Structure of an unusual complex-type oligosaccharide isolated from Chinese hamster ovary cells

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 199, No. 2, February, pp. 393-399, 1980

Structure

of an Unusual Complex-Type Chinese Hamster EI,LEN

LI, RON GIBSON,

Rewired

Oligosaccharide Ovary Cells

AND STUART

Isolated

from

KORNFELD

July 24, 1979, revised October 4, 1979

An aspal,agine-linked oligosaccharide \vith an unusual stwctuw has been i4atetl fl,om Pronase digests of Chinese hamster ovary cell glycoproteins using gel filtration, ion exchange chromatography. and affinity chromatography on lectin-Sepharose columns;. The oligo~accharitle contained approximately 7.5% of the total cellular glycopeptide galactose and I.:<% of the mammae. The structure of the oligosaccharitle ~vaa determined by the combination of methglation analgsi:: and digestion 14ith e.cw ;md e,ldo-glq-cositla~es. The oligoxacch;u%le has predominantly a triantennary structure consisting of repeating [(;alpl + -1GlcS.4cfil + :3] units in the outer branches linketl to a tlimannosgl-tli-N,AT’-acet).lchitohiose cow. It resembles oligosaccharides present on human cargthrocyte glycoplvtrins and w~neal ker&an sulfate. I/ml Sepharose 4R) was kindly provitled by 31~. (ii1 Mintz of this institution. The oligo~;accharitle stantlxrd was Man(~1 --) S[Mancul --) G]MManjjl --) 4GlcNAc prepwet a~ described elsewhere (6). The compound was retlucecl with NaBH, to form Manal + :j[Manal -f GlMan~l + 4 ,~‘-acetylglucosaminitol. The wtlucetl oligosaccha~~itle KXS treutetl with jack bean wmannositlaw to gc’ner’ate Zjlan~l + 4,X’-;lCetylglUcosaminitol.

We have been intelwtetl in determining the laepertoire of oligosaecharitle structures present on the surface of Chinese hamster oval~g cells (1, 3). During the course of this \\.oyk \ve isolated an unusual aspwaginelinked complex-type oligosaccharitle which hat1 the typical trimannosyl core but \vhich contain4 outer branches \vith [Gal@ + 4GlcXAcpl -+ 31’ repeating units. Oligosaccha~~itles\vith similar structures have l~ecently been identified on the band :3glycopwtein of human erythwcytes (S.5). In this col?llnunicatioii I\-e describe the characterization of the oligxxxchwitle.

Isolrftio,c

ccird,~ccctioirntio,l

c~f’c~ll~tlfft~g1~~co;orptide.s.

The glycopeptides \+YW ixolatc~d from Chinese hamstw ovary cell:: kvo\vn in suspension culturr~ in the prewnw of r>-[Ii-“H]fucose and D-[1-l-‘CIglucosaminc, ;l~ de~wibetl previowly (1). The ,
0003-98Gl/XO/O20393-O’i$O2.00/0 C:opyright C 1980 hy Academic Press, Inc. X11rights of reproduction in any form reserved.

IA, GIBSON, AND KORNFELD

FIG. 1. Isolation of glycopeptitle Gal-la. Neutral glycopeptides (containing a total of 20 pmol of adlactose) which had passed through a DEAE-cellulose column were concentrated anal fractionated as described under Materials and Methotls. (.4) RCA I-Sepharosr column; (B) Bio-Gel P-6 column; (C) Con A-Sepharwe column. Aliquots were countecl to tleterminr ‘“C (0) and “H (0) ratlioactivit!;.

