The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation.

Human immunoglobulin G isotype 4 (IgG4) antibodies (Abs) are potential candidates for immunotherapy when reduced effector functions are desirable. IgG4 Abs are dynamic molecules able to undergo a process known as Fab arm exchange (FAE). This results in functionally monovalent, bispecific antibodies (bsAbs) with unknown specificity and hence, potentially, reduced therapeutic efficacy. IgG4 FAE is suggested to be an important biological mechanism that provides the basis for the anti-inflammatory activity attributed to IgG4 Abs. To date, the mechanism of FAE is not entirely understood and studies measuring FAE in ex vivo matrices have been hampered by the presence and abundance of endogenous IgG4 wild-type (WT) Abs. Using representative humanized WT IgG4 monoclonal Abs, namely, anti-IL-6 and anti-TNF, and a core-hinge stabilized serine 228 to proline (S228P) anti-IL-6 IgG4 mutant, it is demonstrated for the first time how anti-IgG4 affinity chromatography can be used to prepare physiologically relevant matrices for assessing and quantifying FAE. A novel method for quantifying FAE using a single MSD immunoassay is also reported and confirms previous findings that, dependent on the redox conditions, the S228P mutation can prevent IgG4 FAE to undetectable levels both in vitro and in vivo. Together, the findings and novel methodologies will allow researchers to monitor and quantify FAE of their own IgG4 molecules in physiologically relevant matrices.

Latrophilin 1 and its endogenous ligand Lasso/teneurin-2 form a high-affinity transsynaptic receptor pair with signaling capabilities.

Latrophilin 1 (LPH1), a neuronal receptor of α-latrotoxin, is implicated in neurotransmitter release and control of presynaptic Ca(2+). As an "adhesion G-protein-coupled receptor," LPH1 can convert cell surface interactions into intracellular signaling. To examine the physiological functions of LPH1, we used LPH1's extracellular domain to purify its endogenous ligand. A single protein of ∼275 kDa was isolated from rat brain and termed Lasso. Peptide sequencing and molecular cloning have shown that Lasso is a splice variant of teneurin-2, a brain-specific orphan cell surface receptor with a function in neuronal pathfinding and synaptogenesis. We show that LPH1 and Lasso interact strongly and specifically. They are always copurified from rat brain extracts. Coculturing cells expressing LPH1 with cells expressing Lasso leads to their mutual attraction and formation of multiple junctions to which both proteins are recruited. Cells expressing LPH1 form chimerical synapses with hippocampal neurons in cocultures; LPH1 and postsynaptic neuronal protein PSD-95 accumulate on opposite sides of these structures. Immunoblotting and immunoelectron microscopy of purified synapses and immunostaining of cultured hippocampal neurons show that LPH1 and Lasso are enriched in synapses; in both systems, LPH1 is presynaptic, whereas Lasso is postsynaptic. A C-terminal fragment of Lasso interacts with LPH1 and induces Ca(2+) signals in presynaptic boutons of hippocampal neurons and in neuroblastoma cells expressing LPH1. Thus, LPH1 and Lasso can form transsynaptic complexes capable of inducing presynaptic Ca(2+) signals, which might affect synaptic functions.

Anti-AMPA Receptor Autoantibodies Reduce Excitatory Currents in Rat Hippocampal Neurons.

The GluR3 subunit of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) has been identified as a target for autoantibodies (Aabs) in autoimmune encephalopathy and other diseases. Recent studies have proposed mechanisms by which these Aabs act, but their exact role in neuronal excitability is yet to be established. Patient Aabs have been shown to bind to specific regions within the GluR3 subunit. GLUR3B peptides were designed based on described (ELISA) immunogenic epitopes for Aabs and an immunisation strategy was used to generate novel anti-AMPAR Aabs. Target-specific binding and specificity of affinity-purified anti-AMPAR Aabs was confirmed using enzyme-linked immunosorbent assay, immunocytochemistry and Western blot. Functional anti-AMPAR Aab effects were determined on excitatory postsynaptic currents (EPSCs) from primary hippocampal neurons using whole-cell patch-clamp electrophysiology. Acute (10 or 30 min) or longer-term (24 h) application of anti-AMPAR Aabs caused a significant reduction in the mean frequency of spontaneous and miniature EPSCs in hippocampal neurons. Our data demonstrate that anti-AMPAR Aabs targeting peptides linked to auto-immune diseases mediate inhibitory effects on neuronal excitability at the synaptic level, such effects may lead to disruption of the excitatory/inhibitory balance at a network level.

