1H NMR spectroscopy of carbohydrates from the G glycoprotein of vesicular stomatitis virus grown in...

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 230, No. 1, April, pp. 363-374, 1984

‘H NMR Spectroscopy of Carbohydrates from the G Glycoprotein of Vesicular Stomatitis Virus Grown in Parental and

Lec4 Chinese Hamster Ovary Cells

PAMELA STANLEY,’ GRACE VIVONA, AND PAUL H. ATKINSON*

Departnum~ of Cell Biology and. *Develqmental BidoQy and Cancer, Albert Einstein College of Medicine, Bmnx, New York 10661

Received September 2,1983

Carbohydrate moieties derived from the G glycoprotein of Vesicular Stomatitis Virus (VSV) grown in parental Chinese hamster ovary (CHO) cells and the glycosylation mutant Lec4 have been analyzed by high-field ‘H NMR spectroscopy. The major glycopeptides of CHO/VSV and Lec4/VSV were purified by their ability to bind to concanavalin A-Sepharose. The carbohydrates in this fraction are of the biantennary, complex type with heterogeneity in the presence of cu(2,3)-linked sialic acid and cy(1,6)- linked fucose residues. A minor CHO/VSV glycopeptide fraction, which does not bind to concanavalin A-Sepharose but which binds to pea lectin-agarose, was also investigated by ‘H NMR spectroscopy. These carbohydrates are complex moieties which appear to contain N-acetylglucosamine in /3(1,6) linkage. Their spectral properties are most similar to those of a triantennary complex oligosaccharide containing a 2,6-disubstituted mannose c~(1,6) residue. Carbohydrates of this type are not found among the glycopeptides of VSV grown in the Lec4 CHO glycosylation mutant.

The carbohydrates associated with the G glycoprotein of Vesicular Stomatitis Vi- rus (VSV)2 are determined by the amino acid sequence of G (1) and the glycosylation enzymes and cofactors provided by the host cell (2, 3). By studying G glycopeptides from VSV grown in a family of glycosylation mutants of Chinese hamster ovary (CHO) cells (see (4,5)), it has been possible to identify the different structural carbohydrate defects associated with many of the mutant types (6-10). The use of G sim- plifies the subset of carbohydrate structures to be compared. For example, chro- matographic and glycosidase analyses of

1 To whom correspondence should be addressed. ’ Abbreviations used: VW, vesicular stomatitis vi-

rus; CHO, Chinese hamster ovary; FCS, fetal calf serum; SDS, sodium dodecyl sulfate; Con-A, concanavalin-A, PSA, pea lectin; GlcNAc. N-acetylglucosamine; L-PHA, leukoagglutinin from P. vulgaris

radiolabeled G glycopeptides from VSV grown in the Lec4 glycosylation mutant revealed a lack of branched carbohydrate structures which bind to pea lectin (PSA)- agarose (7, 8). Such carbohydrates are found as a minor component amongst the glycopeptides of VSV grown in parental CHO cells. The lectin-binding behavior of these carbohydrates is characteristic of triantennary moieties which contain a 2,6- disubstituted mannose residue as well as fucose linked (~(1,6) to the N-acetylglucosamine attached to asparagine (11, 12). However, alternative structures might display the same lectin-binding properties. Therefore, it was critical to obtain direct carbohydrate structural information so that the Lec4 glycosylation lesion could be precisely defined. High-field ‘H NMR spectroscopy can be used to determine the complete sugar composition and the linkage relationships between sugars in an un-

363 0003-9861/84 33.00 Copyright Q 1994 by Academic Press, Inc. All rights of reproduction in my form reserved.

known glycopeptide because of the large number of carbohydrates for which spectra have already been assigned (13,14). In this paper, we present the ‘H NMR spectral properties of the major carbohydrates associated with the G glycoprotein from CHO/VSV and Lec4/VSV. In addition, the minor glycopeptide fraction from CHO/ VSV, which is not synthesized by the Lec4 glycosylation mutant, is partially char- acterized. A preliminary report of this work has appeared.3

MATERIALS AND METHODS

Preparation of pur$ed &-US. Pro-5 (parental) and Pro-5Lec4.12-2 (a glycosylation mutant from com- plementation group 4 (4, 5)) CHO cells were grown in suspension to approximately 8 X 105 cells/ml in alpha medium containing 10% horse serum and 2% fetal calf serum (FCS). Fifteen liters of cells were concentrated by centrifugation (2000 rpm for 15 min) and resuspended in 250 ml alpha medium containing VSV (Indiana strain) at 0.5 to 1.0 pfu per cell. After 1 hr of gentle stirring at 37°C. the cells were diluted to approximately 1.25 X lo6 cells/ml in alpha medium containing 2% heat-inactivated FCS. To allow for the increase in acidity of the culture due to cellular me- tabolism during incubation, the pH was adjusted at this time with NaOH to pH 8-9. Progeny virus was harvested after incubation of the infected cells for 16 h at 37°C. Cells were removed by centrifugation (3000 rpm for 30 min) and the supernatants were combined in a g-liter flask on ice. The culture fluid was concentrated to approximately 230 ml by Millipore cassette ultrafiltration using 5 ft’of membrane (pTHK 100,000). Viral titers were measured in the original culture fluid, the filtrate, and the concentrate. The yield of infectious virus varied between 1000 and 5000 pfu per cell, A small amount of infectious virus (-0.05%) was found in the filtrate.

