Incomplete assembly of IgA2m(2) in Chinese hamster ovary cells

Molecular Immunology 44 (2007) 3445–3452

Incomplete assembly of IgA2m(2) in Chinese hamster ovary cells Koteswara R. Chintalacharuvu ∗ , Brian Gurbaxani 1 , Sherie L. Morrison Department of Microbiology, Immunology and Molecular Genetics and the Molecular Biology Institute, University of California Los Angeles, 405 Hilgard Avenue, Los Angeles, CA 90095, USA. Received 23 October 2006; accepted 22 December 2006 Available online 27 April 2007

Abstract Myeloma and Chinese hamster ovary (CHO) cells are frequently used for the production of recombinant antibodies. With increasing interest in producing recombinant IgA for protection against infectious agents, it is essential to characterize the IgA produced in these cells. Here we show that while myeloma cells secrete IgA2m(2) predominantly as H2 L2 , CHO cells secrete H2 L and H2 in addition to fully assembled H2 L2 . When the CHO cells also synthesize J chain and secretory component (SC), polymeric IgA and secretory IgA in which SC is disulfide bonded to the polymeric IgA are produced. Blocking cysteines on purified IgA2m(2) protein by alkylating with iodoacetamide stabilizes the disulfide bonds between the H and L chains suggesting that the disulfide bonds between H and L chains are unstable. Taken together our results suggest that the covalent assembly of IgA2m(2) is different in myeloma and CHO cells. © 2007 Published by Elsevier Ltd. Keywords: Expression in CHO cells; IgA; IgA2m(2); Disulfide bond instability

1. Introduction Protein folding and disulfide bond formation are important in the assembly and secretion of functional immunoglobulins (Ig). The Ig heavy (H) and light (L) chains are synthesized on the membrane bound polysomes and translocated into the lumen of the ER where they fold into globular domains of 110 amino acids stabilized by at least one intrachain disulfide bond. The basic immunoglobulin is an H2 L2 molecule; however, both IgM and IgA form higher polymers often with associated J chain. IgM and IgA are also found in the mucosal secretions associated with secretory component (SC), the cleavage product of the polymeric immunoglobulin receptor. There are two subclasses of human IgA, IgA1 and IgA2, with IgA2 existing as three allotypes, IgA2m(1) and IgA2m(2) and IgA2n (Chintalacharuvu et al., 1994). The different forms of human IgA differ in their H and L chain disulfide-bonding

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Corresponding author at: Department of Microbiology, Immunology and Molecular Genetics, University of California Los Angeles, 405 Hilgard Ave, Los Angeles, CA 90095-1489, USA. Tel.: +1 310 206 5124; fax: +1 310 794 5126. E-mail address: [email protected] (K.R. Chintalacharuvu). 1 Present address: Centers for Disease Control and Prevention (CDC), 1600 Clifton Road NE, Bldg 6, MS A-15, Atlanta, GA 30333, USA. 0161-5890/$ – see front matter © 2007 Published by Elsevier Ltd. doi:10.1016/j.molimm.2006.12.030

pattern. In IgA1, IgA2m(2) and IgA2n a disulfide bond links the L chain to the H chains. In contrast, the major form of IgA2m(1) lacks disulfide bonds between H and L chain although a small amount of H–L disulfide linked IgA2m(1) is made (Chintalacharuvu and Morrison, 1996). For IgA1, Cys133 bonds to L chain, Cys241 and 301 to H chain, Cys471 to J chain and Cys311 to SC (Fallgreen-Gebaur et al., 1993). A similar structure has been proposed for IgA2m(1) except there is no disulfide bond with the L chain since Cys133 is now Asp (Tsuzukida et al., 1979). Although IgA2m(2) and IgA2n also lack Cys133, they form covalent bonds with L chain using Cys241 or 242 in CH 2 (Chintalacharuvu et al., 2002). Murine myeloma cells that do not produce endogenous heavy and light chains and Chinese hamster ovary (CHO) cells comprise the two most frequently used expression systems for the production of recombinant immunoglobulins. It is generally expected that the immunoglobulins produced by the two systems will be equivalent although the structure of the attached carbohydrate varies. Murine cells but not CHO cells can attach Gal ␣1 → 3 Gal, a structure abundant on glycoconjugates of non-primate mammals, prosimians, and New World monkey but absent from Old World monkeys, apes and man which do not express the necessary glycosyltransferases (Galili, 1989). CHO cells also appear to lack the glycosyltransferases necessary for generating the bisecting GlcNAc as well as ␣2 → 6