CD1a promotes systemic manifestations of skin inflammation.

Inflammatory skin conditions are increasingly recognised as being associated with systemic inflammation. The mechanisms connecting the cutaneous and systemic disease are not well understood. CD1a is a virtually monomorphic major histocompatibility complex (MHC) class I-like molecule, highly expressed by skin and mucosal Langerhans cells, and presents lipid antigens to T-cells. Here we show an important role for CD1a in linking cutaneous and systemic inflammation in two experimental disease models. In human CD1a transgenic mice, the toll-like receptor (TLR)7 agonist imiquimod induces more pronounced splenomegaly, expansion of the peripheral blood and spleen T cell compartments, and enhanced neutrophil and eosinophil responses compared to the wild-type, accompanied by elevated skin and plasma cytokine levels, including IL-23, IL-1α, IL-1ß, MCP-1 and IL-17A. Similar systemic escalation is shown in MC903-induced skin inflammation. The exacerbated inflammation could be counter-acted by CD1a-blocking antibodies, developed and screened in our laboratories. The beneficial effect is epitope dependent, and we further characterise the five best-performing antibodies for their capacity to modulate CD1a-expressing cells and ameliorate CD1a-dependent systemic inflammatory responses. In summary, we show that a therapeutically targetable CD1a-dependent pathway may play a role in the systemic spread of cutaneous inflammation.

The latrophilins, "split-personality" receptors.

Latrophilin, a neuronal "adhesion-G protein-coupled receptor", is the major brain receptor for alpha-latrotoxin, a black widow spidertoxin which stimulates strong neuronal exocytosis in vertebrates. Latrophilin has an unusual structure consisting of two fragments that are produced by the proteolytic cleavage of the parental molecule and that behave independently in the plasma membrane. On binding an agonist, the fragments reassociate and send an intracellular signal. This signal, transduced by a heterotrimeric G protein, causes release of calcium from intracellular stores and massive release of neurotransmitters. Latrophilin represents a phylogenetically conserved family of receptors, with orthologues found in all animals and up to three homologues present in most chordate species. From mammalian homologues, latrophilins 1 and 3 are expressed in neurons, while latrophilin 2 is ubiquitous. Latrophilin 1 may control synapse maturation and exocytosis, whereas latrophilin 2 may be involved in breast cancer. Latrophilins may play different roles during development and in adult animals: thus, LAT-1 determines cell fate in early embryogenesis in Caenorhabditis elegans and controls neurotransmitter release in adult nematodes. This diversity suggests that the functions of latrophilins may be determined by their interactions with respective ligands. The finding of the ligand of latrophilin 1, the large postsynaptic protein lasso, is the first step in the quest for the physiological functions of latrophilins.

Penelope's web: using alpha-latrotoxin to untangle the mysteries of exocytosis.

For more than three decades, the venom of the black widow spider and its principal active components, latrotoxins, have been used to induce release of neurotransmitters and hormones and to study the mechanisms of exocytosis. Given the complex nature of alpha--latrotoxin (alpha-LTX) actions, this research has been continuously overshadowed by many enigmas, misconceptions and perpetual changes of the underlying hypotheses. Some of the toxin's mechanisms of action are still not completely understood. Despite all these difficulties, the extensive work of several generations of neurobiologists has brought about a great deal of fascinating insights into pre-synaptic processes and has led to the discovery of several novel proteins and synaptic systems. For example, alpha-LTX studies have contributed to the widespread acceptance of the vesicular theory of transmitter release. Pre-synaptic receptors for alpha-LTX--neurexins, latrophilins and protein tyrosine phosphatase sigma--and their endogenous ligands have now become centrepieces of their own areas of research, with a potential of uncovering new mechanisms of synapse formation and regulation that may have medical implications. However, any future success of alpha-LTX research will require a better understanding of this unusual natural tool and a more precise dissection of its multiple mechanisms.

C-terminal phosphorylation of latrophilin-1/ADGRL1 affects the interaction between its fragments.

