Folding and oligomerization properties of a soluble and secreted form of the paramyxovirus hemagglutinin-neuraminidase glycoprotein

VIROLOGY

178,499508

(1990)

Folding and Oligomerization Properties of a Soluble and Secreted Form of the Paramyxovirus Hemagglutinin-Neuraminidase Glycoprotein GRIFFITH Department

of Biochemistry,

Molecular

Biology Received

D. PARKS

AND

and Cell Biology,

ROBERT A. LAMB’ Northwestern

April 5, 1990; accepted

University,

Evanston,

Illinois

60208-3500

June 5, 1990

The paramyxovirus SV5 hemagglutinin-neuraminidase (HN) glycoprotein (a type II integral membrane protein) was converted into a soluble and secreted form (HN-F) by replacing the HN signal/anchor domain with a hydrophobic domain that can act as a cleavable signal sequence. Approximately 40% of the HN-F synthesized was secreted from cells (flla - 2.5-3 hr). The extracellular HN-F molecules were identified as disulfide-linked dimers and the majority of the population of molecules were resistant to endoglycosidase H digestion. Examination of the oligomeric form of the secreted HN-F, by sucrose density gradient sedimentation, indicated that under conditions where HN was a tetramer, HN-F was found to be a dimer, and no extracellular HN-F monomeric species could be detected. Secreted HN-F was fully reactive with conformation-specific monoclonal antibodies and was enzymatically active as shown by HN-F having neuraminidase activity. Examination of the intracellular HN-F species indicated that HN-F monomers were slowly converted to the disulfide-linked form and that under the sucrose density gradient sedimentation conditions used the HNF monomers aggregated. Some of the HN-F monomers were degraded intracellularly. These data are discussed in relationship to the seemingly different folding and oligomerization requirements for the intracellular transport of soluble and membrane bound forms of a glycoprotein. The soluble and biologically active form of HN may be suitable for further structural and enzymatic studies. 0 199OAcademic Press, I~C

INTRODUCTION

We are interested in determining whether the same constraints on folding and assembly found with anchored membrane proteins apply to soluble proteins. We have recently shown that a soluble form of the normally tetrameric neuraminidase (NA) membrane protein of influenza virus was efficiently secreted from cells (h - 90 min) as a mixture of monomers, dimers, and tetramers and only a small portion of the secreted NA was folded into a native conformation (Paterson and Lamb, 1990). In addition, in recent studies it has been found that when HA is converted from a membranebound to a soluble form, by deletion of its anchor domain, that the soluble HA can be secreted from cells in a monomeric form (Singh et al., 1990) implying that the requirement for oligomerization for intracellular transport applies only to the anchored form. However, the transport rate to the cell surface for the soluble HA (tl12 - 60 min) was considerably slower than for the membrane-bound form (t,,P - 25 min) (Copeland et al., 1986; Singh et a/., 1990). We have been studying the intracellular transport of the hemagglutinin-neuraminidase (HN) glycoprotein of the paramyxovirus simian virus 5 (SV5), which is a type II integral membrane protein that is expressed at the surface of infected cells. The SV5 HN contains a 17residue cytoplasmic tail, a 19-residue internal uncleaved signal/anchor (S/A) domain, and a 529-residue C-terminal ectodomain (Hieber-t et al., 1985; Paterson

Viral membrane glycoproteins have proven to be useful models for studying the intracellular transport of integral membrane proteins in the exocytotic pathway (reviewed by Rose and Doms, 1988). For example, studies with influenza virus hemagglutinin (HA), Rous sarcoma virus envelope glycoprotein (env), and vesicular stomatitis virus (VSV) G protein have indicated that proper folding toward a native conformation and oligomerization are sequential events and are prerequisite for transport of these proteins out of the endoplasmic reticulum (ER) (Gething et a/., 1986; Kreis and Lodish, 1986; Copeland et al., 1988; Doms et al., 1988; Einfeld and Hunter, 1988). Many mutant forms of these viral integral membrane proteins which do not fold or oligomerize properly exhibit a prolonged association with the resident ER protein GRP78-BiP, which prevents transport of the defective proteins from the ER (Pelham, 1986; Gething et a/., 1986; Hurtley et a/., 1989). Thus, the mechanisms controlling intracellular transport are very selective and it appears that integral membrane proteins must meet certain criteria to be competent for transport from the ER (Rose and Doms, 1988). ’ To whom correspondence and requests for reprints should be addressed: Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, 2153 Sheridan Road, Evanston, IL 60208-3500.

0042.6822/90

$3.00

CopyrIght 0 1990 by Academic Press, Inc. All rights of reproduction I” any form reserved.

498

SOLUBLE

AND

SECRETED

et a/., 1985). HN exists as a tetramer (Markwell and Fox, 1980) consisting of a pair of disulfide-linked dimers that form a mixture of noncovalently linked tetramers and disulfide-linked tetramers (Ng et al., 1989). It has been shown that the folding of HN toward a native structure and its assembly into a homooligomer occurs relatively slowly (tl12 - 25-30 min) and these processes preceed its transport from the ER (Ng et al., 1989). Mutants of HN with alterations in the ectodomain which disrupt native folding and oligomerization are blocked in intracellular transport in the ER (Ng et al., 1990) suggesting that native folding and assembly may be a prerequisite for the transport of HN out of the ER. We describe here properties of a soluble form of HN in which the uncleaved S/A domain was replaced by a cleavable signal sequence. MATERIALS

