©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Heavy Chain Dimers as Well as Complete Antibodies Are Efficiently Formed and Secreted from Drosophila via a BiP-mediated Pathway (*)

(Received for publication, March 16, 1995; and in revised form, May 17, 1995)

Robert B. Kirkpatrick (1)(§)(¶) Subinay Ganguly (1)(§) Monica Angelichio (1) Sandra Griego (3) Allan Shatzman (1) Carol Silverman (2) Martin Rosenberg (1) (2)

From the  (1)Departments of Gene Expression Sciences, (2)Protein Biochemistry, and (3)Molecular Virology and Host Defense, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have constructed a stable Drosophila cell line co-expressing heavy chain (HC) and light chain (LC) immunoglobulins of a humanized monoclonal antibody (mAb) that recognizes the F antigen of respiratory syncytial virus (Tempest, P. R., Bremmer, P., Lambert, M., Taylor, G., Furze, J. M., Carr, F. J., and Harris, W. J.(1991) Bio/Technology 9, 266-271. These cells efficiently secrete antibody with substrate binding activity indistinguishable from that produced from vertebrate cell lines. Significantly, the Drosophila homologue of the immunoglobulin binding chaperone protein (BiP), hsc72, was found to interact specifically with the immunoglobulin HC in an ATP-dependent fashion, similar to the BiP-HC interaction known to occur in vertebrate cells. This is, in fact, the first substrate ever shown to interact specifically with Drosophila hsc72. Most surprisingly, expression of heavy chains in the absence of LC led to the efficient secretion of heavy chain dimers. Moreover, this secretion occurred in association with hsc72. This dramatically contrasts with what is seen in vertebrate cells where in the absence of LC, HC remains sequestered inside the cell in stable association with BiP. Our results clearly suggest that Drosophila BiP can substitute for its mammalian counterpart and chaperone the secretion of active IgG. However, the finding that Drosophila BiP can also uniquely chaperone heavy chain dimers indicates mechanistic differences that may relate to the evolved need for retaining immature IgGs in vertebrates.


INTRODUCTION

Immunoglobulin heavy chain binding protein (BiP) is the only member of the stress 70 protein family that is localized to the endoplasmic reticulum (ER) (^1)of eucaryotic cells, where its functions as a chaperone are believed to support proper protein folding and protein translocation into the ER lumen (Gething and Sambrook, 1992). In vertebrate cells, BiP has been identified in association with various secretory proteins traversing the ER, in particular immunoglobulin heavy chain (HC) polypeptides (Haas and Wabl, 1983). The most stable of these interactions are formed with proteins that are incompletely folded or aberrantly glycosylated (Bole etal., 1986; Gething, et al., 1986; Dorner et al., 1987), suggesting that BiP may promote proper protein folding and/or prevent immature proteins from leaving the cell.

In vertebrate B lymphocytes, BiP associates stably with unassembled HC. BiP dissociation is triggered after LC is added to HC and the maturation of the immunoglobulin (Ig) structure is completed. This is exemplified in myeloma and pre-B cell lines expressing HC but not LC, where BiP remains tightly associated with HC immunoglobulins, allowing them to form dimers intracellularly, which are not secreted in the absence of LC association (Morrison and Scharff, 1975; McCune and Fu, 1981; Haas and Meo, 1988). This situation can be reversed by forming hybrids between these cell lines and lines expressing LC only, leading to the synthesis and secretion of complete HCbulletLC heterodimers (McCune and Fu, 1981; Mains and Sibley, 1982; Bole et al., 1986). Moreover, the HC dimers, which are bound irreversibly by BiP prior to such cell fusions, are released from BiP following fusion. Apparently, BiP sequesters the incompletely assembled antibodies in the ER, and LC incorporation participates in the release of BiP from the newly folded antibody (Hendershot, 1990).

The release of antibody from BiP is known to be an ATP-dependent process (Munro and Pelham, 1986; Gaut and Hendershot, 1993; Dorner et al., 1990). This has been shown by the fact that antibody-BiP complexes can be dissociated in vitro by the addition of ATP (Munro and Pelham, 1986). Furthermore, mutations within the nucleoside binding site of BiP inhibit ATP hydrolysis and, in turn, the in vivo release of immunoglobulin heavy chain from BiP complexes (Gaut and Hendershot, 1993). Dissociation from BiP and secretion can also be blocked by depleting cellular ATP levels (Dorner et al., 1990). Therefore, ATP binding and hydrolysis are required for the release of HC from BiP.