was resuspended in about 2 ml of 5 mM Tris-HCl, pH 8.5, and stored at -100°C. The bottom band (a mixture of virus and membrane fragments) was harvested, concentrated by centrifugation through ET buffer, and resuspended in 6 ml ET buffer. After a brief sonication, the pellets were distributed between six, 38-ml K-tartrate gradients (15-33% (w/w) in ET buffer) and centrifuged at 25,000 rpm for 2 h at 4°C. The top (virus) bands were harvested into ET buffer and concentrated by centrifugation. The pellets were resuspended in about 2 ml of 5 mM Tris-HCl, pH 8.5, and stored at -100°C. Both virus bands were analyzed by SDS-polyacrylamide gel electrophoresis to ensure that the major protein components were VSV structural proteins (see Fig. 1). Protein was determined by the method of Markwell et al (15). Yields of purified virus typically varied from 5 to 20 mg protein per 10”’ infected cells.

Gel electrophoresis. Slab gels of 1 mm thickness containing 10% acrylamide and 0.1% SDS were run using a discontinuous buffer system. Samples were dissolved in 0.0625 M Tris-HCl, pH 6.8, containing 5%

The concentrate obtained by ultrafiltration was centrifuged at 25,000 rpm for 1 h at 4°C. Pellets were resuspended in a total of 6 ml ET buffer (0.1 M Tris- HCl, 0.01 M EDTA, pH 8.0) sonicated briefly, and loaded onto six, 38ml linear gradients of K-tartrate (15-3396 (w/w) in ET buffer). After centrifugation at 4°C overnight at 22,500 rpm, two major bands were formed. The top band (highly purified virus) was harvested, diluted into ET buffer, and concentrated by centrifugation for 1 h at 25,000 rpm. The virus pellet

25.7

FIG. 1. SDS-Gel electrophoresis of pooled CHO/ VSV and Lec4/VSV preparations. Approximately 10 pg protein was loaded per well and the gel was stained with silver stain. (A) CHO/VSV; (B) Lec41VSV. Mo- lecular weights were determined from standards run

a P. Stanley and P. H. Atkinson, (1982) in Proceed- ings, 12th International Congress of Biochemistry, pp. 343. in the same gel.

364 STANLEY, VIVONA, AND ATKINSON

A B

N, NS

M

‘H NMR SPECTROSCOPY OF VESICULAR STOMATITIS VIRUS CARBOHYDRATES 365

SDS, 2% 2-mercaptoethanol, and 10% glycerol, and boiled for 2 min. After the addition of 4 ~1 bromphenol blue, 50 rl was loaded per well and the gel was run at 15 mA for approximately 7 h. Gels were fixed and stained with Coomassie blue (100 pg protein per well) or with silver stain (2-10 pg protein per well) by the following modification of the method of Dubray and Bezard (16): the incubation with 0.2% periodic acid was for 5 min at room temperature and, after removal of the gel from ammoniacal silver, it was given three, lo-min washes. A mixture of protein standards was included in each gel: a-chymotrypsinogen (df, 25,700), ovalbumin (A& 43,000), bovine serum albumin (M, SS,OOO), and phosphorylase b (A& 92,500).

l&par&an of’ Pramwe glyeqpeptides Purified virus in 5 mM Tris-HCl, pH 8.5, was pooled and Pronase and CaClr were added to 1 mg/ml and 3 mM final concentrations, respectively. The mixture was incu- bated under toluene at 37°C for 24 h. Pronase and CaCl, were subsequently added at 24-h intervals for 5 days. On the sixth day, the samples were centrifuged at 25,000 rpm for 2 h to remove insoluble material. The supernatants were boiled for 5 min and their neutral sugar contents were determined by the phenol-sulfuric acid assay (17). Glycopeptides were purified from this material for ‘H NMR spectroscopy.

Pectin a&& chromatography. Pronase glycopeptides (30 to 50 ml) in Con A buffer (0.1 M sodium acetate, 0.01 M MgCl,, 0.01 M CaCl,, 0.01 M MnC12, 0.02% sodium aside, pH 7.3) were mixed with 1 X lo6 cpm ‘%I and loaded directly onto an affinity column (1.5 X 25 cm) of Con A-Sepharose equilibrated in Con A buffer. Unretarded material (Con A-T glycopeptides) was pooled on the basis of the known column parameters (determined using radiolabeled CHO/VSV and Lec4/VSV F’ronase glycopeptides and mI; see Fig. 2). After elution of the Con A-T glycopeptides, the column was washed with 165 ml Con A buffer before the addition of 50 mM cY-methylmannoside in Con A buffer. Bound glycopeptides which eluted with sugar (Con A-B glycopeptides) were pooled according to the known column parameters (see Fig. 2). The Con A-B glycopeptides were desalted twice on Bio-Gel-P2 (1.5 X 44 cm) and neutral sugars were determined by the phenol-sulfuric acid assay (17).