AND

METHODS

Plasmid construction and preparation of recombinant Sv40 virus. pSV103HN-G3Y and pSV103NAF were used as sources of DNA segments for the construction of pSVl03HN-F. In pSVl03HN-G3Y, the HNspecific cDNA insert had been altered by the conversion of bases 169-l 77, which encode residues 35-37 at the C-terminal end of the HN membrane spanning domain, to a Xhol site (Parks et al., 1989). pSVl03NAF contains a cDNA encoding the influenza A/Tokyo/3/67 (H2N2) virus neuraminidase (NA) protein which was altered by the replacement of the membrane spanning domain of NA with the fusion-related external domain (FRED) of the SV5 virus F protein as described (Paterson and Lamb, 1990). To construct the HN-F gene, two complementary oligonucleotides were used as a BamHI-Xhol linkeradaptor to join the Asp7 188BamHI DNA fragment encoding the NAF N-terminal and hydrophobic domains (bases l-l 18) to the Xhol-Pstl DNA segment encoding a portion of the HN ectodomain (bases 169-301) and these molecules were cloned into the Asp7 18 and Pstl sites of pGEM3. The resulting insert DNA encoded NAF residues l-38, three amino acids specified by the linker-adaptor (Leu-Asp-Val), and HN residues 3781. After confirmation of the correct construction by dideoxynucleotide chain-terminating sequencing (Sanger et a/., 1977), the NAF/HN hybrid DNA segment was excised and joined to a cDNA fragment encoding the remaining HN ectodomain residues (82-565) in the SV40 late region replacement vector pSV103 (Paterson eta/., 1985) to generate pSVl03HN-F. Oligonucleotrdes were synthesized by the Northwestern University Biotechnology Facility on a DNA synthesizer (Model 380B, Applied Biosystems Inc., Foster City, CA). pSV1 13LDH was constructed by inserting the en-

HN GLYCOPROTEIN

499

tire Smal fragment encoding the lactate dehydrogenase C4 open reading frame (Millan et al., 1987) into pSV1 13. Recombinant SV40 virus (designated SV-HNF) was produced by DEAE-dextran-mediated cotransfection of CV-1 cells with pSV103HN-F and DNA from an SV40 early region mutant (Pipas et a/., 1983). In vitro transcription and translation. The HN-F gene was subcloned into pGEM3 to yield pGEM3HN-F such that mRNA-sense transcripts could be generated using bacteriophage T, RNA polymerase promoter and T, RNA polymerase. DNA was linearized by HindIll digestion and synthetic RNA was synthesized using T, RNA polymerase in the presence of the cap analog 7mG(5’)ppp(5’)G as described (Hull et al., 1988). The resulting RNA was used to program translation in reticulocyte extracts as described previously (Thomas et al., 1988) and a fraction of the resulting sample was immunoprecipitated and analyzed by SDSPAGE as described below. Cells. Monolayer cultures of CV-1 cells were grown in Dulbecco’s modified Eagle’s medium (DME) con taining 10% fetal calf serum, as described previously (Lamb and Lai, 1982). Isotopic labeling of polypeptides, immunoprecipitation, endoglycosidase analysis, tunicamycin treatment, and polyacrylamide gel electrophoresis. CV-1 cells in fected with recombinant SV40 virus were radiolabeled between 40 and 48 hr postinfection with 100-400 &iI ml Tran[35S]-label (ICN Radiochemicals Inc., Irvine, CA) in DME lacking methionine and cysteine. Where indicated, cells were incubated in chase medium consisting of DME supplemented with 5 mM nonradioactive methronrne. Cells were solubilized by the addition of cold RIPA buffer (Lamb et a/., 1978). Immunoprecipitation of cell extracts using monoclonal antibodies (mAb) specific for SV5 HN protein (Randall et al., 1987) or polyclonal rabbit sera to denatured HN (HN antisera) was carried out as described (Ng er a/., 1989, 1990; Erickson and Blobel, 1979). lV-Acetyl-B-endoglucosaminidase H (endo H) digestion was as described previously (Parks and Lamb, 1990). For tunicamycin treatment, the drug (1 pg/mI) (Calbiochem-Behring Corp., La Jolla, CA) was added to cells 2 hr before and marntained during the labeling period. All samples were analyzed on 10% polyacrylamide gels followed by fluorography (Lamb and Choppin, 1976). Trypsin digestion and alkali extraction of microsomal membranes. Recombinant SV40 virus-infected cells were radiolabeled with 200 &i/ml Tran[35S]-label and crude microsomes isolated (Adams and Rose, 1985) and digested with trypsin or extracted under alkali conditions as described previously (Paterson and Lamb, 1987; Parks et al., 1989).

500

PARKS

Sucrose gradient sedimentation analysis of HIV-F. Proteins were analyzed by sucrose gradient centrifugation as previously described (Ng et al., 1989). For secreted proteins, CV-1 cells infected with recombinant SV40 virus expressing NAF (Paterson and Lamb, 1990) or HN-F were radiolabeled with Tran[35S]-label for 30 min and incubated in chase medium for 3 (NAF) or 6 hr (HN-F). Media (1 ml) was clarified (2 min, 14,000 g), adjusted to gradient conditions (30 mM Tris-HCI, 20 mM Mes, pH 5.0, 100 mM NaCI, 0.1% Triton X-l 00) by the addition of 1 OX buffer, and analyzed by sedimentation on a linear 7.5-22.50/o sucrose gradient as described previously (Ng et al., 1989; Parks and Lamb, 1990). For the analysis of intracellular HN and HN-F recombinant SV40 virus-infected cells were radiolabeled for 30 min with Tran[35S]-label and incubated for 2 hr in chase medium. Cells were lysed in gradient buffer supplemented with 1% Triton X-l 00 and 1% aprotinin (Sigma Chemical Co., St. Louis, MO), clarified by centrifugation (5 min, 14,000 g) and analyzed as described above. Assay for neuraminidase activity. Neuraminidase activity was determined essentially as described (Lentz et a/., 1987). Briefly, media was collected from mockinfected or SV-HN-F-infected cells between 48 and 72 hr postinfection. After clarification by centrifugation (5 min at 12,000 g), samples were dialyzed at 4” against 25 mM Na acetate, pH 4.5, concentrated approximately 1O-fold against solid polyethylene glycol 8000 and stored at 4”. Neuraminidase activity was assayed using the thiobarbituric acid method (Aminoff, 1961). Optimal conditions for extracellular HN-F neuraminidase activity included 20 mM Na acetate, pH 5.0 (Scheid eta/., 1972), and 100 pglml BSA. For mAb inhibition studies, concentrated medium (160 ~1) was incubated for 30 min at 37” in buffer (50 mM Na acetate, pH 5.0) containing 0.5 ~1 of mAb ascites fluid before assaying. RESULTS Construction

