THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1985 by The American Society of Biological Chemists, Inc.

Vol ,260, No. 27, Issue of November 25, pp. 14771-14774,1985 Printed in U.S.A.

Oligosaccharide Composition of an Influenza Virus Hemagglutinin with Host-determined Binding Properties*

(Received for publication, April 26, 1985)

Carl M. Deom and Irene T. Schulzel From the Department of Microbwbgy, St. Louis University School of Medicine, St. Louis, Missouri 63104

We have previously reported that the binding prop- erties 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 Schulze, I. T. (1984) Virology 139, 164-177); at 37 “C 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 put onto the viral HA by these two cells. Experiments using tunicamycin 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 oli- gosaccharides on the HA subunits, HA1 and HA2, de- 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- and tetraantennary) complex oligosaccharides than did the subunits from chick embryo fibroblasts. In addi- tion, the HA subunits from the two sources differed in the amount of galactose-containing “bisected” complex oligosaccharides and in the presence of certain fuco- sylated triantennary oligosaccharides. Profiles of the asparagine-linked oligosaccharides from the host cells did not show these differences, indicating that the HA subunit profiles were not necessarily representative of the structures found on the cellular glycoproteins. The data support the conclusion that bulky oligosaccha- rides 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

* This work was supported in part by Grants AI-10097 and AI- 14590 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 article must 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 chick embryo fibroblasts; MDBK, Madin-Darby bovine kidney cells; ConA-Sepharose, concanavalin A-Sepharose; E-PHA-agarose, ery- throagglutinating phytohemagglutinin-agarose; L-PHA-agarose, leu- koagglutinating phytohemagglutinin-agarose; MEM, minimal essen- tial media; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; NP40, Nonidet P- 40; TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone; PUK, proteolytic units Kaken.

antigen to which neutralizing antibodies are produced. Through its antigenic variability, the HA is responsible for the continued appearance in the human population of epi- demic strains of influenza A virus.

The biologically active HA is a trimer of identical glycopro- teins each of which is cleaved into two subunits, designated HA1 and HA, (1 ,Z) . Carbohydrate side chains are attached to both subunits through asparagine linkages (N-linkages) (3) and are of the complex, high mannose, or hybrid types (4, 5). These oligosaccharides can vary in number and structure 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 oligosac- charides (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 influ- enza 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 erythrocyte binding 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 but poorly in MDBK cells. In addition, binding studies have indicated that the F 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 FCEF in the opposite 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 chro- matography (lo), we have obtained information about the structure of the oligosaccharides put onto the two 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 addi- tion, we present evidence that the N-linked oligosaccharides on the virus are not necessarily representative of those found on the host cell glycoproteins.

EXPERIMENTAL PROCEDURES AND RESULTS~

DISCUSSION

The lectin affinity analysis presented here clearly demon- strates a number of differences between the complex oligosac-

Portions of this paper (including “Experimental Procedures,” “Results,” Figs. 1-4, and Footnotes 3-6) are presented in miniprint

