Immunoglobulin heavy chain binding protein (BiP) is the only member of the stress 70 protein family that is localized to the endoplasmic reticulum (ER) ()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 HCLC 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

()

()

Cell Culture and Cell Lines

Antigen Binding ELISA Assay

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RNA Analysis

Precipitation of hsc72HC Complexes

Metabolic Labeling

Purification of RSHZ19 mAb

Protein Analysis

RESULTS

Production of Active Human IgG1 Antibodies from Drosophila Cells

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. 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 HCLC 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 1.0 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

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 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 (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 hsc72HC interaction in Drosophila cells that is analogous to the BiPHC interaction in vertebrates.

HC Dimers Are Secreted in the Absence of LC

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 (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 C1 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.

()

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.

()

P.R. Tempest, personal communication.

()

J. Trill, S. Ganguly, and C. Silverman, unpublished data.

()

D. Sindoni and C. Silverman, unpublished data.

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