of the HN-F molecule

To examine the assembly and intracellular transport of a soluble form of the SV5 HN protein, an altered HN molecule was constructed in which the internal uncleaved S/A domain was replaced by a cleavable signal sequence. The hydrophobic domain (FRED) from the N-terminal region of the F,-subunit of the paramyxovirus fusion protein was used as a signal sequence because when this domain replaced the S/A domain of influenza virus neuraminidase (NA), the hybrid protein (NAF) was targeted to the lumen of the ER. The FRED was cleaved at a site predicted to be recognized by signal peptidase to generate a soluble form of NA that

AND

LAMS

HN

HN-F

NN

Nj&&, FRED

FIG. 1. Structure of HN and HN-F. The HN and HN-F polypeptides are shown schematically. The HN membrane spanning domain is indicated as a light-shaded box. For HN-F, the filled-in box represents the N-terminal six residues of influenza virus neuraminidase and the cross-hatched box represents the fusion-related external domain (FRED) from the hydrophobic N-terminus of the F, subunit of the SV5 F protein. The arrow indicates the predicted site of cleavage within the FRED sequence by signal peptidase, (or similar actrvity). which has been shown to be used in another related hybrid molecule (Paterson and Lamb, 1990). Vertical forks indicate known sites for Nlrnked glycosylatron within the HN ectodomain (Ng eta/., 1990).

was secreted from the cell (Paterson and Lamb, 1990). Thus, we anticipated that the FRED would function in a similar manner when joined to the HN ectodomain and would target HN to the ER lumen and yield a soluble form of HN. A cDNA molecule was constructed (HN-F, Fig. 1) that encodes the first 38 residues of NAF (including the FRED hydrophobic domain) linked to the ectodomain of HN. Three additional residues (Leu-Asp-Val) which are encoded by the linker used at the junction of the construction are not found in the parental molecules. The resulting HN-F gene was inserted into an SV40 late region replacement vector pSV103 (Paterson et a/., 1985) and recombinant SV40 virus (designated SV-HNF) was produced. Expression of HN-F

and biochemical

characterization

To examine for the expression of HN-F and to determine if the molecule was properly targeted to the ER membrane, CV-1 cells were infected with SV-HN-F or SV-HNm (expressing HN, Paterson et a/., 1985). The cells were labeled with Tran[35S]-label and a crude preparation of microsomes isolated and digested with trypsin. If HN-F was translocated across the ER membrane it would be expected to be protected from digestion by exogenously added protease. As expected, the control HN polypeptide was not digested by trypsin (Fig. 2A, HN, lanes 1 and 2) unless the membranes were disrupted by addition of detergent (lane 3). The HN-F protein was synthesized as a single polypeptide species with a slightly increased electrophoretic mobility in comparison to HN (Fig. 2A). The majority of HN-F was found to be protected from protease digestion of microsomes unless detergent was present (HN-F, lane

SOLUBLE

AND

SECRETED

HN

GLYCOPROTEIN

C.

B.

A. HN 123

H N-F 1

2

3

M

501

HN-F --- HN PSPSPS

LDH

HN-F 1

2

FIG. 2. Expressron and brochemrcal characterizatron of HN-F. (A) Trypsrn treatment of mrcrosomal membranes. Recombinant SV40 virus Infected cells expressrng HN or HN-F were radiolabeled with Tran[35S]-label (200 &/ml) for 30 mm and mrcrosomes were prepared. Samples were treated with buffer (lane l), 11 fig/ml trypsin (lane 2) or trypsin plus 1% NP40 (lane 3). After 45 mm at 37”, membranes were Isolated by centnfugatron and the proteins were rmmunopreciprtated with HN antisera and analyzed by SDS-PAGE. The arrow rndrcates the posrtron of HN. (Lane M) SV5 virus-Infected cell polypeptrdes as molecular werght markers. (B) Alkali fractronatron of mrcrosomal membranes Recombrnant SV40 vrrus-Infected cells expressing HN, HN-F, or LDH were radiolabeled for 1 hr wrth Tran[%-label and mrcrosomes were prepared as described under Materials and Methods Samples were Incubated for 30 mm at 0” In pH 11 0 buffer and fractronated by centnfugatron. Equal portrons of the resultrng pellet (P) and supernatant (S) were neutralized and rmmunoprecrprtated usrng HN- or LDH-specrfrc antisera and the polypeptrdes were analyzed by SDS-PAGE. (C)Comparison of the electrophoretrc mobility of HN-F synthesized In tunrcamycrn-treated cells and HN-F synthesized in vitro. (Lane 1) Synthetic HN-F mRNA was transcribed with T, RNA polymerase from pGEM3 HN-F and translated in vmo In a retrculocyte extract as described under Materials and Methods and the resulting products were rmmunopr-ecrprtated with HN antisera. (Lane 2) CV-1 cells were Infected with SVHN-F, treated with tunrcamycrn (1 pg/ml). and radiolabeled with Tran[%-label for 2 hr In the presence of the drug and then rmmunoprecrpitated with HN antisera and the polypeptrdes were analyzed by SDS -PAGE