Latrophilin-1 is an adhesion G protein-coupled receptor that mediates the effect of α-latrotoxin, causing massive release of neurotransmitters from nerve terminals and endocrine cells. Autoproteolysis cleaves latrophilin-1 into two parts: the extracellular N-terminal fragment (NTF) and the heptahelical C-terminal fragment (CTF). NTF and CTF can exist as independent proteins in the plasma membrane, but α-latrotoxin binding to NTF induces their association and G protein-mediated signaling. We demonstrate here that CTF in synapses is phosphorylated on multiple sites. Phosphorylated CTF has a high affinity for NTF and copurifies with it on affinity columns and sucrose density gradients. Dephosphorylated CTF has a lower affinity for NTF and can behave as a separate protein. α-Latrotoxin (and possibly other ligands of latrophilin-1) binds both to the NTF-CTF complex and receptor-like protein tyrosine phosphatase σ, bringing them together. This leads to CTF dephosphorylation and facilitates CTF release from the complex. We propose that ligand-dependent phosphorylation-dephosphorylation of latrophilin-1 could affect the interaction between its fragments and functions as a G protein-coupled receptor.

Proteolytically released Lasso/teneurin-2 induces axonal attraction by interacting with latrophilin-1 on axonal growth cones.

A presynaptic adhesion G-protein-coupled receptor, latrophilin-1, and a postsynaptic transmembrane protein, Lasso/teneurin-2, are implicated in trans-synaptic interaction that contributes to synapse formation. Surprisingly, during neuronal development, a substantial proportion of Lasso is released into the intercellular space by regulated proteolysis, potentially precluding its function in synaptogenesis. We found that released Lasso binds to cell-surface latrophilin-1 on axonal growth cones. Using microfluidic devices to create stable gradients of soluble Lasso, we show that it induces axonal attraction, without increasing neurite outgrowth. Using latrophilin-1 knockout in mice, we demonstrate that latrophilin-1 is required for this effect. After binding latrophilin-1, Lasso causes downstream signaling, which leads to an increase in cytosolic calcium and enhanced exocytosis, processes that are known to mediate growth cone steering. These findings reveal a novel mechanism of axonal pathfinding, whereby latrophilin-1 and Lasso mediate both short-range interaction that supports synaptogenesis, and long-range signaling that induces axonal attraction.

Neurexin Ibeta and neuroligin are localized on opposite membranes in mature central synapses.

Synaptogenesis requires formation of trans-synaptic complexes between neuronal cell-adhesion receptors. Heterophilic receptor pairs, such as neurexin Ibeta and neuroligin, can mediate distinct intracellular signals and form different cytoplasmic scaffolds in the pre- and post-synaptic neuron, and may be particularly important for synaptogenesis. However, the functions of neurexin and neuroligin depend on their distribution in the synapse. Neuroligin has been experimentally assigned to the post-synaptic membrane, while the localization of neurexin remains unclear. To study the subcellular distribution of neurexin Ibeta and neuroligin in mature cerebrocortical synapses, we have developed a novel method for the physical separation of junctional membranes and their direct analysis by western blotting. Using urea and dithiothreitol, we disrupted trans-synaptic protein links, without dissolving the lipid phase, and fractionated the pre- and post-synaptic membranes. The purity of these fractions was validated by electron microscopy and western blotting using multiple synaptic markers. A quantitative analysis has confirmed that neuroligin is localized strictly in the post-synaptic membrane. We have also demonstrated that neurexin Ibeta is largely (96%) pre-synaptic. Thus, neurexin Ibeta and neuroligin normally form trans-synaptic complexes and can transduce bidirectional signals.

Improving the sensitivity and specificity of a bioanalytical assay for the measurement of certolizumab pegol.

AIM: In order to evaluate placental transfer of certolizumab pegol (CZP), a more sensitive and selective bioanalytical assay was required to accurately measure low CZP concentrations in infant and umbilical cord blood. Results & methodology: A new electrochemiluminescence immunoassay was developed to measure CZP levels in human plasma. Validation experiments demonstrated improved selectivity (no matrix interference observed) and a detection range of 0.032-5.0 µg/ml. Accuracy and precision met acceptance criteria (mean total error ≤20.8%). Dilution linearity and sample stability were acceptable and sufficient to support the method. CONCLUSION: The electrochemiluminescence immunoassay was validated for measuring low CZP concentrations in human plasma. The method demonstrated a more than tenfold increase in sensitivity compared with previous assays, and improved selectivity for intact CZP.

The Mechanism of Regulated Release of Lasso/Teneurin-2.

