Oligosaccharide Composition of an Influenza Virus Hemagglutinin ...

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tected a number of host-dependent differences in the complex oligosaccharides. Both HA, and HA2 from. MDBK cells contained more highly branched (i.e. tri-.
Vol ,260,No. 27, Issue of November 25, pp. 14771-14774,1985 Printed in U.S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1985 by The American Society of BiologicalChemists, Inc.

Oligosaccharide Composition of an Influenza Virus Hemagglutinin with Host-determined Binding Properties* (Received for publication, April 26, 1985)

Carl M. Deom and IreneT. Schulzel From theDepartment of Microbwbgy, St. Louis University School of Medicine, St. Louis, Missouri 63104

We have previously reported that thebinding properties of the hemagglutinin (HA) of the WSN-F strain of influenza A are affected by the cells in which the virus is grown (Crecelius, D.M., Deom, C. M., and 164-177); at 37 “C Schulze, I. T. (1984)Virology 139, chick embryo fibroblast-grown F virus has a greater affinity for host cells than does the same virus grown in Madin-Darby bovine kidney (MDBK) cells. In an attempt to explain this host-determined property,we have characterized the carbohydrate onto put the viral HA by these twocells. Experiments usingtunicamycin indicate that the HA made by MDBK cells contains about 4000 daltons of carbohydrate in excess of that on the HA from chick embryo fibroblast.Serial lectin affinity chromatography of the asparagine-linked oligosaccharides on the HA subunits, HA1 and HA2, detected a number of host-dependent differences in the complex oligosaccharides. Both HA, and HA2 from MDBK cells contained more highly branched (i.e. triand tetraantennary)complex oligosaccharides than did the subunits from chick embryo fibroblasts. In addition, the HA subunits from the two sources differed in the amount of galactose-containing “bisected” complex oligosaccharides and in the presence of certain fucosylated triantennary oligosaccharides. Profiles of the asparagine-linked oligosaccharides from thehost cells did not show these differences, indicating that theHA subunit profiles were not necessarily representativeof the structuresfound on the cellularglycoproteins. The data support the conclusion that bulky oligosaccharides on the MDBK-HA subunits of WSN-F reduce the affinity of the virus for cellular receptors.

The hemagglutinin (HA’) is the major glycoprotein on the surface of the influenza viruses. It is responsible for the attachment and entry of the virus into host cells and is the

antigen to which neutralizing antibodiesare produced. Through its antigenic variability, the HA is responsible for the continued appearance in the human population of epidemic strains of influenza A virus. The biologically active HA is a trimerof identical glycoproteins each of which is cleaved into two subunits, designated HA1 and HA, (1,Z). Carbohydrate side chains are attachedto both subunits through asparagine linkages (N-linkages) (3) and areof the complex, high mannose, or hybrid types (4, 5). These oligosaccharides can vary in number andstructure depending on the virus strain and the host cell. Strain-specific variations in the number and type of carbohydrates result from differences in the primary structure of the HA protein (6,7). Host-specific variations in the size of the HA oligosaccharides (4, 6) are assumed to be due to differences in the ability of various cells to process the oligosaccharide side chains. However, the extent to which the host cell determines specific aspects of the oligosaccharide structures of the influenza HA and how these structures influence the biological activities of this glycoprotein are unknown. This study was undertaken to document host-determined differences in carbohydrate composition of the HA using a strain of virus known to have host cell and erythrocytebinding properties which are influenced by the cells in which it is grown (8).This virus, the F HA variant of the WSN strain of influenza A, grows well in CEF butpoorly in MDBK cells. In addition, binding studies have indicated that theF virus from CEF cells (FCEF) has a greater affinity for both MDBK and CEF cells than does the F virus from MDBK cells (FBK). These differences between F B K and FCEF have been shown to be phenotypic rather than genetic; growth of both F B and ~ F C E F in theopposite host cell for one passage changes the size of the HA and the binding properties of the viruses to those expected from the particular host in which the virus is last grown (8, 9). Thus, we are confident that we are examining host-determined differences in the glycosylation of the same HA polypeptide sequence. By using serial lectin affinity chromatography (lo), we have obtained information about the structure of the oligosaccharides put onto thetwo viral HAS by the two host cells and have identified host-dependent differences in oligosaccharide composition which could be responsible for the differences in binding properties. In addition, we present evidence that the N-linked oligosaccharides on the virus are not necessarily representative of those found on the host cell glycoproteins.

