MOLECULAR AND CELLULAR BIOLOGY, May 1990, p. 1989-2001 Vol. 10, No. 50270-7306/90/051989-13$02.00/0Copyright ©D 1990, American Society for Microbiology

Different Roles of Individual N-Linked Oligosaccharide Chains inFolding, Assembly, and Transport of the Simian Virus 5

Hemagglutinin-NeuraminidaseDAVIS T. W. NG, SCOTT W. HIEBERT,t AND ROBERT A. LAMB*

Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University,Evanston, Illinois 60208-3500

Received 2 November 1989/Accepted 8 January 1990

The role of N-linked glycosylation in protein maturation and transport has been studied by using the simianvirus 5 hemagglutinin-neuraminidase (HN) protein, a model class H integral membrane glycoprotein. The sitesof N-linked glycosylation on HN were identified by eliminating each of the potential sites for N-linkedglycosylation by oligonucleotide-directed mutagenesis on a cDNA clone. Expression of the mutant HN proteinsin eucaryotic cells indicated that four sites are used in the HN glycoprotein for the addition of N-linkedoligosaccharide chains. These functional glycosylation sites were systematically eliminated in various combi-nations from HN to form a panel of mutants in which the roles of individual carbohydrate chains and groupsof carbohydrate chains could be analyzed. Alterations in the normal glycosylation pattern resulted in theimpairment of HN protein folding and assembly which, in turn, affected the intracellular transport of HN. Theseverity of the consequences on HN maturation depended on both the number of deleted carbohydrate sites andtheir position in the HN molecule. Analysis of the reactivity pattern of HN conformation-specific monoclonalantibodies with the mutant HN proteins indicated that one specific carbohydrate chain plays a major role inpromoting the correct folding of HN. Another carbohydrate chain, which is not essential for the initial foldingof HN was found to play a role in preventing the aggregation of HN oligomers. The HN molecules which weremisfolded, owing to their altered glycosylation pattern, were retained in the endoplasmic reticulum.Double-label immunofluorescence experiments indicate that misfolded HN and folded HN are segregated in thesame cell. Misfolded HN forms disulfide-linked aggregates and is stably associated with the residentendoplasmic reticulum protein, GRP78-BiP, whereas wild-type HN forms a specific and transient complex withGRP78-BiP during its folding process.

Asparagine-linked (N-linked) glycosylation is one of themost common posttranslational modifications found on pro-teins in the exocytic pathway. A high-mannose carbohydratechain, Glc3Man9GlcNAc2, attached to a dolichol lipid car-rier, is covalently transferred to the Asn residue in theconsensus sequence, Asn-X-Ser/Thr (where X is any aminoacid except possibly proline or aspartic acid) on the nascentpolypeptide chain during its translocation into the lumen ofthe rough endoplasmic reticulum (RER) (reviewed in refer-ence 26). However, not all consensus sites are modified byglycosylation, and it is not clearly understood what othercharacteristics in the protein primary sequence or its foldingintermediates determine or prevent the usage of any givensite. Processing of the high-mannose carbohydrate chains onproteins begins in the RER and continues with a variety ofpossible modifications in the cisternae of the Golgi appara-tus, such that eventually there can be considerable hetero-geneity in the N-linked oligosaccharide molecules on pro-teins (reviewed in references 22 and 26).

Since the majority of proteins transported by or resident ina compartment of the exocytic pathway are modified byN-linked glycosylation, it is of great interest to understandthe possible functions of the carbohydrate chains. A varietyof roles have been suggested, including (i) aiding in proteinfolding, (ii) maintenance of protein stability and solubility,

* Corresponding author.t Present address: Howard Hughes Medical Institute, Depart-

ment of Microbiology-Immunology, Duke University Medical Cen-ter, Durham, NC 27710.

(iii) modulation of biological activity, and (iv) protectionfrom proteolytic degradation (reviewed in references 41 and48). For one specific group of proteins, the lysosomalhydrolases, the addition of phosphomannosyl residues totheir carbohydrate side chains has been shown to be asorting signal that is recognized by the mannose 6-phosphatereceptor (54).Two general approaches have been used to study the

functional role of N-linked carbohydrate addition within thecell. The first involves the use of inhibitors that disrupt theaddition or modification of N-linked oligosaccharides. Stud-ies in which tunicamycin is used to block glycosylation haveyielded varying results. In most cases, the nonglycosylatedmolecules have been found to be defective in their intracel-lular transport and are often rapidly degraded, whereas, insome cases, there is little effect on transport and biologicalactivity (reviewed in reference 11). These variable effectssuggest that the functional contribution of carbohydrate sidechains is intrinsic to a given protein. The second approachinvolves oligonucleotide-directed mutagenesis of cDNAsencoding the protein to either remove or add sites forN-linked glycosylation, which makes it possible to study theconsequences of the change without the secondary effectsassociated with glycosylation inhibitors, such as a reductionin protein synthesis (8, 9, 31-34, 52, 53, 55). From theseinvestigations, it has been suggested that glycosylation isnecessary for folding of proteins into their native structure,which, in turn, affects the stability of the molecules and theirability to undergo intracellular transport. However, theseverity of the defects caused by the mutations depends on

1989

1990 NG ET AL.

the extent of deglycosylation, since the removal of all siteshas usually been found to have a more deleterious effect thanthe removal of a single attachment site. The position of thecarbohydrate chain on the polypeptide backbone may alsobe important, since the creation of novel sites does notnecessarily compensate for the disruption of sites normallyused (32, 33). In addition, the introduction of new sites forN-linked carbohydrate addition in a molecule can disrupt itsability to fold into a native structure (14, 32).The hemagglutinin-neuraminidase (HN) glycoprotein of

the paramyxovirus simian virus 5 (SV5) is a typical class IIintegral membrane protein that has a large carboxy-terminalectodomain, a single amino-proximal hydrophobic regionthat acts as both an uncleaved signal sequence and trans-membrane anchor domain, and a short amino-terminal cyto-plasmic tail (20. 21). HN is an oligomeric protein that isthought to be a homotetramer composed primarily of twodisulfide-linked dimers that are noncovalently associated.From studies with conformation-specific monoclonal anti-bodies and sucrose-density sedimentation analysis, we haverecently shown that HN oligomerizes relatively slowly (01,2,25 to 30 min) (40) compared with other viral glycoproteins,vesicular stomatitis virus (VSV) G and influenza virus hem-agglutinin (HA) (t1/2, 6 to 8 and 7 to 10 min, respectively) (5,15; reviewed in reference 6). Our studies have also shownthat during its normal maturation pathway, HN forms acomplex with the stress-related cellular protein GRP78-BiP(2, 18, 38, 39) in the ER following synthesis and that itdissociates from GRP78-BiP when it reaches a folded con-formation just prior to oligomerization (40). HN is trans-ported to the Golgi apparatus as an oligomer, and some ofthe carbohydrate chains are processed there from the simpleto the complex form before its expression at the cell surface.

In this paper, we examine the role of N-linked glycosyl-ation in the maturation, assembly, and intracellular transportof HN, a model class II integral membrane protein. The sitesused for the attachment of N-linked carbohydrate chainshave been identified by using oligonucleotide-directed muta-genesis on a cDNA that has been previously shown toexpress a biologically active HN protein (21, 42, 44). Fromthe individual mutants a panel of HN mutants has beenassembled that has allowed us to study the contribution ofindividual carbohydrate chains, as well as their synergisticeffects, in promoting the proper biogenesis and transport ofthe HN protein, including the nature of the interaction withGRP78-BiP.