Recently, a BiP homologue in Drosophila melanogaster called heat shock cognate 3 (HSC3) has been cloned and characterized (Rubin et al., 1993). This gene encodes a 72-kDa protein (hsc72) that is 80% identical to human BiP (Haas and Meo, 1988) and one of at least five constitutively expressed heat shock cognate proteins in Drosophila, but the only ER-resident (Munro and Pelham, 1987; Pelham, 1988; Dean and Pelham, 1990). Drosophila BiP (hsc72) also contains the ATP-binding domain that is well conserved within the heat shock protein family (Lindquist and Craig, 1988; Gaut and Hendershot, 1993). The function of hsc72 in Drosophila is therefore likely to be analogous to that of BiP in vertebrates. To date, no substrates have been identified upon which hsc72 acts in Drosophila.

The aim of the present study was to examine the ability of Drosophila cells to support the expression of antibodies and, most importantly, to examine the potential role of Drosophila BiP (hsc72) in the maturation and secretion of these antibodies. Our results indicate that Drosophila cells are capable of efficiently producing IgG molecules and that hsc72 interacts with HC in an ATP-dependent manner that is analogous to the action of BiP in vertebrate cells. Surprisingly, in Drosophila, HC dimers are efficiently secreted after BiP interaction in a manner independent of LC. The fact that LC does not appear to be required suggests mechanistic differences between hsc72 action in Drosophila and BiP in vertebrates.


EXPERIMENTAL PROCEDURES

Plasmids

All plasmid constructions were generated by standard cloning methods (Sambrook et al., 1989). The cDNA clones encoding the heavy chain and kappa light chain of the RSHZ19 mAb, (^2)isolated from the myeloma line expressing this mAb (Tempest et al., 1991), were subcloned separately into mammalian expression vectors to produce RSHZ19 mAb in CHO cells. (^3)The heavy and light chain cDNAs were then subcloned as separate EcoRI fill in/EcoRV restriction fragments into the EcoRV site of the Drosophila expression plasmid, pMtaL (Angelichio et al., 1991), thus creating pMtHC and pMtLC. Both pMtHC and pMtLC were confirmed by restriction analysis and by sequencing across cloning junctions.

Cell Culture and Cell Lines

D. melanogaster S2 cells (Schneider, 1972) were grown in a modified M3 media (Shields and Sang, 1977). Cells were transfected, and stable lines were selected as described previously (Johansen et al., 1989). For line mtHZ19, 19 µg of pMtHC and 19 µg of pMtLC were co-transfected with 1 µg of hygromycin B resistance vector, pCOHygro (van der Straten et al., 1989). For line mtHC, 19 µg of pMtHC was transfected alone with 1 µg of pCOHygro. HC and LC expression under the control of the metallothionein promoter was induced as described previously (Johansen et al., 1989).

Antigen Binding ELISA Assay

Binding of RSHZ19 antibody to recombinant F protein expressed in S2 cells (^4)was performed using a solid phase ELISA. 100 ng (50 µl) of F protein was diluted in PBS, pH 7.0, and adsorbed onto polystyrene round bottom microplates (Dynatech, Immunolon II) for 18 h at 4 °C. Wells were then aspirated and blocked with 0.5% boiled casein in PBS containing 1% Tween (PBS, 0.5% boiled casein) for 2 h. Antibodies (50 µl/well) were diluted to varying concentrations in PBS, 0.5% boiled casein containing 0.025% Tween 20 and incubated in antigen-coated wells for 1 h. Plates were washed three times with PBS containing 0.05% Tween 20, followed by addition of horseradish peroxidase-labeled goat anti-human IgG (50 µl) diluted 1:2500 (Amersham Corp.). TMBlue substrate (TSI, number TM102) was then added, and plates were incubated an additional 5 min. The reaction was stopped by the addition of 1 N H(2)SO(4), and absorbence was read at 450 nm using a BioTek ELISA reader. All incubations were performed at room temperature. Antibody titers were defined by ED values based on regression analysis of the antibody titration curves using RS/1 statistical procedures.