* This work was supported in part by Grants AI-10097 and AI14590 from the National Institute of Allergy and Infectious Diseases The costs of publication of this article were defrayed in part by the payment of page charges. This articlemust therefore be hereby marked “aclvertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 4 To whom correspondence should be addressed Department of Microbiology, St. Louis University School of Medicine, 1402 South Grand Boulevard, St. Louis, MO 63104. ‘The abbreviations used are: HA, hemagglutinin; CEF, primary EXPERIMENTAL PROCEDURES AND RESULTS~ chick embryo fibroblasts; MDBK, Madin-Darby bovine kidney cells; ConA-Sepharose, concanavalin A-Sepharose; E-PHA-agarose, eryDISCUSSION throagglutinating phytohemagglutinin-agarose; L-PHA-agarose, leuThe lectin affinity analysis presented here clearly demonkoagglutinating phytohemagglutinin-agarose; MEM, minimal essenof differences between the complex oligosactial media; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel strates a number electrophoresis; PBS, phosphate-buffered saline; NP40, Nonidet P40; TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone; PUK, Portions of this paper (including “Experimental Procedures,” proteolytic units Kaken. “Results,” Figs. 1-4, and Footnotes 3-6) are presented in miniprint

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Oligosaccharide CompositionInfluenza of anVirus HA

charide side chains put onto the HA subunits by two host cells. First, the MDBK-HA subunits carry tri- and tetraantennary oligosaccharides more frequently than do the CEFHA polypeptides. Second, galactose-containing “bisected” complex oligosaccharides are more abundant on CEF-HA subunits than on MDBK-HA subunits. Third, certain fucosylated triantennary oligosaccharides are found on the HA subunits from CEF but not from MDBK cells. Finally, glycopeptides from MDBK-HA subunits containa class of molecules which interact with leukoagglutinating phytohemagglutinin-agarose with high affinity. These are not found on CEF-HA subunits. Our data indicate that MDBK-HA contains about 4000 daltons of carbohydrate in excess of that on CEF-HA, approximately 3000 of which are in the HA1 subunit. This can be due in part to thelarge number of tri- and tetraantennary oligosaccharides on theMDBK-HA. However, this extracarbohydrate, about 20 monosaccharides distributed overfive complex oligosaccharides on each MDBK-HA monomer, suggests that additional sugar residues are attached to the outer galactoses of many of the complex carbohydrates synthesized by MDBK cells. The datapresented here do not permit us to make comparisons of the number of additional outer sugar residues on the HAS from the two cells, since the behavior of the complex oligosaccharides on this series of lectins does not depend on whether the outer galactoses are substituted.However, other lines of evidence indicate that MDBK-grown virions have relatively few unsubstituted galactose residues as compared to CEF-grown virions. Influenza virions do not contain sialic acid, but these residues can be added to terminal galactoses on the HAS by incubating the virions with sialyltransferase and CMP-sialic acid (18). When this was done, CEF-HA was sialylated at about 20 times the rateof MDBKHA, even though the lectin profiles presented here indicate that the HA subunits from the two sources contain comparable amounts of oligosaccharides with outer galactoses. The data suggest that terminal galactose residues are inabundance on CEF-HA whereas the outer galactoses on MDBK-HA are predominantly substituted with additional sugars. These additional sugar residues together with the highly branched oligosaccharides on the MDBK-HA would easily account for its large size. It is interesting in this regard that tri- and tetraantennary oligosaccharides from a mouse lymphoma cell line frequently have an outer sequence composed of the repeating disaccharide [Galfl1,4GlcNAc/31,3](19). When considering the ways in which these host-related differences in HA oligosaccharides could explain the binding properties of this virus, the three-dimensional structure of the HA (20) promptsthe following speculation. An MDBK-specified oligosaccharide at the distal end of the HA might be expected to restrict access to the receptor-binding pocket located in that area and thereby reduce the affinity of the virus for the cellular receptor. Alternatively, the size of the oligosaccharides on specific asparagine residues may be important indetermining boththe conformation and thestability of the HA. Since oligosaccharides cover approximately 20% of the surface of the HA trimer (20), carbohydrateprotein interactions within the subunits and atsubunit interfaces could be critical to the proper folding of the nascent at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 85M-1406, cite the authors, and include a check or money order for $2.80 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

polypeptide and assembly of the biologically active trimer. It is also possible that the affinity of the HA for cellular receptors could be modulated by specific outer sugar sequences or by modifications such as the sulfation of specific complex oligosaccharides (21). These possibilities, although not ruled out for the virus strain used here, seem unlikely. Neither attachment of sialic acid residues to theHA (18)nor removal of terminal sugars from the HA (22) destroys the infectivity of these particles. In addition, althoughthe amount of sulfate on the HA can be determined by the host, the hemagglutinating activity of the virus appears to be independent of the degree of sulfation (23). The mechanisms proposed here are similar to those suggested by Zhu and Laine (24) to explain their observations that placental fibronectin which contains high molecular weight polylactosamine carbohydrates has a lower binding affinity for gelatin than those forms which contain smaller N-linked complex carbohydrates. In addition, oligosaccharide size has been shown to affect the folding and conformation of the G protein of the San Juan strain of vesicular stomatitis virus (25). This study also shows that although the host clearly determines some of the characteristics of the HA oligosaccharides, the HA glycopeptide lectin profiles are not simple replicas of the cellular lectin profiles. For example, fucosylated triantennary structures are present on CEF-HA subunits and are virtually absent from the CEF glycoproteins. Whether these differences result from control of glycosylationby the primary structure of the HA protein or from selection of a subset of glycosylated HA molecules during assembly of the virions is not known. The work presented here indicates that interaction between the host cell glycosylating system and the HA polypeptide sequence can be important in determining host range. We are presently investigating a mutant of the FBKvirus which has increased host cell binding activity (8) in order to identify molecular changes which can broaden the host range of the influenza viruses. Acknowledgments-We wish to express our gratitude to Dr. Richard D. Cummings for advice and support throughout this work and for critically reading the manuscript. We thank Barbara Macon for her skilled technical assistance and Sharon McKenzie for skilled secretarial assistance. REFERENCES 1. Klenk, H. D., Rott, R., Orlich, M., and Blodorn, J. (1975) Virology 6 8 ,