The Con A-T glycopeptides were concentrated five- fold by rotoevaporation, desalted on Bio-Gel-P2, concentrated by rotoevaporation, and resuspended in 3 ml Con A buffer containing 1 X lo5 cpm ia?. This mixture was loaded on an affinity column of PSA- agarose (0.9 X 29.5 cm) equilibrated in Con A buffer. The column was eluted with 40 ml starting buffer followed by 40 ml of 50 rnrd cu-methylmannoside in the same buffer. Unretarded material was pooled according to the elution of radiolabeled glycopeptides (Fig. 2) and ‘%I. Glycopeptides bound to the column (PSA-B glycopeptides) were pooled according to the known elution position of bound radiolabeled glyco-

3 9-

PSA

J -“i

-4

20 40 60 FRACTION NUMBER

FIG. 2. Lectin-affinity chromatography of radiolabeled glycopeptides. Pronase glycopeptides from CHO/VSV and Lec4/VSV were prepared from virus grown in the presence of PHI- or [“CJglucosamine, respectively (see (8)). The glycopeptides were mixed, diluted to 32 ml in Con A buffer, and chromatographed under the conditions to be used for preparing unla- beled glycopeptides for ‘H NMR spectroscopy. Only the profile obtained with mHO/VSV glycopeptides is shown for simplicity. The [‘*C&ec4/VSV glycopeptides eluted in an identical fashion. Also shown is the elution profile of ‘%I on the Con A-Sepharose column. This was used as a marker for unretarded material on both affinity columns. The Con A-T fraction was concentrated, desalted, and fractionated on PSA- agarose as described under Materials and Methods. The Con-A column was washed with 165 ml Con A buffer beginning at fraction 50 (//). Bound glyeopep- tides were eluted from both columns with buffer eon- taining 50 mM cu-methylmannoside which was applied at the fractions indicated by the arrows. Fraction size was 2.0 ml.

peptides (Fig. 2) and the first appearance of a-methylmannoside. The PSA-B glycopeptides were subsequently desalted twice on Bio-Gel-P2 (1.5 X 44 cm).

‘H NMR spec&o.s~. Samples in distilled, deionized water were passed through a column of Chelex (0.9 X 10 cm) equilibrated with water to pH 8.0, lyophilized, and resuspended in 99.8% deuterium oxide (DrO). Af-


ter 6-16 h at room temperature, each sample was lyophilized, and subsequently exchanged once more in 99.8% DrO. A third exchange was performed with 99.996% DzO. Samples were subsequently dried over P20c in Z)(ICCUO, resuspended in 100-500 ~199.996% DzO in a dry box, and transferred with approximately an equimolar amount of anhydrous acetone to appro- priate NMR tubes (microcells or 5-mm tubes).

High-field ‘H NMR spectroscopy was performed at ambient temperature and at 7080°C using a 360- MHz spectrometer (Mid-Atlantic Regional NMR Fa- cility) or a 500-MHz spectrometer (Northeast Regional NSF-NMR Facility). Acetone was used as an internal marker, which gives a chemical shift at 2.225 ppm compared to 4,4-dimethyl-4-silapentane sulfonate. Spectra were recorded using 16K data blocks, a sweep- width of 3000-5000 Hz, pulses of 90”, and a cycle delay time of 3.1 s (approximately five times T1 for anomeric protons in carbohydrate moieties containing four or more sugars; (18)). Assignments were made based on published spectra, chemical shifts (a), and coupling constant (&) information for Cl-H, C2-H, and certain other protons previously assigned from spectra of biantennary, complex glycopeptides and oligosac- charides which vary with respect to the number and linkage [(u(2,3) or cy(2,6)] of terminal sialic acid residues and fucose residues linked a&6) or a&3) to N-acetylglucosamine (13, 14, 19-21). In addition, direct spectral comparisons of the VSV glycopeptides were made with carbohydrates of known structure available in this laboratory. The latter included the triantennary asparagine (Asn)-linked oligosaccharide from fetuin [a gift from A. Adamany, Albert Einstein Col- lege of Medicine (see (14))], the biantennary Asn- linked complex glycopeptides from Sindbis virus (19), and the biantennary complex glycopeptide from yl IgG (Tern) termed GS (a gift from J. Carver, Uni- versity of Toronto; see (14)).

