Alternative pathways of disulfide bond formation yield secretion-competent, stable and functional immunoglobulins

Abstract

Disulfide bonds within and between proteins are responsible for stabilizing folding and covalent assembly. They are thought to form by an obligatory pathway that leads to a single native structure compatible with secretion. We have previously demonstrated that the intradomain disulfide in the C H 1 domain of the Ig γ2b heavy chains was dispensable for secretion (Elkabetz et al., 2005). Here we show that the heavy chain-light chain interchain disulfide is also dispensable. γ2b with mutated Cys128, which normally disulfide bonds with the light chain, still assembled with λI light chain into a secretion-competent, tetrameric IgG2b. This assembly comprised of a covalent homodimer of mutant heavy chains (C128S 2 ) accompanied non-covalently by a covalent homo-dimer of light chains (λ 2 ). The λ 2 homo-dimer formed only upon association with C128S 2 , through disulfide bonding of the two "orphan" heavy chain-interacting Cys214 in λI. The unique Ig tetramer was secreted efficiently as a functional antibody whose antigen binding capacity resembled that of normal IgG2b. Therefore, disulfide bonding of Ig manifests considerable plasticity and can generate more than one functional structure that is considered native by the cellular quality control system.

Orchestration of secretory protein folding by ER chaperones

“…B and T cell receptors or MHC proteins assemble from 2 to 7 subunits into the biologically active entity before they exit the ER. In either case, there is at least one subunit whose folding is a pre-requisite for assembly: in the case of B cell receptor, the folding of the heavy chain is completed only upon subunit assembly [81, 82] and in the case of the T cell receptor - only upon folding of the CD3 epsilon subunit [[83]. A similar situation is observed for nicotinic acetylcholine receptor and likely many other receptors [84]: by stabilizing and sequestering subunits during assembly, chaperones like calnexin, BiP and ERp57 regulate the levels of assembled functional receptors.…”

“…Unlike BiP binding to many other sites in the Ig molecule [12, 13], its binding to the C H 1 domain is persistent, ensuring that C H 1 remains unoxidized long after other domains have folded [81]. The ‘purpose’ of this built-in delay is to coordinate the formation of the intradomain disulfide with that of the inter-subunit disulfide bond [51, 82], a key step that links completion of the heavy chain subunit folding to the assembly of heavy and light chains [51, 85]. In vivo experiments demonstrate that these steps, including association with a folded C L domain and isomerization of a conserved proline residue, are essential for antibody assembly and secretion in the cell [85].…”

The Role of cis Dimerization of Signal Regulatory Protein α (SIRPα) in Binding to CD47

“…The formation of such intermolecular disulfide bonds is dependent on intimate noncovalent association between monomers before bond formation. In immunoglobulins, for example, noncovalent association of light chains and heavy chains has been shown to occur before interchain covalent linkage (35,36,50). The high degree of homology between SIRP␣ and SIRP␤ led us to hypothesize that SIRP␣ may form noncovalently linked dimers.…”

“…Because the ectodomains of SIRP␣ and SIRP␤ exhibit a very high degree of homology in protein sequences (Fig. 1A) and the general belief that monomeric partners of a dimer must pair noncovalently in a thermodynamically favorable fashion in order to facilitate the formation of an intermolecular disulfide bond (35,36), we hypothesized that SIRP␣ may form a noncovalently linked dimer on the cell surface. To address this possibility, we analyzed cell lysates of HL-60, a human promyelocytic leukemia cell line that is known to express SIRP␣ and SIRP␤ (31,37), by immunoblot for the presence of SIRP␣ dimers by SDS-PAGE.…”

Creation and evaluation of a single‐chain antibody tetramer that targets brain endothelial cells

“…To this end, several approaches have been used to prepare protein and peptide tetramers, such as adjusting the linker length between the heavy and light chains of scFvs, secreting the antibody in a designed tetramer format or expressing as an scFv‐streptavidin fusion that will spontaneously form tetramers via streptavidin (SA) interactions . As an alternative, one can take advantage of the tetrameric nature of avidin or SA along with its strong affinity for biotin, first biotinylating the target protein and then combining with SA to form tetramers .…”