426-439 2. Lazarowitz, S. G., and Choppin, P. W. (1975) Virology 68,440-454 3. Keil, W., Klenk, H.-D., and Schwarz, R. T.(1979) J. Virol. 31, 253-256 4. Schwarz, R. T., Schmidt, M. F. G., Anwer, U., and Klenk, H.-D. (1977) J. Virol. 23, 217-226 5. Nakamura, K., Bhown, A. S., and Compans, R. W. (1980) Virology 107, 20&221 6. Nakamura, K., and Compans, R. W. (1979) Virology 95,s-23 7. Schwarz, R. T., and Klenk,H.-D. (1981) Virology 113,584-593 8. Crecelius, D. M., Deom,C. M., and Schulze, I. T.(1984) Virology 139, 1 fL-177

9. Noronha-Blob, L., and Schulze, I. T. (1976) Virology 69, 314-322 10. Cummings, R. D., andKornfeld, S. (1982) J. Bid. Chem. 257,11235-11240 11. Basak, S., and Compans, R. W. (1983) Virology 128, 77-91 12. Laemmli, U. K. (1970) Nature 227,680-685 13. Schulze, I. T. (1970) Virology 42, 890-904 14. Cowan, E. P., Cummings, R. D., Schwartz, B. D., and Cullen, S. E. (1982) J. Biol. Chem. 257,11241-11248 15. Hiti, A. L., Davis, A. R., and Nayak, D. P. (1981) Virology 111,113-124 16. Nakamura, K., and Compans, R. W. (1978) Virology 86,432-442 17. Narasimhan, S. (1982) J. Biol. Chem. 257,10235-10242 18. Lakshmi, M. V., and Schulze, I. T. (1978) Virology 88,314-324 19. Cummings, R. D.,and Kornfeld, S. (1984) J. Biol. Chem. 259,6253-6260 20. Wilson, I. A., Skehel, J. J., Wiley, D. C. (1981) Nature 289,366-373 21. Compans, R. W., and Pinter, A. (1975) Virology 66,151-160 22. Collins, J. K., and Knight, C. A. (1978) J. Viol. 27,164-171 23. Downie, J. L. (1978) J. Gen. Virol. 41,283-293 24. Zhu, B. C. R., and Laine, R. A. (1985) J. Biol. Chem. 260,4041-4045 25. Gibson, R., Kornfeld, S., and Schlesmnger, S. (1981) J. Bzol. Chem. 256, 456-462

Oligosaccharide CompositionInfluenza of an

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themannose-labeledFvirus H b were chromatographed on Bio-RadBio-Gel P-6 columns t h e Complex glycopeptides fran MOBK-derived HA s u b u n i t s e r e larger than those frm C E F - d e h e d s u b u n i t s( d a t an o t shown). In o r d e r t o o b t a i n more i n f o r m a t i o na b o u tt h es t r u c t w eo f t h e s ec o m p l e xc a r b o h y d r a t e s , we haveused seriallectinaffinity chromatography t o charactevize the oligosaccharides put onto the HA s u b u n i t s by b o t h c e l l s .

SUPPLEMENTARY MATERIAL TO: OLIGOSACCHARIDE CIXPOSITION O AN F HMAGGLUTININ WITH HOSTDETERMINED

Virus HA

INFLUENZAVIRUS BINDING PROPERTIES

by Carl

M. Dean and I r e n e T. Schulze EXPERIMENTALPROCEDURES ~-

Materials. G-25Sephadex andConA-Sepharose were o b t a i n e d from P h a r m a c i aF i n e Chemicals: l e n t i l l e c t i n - a a a r o s e . E-PHA-aqarose andL-PHA-aoarosefrom E-Y Laboratories: pronase(70,000 PUKIgm) B n dt i n i c a m y c i nf r o mC a l b i o c h e m h e h r i n gG o r p . D-[2-3H]Mannos; (10-20Cilmmol)from Amersham C o r p . ;1 3 5 S l n e t h i o n i n e( 1 4 0 0C i l m m o l )i r o m New England Nuclear;TPCK-TrypsinfromWorthingtonBiochemicalCorp;and EN3Hance frm New England Nuclear. C e l l and V i r u sG r o w t h . MDBK c e l l s and CEF weregrownas previously described (8). R a d i o a c t i v e c e l l s were obtained f r a n c u l t u r e s grown t o confluency overaperiodof 2-3 days i n MEM c o n t a i n i n g 10% f e t a l c a l f serum and 50 u c i l n l o f C2-3Hlmannose. A CEF-gmwn stock o ft h e WSN-F s t r a i n( 8 )o fi n f l u e n z aA( H l N 1 )v i r u s was u s e d i n a l l e x p e r i m e n t s . R a d i o a c t i v ev i r u s e sw e r ep u r i f i e df r o mi n f e c t e dc e l lc u l t u r e sg r w ni nt h e presence o f 2 u C i l m l of C2-3Hlmannose as previouslydescribed(8).

