Plant Physiol. (1986) 81, 206-21 10032-0889/86/8 1/0206/06/$01.00/0

Oligosaccharide Side Chains of Glycoproteins that Remain in theHigh-Mannose Form Are Not Accessible to Glycosidases'

Received for publication November 22, 1985 and in revised form January 21, 1986

LOIC FAYE2, KENNETH D. JOHNSON3, AND MAARTEN J. CHRISPEELS*Department ofBiology C-016, University ofCalifornia, San Diego, La Jolla, California 92093

ABSTRACT

Glycoproteins present in the soluble and organelle fractions of devel-oping bean (Phaseolus vulgaris) cotyledons were analyzed by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis, affinoblotting, frac-tionation on immobilized concanavalin A (ConA), and digestion of theoligosaccharide side chains with specific glycosidases before and afterprotein denaturation. These studies led to the following observations. (a)Bean cotyledons contain a large variety of glycoproteins that bind toConA. Binding to ConA can be eliminated by prior digestion of denaturedproteins with a-mannosidase or endoglycosidase H, indicating that bind-ing to ConA is mediated by high-mannose oligosaccharide side chains.(b) Bean cotyledons contain a large variety of fucosylated glycoproteinswhich bind to ConA. Because fucose-containing oligosaccharide sidechains do not bind to ConA, such proteins must have both high-mannoseand modified oligosaccharides. (c) For all the glycoproteins examinedexcept one, the high-mannose oligosaccharides on the undenatured pro-teins are accessible to ConA and partially accessible to jack bean a-mannosidase. (d) Treatment of the native proteins with a-mannosidaseremoves only 1 or 2 mannose residues from the high-mannose oligosac-charides. Similar treatments of sodium dodecyl sulfate-denatured orpronase-digested glycoproteins removes all a-mannose residues. Theresults support the following conclusions: certain side chains remainunmodified as high-mannose oligosaccharides even though the proteinsto which they are attached pass through the Golgi apparatus, whereother oligosaccharide chains are modified. The chains remain unmodifiedbecause they are not accessible to processing enzymes such as the Golgi-localized a-mannosidase.

The asparagine-linked oligosaccharides found on plant glyco-proteins, like those of other eukaryotes, fall into two generalcategories: high-Man4 and modified oligosaccharides. Both orig-inate from a common Glc3Man9(GlcNAc)2 precursor, which istransferred en bloc from a lipid carrier in the RER to specificasparagine residues of nascent polypeptide chains. Mature high-Man oligosaccharides are formed by the removal ofthe 3 glucose

'Supported by grants from the National Science Foundation (Meta-bolic Biology) and the United States Department of Agriculture (Com-petitive Grants).20n leave from the Laboratoire de Photobiologie (CNRS-LA203)

Faculte des Sciences de Rouen, Mont Saint Aignan, F-76 130, FRANCE,and supported by a grant from NATO.30n leave from the Department of Biology, San Diego State Univer-

sity, San Diego, CA.'Abbreviations: Man, mannose; ConA, concanavalin A; Fuc, fucose,

endo H, endoglucosaminidase H; TTBS, Tween Tris buffered saline;PBS, phosphate-buffered saline; PHA, phytohemagglutinin.

units and 1 to 4 Man residues. Modified oligosaccharides arederived from high-Man chains by trimming to a Man5(GlcNac)2chain, which is then modified by the addition of a GIcNAcresidue, the removal of2 more Man residues, and the subsequentadditions of one or more of the following: GIcNAc, Fuc, Gal,sialic acid, and/or xylose residues. All these modifications occurin the Golgi apparatus before the glycoproteins are transportedto their final destinations. Many ofthe details ofthese processingsteps have been worked out for animal cells (see Refs. 9 and 16)and preliminary investigations indicate that processing of glyco-proteins in plant cells is similar in certain respects (5, 6, 12, 13,21, 24), but not others (e.g. plant glycoproteins lack sialic acid).Glycosyltransferases which transfer Fuc and GlcNAc to theoligosaccharide side chains of glycoproteins have been shown tobe associated with the Golgi apparatus of plant cells (5, 21).Proteins with modified oligosaccharide side chains are thereforegenerally assumed to have passed through the Golgi apparatus.This is often measured by determining whether the side chainsare sensitive or resistant to endoglucosaminidase H (endo H).High-Man oligosaccharides on denatured glycoproteins (or gly-copeptides) are cleaved by endo H, while most modified chainsare resistant (14, 22).