RNA Analysis

Total RNA was isolated from 1 10^7 cells at the times indicated after induction using the Tri reagent RNA isolation reagent (Molecular Research Center). Northern analysis was performed by standard methods (Sambrook et al., 1989) using P-labeled BamHI probe fragments from pMtHC and pMtLC, respectively.

Precipitation of hsc72bulletHC Complexes

Cell lines were induced for 6 days as described above for cell culture and cell lines. 1 10^7 cells were isolated from each culture, washed once with 1 ml of ice cold PBS, and lysed for 30 min in 500 µl of ice cold lysis buffer (50 mM Tris, pH 7.5, 0.15 M NaCl, 0.5% Nonidet P-40, 0.2 units/ml aprotinin, 2 mM phenylmethylsulfonyl fluoride). Debris was removed from cell lysates by centrifugation at 12,000 g for 10 min. 40 µl of protein G-Sepharose (Pharmacia Biotech Inc.) was added to each lysate as a 1:1 slurry with 0.5% Nonidet P-40, 0.1% bovine serum albumin in PBS and incubated on an orbital platform for 1.5 h at 4 °C. Protein G-Sepharose complexes were isolated by centrifugation at 2,000 g for 2 min, washed twice with 1.5 ml of ice-cold wash buffer (50 mM Tris, pH 7.5, 0.15 M NaCl, 0.5% Nonidet P-40) and once with 1.5 ml of ice-cold Tris-saline (50 mM Tris, pH 7.5, 0.15 M NaCl, 1 mM MgCl(2)). hsc72bulletHC complexes were dissociated in the last wash by addition of ATP to a final concentration of 1 mM.

Metabolic Labeling

Line mtHZ19 was seeded at 2 10^6 cells/ml at 25 °C and grown to a density of 7 10^6 cells/ml. Cells were then washed twice with labeling medium (methionine, cysteine, and yeastolate-free M3 medium) and resuspended to a density of 2 10^7 cells/ml in prewarmed labeling medium plus 10% heat-inactivated dialyzed FBS and incubated for 90 min at 25 °C to deplete methionine and cysteine from the cells. Cells were recovered again by centrifugation and resuspended in prewarmed labeling medium, 10% heat-inactivated dialyzed FBS. To these cells, 0.5 mM CuSO(4) was added for induction of RSHZ19 expression and 250 µCi/ml TranS-label (ICN) was added for metabolic labeling. Cells were harvested after 16 h at 25 °C.

Purification of RSHZ19 mAb

Secreted RSHZ19 mAb expressed in Drosophila cells was purified from the conditioned medium of Drosophila cultures using protein G-Sepharose (fast flow, Pharmacia) according to manufacturer's specifications.

Protein Analysis

Proteins were resolved by reducing 0.1% SDS, 10% polyacrylamide gel electrophoresis or nonreducing 0.1% SDS, 7.5% polyacrylamide gel electrophoresis and detected either directly by Coomassie Blue staining or by Western analysis after electroblotting onto nitrocellulose. Heavy and light chains were detected using a horseradish peroxidase-conjugated anti-human IgG polyclonal antisera (Amersham Corp.) with the ECL detection system for horseradish peroxidase (Amersham Corp.). Heavy and light chain subunits from purified control RSHZ19 antibody are detected with equal sensitivity using this antisera (data not shown). Detection of hsc72 was performed using a rat mAb raised to flight wing troponin purified from the waterbug, Lethocerus cordofanus as a primary antibody (Bullard et al., 1988). This antibody is reactive only to hsc72 in Drosophila S2 cells. Horseradish peroxidase-conjugated anti-rat IgG (Amersham Corp.) was used as a secondary antibody followed by detection with ECL.


RESULTS

Production of Active Human IgG1 Antibodies from Drosophila Cells

We were interested initially in whether Drosophila cells could stably support the expression and secretion of recombinant antibody. In order to examine this, HC and kappa LC cDNAs encoding a humanized IgG1 mAb, specific for the F antigen of respiratory syncytial virus, were inserted into an expression vector designed for stable introduction into Drosophila Schneider cells (S2) (Johansen et al., 1989). In these vectors the HC and LC genes were placed under the control of the Drosophila metallothionein promoter, which is inducible in response to heavy metal addition (Maroni et al., 1986). A stable cell line was selected, mtHZ19, and inducible expression of immunoglobulin mRNAs was examined by Northern analysis (Fig. 1A). Both genes were transcribed into mRNA in response to metal induction. Furthermore, HC and LC polypeptides were produced and secreted at levels that appeared to be equivalent as monitored by Western analysis of conditioned media (Fig. 1B).