S e r i a lL e c t i nA f f i n i t yC h r m a t o q r a p h y of HA Subunits and HostCellGlycopeptides. The The r o l e o f HA2 s u b u n i t o f AlWSN133 has one oligosaccharidechainpermolecule(15.16). t h ec e l li nd e t e r m i n i n gt h es t r u c t u r eo ft h eo l i g o s a c c h a r i d e sa tt h i ss i n g l ea t t a c h n e n t s i t e i s shown by t h e l e c t i n a f f i n i t y p r o f i l e s presented i n F i g u r e 2. A largefractionof t h e MDBK-HA2 g l y c o p e p t i d e s f a i l e d t o b i n d t o ConA-Sepharose and appeared i n pool I (Fig. 2a) whereas CEF-HA glycopeptides were fractionatedalmostevenlybetweenpools I and I I ( F i b .2 f ) . A m a l ?b u tr e p r o d u c i b l e peak r e p r e s e n t i n g 5% of t h e CEF-HA2 m t e r i a l ( F i g . 2f, pool 1') was s l i g h t l yr e t a r d e db u tn o tr e t a i n e d by ConA-Sepharase. Thispool wds n o tf o u n d o n MDBK-HA2 and was n o t f u r t h e r analyzed. E s s e n t i a l l y no highmannoselhybridglycopeptides w e r ef o u n d o n HA2 f r o me i t h e rc e l l .T h i s was c o n f i r m e db ys h o w i n gt h a t endo-8-N-acetvlulucosminidase H f a i l e d t o a l t e r t h e e l e c t r o p h o r e t i c m b i l i t v o f t h e i n t a c t s u b u n i t s i n SD$-iAGE (datanot shown). Thus, t h e ConA-Sepharbse p r o f i l e s s h o i t h a t t h e HA2 s u b u n i t s f v a n b o t h cells contain only canplex oligosaccharides but that MOBK-HA2 c o n t a i n s a s i g n i f i c a n t l yg r e a t e rp r o p o r t i o no f t r i - and t e t r a a n t e n n a r yg l y c o p e p t i d e st h a nd o e s CEF-HA2.

--

1 2 3 4

G l y c o s y l a t e da n dU n g l y c o s y l a t e d HA. I n f e c t e dc e l lm n o l a y e r s wre pulse-labeledat 6 h o u r sp o s t - i n f e c t i o nf o r 1 h o u ra t3 7 - Ci n HEM c o n t a i n i no n e - f o u r t ht h en o r m a l c o n c e n t r a t i o n ofmethionine, 2%d i a l y z e d f e t a l c a l f serum and [&]methionine a t 50 u~i/ml. F o l l o w i n g a chase p e r i o d of15minutes a t 37OC i n MEM c o n t a i n i n g 2% f e t a l c a l f serum, t h e c e l l s were washed 3 t i m e s i n i c e - c o l d PBS andlysed a t 4'C f o r 30 minutes i n 10 d Tris-HC1 (pH 7.0). 0.15 M N a C l , 0.5% T r i t o n X-100, a n d 1%NP40. N u c l e i were r e m o v e db y c e n t r i f u g a t i o n a t 12,000 g f o r 5 m i n u t e s and t h es u p e r n a t a n tf r a c t i o n wasmade0.1% SOS. When t u n i c a n y c i n ( 2 u g l m l ) was used t o i n h i b i t g l y c o s y l a t i o n . it was added a t 3 h o u r s p o s t - i n f e c t i o na n d was p r e s e n t h r o u g h o u t h ep u l s e - c h a s ep e r i o d T . he HA w a s i m m u n o p r e c i p i t a t e df r o mc e l l u l a rl y s a t e sa sp r e v i o u s l yd e s c r i b e d( 1 1 )u s i n ga n t i - H A monoclonalantibodiesobtained frm Or. WalterGerhard(WistarInstitute,Philadelphia. PA). SOS-PAGE (12) was used t o d e t e r m i n e t h e s i z e o f t h e g l y c o s y l a t e d and unglycosylated HAS.

rr_

P r e p a r a t i o na n dF r a c t i o n a t i o no f Glycopeptides.C3HIMannose-labeled. p u r i f i e dv i r u s t o be used f o r s e r i a l l e c t i n a f f i n i t y chromatography was subjected t o t r y p s i n t r e a t m e n t ( 5 0 37-C. T h i st r e a t m e n t removes neuraminidaseolvcooroteinr 1131 _ ~ ~ r ,.~, w i i c h h a v ee l e c t r o p h o r e t i cm o b i l i t i e s on SOS-PAGE sirnil;; t o HA1. HA; and HA2 s u b u n i t s w e r et h e ns e p a r a t e d O n 17.5% g e l sc o n t a i n i n g 4 Mu r e aG . e l sw e r ep r o c e s s e df o r f l u o r o g r a p hi n EN3Hance d r i e d andplaced i nc o n t a c tw i t h Kadak M R - 5 f i l m a t -7O'C. F l u o r o g r a p x sw e r eu s e d is t e n p l a t e s t o l o c a t e HA1 and HA2 p r o t e i n s . The a p p r o p r i a t e areas I1 mn x 2 mm). and incubated a t 60-C f o r 4 t o 5 ofthe 421s were e x c i s e d . c u t i n t o s e c t i o n s hours i ;l 3 t o 4 ml o f 0.1 M Tris-HC1 (pH B.'O) Containing 10 mM CaC12 and 10 mglml pronase. ( T h i s pronase s o l u t i o n had been preincubatedfor 15 m i n u t e sa t 37'C). The l i q u i d f r a c t i o n c o n t a i n i n gt h e C3H1 m a n n o s e - l a b e l e dg l y c o p e p t i d e s was removed, b o i l e d f o r 5 minutes, c e n t r i f u g e d t o remove i n s o l u b l e m a t e r i a l . andprepared f o r a n a l y s i s as p r e v i o u s l y d e s c r i b e d (14).