3), suggesting that most of the HN-F polypeptide was translocated across the ER membrane. The faster mishown in Fig. 2A is grating species (Ivl, - 45,000) thought to originate from the recombinant SV40 virus and is nonspecifically immunoprecipitated (see also Parks et al., 1989). The identity of the other bands is not known. To determine if HN-F was released into the ER Iumen, microsomal membranes were isolated from cells infected with SV-HN-F and subjected to alkaline fractionation. Under these conditions integral membrane proteins remain associated with the lipid bilayer, and after centrifugation are found in the pellet fraction, while soluble proteins are found in the supernatant (Anderson et al., 1983; Steck and Yu, 1973). As shown in Fig. 26, the control integral membrane protein HN was only detected in the pellet fraction (P). In contrast, the majority of the HN-F polypeptide was found in the supernatant (S) fraction, as was the control soluble polypeptide lactate dehydrogenase (LDH, S fraction). When the FRED domain was used to target the NAF hybrid protein to the lumen of the endoplasmic reticulum, it was found by direct protein sequencing, that the FRED was cleaved internally at a site where it was predicted that signal peptidase could cleave the hydrophobic domain (Paterson and Lamb, 1990). Thus, it

was expected that a similar cleavage event would occur within the FRED domain of HN-F. To provide rndirect evidence for a proteolytrc cleavage event occurring with HN-F, the electrophoretrc mobility of HN-F synthesized in vitro from a synthetic RNA transcript was compared with that of HN-F synthesized in cells in the presence of the glycosylatron Inhibitor tunicamycin. As shown in Fig. 2C, HIV-F synthesized in vitro (lane 1) had a slower electrophoretic mobility than unglycosylated HN-F (lane 2), which suggests that a proteolytic cleavage had occurred within HN-F in cells. Formation of intermolecular disulfide bonds, rate of intracellular transport of HN-F to the medial Golgi apparatus, and the rate of secretion of HN-F To determine if the soluble HN-F formed an intermolecular disulfide linkage and to examine if the soluble HN-F was secreted from cells, SV-HN-F-infected CV-1 cells were radiolabeled with Tran[3”S]-label for 15 min and incubated in chase medium for varying periods. Lysates from the cells and media were separately immunoprecipitated and analyzed on SDSPAGE under nonreducing conditions. As shown in Fig. 3A intracellular HN-F monomers (HNF,) were lost with time, and a low level of intracellular HN-F drsulfide-linked dimers

502

PARKS CELL

MEDIUM

B.

C.

,

0

2

4

6

8

10

CELL

MEDIUM

fR +S

FIG. 3. Time courses of formatron of HN-F disulfide-linked dimers. secretion of HN-F, and acquisition of resistance to endo H digestion. CV-1 cells infected with SV-HN-F were radrolabeled with Tran[35S]label for 15 min and incubated for the times (hr) indicated in chase media. Lysates were made of both the cells and the media and HNF was immunoprecipitated with HN antisera. (A) Polypeptides were analyzed on SDS-PAGE under nonreducing conditions. HNF, and HNFa indicate monomeric and disulfide-linked HN-F species, respectively. The arrow indicates HN-F aggregates that are too large to enter the gel. (B) Immune complexes of total HN-F protein in the combined cell and media were incubated in the presence (+) or absence (-) of endo H and polypeptides were analyzed by SDS-PAGE under reducing conditions. (C) Equal portions of the cell-associated (Cell) or secreted (Medium) lysate were incubated in the presence (+) or absence (-) of endo H. R, endo H resistant form of HN-F; S, endo H sensitive form of HN-F.

(HNFJ could be detected 30 min after the beginning of the chase period and the abundance of the HNF2 species remained relatively constant in comparison to the decrease of HNF, at >l hr after the pulse-label. In contrast, HN-F disulfide-linked dimers could be immunoprecipitated from the media beginning l-2 hr after the pulse-label and these disulfide-linked dimers accumulated with time. The simplest interpretation of these data is that intracellular HN-F monomers are slowly