Figure 1: Inducible expression and secretion of RSHZ19 mAb from Drosophila cells. A, Northern analysis of total RNA extracted from line RSHZ19 at the times indicated in hours after induction with CuSO(4). Heavy and light chain transcripts were detected using gene-specific DNA probes B, Western analysis of conditioned media using anti human Ig polyclonal antisera to detect both HC and LC polypeptides. Conditioned medium was isolated from the same times as in A. C and D, analysis of purified RSHZ19 antibody by nonreducing (C) or reducing SDS-polyacrylamide gel electrophoresis (D) and Coomassie Blue staining. Lane1, Drosophila S2-expressed protein; lane2, CHO-expressed protein.



In order to examine the maturation of the IgG product into complete HCbulletLC heterodimers, the mAb (RSHZ19) was purified using protein G affinity chromatography from the conditioned media and examined under nonreducing conditions. The results indicate that the antibody had the appropriate molecular mass of approximately 200 kDa (Fig. 1C, lanes1 and 2), indicating that proper maturation and disulfide bond formation had occurred. Under reducing conditions, the Drosophila-produced respiratory syncytial virus mAb resolved into separate HC and LC polypeptides of the expected sizes, 55 and 25 kDa, respectively (Fig. 1D). These results indicate that Drosophila cells can support the production of properly folded immunoglobulin HC and LC into complete antibody heterodimers.

To assess the functionality of mAb produced in Drosophila, the binding of RSHZ19 to respiratory syncytial virus envelope glycoprotein F was compared with that of the same antibody produced in CHO cells. The results (Fig. 2) show that the Drosophila-expressed RSHZ19 antibody retained potent binding to respiratory syncytial virus F antigen, identical to its mammalian counterpart. These results confirm that Drosophila cells support the proper folding of immunoglobulin heavy and light chains into functional antibodies.


Figure 2: Antigen binding ELISA assay. RSHZ19 mAb expressed in CHO cells, RSHZ19 mAb produced in Drosophila S2 cells, and a mutant form of RSHZ19 mAb, CMHZ00, expressed in myeloma cells were compared for binding to respiratory syncytial virus F protein. The mAb, RSHZ19, served as a positive control (Ganguly et al., unpublished observations). The mAb, CMHZOO, lacks binding affinity for F protein and was used as a negative control (obtained from Scotgen Biopharmaceuticals, Inc., Aberdeen, Scotland). RSHZ19 and CMHZ00 antibodies are equivalent to the reshaped and nonreshaped mAbs HuRSV19VHFNS/VK and HuRSV19VH/VK, respectively (Tempest, Bremmer et al. 1991). ED = 19 and 13 ng/ml for Drosophila CHO-expressed mAbs, respectively. ED > 300 ng/ml for CMHZ00.



The level of RSHZ19 mAb secreted from Drosophila cells was estimated by both Western analysis and ELISA to be geq1.0 µg/ml (Table 1). These levels are comparable with the levels of this same antibody expressed in a rat myeloma cell line (Tempest et al., 1991).



Drosophila BiP (hsc72) Interacts Specifically with Immunoglobulin HC

The folding and secretion of mature antibodies from mammalian cells is known to require the specific interaction of immunoglobulin binding protein (BiP) with heavy chain. This interaction appears to be necessary for proper HC folding and subsequent interaction with LC (Munro and Pelham, 1986; Hendershot, 1990). Since Drosophila cells also contain a BiP homologue (hsc72) and, as shown above, fold and secrete functional antibody, we examined the potential role of hsc72 in antibody production.

To examine the interaction between hsc72 and HC in cells producing recombinant IgG, cells expressing antibody were S-labeled, and lysates were analyzed using protein G-Sepharose to look for the presence of HC-associated, co-precipitating hsc72. The results (Fig. 3) show that three selective S-labeled protein bands were detected by protein G precipitation (Fig. 3A, lane2). The most prominent of these is consistent with the 55-kDa molecular mass of HC itself brought down directly by protein G precipitation. A second more minor band at 29 kDa is consistent with LC and suggests that some of the HC being precipitated with the lysate is already associated with LC. Most importantly, the prominent band at 72 kDa is precisely the size expected for hsc72. None of these proteins were detected in Drosophila cell lysates prepared from cells not expressing antibody (Fig. 3A, lane1). Thus, Drosophila BiP appears to be selectively interacting with the recombinant HC produced in these cell lines.