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linked sialic acid (Routier et al., 1997). In addition, mutant CHO cell lines derived from the cell line Pro 5 are available with alterations in their functional glycosyl transferases: Lec 2 cells are defective in the transport of CMP-sialic acid and synthesize a complex carbohydrate structure lacking sialic acid. Lec 8 cells fail to transport UDP-galactose, and attach a complex carbohydrate structure lacking galactose (Stanley, 1984, 1987, 1992). The production of recombinant IgA in both murine myelomas and CHO cells has been described. When produced in murine myelomas, the IgA1 and the three allotypes of IgA2 produced show the same covalent structure as serum IgA (Chintalacharuvu and Morrison, 1996; Chintalacharuvu et al., 1994, 2002; Terskikh et al., 1994). Similarly, IgA1 and IgA2m(1) produced in CHO cells have been reported to resemble serum IgA in covalent structure (Berdoz et al., 1999; Johansen et al., 1999; Krugmann et al., 1997; Morton et al., 1993; Preston et al., 1998). In contrast, we now show that IgA2m(2) produced in Pro 5 and in the Lec 2 and Lec 8 CHO cell lines is incompletely assembled. When the CHO cells synthesize H and L chains, they secrete H2 L and H2 in addition to fully assembled H2 L2 . When the CHO cells also synthesize J chain and SC, polymeric IgA is produced, with SC able to form disulfide bonds with the polymeric IgA, although significant quantities of non-covalently attached SC are also observed in the secretions. Blocking cysteines on purified IgA2m(2) protein with iodoacetamide stabilized the disulfide bonds between the H and L chains suggesting that the disulfide bonds between H and L chains are unstable. 2. Materials and methods 2.1. Cell lines The CHO cell lines Pro 5, Lec 2 and Lec 8, originally derived by Dr. Pamela Stanley (Stanley, 1992) were obtained from the American Type Culture Collection (Rockwell, MD). The cells were grown at 37 ◦ C under 5% CO2 in Iscove’s modified Dulbecco’s medium (IMDM; Irvine Scientific, Irvine, CA) supplemented with 5% fetal calf serum (FCS; Hyclone Laboratories, Logan, UT). The cells were maintained in tissue culture-treated Petri dishes (Falcon Labware, Lincoln Park, NJ) and harvested by treatment with trypsin (Gibco, Grand Island, NY). 2.2. Transfection of CHO cells The CHO cells were transfected using Lipofectin (Bethesda Research Laboratories, Bethesda, MD) as previously described (Wright and Morrison, 1994). Chimeric immunoglobulins with human ␣2m(2) and ␬ constant regions and murine variable regions specific for the hapten dansyl were under the control of the SV40 promoter and enhancer (Wright and Morrison, 1994) with his and gpt providing the selectable markers. The expression of human SC was under the control of the SV40 promoter and the vector contained the neo gene. The CMV promoter was used for expression of human J chain with zeocin resistance