uqlrnll f o v2 0m i n u t e sa t

Cellularglycopeptides

~

e r e preparedasdescribed

~

~

~

- HA+ 1

by Cummings and Kornfeld (10).

B o t h t h e HA and t h e c e l l u l a r g l y c o p e p t i d e s were analyzed by t h e s e r i a l l e c t i n a f f i n i t y chromatographyprocedure of Cwmings and Kornfeld(10)asdescribedby Cowan g . (14). T h i so r o c e d u r ei d e n t i f i e st h em i n i m a lS t r u c t u r a lf e a t u r e sP e a u i r e df o rt h ei n t e r a c t i o no f g l y c o p e p t i d e sw i t ht h ev a r i o u sl e c t i n s .A d d i t i o n a ls u g a r s ,s u c ha st h es i a l i ca c i d residueswhicharepresent on t h e c e l l u l a r g l y c o p r o t e i n s b u t n o t on t h e HA g l y c o p r o t e i n s , do n o t a f f e c t t h e e l u t i o n o r o f i l e s . An e f f e c t on t h e l e c t i n o r a f i l e s o f m d i f i c a t i o n s s u c h as s u l f a t i o n o r p h o s p h o r y l i t i o n ofcanplexoligosaccharides h i s n o t been documented.

et

F i g u r e 1. SDS-PAGE a n a l y s i so fi m m u n o p r e c i p i t a t e dC 3 5 S l ~ t h i o n i n e - l a b e l e d HA fran untreated and t u n i c a m y c i n - t r e a t e dc e l l s .Fv i r u s HA f r o m l: a n e 1. CEF; l a n e MDBK 2, c e l l s ; lane 3, t u n i c m y c i n - t r e a t e d CEF; l a n e 4. t u n i c m y c i n - t r e a t e d MOBK c e l l s .

G l y c o p e p t i d e s were a p p l i e d t o ConA-Sepharosecolumnsand t h r e e f r a c t i o n s were r e c o v e r e d .T r i -a n dt e t r a a n t e n n a r yN - l i n k e do l i g o s a c c h a r i d e sa sw e l la s" b i s e c t e d I. b i a n t e n n a r yo l i g o s a c c h a r i d e sf a i lt ob i n dt o ConA-Sepharose and aredesignatedpool B i a n t e n n a r yN - l i n k e do l i g o s a c c h a r i d e sc o n s t i t u t ep o o l 11. These g l y c o p e p t i d e sb i n dt o ConA-Sepharoseandareelutedwitha-methyl-0-glucoside.High mannose and hybridN-linked o l i g o s a c c h a r i d e s are designatedpool 111. They b i n dt o ConA-Sepharose and are e l u t e d w i t h a-methyl-0-mannoside.Pool I g l y c o p e p t i d e sa r et h e na p p l l e dt o an E-PHA-agarose column whichretards"bisected"glycopeptidescontainingoutergalactoseresidues.Glycopeptides thatdonotinteractwith E-PHA-agaroseareapplied t o l e n t i l l e c t i n - a g a r o s e whichbinds c e r t a i nt r i a n t e n n a r yg l y c o p e p t i d e st h a tc o n t a i naC o r ef u c o s e a n da n" l i n k e d mannose s u b s t i t u t e dw i t hN - a c e t y l g l u c o s a m n ea t C-2 and C-6. These bwndglycopeptidesvhichare e l u t e dw i t ho - m e t h y l - P - m a n n o s i d e .a sw e l la st h o s eg l y c o p e p t i d e st h a tf a i lt ob i n dt o l e n t i ll e c t i n - a g a r o s e . are f u r t h e rf r a c t i o n a t e d an L-PHA-agarose. T h i sl e c t i ni n t e r a c t s w i t h tri- and t e t r a a n t e n n a r y g l y c o p e p t i d e s t h a t c o n t a i n o u t e r g a l a c t o s e r e s i d u e s a n d a n a-linked mnnose substituted with N-acetylglucosamine at C-2 and C-6. T h eb i a n t e n n a r yo l i g o s a c c h a r i d e si np o o l I I a r ef u r t h e ra n a l y z e d on l e n t i l lectin-agarose. Those glycopeptideswhich f a i lt ob i n dt ol e n t i ll e c t i n - a g a r o s el a c ka c o r ef u c o s er e s i d u ew h e r e a st h o s et h a tb i n dc o n t a i nac o r e fucoseresidue and a r e e l u t e d w i t h o-wthyl-D-mannos?de.

i

I

The l e c t i n a f f i n i t y p r o f i l e s o b t a i n e d f r o m d u p l i c a t e o r triplicatepreparationsof v i r u s or of c e l l u l a r g l y c o p r o t e i n s showed e s s e n t i a l l y t h e same d i s t r i b u t i o n o f l a b e l e d mannose r e s i d u e s .T h e s ep r o f i l e s were used t o assignminimalstructuralfeaturestothe oligosaccharides and t o c m p a r e t h e o l i g o s a c c h a r i d e s a t t a c h e d t o t h e HA s u b u n i t s b y t w o h o s tc e l l s .