NaCl-10 mM Tris-HCl, pH i.5 at 4°C: (Fig. 1A). Aftrr waiting 1 h, the column \var washed \vith 160 ml of the equilibrating buffer ant1 Z-ml fractions were collectetl. These fractions contained the high mannose-type glycopeptides (1). The column \~a:: then elutetl with 0.1 M lactose in the same buffer to recover the neutral galactose-containing glycopeptitles. The lactose-elutetl glycopeptides were concentrateil and applietl to a column (1.5 x 90 cm) of Bio-Gel P-6 (200 to 400 mesh) equilibratetl with 0.1 M NH,HCO,,, pH 8.0 (Fig. IR). Fractions, 1 ml, were collectetl ant1 the 1arge.h-t glycopeptide material was poolecl as intlicatetl in the

figure. The pooletl fractions were Igophilizetl, tlissolvrtl in 1 ml of 10 mM Tri-HCl, pH 7.5, containing 0.1 M NaCl, 1 rnhl CaCI,. 1 IBM MnCl,, and 1 mM MgCl,. ant1 applietl to a column (2 X 30 cm) of C’on A-Sephdrose (Fig. lc’). After30 min at room temperature, the column was elutetl with 70 ml of buffer ant1 then with $0 ml of buffer containin,< 0.1 M cY-meth~lglu~ositl~~. Fractions. 4 ml. \r-ere collectetl. The glycopeptitles \vere poolwl ah shown in the figure ant1 tlesaltrtl hy ,gcl filtration through Hio-Gel P-6. Axrt/,~firctl wethods. The carbohytlrate composition I+ as dcterminetl by ,ca-liquitl chromatography as tleawibctl pw,.iously (11). Sialic a&l was tletermine(l after hytlrolysis in 1 N HCl for 90 5 at 100°C hg the thiobarhituric assa,v (12). Free xdlactose ant1 mannosr 8w.e tleterminetl enzymatic&v as tlescribetl previously (1:3). Free iV-acetylglucosamine \vas tlet~rminetl by the Reissig methotl (14). Methylation analysis \vas performed as described previously (1). The oligosaccharitle \vas retlucetl with Na WH, as tlexribetl previously (1). Descrntling paper chromatography on Whatman No. 1 \+as performetl wing ethyl acetate! pyriclinr/acetic acid/H,0 (5151113). E’t~z~~rrcc~ cli,qcstio/cs. The sample was tligestetl with 10 munit of E. j%/ci/rlii r?~do-B-galactositla~(, in 0.0’5 ml of O.Oj M Na-acetate, pH .5.X for 24 h. The sample LVWStligestctl \vith .j munit of D. ,DUP,OUOUI’W B-S-;icet,lglu~osaminitl;rse ant1 0.6 munit of P!!do~--S-acet~lgluco~aminitlaar I) in 0.03 ml of 0.0.5 11 cacotlylate buffer, pH 6.0 for 48 h. The samples i~err tligestctl with either 2 units for ,jack twan Ly-milnnw sitlaw. 30 munit of ,jack bean ij’-g~~lacto~itI;l.ie. or .X munit of jack bean P-S-a~rt~l,~lu~l)~arnirli(l~~~~~ in 0.02 ml of 0.03 M Na-acrtate. pH 4.a for 16 h (umanno~itla~e) or 4X h fp-galactwidaw and fi-,V~~~et~lglu~os~~n~i~~i~l~~~e~, The sample 11ah tligestrd \vith 7 munit of hen oviduct /3-mannosiclase in 0.04 ml of 0.05 M citrate/phosphate, pH 5, for 48 h. All incubations were performed at 37°C under a toluene atmosphere.

The neutlxl glycopeptitles which passed through DEAE-cellulose \vere separated into a high mannose glycopeptitle fraction \vhieh failed to atlsorb to RCA4 I-Sepharose and a flxtion that adsorbed and \vas then elutetl with 0.1 AI lactose (Fig. 1.4). The characterization of the high mannose glycopeptitles has been tlescribetl (1. 2). The neutral glycopeptitles \\-hich bound to RC.1 I - Sepharose were heterogenous in size as shown by gel filtration on Bio-Gel P-G (Fig. 1B). When fraction Gal-l, the large molecular weight fraction, was applied to a Con