Teneurins are large cell-surface receptors involved in axon guidance. Teneurin-2 (also known as latrophilin-1-associated synaptic surface organizer (Lasso)) interacts across the synaptic cleft with presynaptic latrophilin-1, an adhesion G-protein-coupled receptor that participates in regulating neurotransmitter release. Lasso-latrophilin-1 interaction mediates synapse formation and calcium signaling, highlighting the important role of this trans-synaptic receptor pair. However, Lasso is thought to be proteolytically cleaved within its ectodomain and released into the medium, making it unclear whether it acts as a proper cell-surface receptor or a soluble protein. We demonstrate here that during its intracellular processing Lasso is constitutively cleaved at a furin site within its ectodomain. The cleaved fragment, which encompasses almost the entire ectodomain of Lasso, is potentially soluble; however, it remains anchored on the cell surface via its non-covalent interaction with the transmembrane fragment of Lasso. Lasso is also constitutively cleaved within the intracellular domain (ICD). Finally, Lasso can be further proteolytically cleaved within the transmembrane domain. The third cleavage is regulated and releases the entire ectodomain of Lasso into the medium. The released ectodomain of Lasso retains its functional properties and binds latrophilin-1 expressed on other cells; this binding stimulates intracellular Ca(2+) signaling in the target cells. Thus, Lasso not only serves as a bona fide cell-surface receptor, but also as a partially released target-derived signaling factor.

Functional cross-interaction of the fragments produced by the cleavage of distinct adhesion G-protein-coupled receptors.

The unusual adhesion G-protein-coupled receptors (aGPCRs) contain large extracellular N-terminal domains, which resemble cell-adhesion receptors, and C-terminal heptahelical domains, which may couple to G-proteins. These receptors are cleaved post-translationally between these domains into two fragments (NTF and CTF). Using the aGPCR latrophilin 1, we previously demonstrated that the fragments behave as independent cell-surface proteins. Upon binding the agonist, alpha-latrotoxin (LTX), latrophilin fragments reassemble and induce intracellular signaling. Our observations raised important questions: is the aGPCR signaling mediated by reassembled fragments or by any non-cleaved receptors? Also, can the fragments originating from distinct aGPCRs form hybrid complexes? To answer these questions, we created two types of chimerical constructs. One contained the CTF of latrophilin joined to the NTF of another aGPCR, EMR2; the resulting protein did not bind LTX but, similar to latrophilin, could couple to G-proteins. In another construct, the NTF of latrophilin was fused with the C terminus of neurexin; this chimera bound LTX but could not signal via G-proteins. Both constructs were efficiently cleaved in cells. When the two constructs were co-expressed, their fragments could cross-interact, as shown by immunoprecipitation. Furthermore, LTX(N4C) induced intracellular Ca2+ signaling only in cells expressing both constructs but not each individual construct. Finally, we demonstrated that fragments of unrelated aGPCRs can be cross-immunoprecipitated from live tissues. Thus, (i) aGPCR fragments behave as independent proteins, (ii) the complementary fragments from distinct aGPCRs can cross-interact, and (iii) these cross-complexes are functionally active. This unusual cross-assembly of aGPCR fragments could couple cell-surface interactions to multiple signaling pathways.

Latrophilin fragments behave as independent proteins that associate and signal on binding of LTX(N4C).

Heptahelical, or G-protein-coupled, receptors control many cellular functions and normally consist of one polypeptide chain. In contrast, heptahelical receptors that belong to the long N-terminus, group B (LNB) family are cleaved constitutively into two fragments. The N-terminal fragments (NTFs) resemble cell-adhesion proteins and the C-terminal fragments (CTFs) are typical G-protein-coupled receptors (GPCRs) with seven transmembrane regions. However, the functional roles of this cleavage and of any subsequent NTF-CTF interactions remain to be identified. Using latrophilin, a well-studied member of the LNB family, we now demonstrate that cleavage is critical for delivery of this receptor to the cell surface. On the plasma membrane, NTF and CTF behave as separate membrane proteins involved, respectively, in cell-surface reception and signalling. The two fragments can also internalise independently. However, separated NTF and CTF can re-associate on solubilisation. Agonist binding to NTF on the cell surface also induces re-association of fragments and provokes signal transduction via CTF. These findings define a novel principle of structural and functional organisation of the cleaved, two-subunit GPCRs.