MATERIALS AND METHODS

Cell culture. The TC7 subclone of CV-1 cells and a variantof the Madin-Darby bovine kidney (MDBK) cell line weregrown and maintained in Dulbecco modified Eagle medium(DME) supplemented with 10% fetal calf serum as describedpreviously (42).

Generation of site-specific mutants. The plasmid pSV103HN (42), which contains the cDNA encoding the HN proteinofSV5 (21), was digested with XhoI, and the released HNinsert was isolated and ligated into the XhoI site of thereplicative form of bacteriophage M13mpl9X (43). Theligated molecules were used to transfect Escherichia coliTG1 cells. Recombinant phage were selected, plaque puri-fied, and grown to isolate single-stranded phage DNA asdescribed previously (57). Oligonucleotide-directed muta-genesis was performed by the method of Zoller and Smith(57). Six oligonucleotides were synthesized by the North-western University Biotechnology Facility by using a DNA

synthesizer (model 380B; Applied Biosystems, Inc., FosterCity, Calif.) such that they would bring about the substitu-tion of Asn-encoding codons for Ser-encoding codons ateach of the six sites containing the consensus sequence forN-linked glycosylation, Asn-X-Ser/Thr, found in the HNcoding sequence. Mutants were verified by the dideoxynu-cleotide chain-terminating method (51). The HN codingsequences of each mutant were subcloned into the SV40-based late-region expression vector, pSV103 (42), and themutant proteins were expressed as described below. Thefour sites used for N-linked glycosylation (see Results) aredesignated gl, g2, g3, and g4, corresponding to Asn found atHN residues 110, 139, 267, and 504, respectively (21). Adouble mutant was generated by oligonucleotide-directedmutagenesis on site g2 by using the site gl single-mutantDNA as the template. The coding sequences of the gl-g2double mutant was subcloned into pSV103. The generationof the double mutant made it possible to construct mutantswith any combination of deleted glycosylation sites byligating restriction fragments containing the appropriatewild-type or mutant site(s). A total of 11 N-linked glycosyl-ation mutants were constructed, each designated HNg(n),where n is a set of numbers defining the N-linked glycosyl-ation sites that are used: e.g., wild-type HN is HNg1234, amutant lacking the g3 site is HNg124, and the nonglycosy-lated mutant is HNgO (see Fig. 2).

Antibodies. A rabbit polyclonal antiserum was raisedagainst sodium dodecyl sulfate (SDS)-denatured HN (anti-HNSDS). Briefly, SV5 virus was grown in MDBK cells indishes (100 mm), and virions were purified as describedpreviously (46). Viral polypeptides were separated by SDS-polyacrylamide gel electrophoresis (PAGE), and the gel wasstained with Coomassie blue. The HN-containing band wasexcised from the gel, and the protein was eluted in anelectroseparation chamber (Elutrap; Schleicher & Schuell,Inc., Keene, N.H.) as specified by the manufacturer. Thisgel-purified HN protein was used for immunization of rabbitsfor antibody production as described previously (50). Thepurified immunoglobulin G (IgG) fraction of rabbit poly-clonal antisera raised against fetuin-Sepharose affinity-purified SV5 HN (anti-HN IgG) was that used previously(35, 36). Conformation-specific mouse monoclonal antibod-ies (MAbs) specific to HN (HN-lb and HN-4b) were thosedescribed previously (40, 49) and were generously madeavailable by Rick E. Randall, University of St. Andrews, St.Andrews, Scotland. A rat monoclonal antibody (anti-BiP),specific for the cellular heavy-chain binding protein (BiP),was kindly provided by Linda Hendershot, St. Jude Chil-dren's Research Hospital, Memphis, Tenn., and John F.Kearney, University of Alabama, Birmingham (2). All anti-sera described were titrated by using an immunoprecipita-tion assay and were used in all experiments under conditionsof antibody excess.

Expression of mutant proteins and metabolic labeling ofcells. Recombinant SV40-infected CV-1 cells were used in allbiochemical studies unless otherwise stated. Recombinantvirus stocks were prepared essentially as described previ-ously (42). For virus infections, subconfluent monolayers ofCV-1 cells were washed in phosphate-buffered saline andinfected with recombinant virus in DME for 2 h at 370C. Thecells were then washed with DME, and the medium wasreplaced with DME supplemented with 2% FCS and main-tained at 37°C in an atmosphere containing 5% CO2. Meta-bolic labeling was typically carried out 42 to 44 h postinfec-tion (p.i.). Cells were washed twice in phosphate-bufferedsaline, incubated with cysteine- and methionine-deficient

MOL. CELL. BIOL.

ROLE OF HN GLYCOSYLATION 1991

DME (DME Cys-/Met-) for 30 min, labeled with 25 to 50,uCi of Tran[35S]-label (ICN Radiochemicals, Irvine, Calif.)in DME Cys-/Met-, and incubated at 37°C for the timesindicated. In procedures requiring a cold chase, the labelingmedium was removed and replaced with prewarmed chasemedium consisting of DME, 2 mM unlabeled L-cysteine andL-methionine, and 2% fetal calf serum. The chase period wasterminated by washing cells in ice-cold phosphate-bufferedsaline followed by lysis in an appropriate detergent buffer forimmunoprecipitation. In experiments in which expression ofmutant proteins was performed at 30°C, recombinant SV40-infected cells were shifted from 37 to 30°C at 24 h p.i.Metabolic labeling was done as described above, except thatthe cells were incubated at 30°C.Tunicamycin treatment of cells and endo H digestion. For

tunicamycin treatment, recombinant SV40-infected cellswere incubated with DME Cys-/Met- containing 2.5 ,ug oftunicamycin per ml for 30 min prior to and during radioactivelabeling. For endo-o-N-acetylglucosaminidase H (endo H)digestions, recombinant SV40-infected cells were labeled for30 min and incubated with chase medium for 2 h. The cellswere then lysed with 1% SDS and immunoprecipitated withanti-HNSDs as described above. Endo H digestions weredone as described previously (43).

Sedimentation velocity centrifugation of HN glycosylationmutants. Recombinant SV40-infected cells were labeled for30 min with Tran[35S]-label and then incubated in chasemedium for 2 h. The cells were lysed in detergent buffer andsubjected to sedimentation velocity centrifugation as de-scribed previously (40).

Immunoprecipitation, SDS-PAGE, and quantitation of au-toradiograms. HN was immunoprecipitated from radioac-tively labeled recombinant SV40-infected cell lysates undernondenaturing (29) or denaturing (1) conditions as describedpreviously. HN was immunoprecipitated from sucrose gra-dient fractions and HN-GRP78-BiP complexes were copre-cipitated as described previously (40). SDS-PAGE was doneas described previously (28). Autoradiograms were quanti-tated by laser scanning densitometry on variable exposuresof each gel to ensure that they were within the linear range ofthe film. Densitometry and integration were performed asdescribed previously (20).

Indirect immunofluorescence. Indirect immunofluores-cence was done by using cover slips of recombinant SV40-infected CV-1 cells at 42 to 48 h p.i. as described previously(42).