providing the selectable marker. Transfectants expressing the desired proteins were identified by ELISA. For transfectants producing H and L chains, plates were coated with dansylBSA and bound Ig detected using anti-␬. To produce secretory IgA (S-IgA), transfectants expressing H and L chains were co-transfected with vectors for J chain and SC, selected with zeocin, and S-IgA bound to dansyl-BSA coated plates detected with anti-SC. Synthesis of J chain was confirmed following SDS-PAGE and either biosynthetic labeling or Western blotting using rabbit anti-J chain (Nordic Immunology, Capistrano Beach, CA). 2.3. Large-scale cell culture and protein purification Transfectomas were cultured in roller bottles (Fisher Scientific, Pittsburgh, PA) in IMDM supplemented with 2% ␣-calf serum (Hyclone). Proteins were purified by affinity chromatography as previously described (Dangl et al., 1988). Protein concentration was determined using the bicinchoninic acid (BCA) assay (Pierce Chemical Co., Rockford, IL) and confirmed by Coomasie blue staining of proteins separated on SDS-polyacrylamide gel. Purified antibodies were analyzed by SDS-PAGE, unreduced in either 4% tris-glycine or 5% phosphate gels and reduced in 12.5% tris-glycine gels. 2.4. Biosynthetic labeling and pulse-chase analysis Transfectomas were washed twice in methionine-free Dulbecco’s modified Eagle’s medium (DME, Irvine Scientific) supplemented with non-essential amino acids (Gibco) and glutamine (29.2 ␮g/ml). Cells were then incubated for 16 h with 1 ml of methionine-free medium containing 1% (v/v) FCS and 12.5 mCi of [35 S]-methionine (Amersham, Arlington Heights, IL). Pulse-chase experiments were performed as previously described (Shin et al., 1992). Briefly, 2 × 107 cells were washed twice and incubated in 0.5 ml of methionine-free medium at 37 ◦ C for 30 min. Cells were pulsed by adding 125 ␮Ci of [35 S]methionine and incubating at 37 ◦ C for five more minutes. Chase was initiated by adding 10 ml of medium containing 10% bovine calf serum and 3 mg/ml (100×) of unlabeled methionine. At various times after the initiation of the chase, 1 ml aliquots of cells were removed and cooled on ice. Cells were pelleted by centrifuging at 13,000 × g for 30 s in the cold and lysed by incubating on ice for 30 min in 0.5 ml of 10 mM Tris buffer, pH 7.4 containing 1% (v/v) NP-40, 0.4% (v/v) deoxycholate and 66 mM EDTA. The lysates were centrifuged at 13,000 × g for 5 min in the cold to remove any unlysed cells and cell debris. To immunoprecipitate cellular IgA, lysates were incubated on ice for 1 h with 5 ␮l of rabbit anti-human ␣ chain (Sigma Immunochemicals, St. Louis, MO) and 2.5 ␮l of rabbit anti-human Fab (Shin et al., 1992) followed by 10 min with 75 ␮l of 10% fixed Staphylococcus aureus cells with surface protein A (IgG Sorb, Enzyme Center Inc., Malden, MA, USA). The bacteria with associated immune complexes was centrifuged at 13,000 × g for 1 min through a 1 ml layer of 30% sucrose in 10 mM Tris containing 0.5% NP-40, 0.2% deoxycholate, 33 mM EDTA and 0.15% SDS, pH 7.4. The pellet was washed twice with 10 mM

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Tris containing 1% NP-40, 0.4% deoxycholate, 66 mM EDTA and 0.3% SDS, pH 7.4 and once with H2 O. The precipitate was resuspended in electrophoresis sample buffer, boiled for 2 min, centrifuged at 13,000 × g for 2 min and the supernatants analyzed by SDS-PAGE. 2.5. Western blotting Four micrograms of protein were separated by SDS-PAGE in phosphate-buffered 5% gels and transferred to Bioblot-NC nitrocellulose (Costar, Cambridge, MA) according to the method of Towbin et al. (1979). Non-specific sites were blocked by incubating the blot for 2 h at room temperature in PBS containing 0.1% (v/v) Tween-20 and 5% (w/v) dried milk. The H or L chains were detected using rabbit anti-human ␣ or anti-human ␬ (Sigma Immunochemicals). The bound rabbit antibodies were detected using HRP conjugated donkey anti-rabbit and the ECL reagent (Amersham, Buchingham, UK). Nitrocellulose was exposed to Kodak XAR film. To re-probe with antibodies of different specificities, the blot associated antibodies were removed by incubating for 30 min at 50 ◦ C in 62.5 mM Tris–HCl, pH 6.7 containing 100 mM ␤-mercaptoethanol and 2% SDS. The efficiency of removal of the antibodies was confirmed by incubating the blot with ECL substrate and exposing to film. 2.6. Gel filtration chromatography Chromatography of purified proteins and their complexes was performed at 4 ◦ C on a Superose 6 10/300 GL column (Amersham Biosciences, Piscataway, NJ) at a flow rate of 0.25 ml/min with PBS (pH 7.2) containing 0.05% sodium azide. The total amount of protein applied to the column was ∼150 ␮g in 200 ␮l. Proteins were detected by absorbance at 280 nm. 2.7. Alkylation of cysteine residues by reaction with iodoacetamide A M stock of iodoacetamide (Sigma–Aldrich, Inc., St. Louis, MO) was prepared in 1 M Tris pH 8.0. Biosynthetically labeled IgA2m(2) in culture medium or immunoprecipitated and resuspended in SDS-PAGE sample buffer was incubated with 25 mM iodoacetamide at room temperature in the dark for 1 h. Affinity purified IgA2m(2) was incubated with 25 mM iodoacetamide at room temperature in the dark for 1 h. When required, unbound iodoacetamide was removed from the protein mixtures by centrifuging three times in Amicon Ultra centrifugal filter devices (Millipore corporation, Bedford, MA) with 5 K MW cut off using PBS, pH 7.4. 3. Results 3.1. Expression of IgA2m(2) in CHO cells Several studies have reported the production of IgA1 and IgA2m(1) in CHO cells (Berdoz et al., 1999; Johansen et al., 1999; Morton et al., 1993). Here we have expressed IgA2m(2)