Previous work from our laboratory indicates that the glycopro-tein PHA, a lectin found in large amounts in the cotyledons ofPhaseolus vulgaris, has one high-Man and one complex sidechain per-polypeptide (25). This finding raises a number ofquestions. First, is the pattern of oligosaccharide processingunique for PHA, or are there other glycoproteins in bean coty-ledons that contain both types ofoligosaccharide chains? Second,why are certain oligosaccharides processed to form modifiedchains while others remain in the high-Man form?

In an effort to understand the oligosaccharide processing mech-anisms and their control in plants, we have begun a systematicinvestigation of the oligosaccharide side chains present on thesoluble and organellar glycoproteins of bean cotyledons. In thisstudy we report that PHA is not unique among been cotyledonglycoproteins in having both high-Man and complex oligosac-charides side chains, indicating that many glycoproteins passthrough the Golgi apparatus without having their high-Manchains modified. We show, furthermore, that the lack of modi-fication is probably due to the inaccessibility of these high-Manchains to a-mannosidase and other processing enzymes.

MATERIALS AND METHODSMaterials. Plants of Phaseolus vulgaris L. cv Greensleeves

were grown in a greenhouse. Organic chemicals were purchasedfrom Sigma Chemical Co. unless otherwise indicated. D-[6-3H]-Glucosamine hydrochloride (22 Ci/mmol) was purchased fromAmersham Co.; L-[6-3H]fucose (30 Ci/mmol) was obtained fromICN Radiochemical.

Tissue Labeling and Cell Fractionation. Cotyledons from de-veloping seeds were incubated with [3H]glucosamine or [3H]-

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OLIGOSACCHARIDE SIDE CHAINS OF GLYCOPROTEINS

fucose (10 gCi/cotyledon) as described (18) for 24 h at 20°C.After the labeling period, a 1-mm slice from the radiolabeledsurface of each cotyledon was made with a razor blade, and suchradioactive slices from typically 4 to 8 cotyledons were combinedand ground in a small mortar with 3 ml of 100 mm Tris-HCl(pH 7.8) containing 1 mM EDTA and 12% (w/w) sucrose. Thishomogenization procedure disrupts the protein bodies, whosestored proteins (mostly phaseolin and PHA) thus become partof the soluble fraction of the homogenate. The homogenate wascentrifuged for 5 min at lOOOg to remove cell walls, and thesupernatant was layered onto a discontinuous sucrose gradientconsisting of 1 ml of 35% (w/w) sucrose under 8 ml of 16%(w/w) sucrose in the same Tris-EDTA buffer noted above. Aftercentrifugation for 90 min at 150,000g, the sample layer (3 ml)plus the uppermost 2 ml of the 16% sucrose layer were collectedas the soluble protein fraction. The organelle fraction was man-ifest as an opaque band at the interface ofthe 16 and 35% sucroselayers; it was collected and adjusted to 1% Triton X-100, thenincubated for I h at 5°C to ensure solubilization of the mem-branes.PAGE, Fluorography, and Affinoblotting. Separation of poly-

peptides by SDS-PAGE was performed as described (17). Fluo-rographs were prepared according to Bonner and Laskey (1).Affinoblotting with the ConA/peroxidase procedure has beendescribed (10).

Precipitation of Glycoproteins with Immobilized ConA. Pro-tein fractions were dialyzed against TTBS (20 mm Tris-HCl [pH7.4], containing 500 mm NaCl and 0.1% Tween 20), mixed withincreasing amounts of ConA-agarose, and tumbled for 2 h at5C. Controls were tumbled in the presence of 500 mm a-methyl-mannoside. The unbound proteins were recovered and combinedwith the first 4 volumes of column washings of TTBS. The gelwas washed with another 10 volumes of TTBS, which werediscarded. The bound proteins were eluted with 4 column vol-umes of TTBS containing 500 mM a-methyl-mannoside. Pro-teins were precipitated with cold TCA (12.5% final concentra-tion), washed with 90% aqueous acetone at 0°C, and resolubilizedovernight at 37°C in a small volume ofSDS-PAGE sample buffer(20 mm Tris-HCl [pH 8.6], with 1% SDS, 0.24% ,3-mercaptoeth-anol, and 8% glycerol).Treatment of Glycoproteins with a-Mannosidase and Analysis