M&e&&. Pronase (Grade B, obtained from Cal- biochem Corporation, La Jolla, Calif.) was dissolved in 50 mM Tris-HCl, pH 8.5, at 10 mg/ml and heated at 50” for 1 h before use. Con A-Sepharose was from Pharmacia Fine Chemicals, Uppsala, Sweden, and PSA-agarose was from Vector Laboratories, Burlin- game, California. lzsI (100 mCi/ml) was purchased from the Amersham Radiochemical Corporation, Ar- lington Heights, Illinois. Bio-Gel-P2 (-400 mesh) and Chelex-100 (200-400 mesh, Na+ form) were obtained from Bio-Rad Laboratories, Richmond, California. Deuterium oxide 99.8% and 99.996% in 0.5-ml sealed anhydrous ampoules were obtained from Stohler Iso- tope Chemicals. Tubes for ‘H NMR spectroscopy (microcells, 508-cp; or 5-mm tubes, 528 pp) were obtained from Wilmad Chemical Company, sodium dodecyl sulfate (sequanal grade) was from Pierce Chemical Company, and protein standards for gel electrophoresis were from Bethesda Research Laboratories. All other chemicals were reagent grade. Media and sera

were from Gibco Laboratories, Grand Island, New York.

RESULTS AND DISCUSSION

PuriJicatim of Viral Glycopeptides

SDS-gel electrophoresis of the pooled samples used to generate Pronase glycopeptides showed that the major proteins corresponded to the VSV structural proteins L, G, N, NS, and M (Fig. 1). Minor bands were equally represented in both CHO/VSV and Lec4/VSV preparations. G is the only viral protein which is glyco- sylated (see (6)).

Pronase glycopeptides derived from CHO/VSV and Lec4/VSV were fractionated by lectin-affinity chromatography (Scheme 1). Glycopeptides which bound to Con A-Sepharose (Con A-B glycopeptides) or which passed through Con A-Sepharose and bound to PSA-agarose (Con A-T/ PSA-B glycopeptides) were pooled as shown in Fig. 2. The Con A-B glycopeptides were the major carbohydrate fraction from both CHO/VSV and Lec4/VSV. By com- parison, the Con A-T/PSA-B glycopeptides of CHO/VSV represented only about 5% of the total carbohydrates (Scheme 1).

The Cm A-B Glycopeptides of CHO/VSV and L.ec.J/VSV

After several exchanges in D20, the Con A-B glycopeptides of CHO/VSV and Lec4/ VSV were subjected to high-field ‘H NMR spectroscopy. Spectra were obtained at 23 and 70°C to facilitate the assignment of resonances in both the anomeric and C2- H regions of the spectrum. Increased temperature causes a shift in the HDO peak (a major component of the spectrum) to higher field and results in several other characteristic shifts for protons in the anomeric region (19).

The complete spectrum of the Con A-B glycopeptides of CHO/VSV obtained at 23°C is typical of a biantennary, complex carbohydrate structure (Fig. 3). Expanded anomeric and N-acetyl regions of spectra obtained at 70 and 23°C are consistent with this interpretation (Figs. 4 and 5). These expansions also show that the Con A-B


Purified CHO/VSV (~lOOmg)+ or Lec4/VSV (WJng)t

+Pronase (lmg/ml X 5; 5 days at 37')

25,000 rpm/2 hr.

r I Glycopeptides Insoluble

*(Q-3 mg) Material

CON A-sepharose

rL1 CON A-T pikJ - 'H-NMR

J-l

*(l-2 mg; CHO/VSV and Desalt PSA-agarose -1

PSA-T Desalt ) 'H-NMR *(O-l-0.2 mg; cHO/VSV only)

CON A-T/PSA-B glycopeptides

SCHEME 1. Glycopeptide purification. Pronase glycopeptides were purified by lectin-affinity chromatography as described under Materials and Methods and Fig. 2. t, milligrams of protein; *, milligrams of carbohydrate; LuMM, a-methylmannoside.

glycopeptides from Lec4/VSV are struc- (u(2,6) linkage which would result in char- turally very similar to those from CHO/ acteristic C3-H resonances at room tem- VSV. The chemical shifts (6) for charac- perature of 6 = 1.716 and 6 = 2.67 ppm (13). teristic protons from both glycopeptide Fucose is attached to approximately 15- preparations are given in Table I. 30% of the molecules and is linked a(1,6)

The ‘H NMR spectral properties of the to the GlcNAc residue attached to aspar- Con A-B glycopeptides from CHO/VSV and agine. A chemical shift for the anomeric Lec4/VSV show that they are biantennary, proton of the core GlcNAc residue in bian- complex carbohydrates which exhibit het- tennary compounds in which the Asn- erogeneity with respect to the presence of GlcNAc residue is not fucosylated, has not sialic acid and fucose residues. Sialic acid, previously been reported at 70°C. This res- when present, is linked a(2,3) to galactose. onance exhibits a shift to lower field with There is no evidence for sialic acid in the increased temperature, in contrast to the

1, ’ c 0 ” t “I * 0 ” 0 I,, 1, I, b c 5.6 6.4 5.2.&O 4.S 4.6 4.4 4.2 4.0 36 3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8

ChemicalShift,ppm

FIG. 3. Complete spectrum of the Con A-B glycopeptides from CHONSV. This spectrum was obtained at ambient temperature at 500 MHz and was accumulated from 434 scans.


