Role of Tyrosine Phosphorylation in Ligand-induced Regulation of Transcytosis of the Polymeric Ig Receptor

The polymeric Ig receptor (pIgR) transcytoses its ligand, dimeric IgA (dIgA), from the basolateral to the apical surface of epithelial cells. Although the pIgR is constitutively transcytosed in the absence of ligand, binding of dIgA stimulates transcytosis of the pIgR. We recently reported that dIgA binding to the pIgR induces translocation of protein kinase C, production of inositol triphosphate, and elevation of intracellular free calcium. We now report that dIgA binding causes rapid, transient tyrosine phosphorylation of several proteins, including phosphatidyl inositol-specific phospholipase C-γl. Protein tyrosine kinase inhibitors or deletion of the last 30 amino acids of pIgR cytoplasmic tail prevents IgA-stimulated protein tyrosine kinase activation, tyrosine phosphorylation of phospholipase C-γl, production of inositol triphosphate, and the stimulation of transcytosis by dIgA. Analysis of pIgR deletion mutants reveals that the same discrete portion of the cytoplasmic domain, residues 727–736 (but not the Tyr734), controls both the ability of pIgR to cause dIgA-induced tyrosine phosphorylation of the phospholipase C-γl and to undergo dIgA-stimulated transcytosis. In addition, dIgA transcytosis can be strongly stimulated by mimicking phospholipase C-γl activation. In combination with our previous results, we conclude that the protein tyrosine kinase(s) and phospholipase C-γl that are activated upon dIgA binding to the pIgR control dIgA-stimulated pIgR transcytosis.     

Multivalent immune complexes divert FcRn to lysosomes by exclusion from recycling sorting tubules

The neonatal receptor for immunoglobulin G (IgG; FcRn) prevents IgG degradation by efficiently sorting IgG into recycling endosomes and away from lysosomes. When bound to IgG-opsonized antigen complexes, however, FcRn traffics cargo into lysosomes, where antigen processing can occur. Here we address the mechanism of sorting when FcRn is bound to multivalent IgG-opsonized antigens. We find that only the unbound receptor or FcRn bound to monomeric IgG is sorted into recycling tubules emerging from early endosomes. Cross-linked FcRn is never visualized in tubules containing the unbound receptor. Similar results are found for transferrin receptor, suggesting a general mechanism of action. Deletion or replacement of the FcRn cytoplasmic tail does not prevent diversion of trafficking to lysosomes upon cross-linking. Thus physical properties of the lumenal ligand–receptor complex appear to act as key determinants for sorting between the recycling and lysosomal pathways by regulating FcRn entry into recycling tubules.     

Monomeric IgG1 Fc molecules displaying unique Fc receptor interactions that are exploitable to treat inflammation-mediated diseases

The IgG1 Fc is a dimeric protein that mediates important antibody effector functions by interacting with Fcγ receptors (FcγRs) and the neonatal Fc receptor (FcRn). Here, we report the discovery of a monomeric IgG1 Fc (mFc) that bound to FcγRI with very high affinity, but not to FcγRIIIa, in contrast to wild-type (dimeric) Fc. The binding of mFc to FcRn was the same as that of dimeric Fc. To test whether the high-affinity binding to FcγRI can be used for targeting of toxins, a fusion protein of mFc with a 38 kDa Pseudomonas exotoxin A fragment (PE38), was generated. This fusion protein killed FcγRI-positive macrophage-like U937 cells but not FcγRI-negative cells, and mFc or PE38 alone had no killing activity. The lack of binding to FcγRIIIa resulted in the absence of Fc-mediated cytotoxicity of a scFv-mFc fusion protein targeting mesothelin. The pharmacokinetics of mFc in mice was very similar to that of dimeric Fc. The mFc''s unique FcγRs binding pattern and related functionality, combined with its small size, monovalency and the preservation of FcRn binding which results in relatively long half-life in vivo, suggests that mFc has great potential as a component of therapeutics targeting inflammation mediated by activated macrophages overexpressing FcγRI and related diseases, including cancer.     

Cytoplasmic Tail Regulates the Intercellular Adhesion Function of the Epithelial Cell Adhesion Molecule

