Recombinant human IgA expressed in insect cells.

Abstract

IgA serves as the fwst line of humoral defense at all mucosal surfaces and is present in large quantities in serum. To map the sites of interaction of immune effector molecules with the IgA constant region (C.), we have expressed soluble, chimeric human IgA in insect cells using recombinant baculoviruses. This antibody is correctly assembled into heavy chain/light chain heterodimers, N-glycosylated Consequently, it is also the target of specific receptors and proteases produced by a number of pathogenic bacteria (6). Blood also contains a large quantity [average, 2 mg/ml (7)] of predominantly monomeric IgA; this circulating pool is largely independent ofthe mucosal pool in humans (8).(ii) IgA binds to an Fca receptor [monocyte/macrophage (mFcaR) (9-11)] on the surface of eosinophils (12), neutrophils (13), and monocytes/macrophages (9), triggering effector responses in so doing. (iii) B lymphocytes and T lymphocytes possess surface receptors for Fca through which immunoregulatory signals are thought to be transmitted (14). (iv) IgA is thought to activate complement through the alternative pathway (15). Thus, IgA not only plays a role in host defense against extracellular viruses and bacteria but is also potentially critical for neutralization of intracellular viruses in tissues expressing pIgR (16) and for destruction of helminths, protozoans, and other eukaryotic parasites (17).Several immunologic disease processes are mediated by IgA, including IgA glomerulonephritis (18) and possibly the exacerbation of allergic asthma (19,20). Each of these properties of IgA depends on the ability of effector molecules such as complement component C3 and mFcaR to recognize specific sites on the surface of the IgA heavy-chain constant region (CJ; however, none of these sites has been well defined.To investigate the above-mentioned functions of IgA, we have established its production in insect cells utilizing recombinant baculoviruses (21). The baculovirus system allows production of mutant antibodies as well as combinatorial expression of immunoglobulin with other polypeptides (J chain, chaperoning, etc.) much more rapidly than with stably transfected mammalian lines. Here we describe production of immunologically and functionally authentic human IgA with hapten specificity and explore the utility of this expression system in addressing structure-function correlations in Ca. MATERIALS AND METHODSSynthesis of Ig Coding Regions. DNA fragments corresponding to the heavy-and light-chain variable (V) regions of monoclonal antibody (mAb) 93G7 were amplified by PCR from plasmids pHyl-360E and pHK-360E (22), respectively. The oligonucleotide primers for these amplifications included a 5' Nco I site at the initiation codon and a 12-nt antisense overlap with Cca at the 3' end. The coding regions of human Cal and CK were obtained from human peripheral-blood leukocyte RNA by reverse transcription-PCR (23); in this case, the primers included a 12-nt sense overlap with the appropriate V region at the 5' end and an Xba I site at the...

Characterization of Human Class-Switched Polymeric (Immunoglobulin M [IgM] and IgA) Anti-Human Immunodeficiency Virus Type 1 Antibodies 2F5 and 2G12

“…1). The J-chain has been suggested to be crucial for pentamer formation of IgM (16) and dimer formation of IgA (4,10). Since a suitable antibody to detect incorporated human J-chain was not available, we did not determine J-chain in the immunoblots.…”

The Carboxyl-terminal Domains of IgA and IgM Direct Isotype-specific Polymerization and Interaction with the Polymeric Immunoglobulin Receptor

“…Mucosally produced IgA consists predominantly of dimers and some larger polymers, collectively called polymeric IgA (pIgA). 1 IgA polymerization is regulated by the incorporation of the joining chain (J chain) in that its presence greatly stimulates polymerization (3)(4)(5)(6)(7)(8)(9). J chain is also essential in forming a docking site on pIgA for the polymeric immunoglobulin receptor (pIgR) (5, 10 -14).…”

The joining (J) chain is present in invertebrates that do not express immunoglobulins.

“…In vertebrates, the J chain is found exclusively in association with polymeric Ig and participates in Ig polymerization (1)(2)(3)14). Randoll et al (34) demonstrated that transfection of a J-chain cDNA into antibody-secreting B cells lacking J-chain expression resulted predominantly in the secretion of the pentameric form of IgM.…”