of the Oligosaccharide Side Chains. Proteins from organelle andsoluble fractions obtained from cotyledons labeled with [3H]GlcN were diluted in 0.5 ml of 50 mm Na-acetate (pH 5.8),containing 5 mM ZnSO4 and incubated for 48 h at 37°C with orwithout 10 units of jack bean a-mannosidase under a tolueneatmosphere. The samples were then boiled for 2 min to inactivatethe a-mannosidase and digested with 1 mg of pronase for 48 hat 37C after adjusting the pH to 8.5 with 100 mM Tris-HCI. Thedigest was boiled for 2 min and chromatographed on a 1-mlcolumn of ConA-agarose to separate the high-Man chains fromthe modified chains. The latter do not bind to ConA. Theretained glycopeptides were eluted with 500 mm a-methyl-man-noside as described above, and both glycopeptide fractions weredesalted on a short (1.5 x 18 cm) Bio-Gel P4 (minus 400 mesh,BioRad, Oakland, CA) column equilibrated with 100 mm aceticacid. The glycopeptide fractions were lyophilized and analyzedby gel filtration on a long (1.0 x 100 cm) Bio-Gel PA columnas described (25). The positions of glycopeptides with oligosac-charide chains ofknown size are indicated on the elution profiles.Treatment of Glycopeptides with a-Mannosidase and Analysis

of the Hydrolysis Products. In some experiments the digestionsdescribed above were reversed in order. [3H]GlcNAc-labeledproteins were first digested with pronase, then the glycopeptideswere fractionated on ConA agarose, purified with a short Bio-Gel P4 column, and finally digested with a-mannosidase asdescribed above. The proteins from the soluble fraction of the

homogenate were precipitated with 1.5 volumes ofmethanol and0.06 volumes of glacial acetic acid (2 h at -20°C) and washedwith 90% aqueous acetone at 0°C. The proteins from the orga-nelle fraction were extracted with organic solvents to removeglycolipids, as described by Davies and Delmer (7).

RESULTS

Analysis of Glycoproteins by SDS-PAGE and Affmoblotting.The proteins present in the soluble and organelle fractions ofdeveloping Phaseolus vulgaris cotyledons were fractionated bySDS-PAGE and transferred onto nitrocellulose paper. The poly-peptides were stained with amidoblack, and all the glycoproteinsthat bind ConA were visualized by affinodetection (Fig. 1). Themost abundant proteins in the soluble and organelle fractionsare phaseolin and PHA, which are both ConA-binding proteins.Both are known to contain high-Man oligosaccharide chains (7,

z

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wo

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JJ :^i8#* Or_ so:-11" u _s

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s o s o s o soFIG. 1. Analysis of proteins and glycoproteins by SDS-PAGE and

affinoblotting with ConA-peroxidase. Proteins from the soluble (S) andorganelle (0) fractions of bean cotyledons were separated by SDS-PAGEand transferred to nitrocellulose paper. The paper was stained withAmido black, or the ConA-binding glycoproteins visualized by affino-blotting with ConA and peroxidase. Alternatively, the cotyledons werelabeled for 24 h with [3H]GIcN or [3H]Fuc, and a fluorograph preparedafter separation by SDS-PAGE. Three arrowheads, phaseolin; two arrow-heads, PHA.

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Plant Physiol. Vol. 81, 1986

25). (It should be emphasized that the contents of the proteinbodies becomes part of the soluble fraction during the homoge-nization).When cotyledons were labeled for 24 h with [3H]GlcN and the

labeled proteins visualized by fluorography after SDS-PAGE,phaseolin and PHA were again the most abundant proteins (Fig.1). The correspondence between the [3H]GlcNAc-labeled pro-teins and the ConA-binding proteins is obvious. When cotyle-dons were labeled for 24 h with [3H]Fuc and the labeled proteinsvisualized by fluorography after SDS-PAGE, PHA was the mostabundantly labeled protein (Fig. 1), but many other fucosylatedproteins were present in the organelle fraction. We used tunica-mycin to demonstrate that Fuc is part of N-linked rather than0-linked oligosaccharide chains. When applied to bean cotyle-dons as a 2 h pretreatment at 500 ,ug/ml, tunicamycin is only 50to 60% effective (3). Incorporation of [3H]GlcNAc and [3H]Fucinto glycoproteins (soluble and organelles) was inhibited 50% bya similar tunicamycin treatment (data not shown), indicatingthat most, if not all, of the incorporation is into N-linkedoligosaccharides.