Figure 3: Association of hsc72 with HC immunoglobulins in Drosophila cells. A, lanes1 and 2, protein G precipitation of S-labeled cell lysates from line RSHZ19 in the absence or in the presence of CuSO(4) induction. Faintbands seen in lanes1 and 2 at approximately 70 and 20 kDa represent nonspecific co- precipitates. B, Western detection of hsc72 in cell lysates in the absence (lane1) or in the presence of CuSO(4) (lane2). C, Western detection of hsc72 (toppanel) or IgG (bottompanel) from mtHZ19 cell lysates precipitated with protein G. Lane1, nonrecombinant S2 cells; lane2, uninduced mtRZ19 cells; lanes3 and 4, induced mtHZ19 cells washed in the absence (lane3) or in the presence of 1 mM ATP (lane4); lanes5 and 6, protein released from the final wash of protein G precipitations in the absence (lane5) or in the presence of 1 mM ATP (lane6).



We next examined Drosophila cell lysates prepared in the absence of S label for the presence of hsc72 using standard Western analysis. The results (Fig. 3B) show that hsc72 is detected readily in cells either induced (lane1) or uninduced (lane2) for antibody expression. These lysates were then incubated with protein G, and the resulting precipitates were analyzed for the presence of hsc72, HC, and LC. In the absence of IgG expression, no hsc72 was detected in the protein G precipitate (Fig. 3C, lanes1 and 2). In contrast, when cells were induced to express antibody, protein G precipitates clearly contained significant levels of hsc72 (Fig. 3C, lane3), again indicating a selective interaction between hsc72 and HC.

Finally, as a direct measure of the specificity of this interaction, we examined the effect of ATP on the release of HC from hsc72. In vertebrate cells BiP is known to undergo ATP hydrolysis during release of bound protein (Munro and Pelham, 1986). If the binding of hsc72 to immunoglobulin HC mimics that of mammalian BiP, then the same ATP-dependent substrate release should be observed. To test this hypothesis, Protein G precipitates were treated with ATP, and the release of hsc72 into the supernatant was monitored. The results indicate that after ATP treatment, approximately 90% of the hsc72 was dissociated from heavy chain (Fig. 3C, lane4; toppanel) and subsequently appeared in the supernatant (Fig. 3B, lane6; toppanel). In the absence of ATP, all of the hsc72 remained associated with HC (Fig. 3C, lane3; toppanel), and none was released into the supernatant (Fig. 3C, lane5; toppanel). As an internal control, we measured HC directly and showed it to be equivalent in both ATP-treated and untreated samples (Fig. 3C, lanes3 and 4; bottompanel). In addition, no HC was found in the supernatant in response to ATP addition (Fig. 3C, lanes5 and 6; bottompanel). These results demonstrate a specific, ATP-dependent hsc72bulletHC interaction in Drosophila cells that is analogous to the BiPbulletHC interaction in vertebrates.

HC Dimers Are Secreted in the Absence of LC

In vertebrate cells, the interaction of LC with HC appears to be required for antibody secretion. LC interaction is necessary for the efficient dissociation of BiP from HC. Thus, HC dimers alone are not secreted from mammalian cells (Bole et al., 1986) except in rare circumstances where specific mutations in the HC constant region enable it to bypass this process, presumably by allowing BiPbulletHC dissociation in the absence of LC (Hendershot et al., 1987).

To examine and compare the role of hsc72 in immunoglobulin secretion from Drosophila cells, we tested the need for LC synthesis for HC dimer production. We constructed a stable Drosophila cell line, mtHC, containing only the HC gene construct in the absence of the LC gene, and analyzed HC expression from these cells. Surprisingly, and in contrast to what has been found in mammalian systems, the heavy chains were efficiently expressed and secreted from this cell line (Fig. 4, lane2) (Table 1). Furthermore, the apparent molecular weight of the HC produced from this line was found to be identical to that expected for an intact HC dimer, indicating that HC folding and association had occurred in the absence of LC. We next examined whether hsc72 interacts with these HC molecules during synthesis and secretion from this cell line. Lysates from these cells were precipitated with protein G and analyzed by Western blotting. The results show that hsc72 was co-precipitated selectively with HC (Fig. 5, lane3). No hsc72 was precipitated from control lysates that did not contain HC (lanes1 and 2). Clearly, hsc72 specifically interacts with immunoglobulin HC in this cell line, just as was found in the Drosophila cells producing complete antibodies. Apparently, the secretion of HC dimers from this cell line also involves an interaction with hsc72.