14771

14772 Oligosaccharide Composition of an Influenza Virus HA

charide side chains put onto the HA subunits by two host cells. First, the MDBK-HA subunits carry tri- and tetraan- tennary oligosaccharides more frequently than do the CEF- HA polypeptides. Second, galactose-containing “bisected” complex oligosaccharides are more abundant on CEF-HA subunits than on MDBK-HA subunits. Third, certain fuco- sylated triantennary oligosaccharides are found on the HA subunits from CEF but not from MDBK cells. Finally, gly- copeptides from MDBK-HA subunits contain a class of mol- ecules which interact with leukoagglutinating phytohemag- glutinin-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, ap- proximately 3000 of which are in the HA1 subunit. This can be due in part to the large number of tri- and tetraantennary oligosaccharides on the MDBK-HA. However, this extra car- bohydrate, about 20 monosaccharides distributed over five complex oligosaccharides on each MDBK-HA monomer, sug- gests that additional sugar residues are attached to the outer galactoses of many of the complex carbohydrates synthesized by MDBK cells. The data presented 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. How- ever, 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 sialyl- transferase and CMP-sialic acid (18). When this was done, CEF-HA was sialylated at about 20 times the rate of MDBK- HA, even though the lectin profiles presented here indicate that the HA subunits from the two sources contain compa- rable amounts of oligosaccharides with outer galactoses. The data suggest that terminal galactose residues are in abundance on CEF-HA whereas the outer galactoses on MDBK-HA are predominantly substituted with additional sugars. These ad- ditional 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 re- peating 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) prompts the following speculation. An MDBK-spec- ified 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 im- portant in determining both the conformation and the stabil- ity of the HA. Since oligosaccharides cover approximately 20% of the surface of the HA trimer (20), carbohydrate- protein interactions within the subunits and at subunit inter- faces 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 recep- tors 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 the HA (18) nor removal of terminal sugars from the HA (22) destroys the infectivity of these particles. In addition, although the amount of sulfate on the HA can be determined by the host, the hemagglutin- ating activity of the virus appears to be independent of the degree of sulfation (23).

The mechanisms proposed here are similar to those sug- gested 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 deter- mines 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 trianten- nary structures are present on CEF-HA subunits and are virtually absent from the CEF glycoproteins. Whether these differences result from control of glycosylation by 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 FBK virus 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. Rich- ard 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.

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2. Lazarowitz, S. G., and Choppin, P. W. (1975) Virology 68,440-454

4. Schwarz, R. T., Schmidt, M. F. G., Anwer, U., and Klenk, H.-D. (1977) J. 3. Keil, W., Klenk, H.-D., and Schwarz, R. T. (1979) J. Virol. 31, 253-256

5. Nakamura, K., Bhown, A. S., and Compans, R. W. (1980) Virology 107,

426-439

Virol. 23, 217-226

6. 7. 8.

9. 10. 11. 12. 13. 14.

15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.

20&221 Nakamura, K., and Compans, R. W. (1979) Virology 95,s-23 Schwarz, R. T., and Klenk, H.-D. (1981) Virology 113,584-593 Crecelius, D. M., Deom, C. M., and Schulze, I. T. (1984) Virology 139,

1 fL-177 Noronha-Blob, L., and Schulze, I. T. (1976) Virology 69, 314-322 Cummings, R. D., andKornfeld, S. (1982) J. Bid. Chem. 257,11235-11240 Basak, S., and Compans, R. W. (1983) Virology 128, 77-91 Laemmli, U. K. (1970) Nature 227,680-685 Schulze, I. T. (1970) Virology 42, 890-904 Cowan, E. P., Cummings, R. D., Schwartz, B. D., and Cullen, S. E. (1982)

Hiti, A. L., Davis, A. R., and Nayak, D. P. (1981) Virology 111,113-124 Nakamura, K., and Compans, R. W. (1978) Virology 86,432-442 Narasimhan, S. (1982) J. Biol. Chem. 257,10235-10242 Lakshmi, M. V., and Schulze, I. T. (1978) Virology 88,314-324 Cummings, R. D., and Kornfeld, S. (1984) J. Biol. Chem. 259,6253-6260 Wilson, I. A., Skehel, J. J., Wiley, D. C. (1981) Nature 289,366-373 Compans, R. W., and Pinter, A. (1975) Virology 66,151-160

Downie, J. L. (1978) J. Gen. Virol. 41,283-293 Collins, J. K., and Knight, C. A. (1978) J. Viol. 27,164-171

Zhu, B. C. R., and Laine, R. A. (1985) J. Biol. Chem. 260,4041-4045 Gibson, R., Kornfeld, S., and Schlesmnger, S. (1981) J. Bzol. Chem. 256,

J. Biol. Chem. 257,11241-11248

456-462

Oligosaccharide Composition of an Influenza Virus HA 14773 SUPPLEMENTARY MATERIAL TO:

OLIGOSACCHARIDE CIXPOSITION OF AN INFLUENZA VIRUS HMAGGLUTININ WITH HOST DETERMINED BINDING PROPERTIES

by

Car l M. Dean and I rene T. Schulze

~- EXPERIMENTAL PROCEDURES

Chemicals: l e n t i l lec t in-aaarose. E-PHA-aqarose and L-PHA-aoarose from E - Y L a b o r a t o r i e s : M a t e r i a l s . G-25 Sephadex and ConA-Sepharose were o b t a i n e d from Pharmacia F ine

pronase (70,000 PUKIgm) Bnd t i n i camyc in f rom Ca lb iochemhehr ing Gorp . D-[2-3H]Mannos; (10-20 Cilmmol) from Amersham Corp. ; 135Slneth ion ine (1400 Ci lmmol) i rom New England Nuclear; TPCK-Trypsin f rom Worthington Biochemical Corp; and EN3Hance frm New England Nuclear.

Radioact ive ce l ls were obtained f r a n cu l tu res grown t o confluency over a per iod of 2-3 days C e l l and Vi rus Growth. MDBK c e l l s and CEF were grown as previously descr ibed (8).

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 f t h e WSN-F s t r a i n ( 8 ) o f i n f l u e n z a A ( H l N 1 ) v i r u s was used 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 e v i r u s e s w e r e p u r i f i e d f r o m i n f e c t e d c e l l c u l t u r e s g r w n i n t h e presence o f 2 uCi lml of C2-3Hlmannose as previously descr ibed (8).

G lycosy la ted and Ung lycosy la ted HA. I n f e c t e d c e l l m n o l a y e r s wre pulse- labeled at 6 h o u r s p o s t - i n f e c t i o n f o r 1 h o u r a t 3 7 - C i n HEM c o n t a i n i n o n e - f o u r t h t h e n o r m a l c o n c e n t r a t i o n of methionine, 2% d ia lyzed fe ta l ca l f serum and [&]methionine a t 50 u~i/ml. Fol lowing a chase per iod of 15 minutes 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 and lysed a t 4'C f o r 30 minutes i n 10 d Tris-HC1 (pH 7.0). 0.15 M NaCl , 0.5% T r i t o n X-100, and 1% NP40. N u c l e i were removed by c e n t r i f u g a t i o n a t 12,000 g f o r 5 m inu tes and the supernatant f ract ion was made 0.1% SOS. When tun i canyc in ( 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 hours p o s t - i n f e c t i o n a n d was p r e s e n t t h r o u g h o u t t h e p u l s e - c h a s e p e r i o d . T h e HA w a s i m m u n o p r e c i p i t a t e d f r o m c e l l u l a r l y s a t e s a s p r e v i o u s l y d e s c r i b e d ( 1 1 ) u s i n g a n t i - H A monoclonal antibodies obtained frm Or. Walter Gerhard (Wistar Inst i tute, Phi ladelphia. PA). SOS-PAGE (12) was used t o determine the s ize o f the g lycosy lated and unglycosylated HAS.

P r e p a r a t i o n a n d F r a c t i o n a t i o n o f Glycopeptides. C3HIMannose-labeled. p u r i f i e d v 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 uqlrnl l f o v 2 0 m i n u t e s a t 37-C. T h i s t r e a t m e n t removes neuraminidase olvcooroteinr 1131

w e r e t h e n s e p a r a t e d On 17.5% g e l s c o n t a i n i n g 4 M urea. Gels were processed for w i i c h h a v e e l e c t r o p h o r e t i c m 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

F luorograpxs were used is t e n p l a t e s t o l o c a t e HA1 and HA2 prote ins. The appropr ia te areas f l u o r o g r a p h i n EN3Hance d r i e d and placed i n c o n t a c t w i t h Kadak MR-5 f i l m a t -7O'C.

of the 421s were exc ised. cu t in to sec t ions I1 mn x 2 mm). and incubated a t 60-C f o r 4 t o 5

~ _ ~ ~ r ~ ~ ~ ,.~,

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.

c o n t a i n i n g t h e C3H1 mannose- labe led g lycopept ides was removed, b o i l e d f o r 5 minutes, (This pronase so lu t i on had been preincubated for 15 minu tes a t 37'C). The l i q u i d f r a c t i o n

c e n t r i f u g e d t o remove inso lub le mater ia l . and prepared fo r ana lys i s as p rev ious ly descr ibed (14) .