Binding to ConA Is Mediated by High-Man OligosaccharideSide Chains. To gain information about the structure of theoligosaccharide side chains that mediate the binding ofConA tothese glycoproteins, proteins which had been separated by SDS-PAGE and transferred onto nitrocellulose paper were treatedwith the glycosidases a-mannosidase or endo H. The proteinswere then visualized using the affinodetection method with ConAand peroxidase (Fig. 2). In each treatment series, ovalbumin andtransferrin (lane 1 in each panel of Fig. 2) were included ascontrols. Ovalbumin has high-Man oligosaccharides that bindConA and are sensitive to both glycosidases. Transferrin has abiantennary modified oligosaccharide that binds ConA but isresistant to both enzymes (panels B and C, lane 1). The otherthree lanes in each panel represent different amounts of proteinsfrom a total seed extract. Varying the amount of proteins loadedwas necessary because phaseolin and PHA are much more abun-dant than other glycoproteins. Treatment of the blots with a-mannosidase or with endo H abolished the binding of ConA toall the proteins (lane 2, panels B and C). That PHA and phaseolinare still visible in lanes 3 and 4 of panels B and C is due to theheavy loading of these lanes necessary to visualize the less abun-dant glycoproteins, rather than to the presence of oligosaccha-rides that both bind ConA and resist the glycosidases. Panel Dof Figure 2 shows that the presence of 200 mm a-methyl-mannoside also prevents the binding of ConA to the glycopro-

A B

u~-44.i::4i

C

teins. Together, these results indicate that the oligosaccharideside chains which mediate the binding of ConA to these glyco-proteins are all of the high-Man type. In addition, the resultsobserved in lane 1 after a-mannosidase (panel C) or endo H(panel B) treatment of ovalbumin and transferrin are completelyconsistent with the known structures of the oligosaccharidechains of these glycoproteins. Consequently, the presence ofmodified side chains like that found in transferrin can be ruledout for the plant glycoproteins detected here.We also determined the affinity ofConA for [3H]GlcNAc- and

[3H]Fuc-labeled glycopeptides isolated from pronase digests ofglycoproteins. The purified glycopeptides were passed through asmall ConA-agarose column. For the [3H]GlcNAc-labeled gly-copeptides, 43% of the radioactivity was not retained by ConAwhile 57% bound tightly to the column and could be eluted with200 mm a-methyl-mannoside. In contrast, more than 95% ofthe [3H]Fuc-labeled glycopeptides failed to bind to the ConA-agarose. Thus, the fucosylated oligosaccharides ofthese glycopro-teins are modified in such a way as to preclude binding to ConA.High-Man Side Chains of Native Glycoproteins Are Accessi-

ble to ConA. The affinodetection data in Figures 1 and 2 indicatethere are numerous glycoproteins in the cotyledons having oli-gosaccharide side chains that can interact with ConA when theseproteins have been denatured with SDS. Are these oligosaccha-rides also accessible to ConA when the proteins are in their nativeconformations? To answer this question, proteins from the sol-uble and organelle fractions were incubated with increasingamounts of ConA-agarose, then the bound glycoproteins wereremoved by centrifugation. The unbound glycoproteins in thesupernatant were denatured in SDS buffer, electrophoresed onSDS gels, and finally visualized by ConA-peroxidase affinoblot-ting after transfer to nitrocellulose paper. Figure 3 indicates thatof all the glycoproteins capable of binding to ConA in theirdenatured states, there is only one organelle glycoprotein thatdoes not bind to ConA when in its native conformation. Withthis one exception aside, we conclude that the high-Man oligo-saccharides of the cotyledon glycoproteins are accessible toConA, whether these proteins are native or denatured.Many Glycoproteins Have Both High-Man and Modified 01-

igosaccharides. The polypeptide of PHA in bean cotyledonscontain both modified and high-Man N-linked oligosaccharides(25). To determine whether other glycoproteins in the cotyledonsexhibit this same feature, we labeled cotyledons with [3H]Fuc or[3H]glucosamine for 24 h, then isolated the glycoproteins fromboth soluble and organelle fractions under nondenaturing con-