AND

LAMB

converted to HN-F disulfide-linked dimers and the HNF dimers are then secreted from the cell, and that those HN-F molecules that did not form disulfide-linked dimers were degraded intracellularly. To examine the sensitivity of HN-F carbohydrate chains to digestion with A/-acetyl-fi-endoglycosidase H (endo H), SV-HN-F-infected CV-1 cells were radiolabeled with Tran[35S]-label for 15 min and incubated in chase medium for varying periods. The rate of accumulation of all HN-F molecules acquiring resistance to endo H digestion was determined by immunoprecipitating the combined media and cell-associated HN-F for each time point. Samples were incubated with (+) or without (-) endo H and analyzed by SDS-PAGE. HNF molecules containing carbohydrate residues resistant to endo H digestion were first detected by 1.5 hr after the labeling period (Fig. 3B), and by 5 hr approximately 339/o of the total HN-F contained complex carbohydrate residues. The mobility of the endo H-resistant species is not the same as that of undigested HNF, most likely because only a subset of the four HN carbohydrate residues are converted from a simple (endo H-sensitive) to complex (endo H-resistant) form after transport to the Golgi apparatus (Ng eta/., 1989). Thus, the rate of accumulation of HN-F molecules containing complex carbohydrate residues was found to be considerably slower than that of membrane anchored HN (t1,2 - 90 min, Ng et a/., 1989). In a duplicate experiment, cell-associated and secreted HN-F were examined individually for their acquisition of endo H-resistant carbohydrate residues. As shown in Fig. 3C the vast majority of the cell-associated HN-F molecules contained carbohydrate residues which were always sensitive to endo H digestion and only very small amounts of endo H-resistant HN-F could be detected after 2, 4, and 6 hr of chase period. The carbohydrate residues on the secreted HN-F molecules were to a large extent resistant to endo H digestion although a proportion of endo H-sensitive molecules were found to be secreted. Thus, the acquisition of carbohydrate chains which are resistant to endo H digestion is not a prerequisite for secretion of HN-F. From densitometer scanning of autoradiographs from several experiments it was found that approximately 40% of the HN-F molecules synthesized in a pulse-label were secreted with a half-time of 2.5-3 hr. The relative lack of detectable endo H-resistant HN-F molecules within the cell and the accumulation of extracellular endo H-resistant HN-F molecules support the idea that the rate-limiting step in intracellular transport of HN-F is its transport from the ER to the medial Golgi complex. Oligomeric structure of extracellular HN-F The paramyxovirus HN protein is thought to exist as disulfide-linked dimers that form a mixture of noncova-

SOLUBLE

AND

SECRETED

lently linked tetramers and disulfide-linked tetramers (Markwell and Fox, 1980; Thompson et al., 1988; Ng et al., 1989). To investigate the oligomeric form of the secreted HN-F it was fractionated by centrifugation on sucrose gradients under conditions which stabilize the SV5 HN tetramer (Ng er a/., 1989). Gradient fractions were collected from the bottom of the tube, immunoprecipitated with the HN antisera, and analyzed by SDS-PAGE under nonreducing conditions. The HN-F protein was detected as a single disulfide-linked species (Fig. 4A, HN-F panel, fractions 7-9) which sedimented slower than wild-type HN solubilized from infected cells (HN panel, fractions 4 and 5; only the relevant portion of autoradiograph is shown). To determine indirectly if the sedimentation characteristics of the HN-F species correspond to those of a dimer, the HN-F sedimentation profile was compared to that of influenza virus NA, a protein of known oligomerit structure and with an individual polypeptide chain length that is somewhat similar to that of HN. Influenza NA protein is a homotetramer (Varghese et a/., 1983) and a secreted and soluble form of NA (NAF) that lacks the transmembrane domain was found to consist of monomers, dimers, and tetramers (Paterson and Lamb, 1990). Thus, secreted HN-F and NAF were cosedimented through a sucrose gradient, and after fractionation, equal portions of each sample were immunoprecipitated with either polyclonal antisera to HN or to NA and analyzed by SDS-PAGE under nonreducing conditions. As described previously (Paterson and Lamb, 1990), NAF was detected as three major species on the gradient (Fig. 4B, NAF panel): a rapidly sedimenting disulfide-linked species which probably represents tetramers (fractions 3 and 4), an intermediate sedimenting dimeric disulfide-linked species (fractions 669), and a non-disulfide-linked monomeric form (fractions 9-l 1). The nature of the diffuse NA bands is not known, but may reflect carbohydrate heterogeneity (Paterson and Lamb, 1990). When the same gradient fractions were immunoprecipitated with HN antisera the disulfide-linked HN-F form (Fig. 4B, HN-F panel, fractions 7-9) was found to sediment in the same gradient fractions as the dimeric form of NAF. Thus, although these data do not provide formal proof of the oligomeric structure of HN-F they strongly indicate that the disulfide-linked form of HN-F is a dimer. Neuraminidase of extracellular

activity HN-F

and mAb reactivity

To determine if the secreted HN-F was enzymatically active, media were collected from uninfected and SVHN-F-infected CV-1 cells, concentrated and assayed for neuraminidase activity. As shown in Fig. 5, samples

503

HN GLYCOPROTEIN

A.

bottom

top

HNF,-

HN

B.

HNF

1

2

3

4

5

6

7

a

9

10

11

12

13

14

15

NAF

FIG. 4. Sucrose gradient sedimentation analysis of extracellular HN-F. Recombrnant SV40 vrrus-Infected cells expressing HN-F, HN, or NAF were radiolabeled for 30 mm wrth Tran[35S]-label and incubated in chase medium for 6 hr (HN-F), 2 hr (HN), or 3 hr (NAF). (A) Medra from cells expressrng HN-F (top) or solubilrzed cell lysate from cells expressing HN (bottom) were subjected to sucrose gradient sedrmentatron centnfugatron as described under Materials and Methods. Gradient fractrons were collected from the bottom of the tube, rmmunoprecrprtated with HN antisera, and analyzed by SDSPAGE under nonreducing condrtrons. HNF, and HNF, Indicate mom nomeric and drsulfide-linked HN-F specres. respectively. Onlythe relevant portron of the autoradiographs are shown. (B) Media (0.5 ml) from cells expressrng HN-F and celis expressing NAF were mixed together and cosedrmented as described above. Fractions were collected and equal portions were rmmunoprecipitated wrth HN antisera (HN-F panel) or NAantrsera (NAF panel) and analyzed by SDS-PAGE, under nonreducrng conditrons. Only the relevant portions of the autoradtographs are shown.

from uninfected cells (U) contained little NA activity, while media from SV-HN-F-infected cells (H) contained large amounts of NA activity. The enzyme activity was lost when the secreted HN-F was preincubated with a neutralizing monoclonal antibody (mAb HN-4a) (Randall et al., 1987) but not when preincubated with mAb

PARKS

504 B.

A.

mAb

4a

AND

LAMB

(fractions 14 and 15) and the nature of this is unknown. Approximately 70%~ of the NA activity applied to the gradient was recovered in fractions 5-7, and this enzyme activity was inhibited by preincubation of the fractions with mAb HN-4a (not shown). Taken together, these data suggest that secreted HN-F, although dimerit, has folded to a form that has enzymatic activity.