RESULTS

Tunicamycin inhibition of HN glycosylation prevents theacquisition of reactivity to conformation-specific antibodies.MAbs that recognize conformational epitopes have beenuseful reagents in probing the folded states of proteins (3, 4,15, 37, 56). We have recently described two HN MAbs thatrecognize different folded forms of HN (40). MAb HN-lbbecomes reactive with a folded form of HN just before thepoint in HN maturation when oligomerization occurs. MAbHN-4b reacts only against the oligomeric form of HN. Bothepitopes recognized by the antibodies are found in the fullymature form of the protein, since both antibodies can neu-tralize viral infectivity and can immunoprecipitate HN fromvirions (49).Tunicamycin has been extensively used to study the

effects of synthesizing proteins in the absence of N-linkedglycosylation (reviewed in reference 11). Since the role ofN-linked glycosylation in protein folding has not been pre-

+DTT-TM +TM

I --. -1

-DTT-TM +TM

HN4

mom HN2

Np

HN ___NP

p

M

M 1 2 3 4 5 6 1 2 3 4 5 6 M

FIG. 1. The nonglycosylated form of HN is not recognized byconformation-specific MAbs. SV5-infected CV-1 cells were eitherincubated in the absence (-TM) or presence (+TM) of tunicamycin(2.5 ,ug/ml) for 1 h at 16 h p.i. The cells were then metabolicallylabeled with Tran[35S]-label for 1 h and incubated in chase mediumfor 2 h, in the absence or presence of tunicamycin. Lysates wereimmunoprecipitated with anti-HN IgG (lanes 1 and 4), MAb HN-lb(lanes 2 and 5), or MAb HN-4b (lanes 3 and 6). Polypeptides wereanalyzed by SDS-PAGE under reducing (+DTT) or nonreducing(-DTT) conditions. The arrowhead indicates the top of the gel.Abbreviations: HN1, HN monomers; HN2, HN dimers; HN4, HNtetramers. Lane M, SV5 viral proteins as molecular weight markers:HN, 66,000; NP, 61,000; P, 44,000; M, 38,000. *, position ofunglycosylated HN immunoprecipitated by the anti-HN IgG. In lane4, the band (Mr 44,000) migrating beneath unglycosylated HN isthe unglycosylated SV5 polypeptide Fo, which has a molecularweight of 44,000 (42), and it coprecipitated owing to illegitimateintermolecular disulfide bonds formed during aggregation of theunglycosylated polypeptide (data not shown).

viously reported for the SV5 HN glycoprotein, we wereinterested in the consequences of synthesizing the wild-typeprotein in the absence of carbohydrate addition. CV-1 cellswere infected with SV5 and metabolically labeled for 1 hwith Tran[35S]-label in the absence or presence of tunicamy-cin (2.5 ,ug/ml), and the cells were incubated in chasemedium for 2 h to allow for the maturation of labeled HNmolecules. Detergent lysates were made, and aliquots wereimmunoprecipitated with anti-HN IgG, MAb HN-lb, orMAb HN-4b and analyzed by SDS-PAGE in the absence orpresence of the reducing agent (dithiothreitol). GlycosylatedHN (tunicamycin absent) was fully reactive with bothMAbs, whereas nonglycosylated HN (tunicamycin present)could be immunoprecipitated with the polyclonal anti-HNIgG but not with either of the MAbs, suggesting that thesemolecules were misfolded (Fig. 1). Analysis of the samesamples under nonreducing conditions showed that glycosy-lated HN (Fig. 1, lane 1, -DTT) could be found primarily asdisulfide-linked dimers (HN2) and tetramers (HN4). In com-parison, unglycosylated HN was found only at the top of thegel, suggesting that it was in the form of disulfide-linkedaggregates that were too large to be resolved in the gel (Fig.1, lane 4, -DTT), as was found previously for inappropri-ately disulfide-linked aggregates of the VSV G protein (7, 16,33). Thus, the lack of glycosylation on HN affects its abilityto acquire reactivity to the conformation-specific antibodies

VOL. 10, 1990

1992 NG ET AL.

and prevents normal intermolecular disulfide bond forma-tion.

Determination of functional N-linked glycosylation sites inHN by expression of site-specific mutants: construction of apanel of glycosylation mutants. The results obtained byexpressing HN in the presence of the glycosylation inhibitortunicamycin indicated that N-linked carbohydrate addition isrequired for proper folding, but it could not be inferred howindividual chains contributed to this process. To address thisquestion, we constructed a panel of HN site-directed mu-tants in which consensus sequences for N-linked glycosyl-ation were altered singly and in various combinations.However, before a panel of glycosylation mutants could beconstructed, it was first necessary to determine the sitesnormally used for carbohydrate addition. The deduced poly-peptide sequence of HN predicts a protein of 565 aminoacids that contains six potential sites for the addition ofN-linked glycosylation in the form of Asn-X-Ser/Thr, whereX is any amino acid (Fig. 2, HNWT) (21). Two of these sites,at HN residues 471 and 497, contain proline in the variableposition (X), and sites of this type are generally unused (26).To determine the sites used for N-linked glycosylation inHN, we used oligonucleotide-directed mutagenesis on acDNA clone to eliminate each of the potential sites includingpositions 471 and 497 in the unlikely event that they wereused. The asparagine-encoding codons at each site werereplaced with seine-encoding codons, since an asparagine-to-serine change is fairly conservative owing to the similarityin bulk and polar nature of the side chains. Six single-sitemutants were generated, and the coding sequences of eachwere inserted into the SV40-based expression vector,pSV103, such that HN could be expressed under the controlof the SV40 late-region promoter. Recombinant virus stockswere made for each glycosylation mutant in CV-1 cells, andthe derivation of the nomenclature for each mutant, suchthat the glycosylation sites used are identified, is describedin Materials and Methods (see also Fig. 2).The expression of the HN glycosylation mutants was

examined by labeling recombinant virus-infected CV-1 cellsat 42 to 44 h p.i. with Tran[35S]-label for 2 h in the presenceor absence of the glycosylation inhibitor tunicamycin. Poly-peptides were immunoprecipitated with HN-specific poly-clonal antisera and analyzed by SDS-PAGE and fluorog-raphy (Fig. 3A). The glycosylation-site mutant polypeptidesHNg234, HNg134, HNg124, and HNg123 (asparagine-to-serine change at positions 110, 139, 267, and 504, respec-tively) showed an increased rate of mobility compared withthe wild-type HN (Fig. 3A, -TM). Synthesis of these mutantpolypeptide in the presence of tunicamycin (Fig. 3A, +TM),indicates that the differences in mobility were caused bydifferences in carbohydrate addition, since the mutants allshow the same mobility as unglycosylated wild-type HN inthe absence of glycosylation. This provides evidence thatsites gl, g2, g3, and g4 are normally used for N-linkedglycosylation. Deletion of the carbohydrate attachment sitesat positions 471 and 497, as expected, did not cause a changein mobility of HN on SDS-PAGE compared with wild-typeHN (Fig. 3A, HNgP1- and HNgP2-), suggesting that thosesites are not glycosylated. These HNgPl- and HNgP2-mutants will not be described further in this report, but theywere examined in further assays described here and werefound to be indistinguishable in every way from the parentalHN, which suggests that at least in these positions thechange from asparagine to serine does not have a deleteriouseffect on the integrity of the HN protein. With the knowledgethat four sites in HN are used for N-linked glycosylation,