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in wild-type CHO (Pro 5) and in CHO cell lines Lec 2 and Lec 8 deficient in carbohydrate addition. Transfectants synthesizing chimeric IgA2m(2) were produced by transfecting cells with alpha heavy chains and kappa light chains containing murine variable regions specific for the hapten dansyl. Transfectants synthesizing high levels of protein were identified by ELISA, the secreted immunoglobulins labeled by overnight growth in [35 S]-methionine, immunoprecipitated and analyzed by SDS-PAGE both unreduced and following reduction with ␤-mercaptoethanol. In contrast to what is observed when chimeric IgA2m(2) is produced in murine myeloma cells (Chintalacharuvu et al., 2001), the secreted IgA is incompletely assembled in all three cell lines. A representative SDS-PAGE analysis of proteins secreted by Lec 2 and Pro 5 is shown in Fig. 1. In addition to fully assembled H2 L2 , incomplete assembly intermediates of H2 L and H2 , and L chains not covalently associated with H chain are observed. Pro 5 is the wild-type CHO cell line from which the mutants were isolated, Lec 2 cells fail to attach terminal sialic acid and Lec 8 cells are deficient in the transport of UDP-galactose, and hence possess complex carbohydrates lacking galactose and terminal sialic acid. Heavy chains produced in Pro 5 are seen to migrate more slowly on SDS-PAGE consistent with the more extensive processing of the carbohydrate moieties. These results indicate that the failure to completely assemble the IgA2m(2) is associated with its production in the CHO cell lines, and is not a result of altered processing of the associated glycans. The initial composition of the different peaks was inferred from their relative migration on the SDS-PAGE gels. To confirm the composition, proteins purified by chromatography on dansyl columns were fractionated by SDS-PAGE and analyzed by Western blot using anti-␣ and anti-␬ antibodies (Fig. 2). The results obtained were consistent with the earlier interpretations. Shown for comparison is IgA2m(2) synthesized in Sp2/0 in which only covalently linked H2 L2 and polymers are seen in the Coomassie blue stained proteins. IgA2m(2) synthesized in Sp2/0 is more extensively polymerized, possibly because of the synthesis of J chain by the murine myeloma but not the CHO cell line. 3.2. Incomplete intracellular assembly of IgA2m(2) The secretion of incompletely assembled IgA2m(2) could reflect a failure in assembly. Alternatively, the molecules could initially form the required disulfide bonds and these bonds could be reduced during further protein maturation within the cell. To distinguish between these possibilities, pulse-chase analysis was performed on IgA2m(2) produced by Pro 5, Lec 2 and Lec 8 cells. The results for Lec 2 produced proteins are shown in Fig. 3; similar results are seen for Pro 5 and Lec 8 produced proteins. It is readily apparent that there is a failure to form the disulfide bonds. The incompletely assembled molecules are also seen to appear in the secretions. Interestingly, non-Ig proteins or degraded H chains are immuno-precipitated associated with the H and L chains in the cytoplasm, but only intact H and L chains are apparent in the secretions.

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Fig. 1. Synthesis of IgA2m(2) in Chinese hamster ovary (CHO) cells. Wild type CHO cells (Pro 5) and carbohydrate mutant Lec 2 and Lec 8 cells were stably transfected with IgA2m(2) heavy chains and kappa light chains. Cells were radiolabeled by overnight growth in [35 S]-methionine, the secreted Igs immunoprecipitated with anti-␬ and analyzed either unreduced (A) or following reduction (B). Assembly intermediates and free chains are indicated at the left.