Conformation of intracellular sample: antibody:

U C

u 48

H C

H 4a

Ii 4a’

FIG. 5. Neuraminidase activity and mAb reactivity of extracellular HN-F. (A) Media from uninfected (U) or SV40-HN-F-infected (H) CV-1 cells were dialyzed, concentrated, and assayed for neuraminidase activity using sialolactose as a substrate as described under Materials and Methods. Samples were preincubated with ascites fluids of a nonspecific mAb (C), with mAb HN-4a (4a), or mAb HN-4a that had been boiled previously (4a*). Ach9, absorbance at 549 nm. (B) Sequential serial immunoprecipitations of Tran[%]-labeled extracellular HN-F was carried out with the conformation-specific and neutralizing mAb HN-4a (lanes l-3) followed by HN polyclonal antibody (P). Samples were analyzed by SDS-PAGE.

HN-4a that had been previously denatured by boiling, thus confirming that the NA activity was specific for the HN-F protein. Monoclonal antibodies that recognize conformational epitopes can be very useful reagents to examine the status of folding of HN (Ng et al., 1989). To examine the conformation of the secreted HN-F and to determine indirectly the fraction of HN-F molecules contributing to the NA activity, the medium from SV-HN-F-infected cells was depleted of secreted HN-F molecules reactive with the conformation-specific mAb HN-4a. The supernatant from the third serial immunoprecipitation was then reacted with a polyclonal sera which is specific for HN in all conformations and samples were analyzed by SDS-PAGE. The vast majority of extracellular HN-F was reactive with mAb HN-4a (Fig. 5B, lanes l-3) and only a very small amount of HN-F was recovered by the polyclonal sera (lane P). As mAb HN-4a inhibits 90% of HN-F NA activity and reacts with >90% of the HN-F molecules, these data imply indirectly that the vast majority of secreted HN-F is both folded into a native conformation and is enzymatically active. To provide further evidence that the enzymatically active HN-F correlated with the dimeric form of HN-F rather than an undetected tetrameric form, secreted NAF was fractionated by sucrose gradient sedimentation and the resulting fractions were immunoprecipitated with HN-specific sera or assayed for NA activity. As shown in Fig. 6, the dimeric HN-F species cofractionated with the enzymatic activity. A small amount of NA activity was detected near the top of the gradient

and oligomeric HN-F

forms

For those integral membrane proteins that have been examined it has been found that folding and oligomerization is a prerequisite for transport from the ER to the Golgi complex (e.g., Gething et al., 1986; Kreis and Lodish, 1986; Copeland et a/., 1988; Doms et a/., 1988; Ng eta/., 1989). Although soluble forms of influenza virus NA and HA are secreted in monomeric forms (Paterson and Lamb, 1990; Singh et al., 1990) the slow and inefficient transport of HN-F from cells could reflect a defect in the folding and/or assembly of the molecule. To examine the folded states of intracellular HN-F, the ability of the different species of intracellular HN-F to be immunoprecipitated by the conformationspecific antibody (HN-4b) was investigated. It has previously been shown with HN that this mAb only recognizes the molecule when it oligomerizes (Ng et a/., 1989). SV-HN-F-infected CV-1 cells were labeled for 15

1

2

3

4

6

6

7

Fraction

0

9 ‘10

11

12

13

14

16

16

Number

B.

6

10 Fraction

16

Number

6. Cosedimentation of NA activity with HN-F. CV-1 cells infected with SV-HN-F were labeled for 2 hr with Tran[35S]-label and incubated in chase medium for 22 hr. Media were collected, concentrated, and analyzed by sucrose gradient sedimentation as described under Materials and Methods. Fractions were (A) immunoprecipitated with HN antisera and polypeptides were analyzed by SDS-PAGE under nonreducing conditions, or(B) assayed for NA activity. A549, absorbance at 549 nm. FIG.

SOLUBLE

A.

2 hr 0

1

AND

SECRETED

4 hr

23P123P

B. P12

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7. mAb reactrvity and sucrose gradient sedimentatron analysrs of intracellular HN-F. CV-1 cells infected wrth SV-HN-F were radiolabeled wtth Tran[%]-label for 15 min and incubated rn chase medra for 2 or 4 hr. (A) mAb reactrvity. Cell lysates were prepared and sern ally rmmunoprecrpitated three trmes with mAb HN-4b (lanes l-3) before rmmunoprecipitation with the polyclonal HN antisera (lane P). (Lane 0) Polypeptrdes synthesized during radiolabeltng period and rmmunoprecrpttated with the HN polyclonal antibody. Polypeptrdes were analyzed by SDSPAGE under nonreducing conditrons. HNF, and HNF2 Indicate monomeric and drmenc forms of HN-F, respectively. (B) Sucrose gradient sedrmentatron analysis. Cell lysates were prepared 2 hr after the pulse label and were analyzed by sucrose gradient sedimentation as described under Materials and Methods. Gradient fractions were immunoprecipitated with HN antisera and analyzed by SDSPAGE under nonreducing condrtions. (Lane P) Polypeptrdes rmmunoprecrprtated from the gradient pellet fraction. (Arrow) Aggregated HN-F material too large to enter the gel; (HNF, and HNF,) as above. FIG.