HNWT

HNg234

HN91 34

HNg1 24

HNgl 23

HNg34

HNgl2

HNg1

HNg2

HNg3

HNg4

gl92 g3 g4NH2 y y y COOHI* 11013 - 7 295 I

S/A P1 P2

YY Y

I--___

- l1

Y

HNgOFIG. 2. Schematic representation of HN glycosylation mutants

generated by site-directed mutagenesis. The HN wild-type (HNWT)protein is shown within the box at the top of the figure. Symbols:iz*, 565-amino-acid polypeptide chain; , N-terminal signalsequence/transmembrane anchor (S/A); ', functional N-linked gly-cosylation sites; the residue number indicates the Asn residue in theHN amino acid sequence. The designations of the carbohydratechains (gl, g2, g3, and g4) are indicated above each site. The arrowsmark the unused sites, P1 and P2, at positions 471 and 497,respectively. HN glycosylation mutants are represented in a similarfashion, with the designated name shown at the left of each diagram.The functional glycosylation sites are indicated by ?.

further mutants in which two, three, or all four sites wereinactivated were made as described in Materials and Meth-ods and shown schematically represented in Fig. 2. Thenucleotide sequence of all the mutants was verified bothbefore and after the construction of each respective expres-sion vector. Expression of the double, triple, and quadrupleglycosylation-site mutant polypeptides showed shifts in elec-trophoretic mobility compared with wild-type HN (Fig. 3Band C, -TM) commensurate with the deletion of two, three,or all four carbohydrate chains. As expected, when themutant polypeptides were synthesized in the presence oftunicamycin (Fig. 3B and C, +TM), they all showed thesame gel mobility as nonglycosylated wild-type HN, indicat-ing that the mobility differences seen in the absence oftunicamycin are caused by differences in glycosylation. Theslight differences in electrophoretic mobility seen betweenHNg34 and HNg12 are due to differences in carbohydratetrimming and modification (see Table 1).

Conformation-specific antibody reactivity of the HNglycosylation mutants. To study how individual carbohydrate

MOL. CELL. BIOL.

ROLE OF HN GLYCOSYLATION 1993

A. -TM

-~ ~ ~ ~ T

-~~~~~~~~~~~~~~~~~~~~~~~~~~~~~. N "qZ

B -ThTM +TMA* =

~

*.!40' vA+ #:,pv+i

C - M-TM.

k

+T M

a m so

+TM

FIG. 3. Expression of HN glycosylation mutants. RecombinantSV40-infected cells were metabolically labeled at 42 h p.i. withTran[35S]-label for 2 h in the absence (-TM) or presence (+TM) oftunicamycin (2.5 p.g/ml). Detergent lysates of labeled proteins were

immunoprecipitated with anti-HNSDS serum and analyzed by SDS-PAGE and fluorography. (A) Single-site mutants. (B) Double-sitemutants. (C) Triple- and quadruple-site mutants. Only the relevantsection of the autoradiogram is shown.

chains contribute to the process of folding, HN mutantrecombinant SV40-infected cells were metabolically labeledfor 30 min, further incubated in chase medium for 2 h, andimmunoprecipitated with either polyclonal anti-HN IgG or

MAb HN-lb or HN-4b. The polypeptides were analyzed bySDS-PAGE, and autoradiograms were quantitated by densi-

tometry. To measure the immunological reactivity of themutant proteins, we compared the amount immunoprecipi-tated by the conformation-specific antibodies with that pre-cipitated by HN polyclonal antiserum; the results are shownin Table 1. Inactivation of a single glycosylation site atposition gl, g2, or g4 had no significant effect on the abilityof HN to fold normally, whereas inactivation of glycosyl-ation site g3 (HNg124) prevented about 70% of the mutantmolecules from folding such that they gained full reactivitywith MAb HN-4b. Thus, one of the four carbohydrate chainsappears to play a critical role in HN folding. When thedouble mutant HNg12 was examined, it was found that theHNg12 protein could not be recognized by the MAbs,suggesting that the effect of carbohydrate chains is additive.In comparison, the reciprocal double mutant HNg34 wasfound to gain nearly full MAb reactivity. Analysis of thetriple mutants in which each contained a single functionalglycosylation site indicated that the presence of the gl, g2, or

g4 carbohydrate chain alone was insufficient to gain MAbreactivity. However, in the HNg3 mutant which has only theg3 carbohydrate chain, approximately 10% of the moleculesacquired MAb reactivity. This result further suggests an

important role for the g3 carbohydrate site in folding of HN,but it also indicates that other carbohydrate chains contrib-ute to the folding process. The unglycosylated mutant,HNgO, like HN synthesized in the presence of TM, was notreactive with the MAbs.

Oligomeric form of the engineered HN glycosylation mu-

tants. Recently, it has been shown that HN assemblesintracellularly into homotetramers, made up primarily of twononcovalently associated disulfide-linked dimers, and thatMAb HN-4b is specific for the oligomeric form (40). Al-though the data presented in Table 1 on the reactivity of themutant proteins with MAb HN-4b are indicative of theextent of oligomerization, it was also necessary to examinethe assembly of the molecules biochemically, since it wasnot known whether the mutations may result in oligomericforms potentially reactive to MAb HN-4b other than tetram-ers (e.g., dimers). Recombinant virus-infected cells were

labeled with Tran[35S]-label for 30 min at 37°C and incubated

TABLE 1. MAb reactivity, endo H sensitivity, and cell surface expression of wild-type and mutant proteins37°C 300C

Protein MAb reactivitya Endo H Cell surface MAb reactivity Endo H Cell surface

HN-lb HN-4b sensitivity' expression' HN-lb HN-4b sensitivity expression

HNWT ++++ ++++ R Yes ++++ ++++ R YesHNg234 ++++ ++++ R Yes ++++ ++++ R YesHNg134 ++++ ++++ R Yes ++++ ++++ R YesHNg124 ++ ++ R Yes +++ +++ R YesHNg123 ++++ ++++ R Yes ++++ ++++ R YesHNg34 +++ +++ R Yes +++ +++ R YesHNg12 - - S No + + + + R YesHNgl - - S No + + R YesHNg2 - - S No + + R YesHNg3 + + R Yes + + + + R YesHNg4 - - S No + + S YesHNgO - - NA No - - NA No

a For MAb reactivity assays, recombinant SV40-infected cells were labeled with Tran[35S]-label for 30 min and incubated in chase medium for 2 h. Cells werelysed in RIPA buffer, and aliquots were immunoprecipitated with the polyclonal HNSDs serum or MAb HN-lb or HN-4b. Polypeptides were analyzed bySDS-PAGE, and autoradiograms were quantitated by laser-scanning densitometry. The data are presented as the amount immunoprecipitated by the MAbsrelative to that precipitated by the polyclonal HNSDs serum: + + + +, 70 to 100%; + + +, 40 to 70%o; + +, 20 to 40%o; +, 5 to 20%o; -, <5%.

b To determine the sensitivity of the HN carbohydrate chains to digestion with endo H, recombinant virus-infected cells were metabolically labeled as describedabove and immunoprecipitated HN was digested with endo H as described in Materials and Methods. Abbreviations: S, carbohydrate chains were sensitive toendo H digestion; R, some carbohydrate chains were detected to be resistant to endo H digestion; NA, not applicable.

c Cell surface expression was analyzed by indirect immunofluorescence with both rabbit polyclonal anti-HN IgG and MAb HN-4b.