3.3. IgA2m(2) produced in CHO cells associates to form monomers in solution SDS-PAGE analysis indicates the covalent assembly of proteins. It has been suggested that non-covalent IgA monomers will form in solution (Berdoz et al., 1999; Morton et al., 1993). To address this issue, purified IgA2m(2) produced in Lec 2 (the same preparation analyzed by Western blot in Fig. 3) was analyzed by gel filtration on a Superose 6 10/300 GL column in PBS (Fig. 4). A symmetric peak slightly larger than recombinant IgG2 is seen indicating that the IgA2m(2) associates to form monomers in solution. 3.4. Production of S-IgA2m(2)

Fig. 2. Analysis of IgA2m(2) by Western blotting. IgA2m(2) synthesized in Lec 2 CHO cells (Lanes 1, 3 and 5) and IgA2m(2) synthesized in murine myeloma cells (Lanes 2, 4 and 6) were affinity purified by chromatography on dansyl columns. Proteins were fractionated by SDS-PAGE and visualized by staining with Coomassie blue or following Western blotting with anti-␬ and anti-␣. The Western blot analysis with anti-␬ and anti-␣ was done on the same blot after stripping off the initial antibody. The staining of protein with Coomasie blue was done on a separate gel. The CHO cells made H and L chains but not J chain.

There is considerable interest in the development of expression systems suitable for the production of secretory IgA. To produced transfectants producing S-IgA, genes coding for human SC and J chains were transfected into Lec 8 cells producing of ␣2m(2) H chains and ␬ L chains. H2 L and H2 assembly intermediates continued to be observed when SC and SC and J chain were expressed along with H and L chains in the Lec 8 cells (Fig. 5). When SC was expressed with the H and L chains in the absence of J chain, anti-SC precipitated only free SC and anti-␬ precipitated little SC indicating that there was little association of SC with the H chain in

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Fig. 3. Pulse-chase analysis of the assembly of IgA2m(2) in Lec 2 cells. Cells were pulsed with [35 S]-methionine for 5 min, and then chased with excess unlabeled methioine. At the indicated times, samples were removed, cytoplasmic lysates and secretions prepared and immunoprecipitated with anti-␬. Samples were analyzed either unreduced (panels A and C) or following reduction (panels B and D). Cytoplasmic Igs are shown in panels A and B; secreted Igs in panels C and D. The times following the chase when the sample was harvested is shown at the top of the gels. The size of marker proteins (M) are indicated at the side of the gel. Assembly intermediates as well as free H and L chains are indicated.

the absence of J chain as would be expected. When both SC and J chain were co-expressed with H and L chains, precipitation with anti-␬ brought down H2 L2 , H2 L and H2 as well as covalently assembled polymers and small amounts of free SC. Precipitation with anti-SC brought down free SC, H2 L2 , H2 L and H2 as well as covalently assembled polymers. In contrast to what had been observed with transfected murine myelomas producing secretory IgA (Chintalacharuvu and Morrison, 1997), significant quantities of free SC are secreted, some of which is non-covalently associated and precipitated with anti-␬.

3.5. Treatment with iodoacetamide Alkylation of free cysteine residues stabilizes disulfide bonds (Chintalacharuvu et al., 1993). To determine if alkylation of IgA2m(2) produced by CHO cells stabilizes the disulfide bonds, proteins were treated with iodoacetamide (IA) and analyzed by SDS-PAGE (Figs. 6 and 7). IA treatment either in culture medium or under denaturing conditions in SDS-PAGE sample buffer had no effect on biosynthetically labeled and immunoprecipitated IgA2m(2) (Fig. 6). Surprisingly, when proteins purified by antigen-based affinity chromatography was treated with IA,

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Fig. 4. Gel filtration chromatography of purified proteins. Elution profiles of IgA2m(2) produced in Lec 2 cells and IgG2 produced in murine myeloma cells obtained with Superose 6 10/300 GL column are shown. Proteins were detected by absorbance at 280 nm.