Cel lu lar g lycopept ides e r e prepared as described by Cummings and Kornfeld (10).

B o t h t h e HA and the ce l lu la r g lycopept ides 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 chromatography procedure of Cwmings and Kornfeld (10) as described by Cowan et g . (14). Th is o rocedure i den t i f i es t he m in ima l S t ruc tu ra l f ea tu res Peau i red f o r t he i n te rac t i on o f g l y c o p e p t i d e s w i t h t h e v a r i o u s l e c t i n s . A d d i t i o n a l s u g a r s , s u c h a s t h e s i a l i c a c i d

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 r e s i d u e s w h i c h a r e p r e s e n t on t he ce l l u la r g l ycop ro te ins bu t no t on t he HA g lycoprote ins,

as su l fa t i on o r phosphory l i t i on of canplex oligosaccharides h i s n o t been documented.

G l y c o p e p t i d e s were a p p l i e d t o ConA-Sepharose columns and 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 d t e t r a a n t e n n a r y N - l i n k e d o l i g o s a c c h a r i d e s a s w e l l a s " b i s e c t e d b i a n t e n n a r y o l i g o s a c c h a r i d e s f a i l t o b i n d t o ConA-Sepharose and are designated pool I. Bian tennary N - l i nked o l i gosacchar ides cons t i t u te poo l 11. These g l y c o p e p t i d e s b i n d t o ConA-Sepharose and are eluted with a-methyl-0-glucoside. High mannose and hybrid N-l inked o l igosacchar ides are designated pool 111. They b i n d t 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 s a r e t h e n a p p l l e d t o an E-PHA-agarose column which retards "bisected" g lycopept ides containing outer galactose residues. Glycopept ides t h a t d o n o t i n t e r a c t w i t h E-PHA-agarose are applied t o l e n t i l l e c t i n - a g a r o s e which binds c e r t a i n t r i 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 a C o r e f u c o s e and an " l inked mannose s u b s t i t u t e d w i t h N - a c e t y l g l u c o s a m n e a t C-2 and C-6. These bwnd glycopeptides vhich are e l u t e d w i t h o - m e t h y l - P - m a n n o s i d e . a s w e l l a s t h o s e g l y c o p e p t i d e s t h a t f a i l 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 . are f u r t h e r f r a c t i o n a t e d an L-PHA-agarose. T h i s l e c t i n i n t e r a c t s w i th tri- and te t raan tennary g l ycopep t ides t ha t con ta in ou te r ga lac tose res idues and an a- l inked mnnose subst i tuted wi th N-acetylglucosamine at C-2 and C-6.

lectin-agarose. Those glycopeptides which f a i l 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 l a c k a T h e b i a n t e n n a r y o l i g o s a c c h a r i d e s i n p o o l I I a r e f u r t h e r a n a l y z e d on l e n t i l

co re f ucose res idue whereas t hose t ha t b ind con ta in a co re fucose residue and are e lu ted w i t h o-wthyl-D-mannos?de.

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 t r i p l i c a t e p r e p a r a t i o n s o f v i rus 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 e p r o f i l e s were used t o assign minimal structural features to the 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 subun i t s by two hos t ce l l s .

the HA1 and HA2 subuni ts as wel 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 i n H o s t C e l l Dependent Glycosy lat ion of the Hemaqqlutinin. We have prev ious ly Shown t h a t