min with Tran[35S]-label and then incubated in chase media for 0, 2, or 4 hr. Serial immunoprecipitations were performed first with mAb HN-4b followed by immunoprecipitation with the HN polyclonal Ab and the polypeptides were analyzed by SDS-PAGE under nonreducing conditions (Fig. 7A). At 0 time, the vast majority of the newly synthesized HN-F immunoprecipitated by the polyclonal Ab was in a monomeric form (lane O), which migrated as a diffuse band (HNF,) and there were small amounts of dimeric HN-F (HNF,). The origin of the prominent species which migrated slower than HNFp is unknown but it may represent dimeric HN-F molecules which have not been cleaved in the FRED signal domain after its translocation into the ER but at

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GLYCOPROTEIN

505

the present time this has not been investigated further. After 2 or 4 hr of chase period, dimeric HNF, protein reactive with the conformation specific antibody mAb HN-4b was detected (lanes l-3) but these forms represented only a small portion of the total intracellular HNF molecules. The forms of HN-F which were not reactive with mAb HN-4a were recovered with the polyclonal HN Ab (lane P) and they were found to be predominantly HNF, monomers and a small amount of HNFZ dimers. These latter dimer species are presumably in a different folded form from those recognized by mAb HN-4b. After 4 hr of chase period a similar pattern of polypeptide species was obtained, except that the amount of monomeric HNF, had decreased due to their conversion to dimers followed by secretion. The level of the dimeric form remained low and its amount was fairly constant during the chase period, again suggesting that once formed the majority of dimers are rapidly secreted. To examine the oligomeric form of intracellular HNF, SV-HN-F-infected CV-1 cells were radiolabeled with Tran[35S]-label for 15 min and incubated in chase medium for 2 hr. Cell lysates were then prepared and subjected to sedimentation on sucrose gradients. Fractions were immunoprecipitated with the polyclonal HN Ab and analyzed under nonreducing conditions on SDS-PAGE. As shown in Fig. 7B two major species could be identified. One species had the properties of the disulfide-linked dimer (HNF*, fractions 7-9). The other HN-F species sedimented through the gradient and was recovered in the pellet fraction (lane P). On SDS-PAGE the majority of this HN-related species was found as an aggregate that was too large to be resolved in the gel (arrow, top of lane P) and remained at the origin of the separating gel but there were also small amounts of dimers and monomers. Before centrifugation this aggregated HN-F material consisted of dimeric and monomeric HN-F under nonreducing conditions (Fig. 7A), and these data suggest that a portion of the intracellular HN-F forms inappropriate disulfide bonds after lysis of the cells. This was confirmed by treating cell lysates with the alkylating agent iodoacetamide. On sucrose gradient sedimentation the alkylated HN formed noncovalently linked aggregates which sedimented heterogeneously from the HN-F monomeric position to the bottom of the gradient. On SDS-PAGE these aggregates were dissociated and they were found to migrate on gels as HNF, monomers (data not shown). Thus, together all these data indicate that a significant proportion of HN-F is defective in folding and assembly. DISCUSSION Several studies with naturally existing integral membrane proteins have indicated that native folding of the

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polypeptide chain and oligomerization of the monomers are sequential and prerequisite events for intracellular transport out of the ER (Gething et a/., 1986; Kreis and Lodish, 1986; Copeland eta/., 1988; Doms et al., 1988; Ng eta/., 1989; reviewed by Rose and Doms, 1988). Most malfolded and nonoligomerized integral membrane proteins fail to be transported intracellularly and they exhibit prolonged associations with the resident ER protein GRP78-BiP (Pelham, 1986; Gething et a/., 1986; Hurtley et al., 1989; Ng et a/., 1990). The paramyxovirus SV5 HN glycoprotein exists as a pair of disulfide-linked dimers that form a mixture of noncovalently-linked tetramers and disulfide-linked tetramers (Ng et al,, 1989). Restrictions on the intracellular transport of HN can occur when the ectodomain is malfolded and when alterations are made to the N-terminal cytoplasmic tail, changes which in turn alter the oligomerization process (Ng et al., 1989, 1990; Parks and Lamb, 1990). We have studied the folding and assembly processes of a soluble form (HN-F) of the HN protein to determine if the same transport restrictions applied to HN-F as were found with the membrane-anchored form. The results indicate that the secreted HN-F is biologically active having neuraminidase activity, that almost all of the secreted HN-F molecules are recognized by conformation-specific mAbs, implying as far as can be determined that the molecules are folded in a native conformation, and that almost all the secreted HN-F molecules formed an intermolecular disulfide bond to form a dimer. Analysis of the oligomeric form of secreted HN-F on sucrose density gradients indicated that it was dimeric, under conditions in which the membrane-anchored form is tetrameric. However, sedimentation of detergent-solubilized HN is pH sensitive and at neutral pH the noncovalently linked tetramers dissociate to some extent to disulfide-linked dimers. Thus, it is difficult to determine definitively if the extracellular HN-F was a weakly associated tetramer that dissociated on dilution into the medium or on sucrose gradients, or whether HN-F was secreted in the form of a dimer. We favor the interpretation that HN-F oligomerization did not proceed beyond the dimer stage because with membrane-bound HN some disulfidelinked tetramer is formed and no covalently linked HNF tetramer could be observed. When virions of another paramyxovirus, Sendai virus, were treated with protease to release a water-soluble HN ectodomain, and this molecule was analyzed on sucrose gradients, it was found to sediment as a mixture of biologically active dimers and tetramers (Thompson et al., 1988). However, these data do not necessarily indicate that this HN can be expressed at the cell (virion) surface in a dimer form, because the neutral pH centrifugation