.A-Sepha~wsti colunn~, most of it failetl to atlsol$ to the colunm although it snx~ll amount (lit1 hind and \cas elutetl with 0.1 M tu-iileth~l~lucositle (Fig. 1C). The matwial \vhich passetl through the column \v;w temwtl Gal-la while the mate4 \\-hich bountl \\.iw tctmetl Gal-lb. The cai+wh~c\rate compositions of glycopeldtle fractions Gal-la antI Gal-lb WC~ sho\vn in Tahlr I. Relative to the n~annose content, both ~lycopeptides we gwatl? enrichetl in galactose ant1 LV-acet~lg$ucosamine. The Gal-la flxction contamvl aI,proximately 7.5% of the total cellular glycopeptitle gxlactose ant1 1.:3% of the nxumos~ \\hile Gal-lb containecl 0.6% of the total ,&ctose. Insufficient quantities of Gal-lb mateid (O.:S ~inol galactose) piwlutletl further stutliw on this fraction.

(‘OMPOSITIOK

OF ~;I.YCOPEPTIDES

(;a-la

ASD

‘+ 3 I, A

t -

i‘, _ :\ ,

II’,

,:I 7 -B “. i

: c 15:i

.

. . . . . . . . . . . . . . . . . . . . . . . .. . .. .

*.. *. . . . . . . . . . . . . . . . . . . . . *

\ -....

l-c-

(;a-lb

” Mannohe arbitrarily wt equal to :!.O. Starting bvith 100 ml of packed Chinese hamstcar ovary cells (1). the total ~~o~c~ry of mannosr \vas 1.1 pmol in the &-la fraction and 0.12 Fmol in the Gal-lb fraction. Fucose, mannose, galactose. .V-acetglglucosamine and Nacet~lga1actosamiIle xvere determined by gps-Iiquitl chromatography ofthe trimethylsilyl derivatives of the methylglywaitles. Sialic acid u as tleterminetl by the thiobarbitwic acid assay.

of the lxtlioactivity as lo\\, molt~cula~*\veight oli~osa~ch~ll,i(l~s. Thcl 1~1eas:etl nxrtcl~ial contained ;I trisaccharitle ant1a clisacchaiitle in the ratio of 1:” (Fig. 2X). The tlis;acchal%lr was resistant to jack bean P-galactositlase but released free N-acet~lglucosamille upon tli,gwtion \vith ,jack bean ~-LV-xetyl~lucosaminitlasc (Fi:. 2I3). The trisxchwitle \z’ah lwistant to jack bran P-S-xetylglucosamine but \\‘as converted to a tlisacchalitle upon tligestion I\-ith jack bean &galactosidase (Fig. K). On the basis of thesc enzyme tligestions ant1the km\\-n specificit\ of the c,rdo-p-,~~lactosi(l~s~, the stmctuw

396

LI,

GIBSON,

AND

of the trisaccharide is proposedto be Gal01 --f 4GlcNAcPl -+ 3Gal and the structure of the disaccharide is proposed to be GlcNAcpl + 3Gal. The results of permethylation of Gal-la are consistent with this proposal. L4sshoivn in Table II, the methylation analysis revealed that Gal-la contains predominantly 3substituted galactose and 4-substituted N-acetylglucosamine although disubstitutetl N-acetylglucosamine ~vas not recoveretl from the ECNSS column. In addition there were two terminal galactose residues. Evidence fo18n (Man):I(GlcNAc)~

Core

The residual glycopeptide following E. freundii endo-P-galactosidase treatment was digested successively with jack bean /3-N-acetylglucosaminidase and then with D. pneumouiae /?-N-acetylglucosaminidase plus exdo-fi-N-acetylglucosaminidase D. The two digestions resulted in the release of 62% of the remaining ‘“C radioactivity as free N-acetylglucosamine (44% in the first TABLE RELATIVE

II

PROPORTIONS OF METHYLATED SUGARSIN Gal-la

Methylated

sugars

Galactose 2,3,4,f%TetraMe 2,4,6-T&Me 2,8,4-Tri-Me 2,4-Di-Me Mannose 8,4.6-Tri-Me 3,6-Di-Me 3,4-Di-Me 2,4-D&Me N-Acetylglucosamine 3,6-Di-Me

Molar

ratio”

1.6 10.1 0.1 co. 1

0.7 0.3 0.9 1.1 10.0

” The molar ratios of the alditol acetate derivatives ofthe partially methylated sugars were calculated from the area of the peaks on the gas chromatographic scan obtained on 3% ECNSS-M using a flame detector. The values are expressed relative to the total amount of mannose = 3.0. Under the conditions used the alditol acetate derivative of 2,3,5-tri-0-Me-fucose was obscured by nonsugar contaminants and mono-O-Me-Nacetylglucosamine was not recovered from the column.