D

2 3 4 11 2 3 411 2 3 41

FIG. 2. Characterization of oligosaccharide sidechains which bind ConA. One, 3, or 10 volumes(lanes 2, 3, and 4, respectively) of total proteinsfrom bean cotyledons, or a mixture of humantransferrin and ovalbumin (upper band and lowerband, respectively; lane 1) were separated by SDS-PAGE, transferred to nitrocellulose and fixed withacetic acid and isopropanol. The nitrocellulosesheet was washed in PBS buffer and cut into fourpieces; each piece was incubated for 48 h at 37°Cin a sealed plastic bag containing either buffer alone(panels A and D), endo H (panel B), or a-mannos-idase (panel C). After this incubation, ConA-bind-ing glycoproteins were visualized with ConA/per-oxidase using the affinoblotting method. Panel Dwas treated for ConA/peroxidase staining in thepresence of 200 mm ai-methyl-mannoside. Threearrowheads, phaseolin; two arrowheads, PHA.

208 FAYE ET AL.

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OLIGOSACCHARIDE SIDE CHAINS OF GLYCOPROTEINS

A I B

o 33 10 c o 1 3 10 C

FIG. 3. Accessibility of high-Man oligosaccharide side chains on na-

tive glycoproteins to ConA. Proteins from soluble (panel A) and organelle(panel B) fractions were incubated without (lanes 0) or with 100, 300, or

1000 Ml ofConA-agarose gel (lanes 1, 3, 10, respectively). In lane C, 1000Ml of ConA-agarose gel was used in the presence of 500 mm a-methyl-mannoside. Proteins from the unbound fraction of the ConA-agaroseincubation were separated by SDS-PAGE, then subjected to affinodetec-tion with ConA/peroxidase staining on blot. Three arrowheads, phaseo-lin; two arrowheads, PHA.

ditions. These proteins were incubated with increasing amountsof ConA-agarose as described for the affinoprecipitation proce-due. As seen in Table I, most of the [3HJGlcNAc radioactivityin the soluble (92%) and organelle (75%) glycoproteins is precip-itated by the ConA-agarose. The corresponding figures are 65and 40%, respectively, for the [3H]Fuc-labeled glycoproteins. Noradioactivity bound to the ConA-agarose when 200 mm a-

methyl-mannoside was included as a competitive inhibitor, in-dicating again the specificity of the ConA-glycoprotein interac-tion. Since the Fuc-containing glycopeptides do not bind ConA(see results above), we can conclude that 65 and 40% of the Fuclabel in soluble and organelle glycoproteins, respectively, is foundin proteins that contain both modified (fucosylated) and high-Man (ConA-binding) side chains. These glycoproteins are visu-alized in Figure 4, which is a fluorograph from [3H]Fuc-labeledglycoproteins retained (lane C) and unretained (lane B) on aConA-agarose column. The most abundantly labeled glycopro-tein in the soluble fraction is PHA.High-Man Side Chains of Glycoproteins Are Only Partially

Susceptible to a-Mannosidase. The first stage in the modificationof high-Man oligosaccharides to complex ones is the removal of4 a- 1,2-Man residues, converting (GlcNAc)2Manq toMan5(GlcNAc)2 (16). To determine the extent to which high-Man chains on the native glycoproteins are accessible to jack

Table I. Affinoprecipitation of[3HJGlcNAc and [3H]Fuc-LabeledGlycoproteins with ConA-Agarose

Aliquots from soluble and organelle fractions were incubated withincreasing amounts of ConA-agarose gel to ensure affinoprecipitation ofall glycoproteins with accessible high-Man chains. The radioactivityremaining in the unbound fraction is expressed here as a percentage ofthe total in the initial sample. Control: 1000 ,d of ConA-agarose gel wasincubated with the sample in the presence of 200 mM a-methyl-manno-side.

Percent of UnboundRadioactivity

Radioactive Protein Amount of ConAPrecursor Fraction gel (ofl) Co

gel(1ll)Control100 300 1000

Md[3H]Fuc Soluble 38 35 35 98

Organelles 73 63 60 99[3H]GlcN Soluble 13 10 8 98

Organelles 39 36 25 99

bean a-mannosidase, we incubated [3H]GlcNAc-labeled glyco-proteins from the soluble and organelle fractions in the absenceor presence of this glycosidase. The proteins were then exhaus-tively digested with pronase' and the resulting glycopeptides werefractionated on ConA-agarose columns into unretained (modi-fied) and retained (mostly high-Man) fractions. Treatment of thenative glycoproteins with jack bean a-mannosidase did notchange the distribution of total radioactivity between the ConA-retained and -unretained fractions (data not shown). This indi-cates that none of the high-Man chains that bound to ConA inthe controls was degraded by a-mannosidase to the extent thatthey failed to bind ConA.The high-Man chains from the controls (no a-mannosidase