VOL. 10, 1990

14-,I N 0-,

41:. #.'+ ++"., ++""'0 xx ",

1994 NG ET AL.

A HNWT

.m 2 4 '-.91

t)'4 t

. 11-

HN2-

:4 :- -.-. ;4 NA

B HNg234i.

mHN

'. 2 ,_

*

":- HNg3--M

N -1

t,

_ _

H

NP-FSs1

I 14 r M!I.1

-

Ff_S

ffir

-

It.~-

FIG. 4. Oligomerization of HN glycosylation mutants analyzed by sucrose density gradient velocity sedimentation. Recombinantvirus-infected cells at 42 h p.i. were metabolically labeled for 30 min with Tran[35S]-label incubated in chase medium for 2 h to allow forassembly and maturation of labeled proteins. Detergent lysates were subjected to centrifugation on 7.5 to 22.5% sucrose gradients at 180,000x g for 20 h. Fractions (24 0.5-ml fractions) were collected from the bottom of each gradient, and alternate fractions were immunoprecipitatedwith anti-HNSDS serum. Immunoprecipitated proteins were analyzed by SDS-PAGE under nonreducing conditions. Fraction numbers are

indicated at the top of each autoradiogram. (A) Wild-type HN; (B) HNg234; (C) HNg3; (D) HNgl. Symbols: l, tops of gels; <-, oligomericfraction of the proteins; ¢, positions of the HNg234 aggregates. Lanes M, SV5 protein markers.

with chase medium for 2 h, and the oligomeric form of HNwas analyzed by sucrose density gradient centrifugation.Fractions were collected from the bottom of the gradients,alternate fractions were then immunoprecipitated with anti-HNSDS sera, and polypeptides were analyzed by SDS-PAGEunder nonreducing conditions.The mature HN oligomer sediments largely in a single

peak (Fig. 4, fractions 6 to 8) which is thought to be a

tetramer, and on SDS-PAGE analysis this species consists ofdisulfide-linked dimers and tetramers (Fig. 4A, HN2 andHN4, respectively) (40). A small amodnt ofHN dimer whichresults from the centrifugation conditions sediments in frac-tion 12. Immature HN monomers which are precursors tothe HN oligomer sediment in fractions 12 to 16 (40).When the HN glycosylation mutants were examined for

their oligomeric form by sucrose density sedimentation

analysis, four categories of sedimentation behavior wereidentified; an example of each category is shown in Fig. 4.Mutants HNg134 and HNg123, which were fully reactivewith MAb HN4b at 37°C (Table 1), had a wild-type HNsedimentation pattern (Fig. 4A). However, two mutantswhich lacked the gl carbohydrate chain (HNg234 andHNg34) and were fully reactive with MAb HN-4b exhibiteda bimodal sedimentation pattern. For mutant HNg234, themajority of the protein sedimented as a wild-type oligomer(Fig. 4B, fractions 6 to 8), but some of the protein alsosedimented to the bottom of the gradient. Since this latterspecies exhibited the normal pattern of intermolecular disul-fide bond formation (i.e., it consisted mostly of dimers andtetramers), these data suggest that this fast-sedimenting HNmaterial is a higher multimer of properly folded oligomersthat have formed a noncovalent association. The available

20 2 2?4 MA

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*rIi' N 7o

ROLE OF HN GLYCOSYLATION 1995

evidence suggests that this form of oligomer aggregationoccurs in vivo and is not an artifact of the experimentalprocedure, since the higher-multimer oligomer forms (frac-tion 2) have carbohydrate chains that are largely sensitive toendo H digestion, whereas under the same conditions theoligomer form is largely endo H resistant (data not shown).These data suggest that the gl oligosaccharide chain mayplay a role in preventing the aggregation of HN oligomers inthe ER membrane.Two mutant HN proteins (HNg124 and HNg3), both of

which exhibited a reduced reactivity to MAb HN-4b at 37°C,were found to sediment in two forms (Fig. 4C). One was

identified on SDS-PAGE as a species that sedimented infraction 8 and had formed the usual intermolecular disulfidebonds, and the other species sedimented to the bottom of thegradient and, on gel electrophoresis, behaved as an aggre-

gate which was unable to enter the separating gel. Althoughwild-type HN also showed a small amount of this latterspecies, with HNg3 and HNg124 there was a large change inthe ratio from the amount of the normal oligomer to theincreased amounts of the disulfide-linked aggregate, and thisis the important difference. Mutants HNg12, HNgl, HNg2,HNg4, and HNgO, which did not gain any reactivity with theMAb at 37°C (Table 1), sedimented as a broad peak at thebottom of the gradient, and since this material remained atthe top of the polyacrylamide gel during electrophoresis, thisis again consistent with its being a disulfide-linked aggregate(Fig. 4D).Reduced temperature enhances HN glycosylation mutant

protein folding. It has been found for some proteins that thedeleterious effects of inhibiting glycosylation, either bytunicamycin treatment or by mutagenesis, on protein foldingand intracellular transport can be partially alleviated by a

reduction in temperature (14, 16, 33). This apparent temper-ature dependence may be due in part to an increase ininappropriate hydrophobic interactions at the higher temper-ature, resulting in improper polypeptide folding (25).To examine the effect of temperature on the folding of the

HN glycosylation mutants, we shifted HN mutant recombi-nant virus-infected cells from 37 to 30°C at 24 h p.i. At 44 hp.i. the cells were labeled with Tran[35S]-label for 30 min at30°C and then incubated in chase medium for an additional 2h to allow folding of the labeled molecules. Cell lysates were

immunoprecipitated with the polyclonal anti-HN IgG or

MAbs HN-lb and HN-4b, and the polypeptides were ana-

lyzed on SDS-PAGE. The amount of HN immunoprecipi-tated by the MAbs was compared with that immunoprecip-itated by the polyclonal anti-HN IgG (Table 1, 30°Ccolumns). For the wild-type HN and the mutants proficientin folding at 37°C, there was little change in MAb reactivityat 30°C. However, with the HN glycosylation mutants thatwere considered to be defective in folding there was a

significant increase in MAb reactivity at the reduced temper-ature. HNg124 showed an increase in MAb reactivity ofalmost twofold with both MAbs, such that about half themolecules synthesized at 30°C were properly folded com-

pared with about 30% at 37°C. The reciprocal construct, thetriple mutant HNg3, showed a similar increase in foldingefficiency, with approximately 30% reactive to the MAbs at30°C. The most dramatic effect of the temperature shift camefrom mutants that were unreactive with the MAb whensynthesized at 37°C (Table 1, 37°C, HNg12, HNgl, HNg2,and HNg4). When they were synthesized at 30°C a substan-tial fraction of each mutant protein acquired reactivity to theMAbs. However, the nonglycosylated mutant HNgO, whensynthesized at 30°C, did not acquire any reactivity with the

MAbs, suggesting that it is completely defective in foldingeven at the lower temperature. This temperature-sensitivephenotype of many of the glycosylation mutants is furtherevidence that the role of N-linked carbohydrates on HN is toaid in the proper folding of the molecule.