Fig. 6. Iodoacetamide treatment of biosynthetically labeled and immunoprecipitated IgA2m(2). Lec 8 cells producing IgA2m(2) H and L chain were biosynthetically labeled, immunoprecipitated and analyzed by SDS-PAGE under non-reducing conditions as described in Fig. 5. Proteins were incubated with 25 mM IA in supernatant only (Lane 3), in SDS-PAGE sample buffer only (Lane 4) and both in supernatant and sample buffer (Lane 5). Untreated IgA2m(2) produced in Lec 8 cells (Lane 1) and Sp2/0 (Lane 6) cells are shown as controls.

there was a significant decrease in H2 L and L2 and L with a corresponding increase in H2 L2 in proteins produced by wild type CHO, Lec 2 and Lec 8 cells (Fig. 7). In addition, the H2 band completely disappeared from protein produced by Lec 2 cells. Similar results were obtained when S-IgA2m(2) produced in Lec 8 cells was treated with iodoacetamide (Lanes 7 and 8). These results suggest that IA treatment stabilizes the H–L chain disulfide bonds in purified chimeric IgA2m(2) produced in CHO cells. It should also be noted that fewer assembly intermediates are observed in proteins purified by antigen-based affinity chromatography that in those immunoprecipitated from culture supernatants. Fully assembled IgA with two functional binding sites might be expected to be more efficiently bound by antigen columns. 4. Discussion

Fig. 5. Synthesis of S-IgA2m(2) in Lec 8 CHO cells. Lec 8 cells were stably transfected with IgA2m(2) heavy chain and kappa light chain (Lane 1), IgA2m(2) heavy chains, kappa light chains and SC (Lanes 2 and 3), or IgA2m(2) heavy chains, kappa light chains, SC and J chain (Lanes 4–7). Cells were radiolabeled by overnight growth in [35 S]-methionine, the secreted Igs immunoprecipitated with anti-␬ or anti-SC and analyzed either unreduced (top panel or following reduction (bottom panel). Assembly intermediates and free chains are indicated at the left.

Plasma cells are the normal antibody producing factories within the body. Myeloma cells, which are malignant plasma cells, have been found to be an effective system for the production of recombinant antibodies. The different IgA isotypes and allotypes all appear to be properly assembled in murine myeloma cells. In contrast, CHO cells, the other cell line frequently used for the production of recombinant antibodies, are derived from cells that normally do not produce antibodies. In contrast to IgA1 and IgA2m(1) that have been reported to properly assemble when expressed in CHO cells (Berdoz et al., 1999; Morton et al., 1993), our studies now suggest that IgA2m(2) is not properly assembled when expressed in CHO cells. Surprisingly, the small changes in amino acid sequence that distinguish the isotypes and allotypes of IgA appear to lead to differences in assembly in CHO cells. Anti-NIP IgA1 in which the light

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Fig. 7. Iodoacetamide treatment of purified IgA2m(2). IgA2m(2) produced by Pro 5 (Lanes 1 and 2), Lec 2 (Lanes 3 and 4), Lec 8 (Lanes 5 and 6), Sp2/0 myeloma cells (Lanes 9 and 10) and S-IgA2m(2) produced by Lec 8 cells (Lanes 7 and 8) were affinity purified. Proteins were treated with 25 mM IA and analyzed by SDS-PAGE in 5% phosphate gels under non-reducing conditions (Lanes 1, 3, 5, 7 and 9).