treatment) eluted as Man7 8(GlcNAc)2 and Mang(GlcNAc)2 forthe soluble and organelle proteins, respectively (Fig. 5, panels Aand B). The glycoproteins incubated with a-mannosidase con-tained high-Man chains that eluted as Man7(GlcNAc)2, indicat-ing that only 1 or 2 Man residues were removed by this glycosi-dase.To demonstrate that the high-Man side chains are chemically

degradable by a-mannosidase (as opposed to physically accessi-ble), [3H]GlcNAc-labeled glycoproteins from soluble or organellefractions were first digested with pronase, then the high-Manglycopeptides obtained after purification on ConA-agarose wereincubated with a-mannosidase. Panels C and D (Fig. 5) showthat the digestion of the high-Man glycopeptides is virtuallycomplete, resulting in a peak corresponding to Man(GlcNAc)2Asn after the loss of 6 to 8 Man residues. This stands in contrastto the 1 or 2 Man units removable by a-mannosidase when thesesame oligosaccharides are attached to their native proteins. Anal-ogous results were obtained with endo H. High Man chains couldnot be removed from the glycoproteins by endo H treatmentunless the proteins had been denatured, indicating that theseoligosaccharides are not accessible to endo H when the proteinsare in their native conformation (data not shown).

I Under the conditions used, pronase digestion yields glycopeptidesthat lack all amino acids except the oligosaccharide-linked Asn residue.This is supported by the observation that when [3H]GlcN-labeled, high-mannose glycopeptides isolated from pronase digests of bean glycopro-teins are cleaved by endo H, the low molecular weight product cochro-matographs with authentic Asn-GlcNAc.

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Plant Physiol. Vol. 81, 1986

A B C0)

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FIG. 4. Fluorography of[3H]Fuc-labeled polypeptides fractionated onConA-agarose. A sample of total protein obtained from cotyledonslabeled for 24 h with [3H]Fuc was fractionated by ConA-agarose intobound and unbound proteins, which were then separated by SDS-PAGE.Lane A, total protein; lane B, proteins not bound to ConA-agarose; laneC, proteins eluted from ConA-agarose with 200 mM a-methyl-manno-side. Two arrowheads, PHA.

DISCUSSION

Plant glycoproteins contain both high-Man and modified oli-gosaccharide side chains. For example, soybean agglutinin has ahigh-Man side chain with the formula Man8(GlcNAc)2 (8), limabean lectin has a fucosylated side chain with the formulaMan3Fuc(GlcNAc)2 (19), and PHA has one high-Man and onemodified side chain on each polypeptide (25). It is known fromwork with animal cells (reviewed in Kornfeld and Kornfeld [16]) and plant cells (reviewed in Chrispeels [4]) that modified chainsderive from high-Man chains by the actions of Golgi-localizedprocessing enzymes.The data presented here show that the storage parenchyma

cells of developing bean cotyledons synthesize numerous glyco-proteins with both types of oligosaccharide chains (Table I andFig. 4). This raises the question as to why the high-Man chainsof the mature glycoproteins remain unmodified. In animal cellsthe first stage in oligosaccharide modification is the removal ofthe 4 a- 1,2-linked Man residues of Mang(GlcNAc)2 to yieldMan5(GlcNAc)2 (16). This Man trimming must precede all otherprocessing steps. At the moment there is no reason to suspectthat this is not also the first stage of oligosaccharide modificationin plant cells, since inhibitors ofspecific processing enzymes havesimilar effects on oligosaccharide structures in plant and animalcells (6). The results reported here indicate that the high-Manchains on native glycoproteins of bean cotyledons are onlypartially accessible to a-mannosidase in vitro, as this glycosidasedegrades them to Man7(GlcNAc)2 by removing 1 or 2 Manresidues (Fig. 5). The oligosaccharides can be extensively de-graded by this enzyme ifthe glycoproteins are first denatured (as

