): chased product.

tivity was released by this treatment. Therefore, we concludethat the carbohydrate-peptide link is probably not an O-glycos-idic link to serine or threonine, but probably an N-glycosidic linkbetween glucosamine and asparagine to which mannose residuesare attached.

Transfer of N-acetylglucosamine from UDP-N-acetyl-D-14C]Glucosamine to Endogenous Acceptors. Preliminary re-sults indicate that both a radioactive lipid and an interfaceproduct can be obtained from incubations of extracts with UDP-N-acetyl-D-[14C]glucosamine (Fig. 6). The incorporation intointerface product was dependent upon the addition of GDP-mannose to the reaction mixture. Large amounts of radioactivity(400,000 cpm/reaction mixture) must be added in order toobtain measureable incorporation. Thin layer chromatographyof the radioactive lipid from these experiments in chloroform-methanol-water 65:25:4, v/v/v, showed that there were tworadioactive chloroform-methanol-soluble products. One of theseproducts had an RF. similar to that of the chloroform-methanol-soluble mannolipid; the other moved more slowly in this nonpo-lar solvent system. There were not sufficient amounts of theselipid-soluble products to characterize them more fully, but it ispossible that they represent the mono- and disaccharide deriva-tives of a growing lipid oligosaccharide.

DISCUSSION

A crude enzyme preparation from developing kidney beancotyledons has been shown to catalyze the incorporation ofsugars from nucleoside diphosphate sugars into lipid-solublematerial and into insoluble glycoprotein. Two different types ofmannolipid were found: a chloroform-methanol-soluble lipidwhich has properties similar to a mannosyl phosphoryl poly-prenol, and a lipid soluble in chloroform-methanol-water,1:1:0.3, which has properties similar to an oligosaccharide lipid.The kinetics of formation of the various products described

here indicate a precursor-product relationship as follows:

GDP-D-[14C]mannose mannolipid (probably a mannosyl phosphorylpolyprenol) oligosaccharide lipid - glycoprotein

Because only 42% of the insoluble interface product was pro-

Incubation Time (min)FIG. 6. Formation of chloroform-methanol-soluble and -insoluble

products as a function of time of incubation at room temperature withUDP-N-acetyl-D- ['4C]glucosamine.

nase-digestible, it is probable that in addition to glycoprotein,other product(s), such as a mannan, are synthesized from GDP-D_[14C]mannose. That the sugar residues are being transferred tothe glycoprotein from the oligosaccharide lipid is indicated bythe increased pronase-digestibility of the insoluble product aftera chase. There is no evidence that the nonproteinaceous productis also being made from the lipid intermediate but this possibilitycannot be ruled out. We are currently investigating whether atleast a portion of the glycoprotein, the synthesis of which wehave studied, may be vicilin, the storage glycoprotein of P.vulgaris which is made in large quantity during seed develop-ment. However, the glycoproteins that have been shown to bemade via lipid intermediates in cell-free preparations of henoviduct are different from the ovalbumin which is made in vivoin large quantity in this system (16, 19). In our system, prelimi-nary results indicate that the protein product is, at least initially,membrane-bound. Although this could indicate that our in vitrosystem is making membrane protein(s), it does not rule out thepossibility that it is formation of glycosylated vicilin that weobserve. Because vicilin is stored in membrane-bound organellescalled protein bodies (5), it is possible that our membrane-boundproduct is simply vicilin associated with protein body membranesor is vicilin that is attached to the endoplasmic reticulum.

LITERATURE CITED

1. ALAm, S. S. AND F. W. HEMMING. 1973. Polyprenol phosphates and mannosyl transferasesin Phaseolus aureus. Phytochemistry 12: 1641-1649.

2. BABCZINSKI, P. AND W. TANNER. 1973. Involvement of dolicholmonophosphate in theformation of specific mannosyl-linkages in yeast glycoproteins. Biochem. Biophys. Res.Commun. 54: 1119-1124.

3. BRET-HAUER, R. K. AND S. Wu. 1975. Synthesis of the mannosyl-O-serine (threonine)linkage of glycoproteins from polyisoprenylphosphate mannose in yeast (Hansenula hol-stii). Arch. Biochem. Biophys. 167: 151-160.

4. CHAMBER, J. AND A. D. ELBEIN. 1975. Biosynthesis and characterization of lipid-linkedsugars and glycoproteins in aorta. J. Biol. Chem. 250: 6904-6915.

5. ERICSON, M. C. AND M. J. CRISPEELS. 1973. Isolation and characterization of glucosamine-containing storage glycoproteins from the cotyledons of Phaseolus aureus. Plant Physiol.52: 98-104.

6. FORSEE, W. T. AND A. D. ELBEIN. 1972. Biosynthesis of acidic glycolipids in cotton fibers.Biochem. Biophys. Res. Commun. 49: 930-939.