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conditions used may have caused dissociation of the Sendai virus HN tetramers. Approximately 40% of the HN-F molecules synthesized were secreted with a t,,2 - 2.5-3 hr and were found in the medium as disulfide-linked dimers. The remainder of the intracellular HN-F molecules were found as dimers and monomers that were not folded in a form that could be recognized by the conformation-specific mAbs and with time they were presumably degraded as they were lost from the cell. It is not known if this degradation occurs in the ER or if the molecules are degraded in lysosomes. We have previously shown for HN that its oligomeric form can be detected in the ER (Ng et a/., 1989). Our data suggest that the formation of the HN-F dimer is important for transport of HN-F to the medial Golgi complex. Disulfide bond formation is a convenient means of measuring the formation of oligomers as we have not been able to separate the two events kinetically. However, we do not know if intermolecular disulfide bond formation is the cause or the consequence of dimerization although we favor the latter interpretation. In the cases of both influenza virus neuraminidase (reviewed by Lamb, 1989) and transferrin receptor (Alvarez et a/., 1989) it has been deduced from analysis of different strains and determined experimentally by site-specific muatagenesis, respectively, that intermolecular disulfide bond formation is not needed for oligomer formation and intracellular transport. As intermolecular as well as intramolecular disulfide bonds may be catalyzed by protein disulfide isomerase, a resident component of the ER (Freedman, 1984) the HN-F intermolecular disulfide bond may form in the ER. HN oligomerizes with a t,,2 - 30 min. (Ng et a/., 1989) whereas HN-F dimers could first be detected within 15-30 min after a 15-min labeling period but then the process continued for several hours. The relatively small pool of intracellular disulfide-linked HNF dimers at any one time after a pulse-label and the virtual absence of detectable intracellular HN-F species resistant to endo H digestion, are compatible with the view that formation of HN-F dimers is the rate-limiting step in the secretion process, and that assembly only needs to proceed as far as a dimer for HN-F to be competent for transport. However, the conversion of high mannose and endo H-sensitive carbohydrate chains to complex and endo H-resistant carbohydrate chains in the media/Golgi complex on both polypeptide chains in the dimer is not a prerequisite for secretion of HN-F. In contrast, membrane-bound HN molecules which contain alterations to the cytoplasmic tail mature as far as folded dimers but they do not form detectable tetramers and these molecules fail to be transported to the medial Golgi apparatus (Parks and Lamb, 1990). Given the caveats discussed above concerning the

SOLUBLE

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oligomeric form of HN-F, these data suggest that conversion of the formerly membrane-bound HN to a soluble form has changed the rules for intracellular transport in that the stringent requirement for normal oligomerization has been removed. We do not know when the hydrophobic domain (FRED) that acted as a signal sequence is cleaved with respect to completion of synthesis of the nascent HNF polypeptide chain and folding of HN-F. Therefore, we do not know if normal dimerization is dependent on attachment to the membrane. If cleavage of the HN-F signal sequence with release of HN-F into the ER lumen occurs before folding and/or oligomerization, the soluble HN-F can be expected to have more difficulty finding assembly partners (diffusion in three dimensions) than membrane-anchored HN (diffusion in two dimensions) and this could explain the slow dimerization of soluble HN-F molecules. The folding and oligomerization properties of several soluble and secreted forms of formerly membranebound integral membrane proteins have been examined including the influenza virus HA (Copeland et al., 1986; Gething et a/., 1986; Singh et a/., 1990), influenza virus NA (Paterson and Lamb, 1990), RSV env (Einfeld and Hunter, 1988), and VSV G (Crise et al., 1989). The transport rate of the soluble forms has varied from being the same as that of the membranebound form (RSV env, influenza virus NA) to being considerably slower (influenza virus HA, VSV G, SV5 HN). In addition, the soluble forms have exhibited a spectrum of different folded and oligomerized forms as compared to their membrane-bound counterpart. The most extreme difference has been found with influenza virus NA, as a plethora of different extracellular folded forms and oligomerized forms were identified. With influenza virus HA (strain X31/A/Aichi/68, H3 subtype), the secreted form of the molecule was found to be monomeric and the evidence indicates that the monomer had not dissociated from the trimer. With RSV env the extracellular form was a monomer that is thought to have dissociated from the oligomeric form and with VSV G the secreted molecule was found to be trimeric. Thus, all these data suggest that the stringent requirement for proper folding and oligomerization of naturally existing membrane-bound proteins for transport out of the ER are altered to varying degrees for soluble proteins, but the precise extent of the change depends on the protein under examination. The rate and efficiency of oligomerization of a protein in the ER may depend on its relative concentration and it remains to be determined if higher levels of HN-F expression will promote more efficient oligomerization and transport. If so, HNF may prove to be a useful molecule for the large-scale

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preparation of the HN ectodomain enzymatic studies.

for structural

and

ACKNOWLEDGMENTS We thank Davis 7. W. Ng and Reay G. Paterson for helpful dtscusslons and Reay G. Paterson for providing the NAF cDNA clone and recombinant SV40 virus. We thank Margaret Shaughnessy for excellent technlcal assistance and Susan Z. Domanlco for constructing the HN-F recombinant DNA molecule as a D99 project at Northwestern Unlverstty. G.D.P. was supported by an American Cancer Society postdoctoral fellowship (PF-3 177). This research was supported by Public Health Service Research Grant Al-23 173 from the Natlonal lnstltute of Allergy and lnfectlous Diseases.

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