50 60 30 40Fraction Number

FIG. 5. Sensitivity of high-Man side chains to a-mannosidase. Theelution profiles show the distribution of [3H]GlcNAc in the high-Manglycopeptides ofsoluble (A and C) and organelle (B and D) glycoproteins.In panels A and B, the native glycoproteins were incubated with a-mannosidase and then digested with pronase. The glycopeptides withhigh-Man oligosaccharides were isolated with ConA-agarose and theirsize distribution determined on a long Bio-Gel P4 column. In panels Cand D, the glycoproteins were first digested with pronase and the glyco-peptides with high-Man oligosaccharides recovered with ConA-agarose.These glycopeptides were incubated with a-mannosidase and the prod-ucts sized on Bio-Gel P-4. (-), Control; (- -), a-mannosidase-treated. Glycopeptides of known structure were used as standards andare indicated as G2M9 = Man9(GlcNAc)h, G2M7 = Man7(GlcNAc)2 andG2M = Man(GlcNAc2.

on the nitrocellulose blots) or digested with pronase. We propose,therefore, that the high-Man chains present on mature glycopro-teins (those that have passed through the Golgi complex) haveremained as such because they are not sufficiently accessible tothe Golgi a-mannosidases, which trim accessible chains at leastto Man5(GlcNAc)2 before other sugar residues can be added.The work reported here extends the conclusion reached in

previous studies on animal (13, 26), yeast (23), and viral (15)glycoproteins, that protein conformation is a major determinantin oligosaccharide modification. In those experiments the oligo-saccharide side chains which remain unmodified in vivo are notaccessible to endo H in vitro unless the proteins are first dena-tured. We have used jack bean a-mannosidase as a probe toassess the accessibility of high-Man chains on native glycopro-teins to the Golgi processing enzymes. While neither endo H norjack bean a-mannosidase are Golgi processing enzymes-indeed,there is evidence that acid mannosidases and the Golgi a-man-nosidases in plants are quite different in their properties ( 1)-we feel that use ofjack bean a-mannosidase may provide a moregeneral and quantitative measure ofoligosaccharide accessibility,as compared to the all-or-none action of endo H. Not only doesa-mannosidase remove 1 or 2 terminal Man residues from high-Man oligosaccharides on native glycoproteins (Fig. 5), it alsocleaves terminal mannoses from the fucosylated side chains ofnative PHA (results not shown). This is consistent with thegeneral finding that fucosylated (modified) side chains, thoughstructurally resistant to endo H, are physically accessible tomodifying glycosidases in vitro and, presumably, in vivo.When nondenatured glycoproteins from bean were incubated

with jack bean a-mannosidase, the high-Man chains could notbe reduced to a size smaller than Man7(GlcNAc)2. While thereis as yet no firm evidence for plant cells that Man5(GlcNAc)2 isan obligatory intermediate for the addition of peripheral sugarresidues to the N-linked oligosaccharide chains, our work (KD

210 FAYE ET AL.

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OLIGOSACCHARIDE SIDE CHAINS OF GLYCOPROTEINS

Johnson, MJ Chrispeels, unpublished results) indicates thatMan7(GlcNAc)2 is a very poor in vitro acceptor ofGlcNAc fromUDP-GlcNAc, while Man5(GlcNAc)2 is an excellent acceptor.With the single exception noted, all the bean cotyledon gly-

coproteins that have high-Man chains bind to ConA, even whenthe proteins are not denatured. This indicates that the high-Mangroups are sufficiently exposed to allow an interaction withConA, just as they can interact to a limited extent with a-mannosidase. The exception, a protein of unknown identity,behaves like jack bean a-mannosidase. Although it has a high-Man side chain on its large subunit, jack bean a-mannosidasecannot bind to ConA when in its native conformation (2).

LITERATURE CITED

1. BONNER WM, RA LASKEY 1974 A film detection method for tritium-labelledproteins and nucleic acids in polyacrylamide gels. Eur J Biochem 46: 83-88

2. BOWLES DJ, MF CHAPLIN, SE MARCUS 1983 Interaction of concanavalin Awith native and denatured forms of jack-bean a-D-mannosidase. Eur JBiochem 130: 613-618

3. CHRISPEELS MJ 1983 Incorporation of fucose into the carbohydrate moiety ofphytohemagglutinin in developing cotyledons of Phaseolus vulgaris. Planta157: 454-461

4. CHRISPEELS MJ 1984 Biosynthesis, processing and transport of storage proteinsand lectins in cotyledons ofdeveloping legume seeds. Phil Trans R Soc LondBiol Sci 304: 309-322

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