Figure 4: Secretion of RSHZ19 immunoglobulin HC dimers in the absence of LC. Analysis of conditioned media from uninduced mtHC cells (lane1), and mtHC cells induced with CuSO(4) (lane2) by nonreducing SDS-polyacrylamide gel electrophoresis and Western analysis to detect immunoglobulin heavy chain polypeptides.




Figure 5: Association of hsc72 with RSHZ19 immunoglobulin HC, secreted as dimers. Western detection of hsc72 (toppanel) or HC immunoglobulins (bottompanel) from protein G precipitations of mtHC cell lysates is shown. Lane1, nonrecombinant S2 cells; lane2, uninduced S2 cells; lanes3 and 4, induced mtHC cells washed in the absence (lane3) or in the presence of 1 mM ATP (lane4); lanes 5 and 6, protein released from the final wash of protein G precipitations in the absence (lane5) or in the presence of 1 mM ATP (lane6).



The specificity of this interaction was examined further by testing for the specific ATP-dependent release of hsc72 from HC. Again, protein G precipitates were treated with ATP and monitored by Western analysis for release of hsc72 into the supernatant. In the absence of ATP, all of the hsc72 remained associated with HC, and none was released into the supernatant (Fig. 5, lanes3 and 5, respectively). In contrast, in the presence of ATP more than 80% of the hsc72 was released from HC into the supernatant (Fig. 5, lanes4 and 6, respectively). Again as an internal control, the level of HC precipitating in the presence or in the absence of ATP was found to be equivalent (Fig. 5, lanes3 and 4; bottompanel). These results demonstrate that hsc72 specifically interacts with immunoglobulin HC dimers during their maturation and secretion and that this process occurs in the absence of light chain expression. Moreover, the efficiency of the process is equivalent to that observed for complete antibodies (Table 1).


DISCUSSION

We have created a stable Drosophila melanogaster cell line that can be selectively induced to co-express IgG HC and LC and that efficiently secretes fully folded antibody exhibiting normal substrate binding affinity. Although Drosophila cells do not naturally produce antibodies, they apparently do possess conserved ER components that can recognize immunoglobulins, properly fold them, and secrete them as mature antibodies from the cell. We have demonstrated that Drosophila hsc72, the insect counterpart to mammalian immunoglobulin binding protein (BiP), is selectively associated with the IgG HC and released from HC in an ATP-dependent fashion. This is analogous to the interaction of BiP with HC characterized in vertebrate cells. Further, we find that Drosophila cells can efficiently secrete HC dimers in the absence of LC production. This dramatically contrasts with vertebrate systems, in which LC synthesis and interaction with HC is known to be required for BiP release and the subsequent secretion of IgG. Although hsc72 specifically interacts with HC in Drosophila, the release is independent of LC association, thereby allowing efficient secretion of these HC dimers. Our results indicate that hsc72 interacts with HC immunoglobulins and plays a role in heterologous IgG production in Drosophila cells but that its release mechanism from HC is independent from LC and therefore mechanistically different from that of its mammalian counterpart.

The ability to efficiently express and secrete antibody in Drosophila cells is consistent with the existence of a highly conserved protein folding and secretion mechanism. Insects do not naturally produce antibodies (Marchalonis and Schluter, 1994), although several insect proteins having homology to the Ig superfamily have been identified (Seeger et al., 1988; Bieber et al., 1989; Pulido et al., 1992; Garbe et al., 1993; Kania et al., 1993). Moreover, the expression of recombinant IgA immunoglobulins has been demonstrated in bacculovirus-infected Spodotera cells (Carayannopoulos et al., 1994). However, in these studies the relative contribution of antibody made as a naturally secreted product versus antibody derived from the lytic stage of the bacculovirus infection was not characterized (Carayannopoulos et al., 1994). Furthermore, no attempt was made to identify virus and/or host components that participated in antibody expression and secretion.