H o s tC e l l Dependent G l y c o s y l a t i o n of theHemaqqlutinin. We have p r e v i o u s l y Shown t h a t t h e HA1 and HA2 s u b u n i t s as w e l l as uncleaved HA fran CEF-grown F v i r u s m i g r a t e f a s t e r in SOS-PAGE t h a n do t h e c o r r e s p n d i n g HA p r o t e i n sf r a nt h e MDBK-grwn v i r u s ( 9 ) . To determine i f t h i s r n o b i l i t v d i f f e r e n c e was due t o c a r b a h v d r a t e . t h e i m m m o w e c i o i t a t e d HA n r n t e i n s f r o m u n t r e a t e 2 and t u n i c a m y c i n - t r e a t e d C E ~ ~ a " d ~ ~ 0 B K ~ i n f e c t e ; l aiaiv& SDS-PAGE (Fig. 1). The major bands i n lanes 1 and 2 r e p r e s e n tt h eg l y c o s y l a t e du G l e a v e h HAS f r o m CEF (75.000 Mr) andfrom MOBK c e l l s (79,000 M,.) As seen i n lanes 3 and 4. t h e unglycosylated HA proteins synthesized by t h e t w c e l l s i n t h e presenceof t u n i c a n y c i n w e r e t h e same s i z e : t h e m o l e c u l a r u e i q h t o f t h i s D r o t e i n (63.000 M.I *as i n oood aoreenent w i t h thatpredicted-bythenucleicacidsequence (63,453) o i t h e ' i A gene id N - i t r a ; n i f l n f l u e n z av i r u s (15). T h u s ,ad i f f e r e n c ei nc a r b o h y d r a t ec o n t e n ta p p e a r e dt ob e responsibleforthehost-determineddifference i nt h es i z eo ft h eg l y c o s y l a t e d HAS. The m i n o r bandseen i nl a n e2i sn o ta l w a y sp r e s e n ti ni n f e c t e d MDBK cell e x t r a c t s and has neverbeenfound i nv i r i o n s . It p r e s u m a b l yr e D r e s e n t si n c o m P l e t e l vQ l v c o s v l a t e d HA moleculesthatarenotincorporatedintovirions.

Eiiis-iiF;

6;

6~

Thedata i n F i g u r e 1 w e r e c o n s i s t e n t w i t h e v i d e n c e frm o t h e rl a b o r a t o r i e si nw h i c h g e l f i l t r a t i o n was used t o show t h a t t h e s i z e o f t h e c o m p l e x g l y c o p e p t i d e s o n t h e HA p r o t e i n was h o s t dependent (4.16). As expected frm t h e s er e p o r t s . when pronasedigestsof

b N

P

F i g u r e 2. L e c t i na f f i n i t yp r o f i l e s ofI%]mannose-labeled MOBK-HA ( a e) and CEF-HA2 Procedures". ?he percentages i n (f-k) glycopeptides obtained asdescribed i n "Experimental parentheses indicate the distribution of t h e g l y c o p e p t i d e s a p p l i e d t o t h e p a r t i c u l a r column. Arrows i n d i c a t e t h e s t a v t of e l u t i o n w i t h competingsugars,m-methyl-0-glucoside (MG) and a-methyl-D-mannoside (MU). The E-PHA andL-PHA-agarose columns werecalibratedusing glycopeptidesof known s t r u c t u r e .k i n d l yp r o v i d e db y Or. R i c h a r d Cummings ( U n i v e r s i t y o f G e o r g i a , Athens, GA). I n t h i sc a l i b r a t i o n ,t h eb i a n t e n n a r yg l y c o p e p t i d e I18 which does n o t i n t e r a c t w i t h e i t h e r E-PHA o r L-PHA-agarose e l u t e d i n f r a c t i o n s 7 - 1 1 and 6-9, r e s p e c t i v e l y . The " b i s e c t e d " b i a n t e n n a r y I g A g l y c o p e p t i d e I A was r e t a r d e d on E-PHA-agarose and e l u t e d i n f r a c t i o n s 18-23. The t e t r a a n t e n n a r yg l y c o p e p t i d eI A 2 was r e t a r d e d on L-PHA-agaroseand e l u t e d i n f r a c t i o n s 8-12. Recovery o f r a d i o a c t i v l t y fran a l l columns was 90-101X.