A

4’

5.4 5.2 5.0 4.8 4.6 4.4 4.2

Chemical Shift, ppm

FIG. 4. Expansion of the anomeric regions of spectra from Con A-B glycopeptides of Lec4/VSV and CHO/ WV. These spectra were obtained at 70°C at 500 MHz. (A) Lec4/VSV (404 scans); (B) CHO/VSV (1’7 scans).

lack of a temperature effect on the Cl-H of the same GlcNAc residue in molecules in which the Asn-GlcNAc carries fucose in a(1,6) linkage ((19) and Table I). There is no spectral evidence for fucose linked a(1,3) to a B(l,Z)GlcNAc residue which, if present, would result in characteristic resonances at room temperature of 6 = 4.832 ppm from the C5-H and 6 = 1.176 ppm from the CH3 group (20-22).

The structure deduced for the fucosylated Con A-B glycopeptides (Table I) is identical to that determined by ‘H NMR for the S1, Se, and S3 glycopeptides of Sind- bis virus, and, for this reason, the num- bering of sugar residues conforms to the convention used by Hakimi et aL (19). The discrepancies between the data in Table I and that previously presented are due to errors in reporting (J. Carver, personal communication). These include the resonance at 6 = 4.128 ppm which was the chemical shift assigned by Hakimi et al to the C2-H proton of the Man a(1,6) residue at 23°C (19) and the chemical shifts

assigned at 70°C to the C2-H protons of the Man a(1,3) and Man ~~(1,s) residues by Hakimi et al. (19). The assignments presented in Table I were confirmed by homonuclear decoupling experiments which determined the relationships between the Cl-H and C2-H resonances for both the a(1,3)- and cY(l,G)-linked mannose residues.

Structure of the Cm A-T/PSA-B Gl~copeptides from CHO/ VSV The CHO/VSV glycopeptides which pass

through Con A-Sepharose but which sub-

, 1

I I I I I I I I I I I I I

7 2.09 2.01 1.93 1.85 1.77 1.69 Chemical Shift, ppm

FIG. 5. Expansion of N-acetyl regions of spectra from Con A-B glycopeptides of Lec4/VSV and CHO/ VSV. These spectra were obtained at 500 MHz at ambient temperature. (A) Lec4/VSV (1971 scans); (B) CHO/VSV (434 scans).


TABLE I.

CHEMICAL SHIFTS (6) FOR THE CON A-B GLYCOPEPTIDES OF CHO/VSV AND Lec4/VSV

8 6 5 4 F II c&,6)

7

!

Galfl(1,4)-GlcNAcj3(1,2)-Mana(l,G) I* +SAo(2,3) Z.z== Mar$(1,4)-GlcNAc6(1,4)-Gl c NAc&l-Asn

7’ Gal@(1,4)-GlcNAc/3(1,2)-Man(al,S) 3 2 1 6’ 5’ 4’

Sugar Residue No.

CHO/VSV chemical shift Lec4/VSV chemical shift (mm) (ppm)

Ambient 70°C Ambient 70°C

Cl protons GlcNAcj3,l GlcNAcfl(1,4) GlcNAcfl(1,4) ManS(L4 Mancu(l,G) Man&l) GlcNAcj3(1,2) GlcNAc6(1,2) G&31,4) GaltVA ham

C2 protons ManS(U) Mancu(l,G) MancY(1.3) Unassigned

Sialic acid C3 protons Equatorial Axial

Fucose protons Fuccu(l,G)

N-Acetyl protons GlcNAc& 1 GlcNA@(1,4) GlcNA@(l,l) GlcNAca(l,2) SAo1(2,3)

1 5.064 5.054 5.073 5.068 2 4.617 4.629 4.618 4.630 2 (+Fuc) 4.682 4.686 4.688 4.689 3 Obsc. 4.761 Obsc. 4.759 4 [+SAa(2,3)] 4.927 4.915 4.926 4.918 4’ [+SAu(2,3)] 5.118 5.135;5.130= 5.117 5.134 5 + 5’ 4.581 4.596 4.583 4.598 5 + 5’ [+SAo(2,3)] 4.572 4.596 4.569 4.598 6 + 6’ [+SAa(2,3)] 4.547 4.539 4.549 4.538 6 4.472 4.481 4.472 4.478 6 4.468 4.481 4.467 4.478

3 4.249 4.226 4.247 4.227 4 @A) 4.110;4.115” 4.109,4.115° 4.110;4.116’ 4.698;4.118” 4’ @A) 4.192 4.184 4.191 4.185 - (4.154)b Obsc. 4.155” Obsc.