Our demonstration that the Drosophila hsc72 protein selectively interacts with HC during either IgG or HC dimer production, suggests strong similarities between BiP substrates found in vertebrate and nonvertebrate systems. Although no natural substrates upon which hsc72 interacts have been identified to date, one likely candidate group of proteins is composed of those belonging to the immunoglobulin superfamily (Williams and Barclay, 1988). Several Drosophila members of this family have been characterized as cell surface proteins related to cell adhesion molecules (Seeger et al., 1988; Bieber et al., 1989; Sun, et al., 1990; Pulido et al., 1992; Garbe et al., 1993; Kania et al., 1993).

The mechanism operating in mammalian systems to retain HC dimers in the absence of LC expression may represent a safety function that has evolved to ensure proper immune function. The importance of this mechanism in maintaining homeostasis is best illustrated by its disruption in humans leading to lymphoproliferative diseases (Franklin and Frangione, 1975; Seligmann et al., 1979). In mouse tumor lines, mutant heavy chain molecules containing deletions in the C(H)1 domain can bypass this mechanism leading to the secretion of HC in the absence of LC (Morrison and Scharff, 1975; Dackowsi and Scharff, 1981). These mutant HCs also fail to associate with BiP, suggesting that this specific interaction is important for HC retention by BiP (Hendershot, et al., 1987).

There are at least two possible explanations for the efficient secretion of HC dimers in Drosophila. One possibility is that the inability of Drosophila cells to retain HC dimers may be due to the absence of a specific cellular retention domain in hsc72 that is separate from its substrate-binding domain. This retention domain would be operative in mammalian BiP to retain inappropriately produced heavy chain dimers. Such a hypothesis would be consistent with the fact that specific HC mutations that eliminate BiP interaction in mammalian systems also result in efficient secretion of heavy chain dimers and, subsequently, disease. Thus, the absence of a retention domain in hsc72 would allow for heavy chain dimer secretion. Alternatively, the retention of HC in the absence of LC in vertebrate systems could involve ER components other than BiP that are absent in Drosophila cells. For example, in vertebrates there exist additional protein chaperones localized in the ER lumen that have no known Drosophila counterparts. One of these, GRP94, is a member of the hsp90 protein family and has been shown to associate directly with newly synthesized HC and LC immunoglobulins (Melnick et al., 1992). Like BiP, GRP94 associates more stably with aberrant protein forms that are not secreted than with normal secreted proteins, suggesting that it too could contribute to the retention of immunoglobulins (Melnick, et al., 1992). Thus, the mechanism for the retention of incomplete antibodies in vertebrates could involve other ER chaperones, and the absence of functionally equivalent proteins in Drosophila would lead to the efficient secretion of HC in the absence of LC.

The Drosophila antibody expression systems that we have created could be quite useful in determining the importance of various vertebrate chaperones in the retention of incomplete antibodies. Stable introduction of any of these genes into our HC-secreting Drosophila line could be used to determine if their presence will lead to HC retention. Moreover, the ability of Drosophila cells to secrete active antibodies raises the possibility of the transgenic insertion of immunoglobulin genes for the production of monoclonal antibodies in flies. By targeting mAbs against extracellular and cell surface associated proteins, it may therefore be possible to assess biological function by disrupting specific protein interactions. This would be especially useful in the Drosophila system where site directed knock-out experiments are not easily performed without a means of genetically selecting for the loss of the gene being disrupted.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
These authors contributed equally to the work presented in this paper.

To whom correspondence should be addressed: Dept. of Gene Expression Sciences, SmithKline Beecham Pharmaceuticals, 709 Swedeland Rd., King of Prussia, PA 19406. Tel.: 610-270-7727; Fax: 610-270-5093.

(^1)
The abbreviations used are: ER, endoplasmic reticulum; hsc and HSC, heat shock cognate; HC, heavy chain; LC, light chain; mAb, monoclonal antibody; CHO, Chinese hamster ovary; ELISA, enzyme-linked immunosorbent assay.

(^2)
P.R. Tempest, personal communication.

(^3)
J. Trill, S. Ganguly, and C. Silverman, unpublished data.

(^4)
D. Sindoni and C. Silverman, unpublished data.


ACKNOWLEDGEMENTS

We thank Dr. Belinda Bullard for the generous gift of rat mAb for detection of hsc72. We are also grateful to Drs. Karen Palter and David Rubin for many valuable suggestions in the course of this work.


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