SOS-PAGE than do the co r respnd ing HA pro te ins f ran the MDBK-grwn v i r u s ( 9 ) . To determine if 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 Eiiis-iiF; a i a i v & 6; SDS-PAGE (Fig. 1). The major bands i n lanes 1 and 2 r e p r e s e n t t h e g l y c o s y l a t e d u G l e a v e h HAS f rom CEF (75.000 Mr) and f rom 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 the tw c e l l s i n t h e presence of tun icanyc in were t h e same s i ze : t he mo lecu la r ue iqh t o f t h i s D ro te in (63.000 M.I *as i n oood aoreenent w i t h tha t p red ic ted-by the nuc le ic ac id sequence (63,453) o i t h e ' i A gene 6~ i dN- i t ra ;n i f l n f l u e n z a v i r u s ( 1 5 ) . T h u s , a d i f f e r e n c e i n c a r b o h y d r a t e c o n t e n t a p p e a r e d t o b e

m i n o r band seen i n l a n e 2 i s n o t a l w a y s p r e s e n t i n i n f e c t e d MDBK cell ex t rac ts and has responsible for the host-determined dif ference i n t h e s i z e o f t h e g l y c o s y l a t e d HAS. The

never been found i n v i r i o n s . It presumably reDresents incomPle te lv Q lvcosv la ted HA molecu les tha t a re no t incorpora ted in to v i r ions .

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 The data 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 other labora tor ies in wh ich

p r o t e i n was host dependent (4.16). As expected frm these repor ts . when pronase digests of

the mannose-labeled F virus H b were chromatographed on Bio-Rad Bio-Gel P-6 columns t h e Complex glycopeptides fran MOBK-derived HA subuni ts e r e larger than those frm CEF-dehed subuni ts (data not 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 n a b o u t t h e s t r u c t w e o f t h e s e c o m p l e x c a r b o h y d r a t e s , we have used s e r i a l l e c t i n a f f i n i t y chromatography t o charactevize the o l igosacchar ides put onto the HA subuni ts by bo th ce l l s .

S e r i a l L e c t i n A f f i n i t y C h r m a t o q r a p h y of HA Subunits and Host Cell Glycopeptides. The

t h e c e l l i n d e t e r m i n i n g t h e s t r u c t u r e o f t h e o l i g o s a c c h a r i d e s a t t h i s s i n g l e a t t a c h n e n t HA2 subuni t o f AlWSN133 has one ol igosacchar ide chain per molecule (15.16). The r o l e o f

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 l a r g e f r a c t i o n o f 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 f ract ionated almost evenly between pools I and I I (F ib . 2 f ) . A m a l ? b u t r e p r o d u c i b l e peak represent ing 5% of the CEF-HA2 m t e r i a l ( F i g . 2f, pool 1') was s l i gh t l y re ta rded bu t no t re ta ined by ConA-Sepharase. This pool wds no t f ound o n MDBK-HA2 and was n o t f u r t h e r analyzed. Essen t ia l l y no high mannoselhybrid glycopeptides w e r e f o u n d o n HA2 f r o m e i t h e r c e l l . T h i s was c o n f i r m e d b y s h o w i n g t 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 (data not 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 subun i t s f van bo th cells contain only canplex o l igosacchar ides but that MOBK-HA2 conta ins a s i g n i f i c a n t l y g r e a t e r p r o p o r t i o n o f 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 n d o e s CEF-HA2.

1 2 3 4 - - rr_

- HA+ 1

F i g u r e 1. SDS-PAGE a n a l y s i s o f i m m u n o p r e c i p i t a t e d C 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 d c e l l s . F v i r u s HA f rom: lane 1. CEF; l a n e 2, MDBK c e l l s ; lane 3, t un i cmyc in - t rea ted CEF; lane 4. t un i cmyc in - t rea ted MOBK ce l l s .

i I

b N

P

(f-k) glycopeptides obtained as described i n "Experimental Procedures". ?he percentages i n F i g u r e 2. L e c t i n a f f i n i t y p r o f i l e s of I%]mannose-labeled MOBK-HA (a e) and CEF-HA2

parentheses ind icate the d is t r ibut ion of the g lycopept ides app l ied to the par t i cu la 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 competing sugars, m-methyl-0-glucoside (MG) and a-methyl-D-mannoside (MU). The E-PHA and L-PHA-agarose columns were cal ibrated using glycopept ides of known st ructure. k ind ly prov ided by Or. Richard Cummings ( U n i v e r s i t y o f Georg ia, Athens, GA). I n th is ca l ib ra t ion , the b ian tennary g lycopept ide I18 which does not 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 lu ted in f rac t ions 7 -11 and 6-9, r e s p e c t i v e l y . The "b isected" b iantennary IgA g lycopept ide IIA was retarded on E-PHA-agarose and e l u t e d i n

e l u t e d i n f rac t i ons 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. f rac t i ons 18-23. The te t raantennary g lycopept ide IA2 was r e t a r d e d on L-PHA-agarose and