Intracellular transport of HN glycosylation mutants. Sev-eral studies have provided evidence that proper folding andoligomerization may be prerequisites for transport of integralmembrane proteins from the ER (3, 4, 15, 27). To determinewhether the HN glycosylation mutants acquired resistanceto endo H digestion, indicative of the conversion of carbo-hydrate chains from the simple to the complex form in themedial Golgi complex, recombinant-virus-infected cellswere labeled with Tran[35S]-label for 30 min at 37 or 30°Cand the incubation was continued at these temperatures inchase medium for 3 h to allow maturation and transport ofmutant molecules. HN proteins were immunoprecipitatedwith the anti-HNSDS serum, digested with endo H, andanalyzed by SDS-PAGE. All HN glycosylation mutants thathad a percentage of their molecules converted to the endoH-resistant form were scored as positive. The amount ofendo H-resistant protein and the rate at which it becomesendo H resistant were not quantitated, because not all HNglycan chains become converted to the complex type (40),and HN molecules transported to the cell surface are inter-nalized and degraded (40). Therefore, such an analysis of therates of transport of the different mutants would be mislead-ing. There was a good correlation between the acquisition ofreactivity for the MAbs (representing folding and oligomer-ization [40]) and the conversion of some HN glycan chains tothe endo H-resistant form (Table 1), indicating intracellulartransport from the ER to the medial Golgi complex. Theexception was mutant HNg4, which at 30°C had some MAbreactivity but no endo H-resistant form. However, this doesnot necessarily indicate a lack of intracellular transport,because, as discussed above, this HN carbohydrate chainmay always be the high-mannose form (40).

Subcellular localization of HN glycosylation mutants. Todetermine the subcellular localization of the HN glycosyl-ation mutants, we performed indirect immunofluorescenceon fixed or fixed and permeabilized recombinant virus-infected CV-1 cells by using the polyclonal anti-HN IgG andfluorescein isothiocyanate-conjugated goat anti-rabbit IgG.Wild-type HN staining was readily detectable on the surfaceof cells (Fig. 5A), and the HN internal staining pattern showsGolgi apparatus-like staining (Fig. 5B). In addition, somelocalization to endosomes and lysosomes could be seen (Fig.5B), and we have shown previously that HN expressed atthe cell surface is extensively internalized and degraded (40).In comparison, the unglycosylated mutant HNgO showed nosurface staining (Fig. SC), and the internal staining patternshowed that HNgO was localized to an extensive reticularstructure characteristic of the ER (Fig. SD). The results ofthe indirect-immunofluorescence analysis for the entirepanel of mutants analyzed after incubation of the recombi-nant virus-infected cells for 20 h at 37 or 30°C are presentedin Table 1. If the characteristic punctate surface-stainingpattern of HN was observed, it was scored as positive, butthe intensity of staining was not quantitated owing to theextensive internalization of wild-type HN from the cellsurface (40). The extensive internalization of HN also pre-cluded the determination of a meaningful level of cell surfaceexpression by methods such as iodination of HN or mea-surement of the amount of antibody bound to cell surfaces.We found that HN glycosylation mutants that were synthe-sized at 37°C, that had some molecules recognizable by the

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1996 NG ET AL.

FIG. 5. Indirect immunofluorescence of HN and nonglycosylated HNgO. Recombinant SV40-infected CV-1 cells on cover slips were fixedin 0.5% formaldehyde-phosphate-buffered saline for cell surface fluorescence (A and C) or fixed and then permeabilized in acetone for internalfluorescence (B and D). HN was stained with anti-HN IgG followed by fluorescein isothiocyanate-conjugated goat anti-rabbit IgG. Panels Aand B contain cells infected with SV40-HN. Panels C and D contain cells infected with SV40-HNgO. Bar, 10 ,um.

MAbs (HNg234, HNg134, HNg124, HNg123, HNg34, andHNg3), and that acquired some degree of endo H-resistantcarbohydrate chains could be detected at the cell surface.HN glycosylation mutants that were completely defective infolding at 37°C (HNg12, HNgl, HNg2, HNg4, and HNgO)did not express detectable levels of HN at the cell surface.However, when the temperature was lowered to 30°C, whichincreased the ability of these mutant polypeptides to foldnormally (except HNgO), they could be detected at the cellsurface. These studies indicate that the ability of HN glyco-sylation mutant molecules to be transported through theexocytic pathway is not dependent on the presence of anyspecific carbohydrate chain but on the ability of the mole-cules to acquire a native conformation.

Segregation of folded and misfolded HN proteins in the samecell. From the folding properties of the glycosylation mutants(summarized in Table 1, 37°C columns), the molecules canbe categorized into three groups: folding competent (wild-type HN, HNg234, HNg134, HNg123, and HNg34), partiallyfolding defective (HNg124 and HNg3), and folding defective(HNg12, HNgl, HNg2, HNg4, and HNgO). To determinewhether the subceilular localizations of folded and unfoldedHN molecules of a given mutant could be distinguished in acell, we performed double-label indirect immunofluores-cence by first binding the conformation-specific mouse MAbHN-4b to properly folded molecules and then binding rabbitpolyclonal anti-HN IgG that recognizes all forms of HN. A

mixture of rhodamine-conjugated goat anti-mouse IgG andfluorescein isothiocyanate-conjugated goat anti-rabbit IgGwas then added to the cells to stain the two species of boundantibodies. All the HN glycosylation mutants were exam-ined, and representative examples of molecules that werefolding competent (wild-type HN; Fig. 6A and B), partiallyfolding defective (HNg124; Fig. 6C and D), and foldingdefective (HNgl; Fig. 6E and F) are shown in Fig. 6. Thesubcellular distribution of folded wild-type HN moleculesstained with the MAb (Fig. 6A) and the staining pattern withthe anti-HN IgG (Fig. 6B) were coincident and showedstaining of the Golgi-like structure and endosomal and lysos-omal vesicles (40) (see above). Mutant HNg124 expressedabout 30% of its molecules in the folded form at 37°C (asdetermined by immunoprecipitation with MAb HN-4b), andthe conformation-specific MAb HN-4b staining showed asimilar but less intense pattern to wild-type HN of Golgi-likestaining together with staining of numerous intracellularvesicles (Fig. 6C). In addition to this staining pattern, thepolyclonal anti-HN IgG staining showed an extensive retic-ular structure that is characteristic of the ER (Fig. 6D). Weinterpret the ER pattern to represent misfolded moleculesthat are not recognized by the monoclonal antisera. MutantHNgl, which is folding defective, showed no staining withthe conformation-specific antibody (Fig. 6E) but, as ex-pected, showed extensive ER-like staining with the poly-clonal antibody (Fig. 6F). Thus, these data confirm that only

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ROLE OF HN GLYCOSYLATION 1997

FIG. 6. Localization of folded and nonfolded forms of HN glycosylation mutants by double-label indirect immunofluorescence.Recombinant SV40-infected CV-1 cells grown on cover slips were fixed and permeabilized at 42 h p.i. The oligomer-specific MAb HN-4b wasthen bound to infected cells. After the cells had been washed, the polyclonal anti-HN IgG was added to the cells. The bound antibodies werethen stained with a mixture of fluorescein-conjugated goat anti-rabbit IgG and rhodamine-conjugated goat anti-mouse IgG. (A and B)Wild-type HN; (C and D) HNg124; (E and F) HNgl. Micrographs were taken of the same field by using fluorescein optics (panels A, C, andE) or rhodamine optics (panels B, D, and F). Bar, 10 Fm.

folded HN molecules are transported through the exocyticpathway, and they indicate that both folded and misfoldedmolecules can exist in the same cell. Interestingly, misfoldedmolecules do not seem to prevent transport of the foldedmolecules, since the MAb staining did not show an ERpattern.The extent of HN glycosylation affects the nature of associ-

ation with GRP78-BiP. We have shown previously that theresident ER protein GRP78-BiP associates with SV5 HNconcomitant with or shortly after synthesis and that this

complex dissociates when HN reaches a folded conforma-tion just prior to oligomerization (40). It was proposed thatGRP78-BiP functions to retain HN in the ER until therelatively slow process of folding and oligomerization wascompleted, since it was found that only folded HN oligomerswere transported to the Golgi complex. Another proposedfunction for GRP78-BiP is that it associates stably withmisfolded molecules and retains them in the ER until themolecules can properly refold or be degraded (8, 15, 23, 24,38). Since we have shown above that defects in HN glyco-

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1998 NG ET AL.