KORNFELD

CM

FIG. 3. Paper chromatogram of the products of D. p~~3~no7~iae PN-acetSlglucosaminidase and endo8-Wacetylglucosaminidase D treatment. The residual glycopeptide following E. freundii w&do-/3-galactosidase treatment was digested with jack bean P-Nacetylglucosaminidase for 48 h and then passed over a Sephadex G-25 column. The glycopeptide material in the void volume was treated with D. p?uwt~o~&e P-‘~-acetylglucoadminitlase and e,zdo-P-hT-acetylglucoaaminidase D for 48 h and subjected to descending paper chromatography for 24 h. The chromatogram \vad cut in l-cm strips and counted for radioactivity. The standards are: 1, Manal + S(Mancul + @Man/31 + 4 GlcNAc; 2, GlcNAc.

digestion and 18% in the second). The remainder of the radioactivity incorporated as [‘“Clglucosamine was equally distributed between two larger species which were separated from each other and from the released N-acetylglucosamine by paper chromatography (Fig. 3). The fastest migrating species is free N-acetylglucosamine released by the D. pzeumoniae P-N-acetylglucosaminidase. The species migrating at 17cm was identified as Manal 4 3(Manal+ 6)Manpl + 4GlcNAc on the basis of the follo\ving evidence. The oligosaccharide cochromatographed with authentic Manal + :S(Mancul

4 @Mar@1

+ 4GlcNAc

(Fig.

3).

Methylation analysis revealed the presence of terminal mannose and 3,6disubstituted mannose as the only methylated mannose species. Reduction of the oligosaccharide with NaB”H, resulted in an “H-labeled oligosaccharide that cochromatographetl with authentic Manal + S[Mancul -, 61 Mar@1 + 4 N-acetylglucosaminitol. Digestion of the reduced oligosaccharide with jack bean a-mannosidase resulted in an oligosaccharide that cochromatographed with Mar@1 + 4 N-acetylglucosaminitol. Subsequent digestion with hen oviduct

STRUCTURE

OF UNUSUAL

COMPLEX-TYPE:

p-mannosidase converted 20% of the radioactivity to N-acetylglucosaminitol.’ The slowest migrating species at 10 cm was tentatively identified as Fuc + GlcNAcpeptide on the basis of the known substrate specificity of endo-~-N-acetylglucosaminidase D (16, 17) and that fact that it contained [“Hlfucose as well as [14C]glucosamine (Fig. 3). The methylated mannose species recovered from the intact glycopeptide indicated that most of the molecules have a triantennary structure with one of the outer mannoses monosubstituted at the 2 position and the other disubstituted at the 2 and 6 positions. In addition, some of the glycopeptide molecules have a tetraantennary structure with the outer mannoses disubstituted at the 2 and 6 positions and the 2 and 4 positions (Table II). This is consistent with the failure of the Gal-la molecules to bind to Con A-Sepharose (B-20). Digestion

of Gal-la

mith en-Glycosidases

Digestion of Gal-la with jack bean pN-acetylglucosaminidase failed to release any N-acetylglucosamine, a finding lvhich is consistent with the inability to detect any terminal N-acetylglucosamine by methylation analysis. In contrast, digestion with jack bean P-galactosiclase released 3.G residues of galactose and 0.4 residue of N-acetylglucosamine. The latter sugar ~vas released due to a slight contamination of the enzyme with P-N-acetylhesosaminidase activity. If a correction is made for the Rr-acetylglucosamine release, then 3.2 residues of galactose are at nonreducing termini, consistent with the presence of predominantly triantennary oligosaccharides with lesser amounts of tetraantennary molecules. The reason \vhy only two resitlues of terminal galactose were detected by methylation analysis is not known. On the basis of the methvlation and glycositlase studies, a structureUis proposed ” Hen ovitluct P-mannositlase reacts very slo\vl> \\ith A%mpl + 4GlcNAcitol (in contrast to Manpl -t GlcNAc), making it difficult to get the reaction to go to completion.