chain is murine ␭ (Johansen et al., 2001, 1999; Krugmann et al., 1997; Morton et al., 1993) was found to form monomers in the absence of J chain and dimers when J chain was present. AntiNIP IgA2m(1) and chimeric anti-RSV IgA2m(1) both formed primarily heavy chain dimers without covalently associated L chain dimers in the absence of J chain (Berdoz et al., 1999; Morton et al., 1993; Senior et al., 2000) as had been reported for IgA2m(1) produced in murine myeloma cells (Chintalacharuvu and Morrison, 1996; Morton et al., 1993). When J chain was expressed, CHO cells were found to assemble and polymerize IgA2m(1) to the same extent as most IgA secreting hybridomas (Berdoz et al., 1999). When SC was also expressed, free SC in addition to S-IgA was found in the supernatants of both IgA1 and IgA2m(1) producing CHO cells (Johansen et al., 2001, 1999). Both IgA1 and IgA2m(2) form covalently assembled H2 L2 molecules. However, they use different Cys residues to form the H–L disulfide bond. In IgA1 Cys133 in CH 1 forms the disulfide bond with the Cys residue at the terminus of the light chain. However, Cys133 is lacking in IgA2 and indeed for IgA2m(1) there is very little covalent assembly of the light and heavy chains. IgA2m(1) and IgA2m(2) differ at the residue following Cys220: IgA2m(1) has Pro 221 whereas IgA2m(2) has Arg221. Although mutagenesis studies showed that the polymorphism at position 221 was responsible for the difference in disulfide bond formation between IgA2m(1) and IgA2m(2) (Chintalacharuvu and Morrison, 1996), it is not Cys220 in CH 1 but instead Cys241 at the beginning of the CH 2 domain that was required for the inter-chain disulfide in IgA2m(2) (Chintalacharuvu et al., 2002); although Cys242 cannot be formally excluded from making the H–L disulfide, it most probably makes an intrachain disulfide with Cys299 in both IgA1 and IgA2 (Herr et al., 2003). Solution structures indicate that IgA1 and IgA2 differ significantly from each other (Boehm et al., 1999; Furtado et al., 2004). In IgA1, the Fab and Fc are linked by a glycosylated hinge region of 23 amino acids. The O-linked glycosylation of the hinge causes it to adopt an extended conformation and the presence of

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this hinge results in an extended Fab and Fc arrangement (Boehm et al., 1999). The studies also suggested that the tailpiece extension characteristic of IgA and IgM is folded up against the CH 3 domain so that Cys311 that is thought to form a disulfide bond with SC could also form a bond with Cys471 in the tailpiece. In contrast, modeling indicated monomeric human IgA2m(1) to be significantly more compact than IgA1 (Furtado et al., 2004). The more compact nature of IgA2m(1) can undoubtedly be attributed to its shorter hinge of only 10 amino acids. The spatial arrangement of cysteines is important for the formation of disulfide bonds, and IgA1 and IgA2m(2) have significant differences that could impact the ease of disulfide bond formation. IgA1 has Pro at position 212 in CH 1; there is a Ser at this position in IgA2m(2) resulting in the addition of N-linked carbohydrate at amino acid 211. As discussed above, IgA1 also has a second Pro at position 221 in CH 1. Prolines introduce kinks in the folding of the polypeptide chain and the presence of Pro residues may hinder the accessibility of Cys on the H chain for bonding with the L chain. Alternatively, a chaperone and/or cellular enzymes involved in disulfide bond formation may not be effective when prolines kink the polypeptide. An unresolved issue is why do we observe differences in the assembly of IgA2m(2) produced by CHO and murine myeloma cells? It is well appreciated that chaperones such as heavy chain binding protein (BiP) and protein-disulfide isomerase (PDI) plays important roles in the folding and assembly of antibody molecule (Lee et al., 1999; Lilie et al., 1994; Mayer et al., 2000; Meunier et al., 2002). It is possible that CHO cells differ from myeloma cells in the expression of these or other important chaperones. It is noteworthy that molecules that are not the size of H or L chain precipitate with H and L chains during the pulsechase analysis of antibody assembly in CHO cells (Fig. 3). These bands were not seen when a similar analysis was performed in murine myeloma cells (Chintalacharuvu and Morrison, 1996) and it is possible that they represent proteins that are playing a role in immunoglobulin assembly and may influence disulfide bond formation. However, initial attempts to identify molecules associated with H and L chains within the CHO cells met with failure and no candidate chaperones were identified (data not shown). Acknowledgements This work was supported by NIH grants AI29470, AI39187 and AI51415 to SLM and an NSF pre-doctoral graduate Fellowship to B.G. References Berdoz, J., Blanc, C.T., Reinhardt, M.P.K.J., Corthesy, B., 1999. In vitro comparision of the antigen-binding and stability properties of the various molecular forms of IgA antibodies assembled and produced in CHO cells. Proc. Natl. Acad. Sci. U. S. A. 96, 3029–3034. Boehm, M.K., Woof, J.M., Kerr, M.A., Perkins, S.J., 1999. The Fab and Fc fragments of IgA1 exhibit a different arrangement from that in IgG: a study by X-ray and neutron solution scattering and homology modelling. J. Mol. Biol. 286, 1421–1447.

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