M C 1 2 : 3-:

GHP78-B,P

HNNP _

do

FIG. 7. HN glycosylation mutants are associated with GRP78-BiP. Recombinant virus-infected cells were metabolically steady-state labeled with Tran[35S]-label at 24 h p.i. for 20 h. Cells werelysed under ATP-depleting conditions, and lysates were immuno-precipitated with anti-HN IgG. Immunoprecipitated proteins wereanalyzed by SDS-PAGE. Lanes: M, SV5 protein markers; U, directradioisotopically labeled lysate from untreated cells; I, direct radio-isotopically labeled lysate from cells treated with tunicamycin (2.5,ug/ml) to induce GRP78-BiP expression; 1 to 12; HNWT, HNg234,HNg134, HNg124, HNg123, HNg34, HNg12, HNgl, HNg2, HNg3,HNg4, and HNgO, respectively.

sylation affect the folding and transport of mutant moleculesto various degrees, we were interested in determining howthese mutations affected the interaction with GRP78-BiP.Recombinant virus-infected CV-1 cells were metabolically

labeled with Tran[35S]-label under steady-state conditions at24 h p.i. for 20 h. Labeled cells were lysed in detergent bufferunder ATP-depleting conditions, the HN proteins wereimmunoprecipitated with anti-HN IgG, and the polypeptideswere analyzed by SDS-PAGE. The anti-HN IgG coprecipi-tated GRP78-BiP with all the wild-type and mutant HN

proteins (Fig. 7). This association is specific, since we haveshown previously that the antiserum does not cross-reactwith GRP78-BiP in uninfected cells and that the complex canbe completely disrupted by the addition of 1 mM ATP (40;data not shown).To determine whether the association between GRP78-

BiP and the mutant HN proteins was transient or stable, wemetabolically pulse-labeled recombinant virus-infected cellsfor 15 min and then incubated them in chase medium for 0 or90 min. We have shown previously that in 90 min, 95% ofpulse-labeled wild-type HN molecules dissociate fromGRP78-BiP during maturation (40). Lysates of cells wereimmunoprecipitated with the anti-BiP MAb, and the poly-peptides were analyzed by SDS-PAGE. Wild-type HN co-precipitates with GRP78-BiP following the pulse (Fig. 8, lanelp, arrowhead), and most of HN has dissociated fromGRP78-BiP following the chase period (lane lc). Similarly,HN mutants that are folding competent (HNg234, HNg134,and HNg123; lanes 2, 3, and 5, respectively) showed amarked decrease in their ability to be coprecipitated with theanti-BiP MAb following the chase period (compare lanes pand c in each respective set). HNg124 and HNg3, which areboth partially folding-defective mutants, showed a slightdecrease in association with GRP78-BiP after the chaseperiod (lanes 4 and 10), but it was not as significant as for themolecules that can fold efficiently. HN glycosylation mu-tants that are defective in folding associated stably withGRP78-BiP over the course of the chase period (HNg12,HNgl, HNg2, HNg4, and HNgO; lanes 7, 8, 9, 11, and 12,respectively), and the complexes exist for >3 h (data notshown). These data are consistent with the view that theassociation between HN and GRP78-BiP is transient formolecules that fold properly but stable for mutants that foldimproperly. It is also interesting that the anti-BiP MAbcoprecipitated many cellular proteins that can be identifiedafter the pulse-label but not after the chase period (Fig. 8),suggesting that a transient association of BiP with moleculesin the ER is common to many newly synthesized proteins inthe exocytic pathway. The nature of the prominent polypep-tide (Mr 46,000) that can be seen in all lanes and seemingly

1 2 3 4 5 6j 7M U p - O c p p C t- - p c

6 9 10 11 12

P C p c p c p c p c

FIG. 8. HN is transiently associated with GRP78-BiP, but HN glycosylation mutants differ in the extent of their association. Recombinantvirus-infected CV-1 cells were either pulse-labeled with Tran[35S]-label for 15 min (lanes P) or pulse-labeled for 15 min and incubated in chasemedium for 90 min (lanes C) and lysed in detergent buffer under ATP-depleting conditions. Lysates were immunoprecipitated with anti-BiPMAb, and polypeptides were analyzed by SDS-PAGE. Lanes: M, SV5 protein markers; U, direct labeled-lysate from untreated cells; I, directlabeled lysate from cells treated with tunicamycin; 1 to 12: HNWT, HNg234, HNg134, HNg124, HNg123, HNg34, HNg12, HNg1, HNg2,HNg3, HNg4, and HNgO, respectively. Symbols: --

, position of HN proteins coprecipitated with GRP78-BiP; * position of GRP78-BiP.

GRP78- Bi P

HNNP

p

NI

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_

ROLE OF HN GLYCOSYLATION 1999

has a transient association with GRP78-BiP is not known,but it is unrelated to HN and is thought to be related to theSV40 infection (data not shown).

DISCUSSION

The importance of conformation in determining proteinstructure and function is well established. However, it hasrecently become evident that proper conformation and oli-gomerization of subunits may be prerequisites for directingthe transport of integral membrane proteins out of the ER.Studies involving the use of the viral proteins influenza virusHA, VSV G, and Rous sarcoma virus envelope glycoproteinhave indicated that folding and assembly occur in the ERprior to transport (3, 10, 15, 27). Furthermore, mutations inHA which caused the molecule to fold improperly resulted inits retention in the ER (15).A universal role for N-linked glycosylation has not yet

been identified, but it is becoming clear that one of its majorfunctions is in aiding protein folding. To examine the roles ofN-linked carbohydrates in the maturation of HN, we usedboth the glycosylation inhibitor tunicamycin and site-di-rected mutagenesis to alter the glycosylation patterns of thewild-type protein. The latter approach, which has the advan-tage of permitting the examination of specific oligosaccha-ride chains, has been used previously to study secretedproteins and class I integral membrane proteins includingVSV G (31-33), influenza B virus NB glycoprotein (55),human HLA-A2 heavy chain (52), human chorionic gonad-otropin (17, 34), and mouse IgA heavy chain (53). Theconclusions derived from these studies vary somewhat de-pending on the protein investigated, but the most common

finding was that a defect in transport occurred when carbo-hydrate addition sites were altered. The defects were

thought to be largely due to aberrant polypeptide folding,which caused the protein to be retained in the ER. The bulkyhydrophilic carbohydrate chains may aid protein folding bypreventing inappropriate hydrophobic interactions. How-ever, with HLA-A2 heavy chain the amino acid residues atthe carbohydrate addition site, rather than the glycan itself,are essential for proper transport (52), and some smallintegral membrane proteins, e.g., influenza virus NB, do notseem to need their normal carbohydrate component forintracellular transport and cell surface expression (55).There is also some degree of flexibility in the position on thepolypeptide backbone at which carbohydrate chains are