HA

OligosaccharideComposition of an Influenza Virus

14774

CEF

MDBK HA1

When t h e complexglycopeptides i n pool I and11fromeach HA] source were further fractionated, the lectin profiles (Fig. 3) were highly similar to thoseobtainedfrom their r e s p e c t i v e HA2 s u b u n i t s ( F i g . 2). Thus, t h e host-dependent differences observed w i t h HA2 Were again Ohserved with HA1 despitethedifference i n t h e number of g l y c o s y l a t i o n s i t e s On t h e two subunits.

HA1

We a l s o o" m. e r a~~. tad lectin affinity orofiles of theN-lirkedoliyosaCcharide3obtained .~.. frm bothhostcells.Ac&parison of t i e b e l e c t i n p r o f i l e s ( F i g . 4 ) ~ u i t h t h o s e f r o r t h e HA s u b u n i t s ( F i g s . 2 and 3) indicatedthattheN-linked canplexOligosaccharides found on an t h e h o s t c e l l t h e HA s u b u n i t s Were n o t n e c e s s a r i l y r e p r e s e n t a t i v e o f t h o s e f o u n d g l y c o p r o t e i n s .F i r s t .t h ep r o p o r t i o no fg a l a c t o s ec o n t a i n i n g" b i s e c t e d " complex found On t h e g l y c o p r o t e i n s glycopeptidespresent on t h e HA s u b m i t s d i d n o t r e f l e c t t h a t A small amount O f "Disected' complex f r o me i t h e rc e l l[ F i g . 2b.g; 3b.g and 4b,g). glycopeptideswhich lack outer galactose residues was always found on CEF c e l l s ( F i g . 4 h , p o o lI A 1 ' )b u tn o t on the HR subunits frm eitherceil6. Second. fucosylat.edglycopeppdes glycoproteins from e i t h e r were found t o be inore abundant on the HA S u b u n i t s t h a n o n t h e cell.Essentiallyall of t h eb i a n t e n n a r yg l y c o p e p t i d e sf r o m MDBK- and CEF-HA subumts i and 3 d , i ) ,w h i l eo n l yam i n o rf r a c t i o no ft h e s e Containedcorefucose(Figs2d s t r u c t u r e sf r o mt h ec e l l u l a ;g l i c o p r o t e i n s was f u c o s y l a t e d( F i g 4d i ) in addition, '2h'and3h) which CEF-HA submits contained fucosylated triantennary g l y c o p e p t i d e s( F i g s . were e s s e n t i a l l y a b s e n t from CEF g l y c o p r o t e i n s ( F i g . 4h). F i n a l l y , since the majority (x80%)o f t h e tri- and t e t r a a n t e n n a r y g l y c o p e p t i d e s f r o m t h e CEF-HA s u b u n i t s i n t e r a c t e d w i t h L-PHA-agarose (Figs.2j.k and 3 j , k ) , t h e y c o n t a i n o u t e r g a l a c t o s e s andhave one *-linked mannose r e s i d u e s u b s t i t u t e d w i t h R - a c e t y l g l u c o s a m i n e a t p o s i t i o n C-2 and C-6. Glycopeptides containing these two structural features itre much less abundant on the CEF glycoproteins.Thisdifference,althoughless pronounced, was also observed between t h e HA subuoits and cellular glycoproteins frmn MDBK cells. ~

~

F i g u r e3 .L e c t i na f f i n i t yp r o f i l e s of L3Hhannose-labeled MDBK-HA1 (a-e) and CEF-HA1 (f-k)glycopeptidesobtained a s descyibed i n "Experimental Procedures". The percentages i n parentheses indicate the distribution of the glycopeptides applied to the particular column. For an explanation of t h e a r m u s and t h e c a l i b r a t i o n o f t h e E-PHA and L-PtiA-agarose columns see Figure 1. A d d i t i o n a l d i f f e r e n c e s between MDBK-HRZ and CEF-HA2 weredetected when pool I olvcooeotides were analvred on t h e o t h e r l e c t i n Columns. Dnlv10% o f t h e MDBK-HA? i l j ' c o b e b t i d e s was r e t a i d e d on E-PHA-agarose [Fig. 2b) i n c o n t r i s t t o 28% of the CEF-Mi glycopeptides(Fig. Zg)3. Thus, CEF-HA2 c o n t a i n e dah i g h e rp r o p o r t i o no f" b i s e c t e d c w n p l e xg l y c o p e p t i d e sw i t ho u t e rg a l a c l o s er e s i d u e st h a nd i d MDBK-HA I na d d i t i o n , e s s e n t i a l l ya l lo ft h e MDBK-HA? (IlYCODeDtideS i n pool I A f a i l e d t o t i n d t o l e n t i l l e c t i n - a g a i o s e( F i g . Zc) whereai s t r i k i n g 64% o f t h e CEF-HA, glycopeptidesputonto this column was retained(Fig. 2h, pool IAZ). The data i n d i c a t et h a tt r i a n t e n n a r yg l y c o p e p t i d e s w i t h a c o r e f u c o s e and an o-linked mannose residue substituted with N-acetululucosanine at positions C-2 and C-6 c o n s t i t u t e d a s i g n i f i c a n t f r a c t i o n o f t h e p o o l I glyfopeptidesfrom CEF-HA ( F i g .2 f )b u tw e r ev i r t u a l l ya b s e n t from MUSK-!& (Fig. 2c). Finally, when the MDBK-Hd glycopeptideswhich f a i l e d t o i n t e r a c t w i t h l e n t i l l e c t i n - a g a r o s e were a p p l i e d t o L-PHA-agarose, o n l y a b w t h a l f bound and therefore contained outer galactose residues and an .-linked mannose r e s i d u es u b s t i t u t e d a t p o s i t i o n s C-2 andC-6.Thesebound Ze, p o o l s I A l b and IAlc) on g l y c o p e p t i d e s f r a c t i o n a t e i n t o two a f f i n i t y c l a s s e s ( F i g . L-PHA-agarose. The b a s i s f o r t h i s s e p a r a t i o n i s unknown, b u tt h i sp r o p e r t y of L-PHA-agarosehasbeen p r w i w s l y ohsewed4. E s s e n t i a l l y a l l of the CEF-HA2 glycopeptides which f a i l e d t o b i n d t o l e n t i l l e c t i n - a g a r o s e c o n t a i n e d o u t e r g a l a c t o s e s andan a - l i n k e d mannose r e s i d u e s u b s t i t u t e d a t p o s i t i o n s C-2 and C-6 (Fig. Zj, poolIAlb). I n addition, 71% of t h e f u c o s y l a t e d t r i a n t e n n a r y m o l e c u l e s [ p o o l I A 2 ) on CEF-HA2 c o n t a i n e d O u t e r galactoseresiduessincethey e r e retained byL-PHA-agarose (Fig. 2k, pool IA2b).