14774 Oligosaccharide Composition of an Influenza Virus HA MDBK HA1

CEF HA1

(f-k) glycopeptides obtained a s descyibed i n "Experimental Procedures". The percentages i n F i g u r e 3 . L e c t i n a f f i n i t y p r o f i l e s of L3Hhannose-labeled MDBK-HA1 (a-e) and CEF-HA1

parentheses indicate the d istr ibut ion of the g lycopept ides appl ied to the part icular column.

see Figure 1. For an explanation of the armus and the ca l ib ra t ion o f the E-PHA and L-PtiA-agarose columns

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 were detected when pool I olvcooeot ides were analvred on t h e o t h e r l e c t i n Columns. Dnlv 10% o f t h e MDBK-HA? i l j ' cobeb t ides 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

cwnp lex g l ycopep t ides w i th ou te r ga lac lose res idues t han d id MDBK-HA In add i t i on , glycopeptides (Fig. Zg)3. Thus, CEF-HA2 conta ined a h igher p ropor t ion o f "b isec ted

e s s e n t i a l l y a l l o f t 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 ec t i n -aga iose (F ig . Zc) whereai s t r i k i n g 64% o f t h e CEF-HA, glycopeptides put onto t h i s

w i t h a core fucose and an o- l inked mannose residue substi tuted with N-acetululucosanine at column was retained (Fig. 2h, pool IAZ). The data ind icate that t r iantennary g lycopept ides

posi t ions C-2 and C-6 const i tu ted a s ign i f icant f ract ion o f the pool I g ly fopept ides f rom CEF-HA (F ig . 2 f ) bu t were v i r t ua l l y absen t from MUSK-!& (Fig. 2c). Final ly, when the MDBK-Hd glycopeptides which fa i led to in te rac t w i th len t i l lec t in -agarose 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 =re bound and therefore contained outer galactose residues and an .-linked 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. These bound 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 . Ze, poo ls IA lb and IAlc) on 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 t t h i s p r o p e r t y of L-PHA-agarose has been 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

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. Z j , pool IAlb). I n addition, which fa i led to b ind to len t i l lec t in -agarose conta ined ou ter ga lac toses and an a - l i nked

71% of t he f ucosy la ted t r i an tennary mo lecu les [ poo l IA2 ) on CEF-HA2 con ta ined Ou te r galactose residues since they e r e retained by L-PHA-agarose (Fig. 2k, pool IA2b).

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 . Biantennary g lycopept ides (Pool 11) frm each HA2 source were further fractionated on

Zd and Zi ) , ind ica t ing tha t they are core fucosylated at a high frequency by both ce l ls .

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 obtained from MDBK-HA1 (Fig. 36-e) and CEF-HA1 (Fig. 3f-k). Unlike the HRz pro f i l es which gave in fo rna t i on on t h e s t r u c t u r e of o l igosacchar ides l inked to a single g lycasylat ion s i te. the HA1 p ro f i l es fmm bo th ce l l sources represent an overa l l spct run o f the N- l inked o l igosacchar fdes ob a ined f r o m f i v e g l y c o s y l a t i o n s i t e s , f o u r o f w h i c h c 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 ol igosacchar ides, 37% of t h e mannose

However. t h e d i s t r l b u t i o n o f t h e complex ol igosaccharides between pools 1 and 11 again l a b e l i n t h e HAL s u b u n i t s appeared i n pool 111 upon ConA-Sepharose chromatography.

var ied deoendino on the source of the v i rus. wi th MDBK-HA, cantainina a oreater orooortion o f tri- and tetr iantennary glycopeptides than'did CEF-HAI. A

-~ I .