OIJGOSACCHARIDE

397

FIG. 4. P~vposetl structure of the major glycopeptitlr in fraction Gal-la. The fraction also contained molecules with four outer branches (tetlx antennary) rather than the three shown. The outcl, branches are arbitrarily set tu be equal in length. Thrs portion of the moleculr enclosed in the tlashetl has is identical to that present in many complex-type oligosaccharitles (21).

for fraction Gal-la, as shown in Fig. 4. In this structure the outer chains are arbitrarily set to be the same length although it is equally possible that the outer branches are not uniform in length. In addition, the methylation analysis suggests that as much as 30% of the molecules in fraction Gal-la have an additional outer branch, e.g., are tetraantennary rather than triantennary as shown. Finally, the precise number of repeating disaccharide units may be somewhat different than shown since even a 10% error in the glc or the methylation analysis will alter the galactose and Sacetylglucosamine residue number by a full residue.

The glycoproteins of Chinese hamster ovary cells contain a diverse population of asparagine-linked oligosaccharitles. The most abundant species consist of typical high manose and complex-type units, sim-

398

LI.

GIBSON,

AND

ilar to those founcl on many serum glycoproteins (21). The complex-type molecules contain two, three, or four outer branches consisting of sialic acid 4 galactose -f Nacetylglucosamine units.:% In addition, there are asparagine-linked oligosaccharicles \vith unusual structures. The data presented in this paper demonstrate that glycopeptide fraction Gal-la consists of oligosaccharitles that have repeating [GalPl + 4GlcNAc@ + 31 units linked to the typical trimannosyldi-N,N’-acetylchitobiose core present in asparagine-linked glycopepticles. These molecules, therefore, are variants of the usual complex-type structures, as shown in Fig. 4. Thus instead of having four or more tiers of [Gal/31 + 4GlcNAc@l + 31 units, the typical complex-type unit has a single tier capped by a sialic acid residue in most instances (21). It is also possible that the final tier of [Galpl ---, 4GlcNAcPl ---, 31 in the unusual oligosaccharide is capped by a sialic acid residue in some molecules as occurs in keratan sulfate (22). Such molecules would be expected to bind to DEL4Ecellulose and therefore would not be present in the fraction described here. The repeating disaccharide units have previously been observed in keratan sulfate (23), in glycopeptitles isolated from Pronase digests of human erythrocyte membranes (3, 4) and purified bancl 3 (5), and in gangliositles with blood group I and i activity (24). In fact, we would predict on the basis of studies on the structural requirement for I and i specificities by Watanabe et al. that Gal-la woulcl exhibit i activity (24). Gal-la differs from I active antigens (including the glycopeptitles of adult erythrocytes) in that it contains no disubstitutecl galactose residues. The resemblance of glycopeptide Gal-la to cornea1 keratan sulfate and the erythrocyte glycopeptides extends to the core regions of these molecules, since the latter species also contains cores composed of mannose and N-acetylglucosamine linked to asparagine residues (4, 22, 25). The biosynthesis of the Gal-la oligosaccharide presents an interesting question relative to the regulation of complex ESE. Li, unpublished

observations.

KORNFELD

oligosaccharicle biosynthesis. Thus after the first tier of [Galpl + 4GlcNAc] units has been added to the trimannosyl core, the molecule could either be converted into a typical complex-type unit by the addition of sialic acid residues or extended by the addition of the nest layer of ,V-acetylglucosamine residues. The factors which tletermine the relative activities of the appropriate sialyltransferase and N-acetylglucosaminyltransferase in this situation remain to be elucidated. ACKNOWI,EDGMENTS LVe \~~oultl especially like Roger Laine, and Jacques enzymes usetl in this study.

to thank Baenziger

Drs. for

Y. T. Li, providing

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