7 + 7’ 7 + 7’

2.759 2.758 2.758 Obsc. 1.798 1.786 1.795 1.784

8 4.874 4.874 4.877 4.875 CHs 1.266 Obsc. 1.295 Obsc.

1 2.668 2.016 2.699 2.017 2 2.078 2.076 2.079 2.078 2 (+Fuc) 2.992 2.089 2.693 2.088 5 + 5’ [+SA(o12,3)] 2.647 2.047 2.947 2.049 7 + T 2.036 2.038 2.030 Obsc.

Note. Obsc., obscured by other resonances. ‘Two resonances resolved due to heterogeneity in substitution by sialic acid linked a(2,3); see (13) and

(19). * Minor contaminant. ‘Represents -0./3 sugar residues.

sequently bind to PSA-agarose (The Con The lectin-affinity properties of these gly- A-T/PSA-B glycopeptides; see Fig. 2) are copeptides are indicative of a branched of particular interest because they are carbohydrate moiety which contains a missing from radiolabeled Lec4/VSV (8). GlcNAc residue linked B(l,S) to the (w(1,6)-


linked core mannose residue (11, 12). The /3(1,6)-linked GlcNAc appears to be a re- quirement for the binding of this type of structure to PSA-agarose and also for binding to the leukoagglutinin (L-PHA) from Ph,aseolus tn.&ark (23,24). Since Lec4 CHO cells are highly resistant to L-PHA, exhibit very low levels of [‘251&-PHA binding (‘7), and do not synthesize Con A-T/ PSA-B glycopeptides (8), it seemed likely that the basis of their glycosylation defect might be the lack of a @(l,G)GlcNAc trans- ferase activity. Before attempting to assay for this enzyme (25), which is not readily detected by a conventional assay of CHO cell extracts (26), it seemed important to show that the glycopeptides missing from Lec4/VSV (i.e., the Con A-T/PSA-B glycopeptides of CHO/VSV) in fact contain a GlcNAc residue in the correct configuration.

The Con A-T/PSA-B fraction represents a minor proportion of the total glycopeptides from CHO/VSV (see (8) and Scheme 1). The same fraction was also prepared from Lec4/VSV by lectin affinity chromatography. ‘H NMR spectroscopy of the latter revealed no resonances typical of carbohydrate moieties even in the “enve- lope” region of the spectrum (6 = 4.0 to 3.4 ppm). This result was consistent with pre- vious experiments which showed that radiolabeled Lec4/VSV lacks the Con A-T/ PSA-B glycopeptide fraction (8). Structural information on the Con A-T/PSA-B carbohydrate moieties could therefore be derived solely from VSV grown in parental CHO cells.

Spectra of the Con A-T/PSA-B glycopeptides from CHO/VSV were obtained at ambient temperature and 70-80°C. Ex- panded portions of these spectra are compared with identical regions of spectra from the CHO/VSV Con A-B glycopeptides in Figs. 6 and 7. Although many resonances are shared, it is clear that the carbohydrates differ markedly in structure. Be- cause of the small amount of carbohydrate in the Con A-T/PSA-B fraction and a ten- dency for this preparation to form aggre- gates in DzO, the spectra were difficult to optimize. However, the combined data provide substantial evidence that all Man a(1,6) residue(s) in the Con A-TIPSA-B

carbohydrates are substituted with a B(l,G)-linked lactosamine sequence.

Several features of the spectrum are indicative of the presence of a B(l,6)-linked “branch” GlcNAc residue (Table II). First, the downfield shift at both temperatures of the Man a(1,3) anomeric proton compared with its position in Con A-B glycopeptides (Fig. 6) is characteristic of both tri- and tetraantennary carbohydrates containing a P(l,G)-linked GlcNAc residue (13, 27). It is also found in biantennary carbohydrates which contain cy(2,6)-linked sialic acid (13,14), but the Con A-T/PSA- B glycopeptides contain only a(2,3)-linked sialic acid residues. Additional chemical shifts which identify the P(l,G)GlcNAc were observed in both the anomeric and N-acetyl regions of the spectra (Table II). The resonance at 6 = 4.487 ppm (Fig. 7, Table II) has been assigned at room temperature to the Cl-H of a Gal which sub- stitutes a &1,6)GlcNAc at C4, and the resonance at 6 = 4.551 ppm has been assigned at room temperature to the Cl-H of the

l,~~l~~~l~~~l~,,l,~~l~,ll

5.4 5.2 5.0 4.0 4.6 4.4 4.2

Chemical Shift, ppm

FIG. 6. Expansion of the anomeric regions of spectra from Con A-B and Con A-T/PSA-B glycopeptides of CHO/VSV. (A) Spectrum of Con A-B glycopeptides obtained at 360 MHz at 70°C from 2190 scans. (B) Spectrum of Con A-T/PSA-B glycopeptides obtained at 500 MHz at 80°C from 3304 scans.