31" a dupl icate experiment using a second preparation of each virus, pool IB frrm MDBK-HA;, and CEF-HA2 was 3% and 28%, respectiveiy.

4R.n. Cumolings, personal cmununication.

5C.n. Dem and I.T. Schulze, manuscript i n preparation.

f ract ionated, the lect in prof i les (Fig. 3) were highly s imi lar to those obtained from t h e i r When t h e complex g lycopept ides i n pool I and 11 from each HA] source were further

respec t i ve HA2 subun i t s (F ig . 2). Thus, t h e host-dependent di f ferences observed w i t h HA2

t he two subunits. Were again Ohserved with HA1 despite the difference i n t h e number of g lycosy la t ion s i tes On

We a l s o o m e r a t a d l e c t i n a f f i n i t y o r o f i l e s of the N-lirked oliyosaCcharide3 obtained .~.. ". ~ ~~. ~

frm both host cells. A c&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 subun i ts (F igs . 2 and 3) indicated that the N-l inked canplex Oligosaccharides found on t h e HA subun i t s Were not necessar i ly representat ive o f those found an t h e h o s t c e l l g l y c o p r o t e i n s . F i r s t . t h e p r o p o r t i o n o f g a l a c t o s e c o n t a i n i n g " b i s e c t e d " complex glycopeptides present on t he 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 found On the g l ycop ro te ins f r o m e i t h e r c e l l [ F i g . 2b.g; 3b.g and 4b,g). A smal l amount O f "Disected' complex glycopeptides which lack outer galactose residues was always found on CEF c e l l s ( F i g . 4 h , poo l IA1 ' ) bu t no t on the HR subunits frm either cei l6. Second. fucosylat.ed glycopeppdes were found t o be inore abundant on the HA Subuni ts than on the glycoproteins from e i t h e r c e l l . E s s e n t i a l l y a l l of the b iantennary g lycopept ides f rom MDBK- and CEF-HA subumts Contained core fucose (Figs 2d i and 3 d , i ) , w h i l e o n l y a m i n o r f r a c t i o n o f t h e s e s t r u c t u r e s f r o m t h e c 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,

were essent ia l l y absent from CEF g l ycop ro te ins (F ig . 4h). F ina l l y , since the majori ty CEF-HA submits contained fucosylated triantennary g lycopept ides (F igs. '2h'and 3h) which

(x80%) o f t h e tri- and te t raantennary g lycopept ides f rom the CEF-HA subun i t s i n te rac ted w i t h L-PHA-agarose (Figs. 2j.k and 3 j , k ) , they con ta in ou ter ga lac toses and have 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. Glycopept ides containing these two structural features i t re much less abundant on the CEF glycoproteins. This difference, although less pronounced, was also observed between t h e HA subuoits and cel lular glycoproteins frmn MDBK cel ls.

123s

111' (27%

"H

"1 u I-

Fractio" F.*C,,** riu Fr.r,ion

(f-k) glycopeptides obtained as described i n "Experimntal Procedures". The percentages I n F igu re 4. L e c t i n a f f i n i t y p r o f i l e s o f [3H]m~nnose-laheled MDBK-cell (a-e) and CEF

parentheses ind ica te the d is t r ibu t ion of the glycopeptides appl ied to the part icular column. For an explanation of the arrows and the ca l ibrat ion of the E-PHA and L-PW-agarose columns see Figure 1.

lent i l lec t in-agarwe pools I IA ' [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 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

o r o f i l e s b u t n o t i n t h e HA subun i t p r o f i l e s . When these glycopeptides Were reapplied t o the respect ive lect in c o l m r wi th or & t h a t a d d i t i o n a l pronase treatment, they e luted a s

f o r these interactions have not been detemined. before. Thus, t he i r l ec t i n i n te rac t i ons =re specific although the structural requirevents

6"Bisected" cwnplex glycopept ides which lack outer ga lactose res idues flow through the

c m i n g s , personal c ~ n i c a t i o n ) . E-PHA-agarose column and in te rac t weak ly w i th l en t i l l ec t i n -agarose (17; Dr. Richard 0.