Ep-CAM, an epithelium-specific cell-cell adhesion molecule (CAM) not structurally related to the major families of CAMs, contains a cytoplasmic domain of 26 amino acids. The chemical disruption of the actin microfilaments, but not of the microtubuli or intermediate filaments, affected the localization of Ep-CAM at the cell-cell boundaries, suggesting that the molecule interacts with the actin-based cytoskeleton. Mutated forms of Ep-CAM were generated with the cytoplasmic domain truncated at various lengths. All of the mutants were transported to the cell surface in the transfectants; however, the mutant lacking the complete cytoplasmic domain was not able to localize to the cell-cell boundaries, in contrast to mutants with partial deletions. Both the disruption of the actin microfilaments and a complete truncation of the cytoplasmic tail strongly affected the ability of Ep-CAM to mediate aggregation of L cells. The capability of direct aggregation was reduced for the partially truncated mutants but remained cytochalasin D sensitive. The tail truncation did not affect the ability of the transfectants to adhere to solid-phase-adsorbed Ep-CAM, suggesting that the ability to form stable adhesions and not the ligand specificity of the molecule was affected by the truncation. The formation of intercellular adhesions mediated by Ep-CAM induced a redistribution to the cell-cell boundaries of α-actinin, but not of vinculin, talin, filamin, spectrin, or catenins. Coprecipitation demonstrated direct association of Ep-CAM with α-actinin. Binding of α-actinin to purified mutated and wild-type Ep-CAMs and to peptides representing different domains of the cytoplasmic tail of Ep-CAM demonstrates two binding sites for α-actinin at positions 289 to 296 and 304 to 314 of the amino acid sequence. The results demonstrate that the cytoplasmic domain of Ep-CAM regulates the adhesion function of the molecule through interaction with the actin cytoskeleton via α-actinin.     

IgG transcytosis and recycling by FcRn expressed in MDCK cells reveals ligand-induced redistribution

The neonatal Fc receptor (FcRn) transports IgG across epithelial cells and recycles serum IgG. FcRn binds IgG at the acidic pH of endosomes and releases IgG at the basic pH of blood. We expressed rat FcRn in polarized MDCK cells and demonstrated that it functions in transcytosis and recycling of IgG. In the absence of IgG, FcRn is distributed predominantly apically, but redistributes to basolateral locations upon IgG addition, indicating that ligand binding induces a signal that stimulates transcytosis. FcRn transcytoses IgG more efficiently in the apical to basolateral than the reverse direction when IgG is internalized by receptor-mediated endocytosis at acidic pH or by fluid phase endocytosis at basic pH. The PI 3-kinase inhibitor wortmannin disrupts basolateral recycling and transcytosis in both directions, but only minimally reduces apical recycling. Confocal imaging and quantitative IgG transport studies demonstrate that apically-internalized IgG recycles to the apical surface mainly from wortmannin-insensitive apical early endosomes, whereas FcRn-IgG complexes that transcytose to the basolateral surface pass through downstream Rab11-positive apical recycling endosomes and transferrin-positive common endosomal compartments.     

Nonvectorial surface transport, endocytosis via a Di-leucine-based motif, and bidirectional transcytosis of chimera encoding the cytosolic tail of rat FcRn expressed in Madin-Darby canine kidney cells

Transfer of passive immunity from the mother to the fetus or newborn involves the transport of IgG across several epithelia. Depending on the species, IgG is transported prenatally across the placenta and yolk sac or is absorbed from colostrum and milk by the small intestine of the suckling newborn. In both cases apical to basolateral transepithelial transport of IgG is thought to be mediated by FcRn, an IgG Fc receptor with homology to major histocompatibility class I antigens. Here, we analyzed the intracellular routing of chimera encoding the rat FcRn tail fused to the ecto- and transmembrane domain of the macrophage FcgammaRIIb. Newly synthesized chimera were delivered in a nonvectorial manner to the apical and basolateral cell surface, from where the chimera were able to internalize and transcytose. Apical to basolateral and basolateral to apical transcytosis were differently regulated. This intracellular routing of the chimera is similar to that of the native FcRn, indicating that the cytosolic tail of the receptor is necessary and sufficient to endow an unrelated FcR with the intracellular transport behavior of FcRn. Furthermore, the di-leucine motif in the cytosolic domain of FcRn was required for rapid and efficient endocytosis but not for basolateral sorting of the chimera.     

The MHC class II-associated invariant chain interacts with the neonatal Fc gamma receptor and modulates its trafficking to endosomal/lysosomal compartments

The neonatal Fc receptor for IgG (FcRn) transfers maternal IgG to the offspring and protects IgG from degradation. The FcRn resides in an acidic intracellular compartment, allowing it to bind IgG. In this study, we found the association of FcRn and invariant chain (Ii). The interaction was initiated within the endoplasmic reticulum by Ii binding to either the FcRn H chain alone or FcRn H chain-beta(2)-microglobulin complex and appeared to be maintained throughout the endocytic pathway. The CLIP in Ii was not required for FcRn-Ii association. The interaction was also detected in IFN-gamma-treated THP-1, epithelial and endothelial cells, and immature mouse DCs. A truncated FcRn without the cytoplasmic tail was unable to traffic to early endosomes; however, its location in early endosomes was restored by Ii expression. FcRn was also detected in the late endosome/lysosome only in the presence of Ii or on exposure to IFN-gamma. In immature human or mouse DCs, FcRn was barely detected in the late endosome/lysosome in the absence of Ii. Furthermore, the cytoplasmic tail of Ii conferred tailless FcRn to route to both the early endosome and late endosome/lysosome in a hybrid molecule. Because the FcRn is expressed in macrophages and DCs or epithelial and endothelial cells where Ii is induced under inflammation and infection, these results reveal the complexity of FcRn trafficking in which Ii is capable of expanding the boundary of FcRn trafficking. Taken together, the intracellular trafficking of FcRn is regulated by its intrinsic sorting information and/or an interaction with Ii chain.     