123s 111' (27%

=re

Biantennaryglycopeptides (Pool 11) frm each HA2 source were further fractionated on l e n t i l lectin-agarose. Greater than 90% o f these glycopeptides bnund t o t h e l e c t i n( F i g s . Zd and Z i ) , i n d i c a t i n g t h a t t h e y are core fucosylated at a high frequencyby b o t h c e l l s . We a l s o g e n e r a t e d l e c t i n a f f i n i t y p r o f i l e s o f g l y c o p e p t i d e s obtainedfrom MDBK-HA1 (Fig. 36-e) and CEF-HA1 (Fig.3f-k).Unlikethe HRz p r o f i l e s which gave i n f o r n a t i o n on t h e s t r u c t u r e of o l i g o s a c c h a r i d e s l i n k e d t o a single glycasylation site. the HA1 p r o f i l e s fmm b o t h c e l l sources represent an o v e r a l l s p c t r u n o f t h e N - l i n k e d o l i g o s a c c h a r f d e s ob a i n e d f r o mf i v eg l y c o s y l a t i o ns i t e s ,f o u ro fw h i c hc o n t a i n complex o l i g o s a c c h a r i d e s ~ . ~As expected frm t h i s r a t i o o f h i g h mannose t o complex oligosaccharides, 37% of t h e mannose labelinthe HAL s u b u n i t s appeared i n pool111upon ConA-Sepharosechromatography. 1 and 11 again However. t h e d i s t r l b u t i o n o f t h e complexoligosaccharidesbetweenpools varied deoendino on the source o f t h e v i r u s . w i t h MDBK-HA, cantainina a oreater orooortion o f tri- and tetriantennary glycopeptides than'did CEF-HAI. A

-~

31" a duplicate experiment using a second preparation of each virus, pool IBfrrm and CEF-HA2was 3% and 28%, respectiveiy.

4R.n.

Cumolings, personalcmununication.

5C.n. Dem and I.T.

Schulze, manuscript i n preparation.

I

.

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u

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F.*C,,**

Fr.r,ion

F i g u r e 4. L e c t i na f f i n i t yp r o f i l e s o f [3H]m~nnose-laheled MDBK-cell (a-e) and CEF The percentages I n (f-k) glycopeptides obtained as described i n "Experimntal Procedures". parentheses i n d i c a t e t h e d i s t r i b u t i o n of the glycopeptides applied to the particular column. For an explanation of the arrows and t h e c a l i b r a t i o n of the E-PHA and L-PW-agarose columns see Figure 1. TWO u n i d e n t i f i e d g l y c o p e p t i d e pools, ConA-Sepharose p o l s 11' [Fig. 4a and 4f) and l e n t i ll e c t i n - a g a r w ep o o l sI I A ' [Fig. 4d and 4 i ) e r e c o n s i s t e n t l y found i n t h e c e l l u l a r HA s u b u n i t p r o f i l e s . When theseglycopeptides Were reapplied t o orofilesbutnotinthe t h e r e s p e c t i v e l e c t i n c o l m r w i t h or & t h a t a d d i t i o n a l pronase treatment, they eluted as = r e specificalthoughthestructuralrequirevents before. Thus, t h e i r l e c t i n i n t e r a c t i o n s f o r these interactions have not been detemined.

MDBK-HA;, 6"Bisected" cwnplex g l y c o p e p t i d e s w h i c h l a c k o u t e r g a l a c t o s e r e s i d u e s flowthroughthe (17; Dr. Richard 0. E-PHA-agarose column and i n t e r a c t w e a k l y w i t h l e n t i l l e c t i n - a g a r o s e c m i n g s , personal c ~ n i c a t i o n ) .