A

Gal~(1,4)[GlcNAc~(1.6)Mana(1.6)]

n

I I I I 1 I ~1 I I I I I 1 -- 4.65 4.60 4.55 4.50 4.45 4.40 4.35 4.30 4.25 4.20 4.15 4.10 4.05

Chemical Shift, ppm

FIG. 7. Expansion of portions of the spectra from Con A-B compared with Con A-T/PSA-B glycopeptides from CHO/VSV. These spectra were obtained at ambient temperature at 500 MHz. (A) Con A-T/PSA-B glycopeptides, accumulated from 3100 scans; (B) Con A-B glycopeptides, expansion of portion of spectrum shown in Fig. 3 (434 scans).

/3(1,6)GlcNAc itself (13,27). The resonance at 6 = 2.038 ppm is characteristic of the N-acetyl protons of a GlcNAc in B(l,S) linkage at 23°C (13, 27).

Additional evidence for the presence of a “branch” GlcNAc in /3(1,6) linkage derives from the complete shift of the Cl-H of the Man a(1,6) compared with the position it occupies in spectra of the Con A-B glycopeptides (Fig. 6). No new resonance was clearly resolved in the spectrum of the Con A-T/PSA-B glycopeptides at high temperature. However, in the 23°C spectrum a resonance at 6 = 4.870 ppm was observed, although this was partially obscured by the Cl-H of fueose 01(1,6) at 6 = 4.875 ppm. In both tri- and tetraantennary carbohydrates containing a 2,6-disubstituted Man 1x(1,6), the anomeric proton of the Man (u(1,6) occurs at 6 = 4.866 ppm at ambient temperature (13).

In addition to the presence of the GlcNAc @(l,S), the spectra in Figs. 6 and 7 show that the Con A-T/PSA-B glycopeptides contain GlcNAc /3(1,2) residues substituted with Gal @(1,4) residues, which may or may not be substituted with sialic acid linked

(~(2,3) (Table II). There is no evidence for the presence of cY(2,6)-linked sialic acid. Fucose is present in only one position attached ar(1,6) to the Asn-linked GlcNAc residue. In contrast to the Con A-B glycopeptides, which are only partially fucosylated, the Con A-T/PSA-B glycopeptides are fully substituted with a(1,6) fu- case (Figs. 6 and 7).

The ‘H NMR characteristics of the Con A-T/PSA-B glycopeptides in the anomeric and N-acetyl regions of the spectra agree with the 23°C chemical shifts reported for a /3(1,6)GlcNAc-containing, triantennary oligosaccharide obtained from human urine (27). Certain resonances in the C2- H region are also consistent with this interpretation (Fig. 7, Table II). The line at 6 = 4.253 ppm (Table II) corresponds to that of the C2-H of the Man @(1,4) residue (6 = 4.250 ppm; (27)) and the line at 6 = 4.199 ppm (Table II) is close to 6 = 4.200 ppm which was assigned to the C2-H of the Man a(1,3) in the urine oligosaccharide (27). The C2-H chemical shift for the Man (u(1,6) in the urine oligosaccharide was assigned to 6 = 4.098 ppm (27). Although a

STANLEY, VIVONA, AND ATKINSON

TABLE II

CHEMICAL SHIFTS (6) OF CON A-T/PSA-B GLYCOPEPTIDES OF CHO/VSV

Chemical shift (ppm)

Sugar Residue no. b Ambient 80°C

Cl protons GlcNAc&l GlcNAc/3(1,4) ManSW) Mancu(l,G) MancY(1,3) GlcNAcj3(1,2) GlcNAcS(1.6) GalSUP) GaW,4) Galj3(1,4)[GlcNAc~(l,s)l

C2 protons ManSW) Mana(l,G) Mancu(l,l) Unassigned

Sialic acid C3 protons Equatorial Axial

Fucose protons FuccY(l,G) C&

N-Acetyl protons GlcNAc&l GlcNAc@(1,4) GlcNAc@(l,B) SAcr(2,3) GlcNAc@(l,G)

1 2 (+Fuc) 3 4 4’ [-+SAa(2,3)] 5 + 5’ [fSAo(2,3)] -

6 + 6’ [+SAa(2,3)] 6 + 6’ -

3 4 4’ -

7 + T 7 + 7’

8 CHa

1 2 (+Fuc) 5 + 5’ [+SAo2,3] 7 + 7’ -

5.056 5.055 4.687 4.682 Obsc. 4.759 (4.870)’ Obsc. 5.132 5.148 4.589 4.663

(4.551)’ (4.599)’ 4.547 4.542 4.472 (4.483)’ 4.487 (4.491)O

4.253 Obsc. (4.1)” Obsc. 4.199 Obsc. 4.158 Obsc.

2.760 Obsc. 1.899 Obsc.

4.875 4.877 1.196 Obsc.