7. FORSEE, W. T. AND A. D. ELBEIN. 1973. Biosynthesis of mannosyl- and glucosyl-phos-phoryl-polyprenols in cotton fibers. J. Biol. Chem. 248: 2858-2867.

8. FORSEE, W. T. AND A. D. ELBEIN. 1975. Glycoprotein biosynthesis in plants. Demonstra-tion of lipid-linked oligosaccharides of mannose and N-acetylglucosamine. J. Biol. Chem.250: 9283-9293.

9. HIGASHI, Y., J. L. STROMINGER, AND C. C. SWEELEY. 1967. Structure of a lipid intermedi-




ate in cell wall peptidoglycan synthesis: a derivative of a C., isoprenoid alcohol. Proc. Nat.Acad. Sci. U. S. A. 57: 1878-1884.

10. JUNG, P. AND W. TANNER. 1973. Identification of the lipid intermediate in yeast mannan

biosynthesis. Eur. J. Biochem. 37: 1-6.11. KAUSS, H. 1969. Dolicholmonophosphates: mannosyl acceptors in a particulate in vitro

system of S. cerevisiae. Fed. Eur. Biochem. Soc. Lett. 16: 245-248.12. LENNARZ, W. J. 1975. Lipid-linked sugars in glycoprotein synthesis. Science 188: 986-991.13. LENNARZ, W. J., AND M. G. SCHER. 1973. The role of lipid linked activated sugars in

glycosylation reactions. In: J. Avery, ed., Membrane Structure and Mechanisms ofBiological Energy Transduction. Plenum Press, New York. pp. 441-453.

14. LENNARZ, W. J. AND B. TALAMO. 1966. The chemical characterization and enzymaticsynthesis of mannolipids in Micrococcus lysodeikncus. J. Biol. Chem. 241: 2707-2719.

15. LEZICA, R. P., C. T. BRETT, P. R. MARTINEZ. AND M. A. DANKERT. 1975. A glucoseacceptor in plants with the properties of an ea-saturated polyprenol monophosphate.Biochem. Biophys. Res. Commun. 66: 980-987.

16. LucAs, J. J., C. J. WAECHTER, AND W. J. LENNARZ. 1975. The participation of lipid-linkedoligosaccharide in synthesis of membrane glycoproteins. J. Biol. Chem. 250: 1992-2002.

17. MORTON, R. A. 1972. Polyprenols, their nature, occurrence and biological roles. In: J.Ganguly and R. M. S. Smallie, eds., Current Trends in the Biochemistry of Lipids.Academic Press, New York. pp. 203-217.

18. PARODI, A. J., N. H. BEHRENS. L. F. LELOIR, AND M. DANKERT. 1972. Glucose transfer

347

from dolichol monophosphate glucose: the lipid moiety of the endogenous microsomalacceptor. Biochim. Biophys. Acta 270: 529-536.

19. PLESS, D. 0. AND W. J. LENNARZ. 1975. A lipid-linked oligosaccharide intermediate inglycoprotein synthesis. J. Biol. Chem. 250: 7014-7019.

20. RAcuSEN, D. AND M. FoOTE. 1971. The major glycoprotein in germinating bean seeds.Can. J. Bot. 49: 2107-2111.

21. RicHARDs, J. B. AND F. W. HEMMING. 1972. The transfer of mannose from guanosinediphosphate mannose to dolichol phosphate and protein by pig liver endoplasmic reticu-lum. Biochem. J. 130: 77-93.

22. ROBERTS, R. M. AND W. E. POLLARD. 1975. The incorporation of D-glucosamine intoglycolipids and glycoproteins of membrane preparations from Phaseolus aureus hypocot-yls. Plant Physiol. 55: 431-436.

23. SCHULTZ, J. C. AND K. TAKAYAMA. 1975. The role of mannosyl-phosphorylpolyisoprenol inglycoprotein biosynthesis in Mycobacterium smegmatis. Biochim. Biophys. Acta 381:175-184.

24. TANNER, W. AND P. JUNG. 1972. The role of a lipid intermediate in mannose-polymerbiosynthesis in yeast. In: R. Piras and H. G. Pontis, eds., Biochemistry of the GlycosidicLinkage. Academic Press, New York. pp. 227-235.

25. WELLBURN, A. R. AND F. W. HEMMING. 1966. Gas-liquid chromatography of derivativesof naturally-occurring mixtures of long-chain polyisoprenoid alcohols. J. Chromatogr. 23:51-66.

Plant Physiol. Vol. 59, 1977 GLYCOPROTEIN SYNTHESIS IN PLANTS



glycoprotein synthesis in plants - plant · pdf filethe kinetics ofincorporation of 14c, as...

Documents