required, since it was found that the addition of new sites toa glycosylation-negative mutant of VSV G partially allevi-ated defects in folding and transport (7, 32). However,addition of supernumerary carbohydrate chains, e.g., forVSV G or influenza virus HA, can also have the oppositeeffect, by disrupting folding and transport (7, 14, 32).The data shown here for HN confirm and extend the

principles established for the roles of N-linked oligosaccha-rides discussed above. Deletion of the individual sites in HNfor N-linked glycosylation indicates that four sites are used(asparagine residues 110, 139, 267, and 504) and that aspar-

agine residues 471 and 497 in the sequence Asn-Pro-SerFThr,as expected, were not utilized. An earlier biochemical anal-ysis of the N-linked carbohydrate composition of SV5 HNreported that HN contained three oligosaccharide chains(47), which, for the methods used, is a close approximationto the four chains we found to be added. Both HN synthe-sized in the presence of the glycosylation inhibitor tunicamy-cin and the mutant HNgO that lacked glycosylation site werefound to be molecules that formed disulfide-linked aggre-

gates, that did not fold into a form recognizable by theconformation-specific MAbs, did not oligomerize, accumu-lated in the ER, and were not transported to the cell surface.The panel of glycosylation mutants from which one, two, orthree glycosylation sites were removed showed phenotypesthat could be divided into three major groups: (i) foldingcompetent (HNg234, HNg134, HNg123, and HNg34), (ii)partially folding defective such that a mixture of folded andunfolded forms was observed (HNg124 and HNg3), and (iii)folding defective (HNg12, HNgl, HNg2, and HNg4); how-ever, because of some restoration of folding at 30°C, thedefects do not appear to be as severe as found for unglyco-sylated HN. Since the equivalent phenotype was obtainedfrom HNgO as wild-type HN synthesized in the presence oftunicamycin and because similar phenotypes were obtainedfrom molecules containing different carbohydrate chains,coupled with the observed temperature sensitivity of foldingof many mutants, these data suggest that the presence orabsence of the carbohydrate chain is responsible for thefolding defects, rather than a direct role of the Asn-to-Serchange. By compilation of the phenotypes exhibited by thepanel of mutants, a direct correlation could be shownbetween the acquisition of antibody reactivity, the ability ofthe molecules to oligomerize, the acquisition of endo H-resistant carbohydrates, and their cell surface expression.The individual elimination of glycosylation site gl, g2, or

g4 had little effect on HN folding, which indicates theflexibility in the requirement of glycosylation. However,elimination of the g3 site from HN (HNg124) had a readilydetectable adverse effect on protein folding. The relativeimportance of glycosylation at the g3 site was emphasized bythe finding that when it was the only glycosylation site(HNg3), 10% of the molecules could fold at 37°C, in com-parison with the other mutants containing a single glycosyl-ation site, in which correct folding was not detected. How-ever, it is clear that the individual contribution of thecarbohydrate chains is synergistic in promoting native fold-ing. A differential effect of eliminating specific carbohydrateaddition sites has also been seen in other proteins, includingthe human chorionic gonadotropin a subunit (17, 34) anderythropoietin (9).HN glycosylation mutants that were misfolded were found

as disulfide-linked aggregates that sedimented to the bottomof sucrose density gradients. These types of aggregate havebeen observed previously with misfolded forms of the VSVG protein (7, 33). The misfolded HN aggregates are notthought to be artifacts of the isolation procedure, since aftera short pulse-label they form with time (data not shown), butit is not known whether these inappropriate disulfide bondswhich occur in the ER (13) are a cause or a consequence ofthe prolonged residence of the molecules in the ER.A second class of aggregate that sedimented to the bottom

of the sucrose gradients was observed with HN mutants thatlacked the gl carbohydrate chain (HNg234 and HNg34).These aggregates consisted of properly folded moleculeswhich, under nonreducing conditions on SDS-PAGE, sepa-rated into the usual dimeric and tetrameric forms, thusindicating that this aggregate is a cluster of noncovalentlyassociated oligomers. These data suggest that an additionalrole for the gl carbohydrate chain is to prevent aggregationof HN oligomers in the plane of the membrane, possibly byproviding a large hydrophilic group that prevents inappro-priate polypeptide surfaces from interacting.The data obtained with the partially folding-defective HN

molecules (HNg124 and HNg3), from double-label indirectimmunofluorescence experiments with MAbs to detect prop-

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2000 NG ET AL.

erly folded molecules and the polyclonal antisera to stain allforms of HN, indicated that within the same cell, foldedmolecules are sorted for intracellular transport whereasmisfolded molecules are retained in the ER. The detailedmechanism by which folded molecules are sorted for trans-port from immature and misfolded molecules in the ER isunknown, but some general principles are becoming clearer.The resident ER protein GRP78-BiP has been implicated as

having a role in the maturation and assembly of proteins (2,19, 38, 45) or in binding proteins that are aberrantly folded(8, 15, 23, 24, 30). GRP78-BiP was originally found associ-ated with immunoglobulin heavy chains in nonsecretingpre-B cell lines and with immunoglobulin precursors insecreting hybridomas and myelomas, but not with the com-

pletely assembled immunoglobulin (2, 18), which led to thehypothesis that GRP78-BiP prevented the transport of in-completely assembled immunoglobulin and ensured thatonly fully assembled proteins were transported (2, 19).However, there has been a paucity of data supporting a rolefor GRP78-BiP in the assembly of other molecules. Incontrast, several examples of a role of GRP78-BiP in bindingto aberrantly folded molecules have been identified; e.g.,GRP78-BiP associates with malfolded mutant molecules ofinfluenza virus HA (15), and these molecules are retained inthe ER until the HA molecule is slowly degraded (23).

Recently, we have shown that GRP78-BiP forms a com-

plex with immature HN molecules during or shortly aftertheir translocation across the ER membrane and that thecomplex dissociated when HN attains a folded conformationjust prior to oligomerization (40). Since only HN oligomersare transported intracellularly, we hypothesized that thespecific and transient interaction ofHN with GRP78-BiP was

analogous to that proposed for immunoglobulin. Thus,GRP78-BiP fulfills the criteria defined for a molecular chap-erone (12). Examination of the association of the HN glyco-sylation mutants with GRP78-BiP showed that the nature ofthe association was dependent on the ability of the moleculesto acquire MAb reactivity. Folding-competent HN mutants,as well as wild-type HN, had a transient association withGRP78-BiP, whereas folding-defective HN mutants formedan association with GRP78-BiP that was stable for longperiods (>3 h) (data not shown). Thus, these data indicatethat folding must be a primary factor in the release ofGRP78-BiP from HN and, conversely, that malfolded HNmolecules remain associated with GRP78-BiP.

ACKNOWLEDGMENTS

We thank Margaret Shaughnessy for excellent technical assis-

tance, Rick Randall for providing the HN-specific monoclonal,antibodies and Linda Hendershot and John Kearney for providingthe BiP-specific monoclonal antibody. We are grateful to Griffith D.

Parks and Reay G. Paterson for helpful discussions.This research was supported by Public Health Service research

award AI-23173 from the National Institute of Allergy and Infectious

Diseases. D.T.W.N. was supported by a National Institutes of

Health Training Program in Cell and Molecular Biology (GM-08061),and S.W.H. held an Abbott Laboratories Inc. Fellowship at North-

western University.

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