2.013 2.023 2.096 2.088 2.052 (2.944)” 2.032 Obsc. 2.038 2.039

Note. Obsc., obscured by other resonances. ’ Tentative assignments. bNo. of residue in structure at top of Table I.

chemical shift close to this value (6 = 4.1 ppm) was present in the spectrum of the Con A-T/PSA-B glycopeptides (Fig. 7), this region of the spectrum is complicated by protons from the C3 of galactose and C5 of fucose (13). In addition to this com- plexity, the spectrum of the Con A-T/ PSA-B glycopeptides contains a prominent resonance at 6 = 4.158 ppm (Fig. 7) which has not previously been reported in spectra of other tri- or tetraantennary carbohydrates containing GlcNAc in /3(1,6) linkage (13,27). Attempts to assign this resonance by homonuclear decoupling experiments

were not successful, although this approach may prove fruitful when larger amounts of material are available.

A number of oligomannosyl carbohydrates give rise to a resonance at 6 = 4.158 -t 0.003 ppm due to the C2-H of a 3,6-disubstituted Man (u(1,6) residue (13, 14). A second type of configuration which results in this line for the C2-H of the Man a(1,6) is the substitution of a B-linked GlcNAc at C4 of the Man 8(1,4) residue (14,28,29). However, the presence of this “bisecting” GlcNAc has profound effects on the Cl-H and C2-H chemical shifts of all three Man


residues as well as giving rise to characteristic resonances from the “bisecting” GlcNAc itself (at 6 = 3.26 ppm due to the C4-H, at 6 == 3.403 ppm due to the C5-H (30) and at 6 = 2.066 ppm due to the N-acetyl protons (29)). The combined features diagnostic for the presence of a “bisecting” GlcNAc or a 3,6-disubstituted Man ~~(1,s) were not evident in the Con A-T/ PSA-B glycopeptide spectra. The line at 6 = 4.158 ppm is the Con A-T/PSA-B gly-

copeptides may be due to the presence or influence of a residue (amino acid, sialic acid or fucose) which is not present in the only triantennary compound of similar structure available as a model (27). The latter is an oligosaccharide and therefore significantly different in composition from the corresponding glycopeptide.

Except for the line at 6 = 4.158 ppm, the spectral properties of the Con A-T/PSA-B glycopeptides may be summarized by the following structure

Gal u GlcNAc 4

Gal m GlcNAc u?$l ” 6 Fuc

+sAaG2 k B14 1 al.6 Man - GlcNAc m GlcNAc-Asn

Gal m GlcNAc u Man4 a'

CONCLUSIONS

The majority of the carbohydrate structures associated with G from CHO/VSV are complex, biantennary moieties which may or may not contain a(2,3)-linked sialic acid and a(l,6)-linked fucose residues. They do not contain sialic acid in (~(2,6) linkage nor fucose in a(1,3) linkage nor the “bisecting” GlcNAc. An essentially identical set of fucosylated structures is found associated wit,h the El and E2 glycoproteins of Sindbis virus grown in chick embryo fibroblasts (19). Because so many biantennary carbohydrate moieties have already been analyzed by ‘H-NMR spectroscopy, the structure deduced for the Con A-B glycopeptides of CHO/VSV may be considered unambiguous (Table I). The Con A-B glycopeptides of Lec4/VSV comprise a very similar mixture of biantennary carbohydrates (Table I).

The Con A-T/PSA-B glycopeptides of CHO/VSV possess a structure typical of Asn-linked complex carbohydrates (Table II). Several lines of evidence indicate that they contain GlcNAc in j3(1,6) linkage substituted with a Gal 8(1,4) residue and attached to the Man a(1,6) of the core region. These structural characteristics and the presence of fucose a(1,6) attached to the Asn-GlcNAc are consistent with the lectin- affinity properties of the Con A-T/PSA-B glycopeptides (11, 12). Although their spectral properties are most similar to those of a triantennary carbohydrate con-

taining a P(l,G)GlcNAc “branch” (27), a complete structure cannot be deduced for the Con A-T/PSA-B glycopeptides solely on the basis of the chemical shift information obtained because no compound with identical spectral properties in the C2-H region has so far been reported in the lit- erature.

ACKNOWLEDGMENTS

This work was supported by the American Cancer Society Grant BC-322-B to P.S. and Grant CA13402- 11 from the National Institutes of Health to P.H.A. Additional support was provided by the Core Cancer Grant (1POl CA 13330). P.S. is the recipient of an American Cancer Society Faculty Award. The use of the NSF Northeast Regional NMR facility at Yale University (Grant CHE-7916210) and the Mid-Atlan- tic NMR facility at the University of Pennsylvania are gratefully acknowledged. Thanks are extended to Johannes Vliegenthart, Jeremy Carver, and Christine Campbell for helpful discussions or for comments on the manuscript.

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