The recycling and transcytotic pathways for IgG transport by FcRn are distinct and display an inherent polarity

The Fc receptor FcRn traffics immunoglobulin G (IgG) in both directions across polarized epithelial cells that line mucosal surfaces, contributing to host defense. We show that FcRn traffics IgG from either apical or basolateral membranes into the recycling endosome (RE), after which the actin motor myosin Vb and the GTPase Rab25 regulate a sorting step that specifies transcytosis without affecting recycling. Another regulatory component of the RE, Rab11a, is dispensable for transcytosis, but regulates recycling to the basolateral membrane only. None of these proteins affect FcRn trafficking away from lysosomes. Thus, FcRn transcytotic and recycling sorting steps are distinct. These results are consistent with a single structurally and functionally heterogeneous RE compartment that traffics FcRn to both cell surfaces while discriminating between recycling and transcytosis pathways polarized in their direction of transport.     

Tryptophan- and dileucine-based endocytosis signals in the neonatal Fc receptor

The neonatal Fc receptor, FcRn, transports immunoglobulin G across intestinal cells in suckling rats. FcRn enters these cells by endocytosis and is present on the apical and basolateral surfaces. We investigated the roles of aromatic amino acids and a dileucine motif in the cytoplasmic domain of rat FcRn. We expressed mutant FcRn in which alanine replaced Trp-311, Leu-322, and Leu-323, or Phe-340 in the inner medullary collecting duct cell line IMCD. Individual replacement of the aromatic amino acids or the dileucine motif only partially blocked endocytosis of (125)I-Fc, whereas uptake by FcRn containing alanine residues in place of both Trp-311 and the dileucine motif was reduced to the level obtained with the tailless receptor. Leu-314 was required for the function of the tryptophan-based endocytosis signal, and Asp-317 and Asp-318 were required for the dileucine-based signal. Nonvectorial delivery of newly synthesized FcRn to the two cell surfaces was unaffected by loss of the endocytosis signals. However, the steady-state distribution of endocytosis mutants was predominantly apical, unlike wild-type FcRn, which was predominantly basolateral. This shift appeared to arise because the loss of endocytosis signals inhibited apical to basolateral transcytosis of FcRn more than basolateral to apical transcytosis.     

Dimerization of the Polymeric Immunoglobulin Receptor Controls Its Transcytotic Trafficking

Binding of dimeric immunoglobulin (Ig)A to the polymeric Ig receptor (pIgR) stimulates transcytosis of pIgR across epithelial cells. Through the generation of a series of pIgR chimeric constructs, we have tested the ability of ligand to promote receptor dimerization and the subsequent role of receptor dimerization on its intracellular trafficking. Using the cytoplasmic domain of the T cell receptor-ζ chain as a sensitive indicator of receptor oligomerization, we show that a pIgR:ζ chimeric receptor expressed in Jurkat cells initiates a ζ-specific signal transduction cascade when exposed to dimeric or tetrameric IgA, but not when exposed to monomeric IgA. In addition, we replaced the pIgR’s transmembrane domain with that of glycophorin A to force dimerization or with a mutant glycophorin transmembrane domain to prevent dimerization. Forcing dimerization stimulated transcytosis of the chimera, whereas preventing dimerization abolished ligand-stimulated transcytosis. We conclude that binding of dimeric IgA to the pIgR induces its dimerization and that this dimerization is necessary and sufficient to stimulate pIgR transcytosis.     

The neonatal Fc receptor (FcRn) binds independently to both sites of the IgG homodimer with identical affinity

The neonatal Fc receptor (FcRn) is expressed by cells of epithelial, endothelial and myeloid lineages and performs multiple roles in adaptive immunity. Characterizing the FcRn/IgG interaction is fundamental to designing therapeutic antibodies because IgGs with moderately increased binding affinities for FcRn exhibit superior serum half-lives and efficacy. It has been hypothesized that 2 FcRn molecules bind an IgG homodimer with disparate affinities, yet their affinity constants are inconsistent across the literature. Using surface plasmon resonance biosensor assays that eliminated confounding experimental artifacts, we present data supporting an alternate hypothesis: 2 FcRn molecules saturate an IgG homodimer with identical affinities at independent sites, consistent with the symmetrical arrangement of the FcRn/Fc complex observed in the crystal structure published by Burmeister et al. in 1994. We find that human FcRn binds human IgG1 with an equilibrium dissociation constant (K_D) of 760 ± 60 nM (N = 14) at 25°C and pH 5.8, and shows less than 25% variation across the other human subtypes. Human IgG1 binds cynomolgus monkey FcRn with a 2-fold higher affinity than human FcRn, and binds both mouse and rat FcRn with a 10-fold higher affinity than human FcRn. FcRn/IgG interactions from multiple species show less than a 2-fold weaker affinity at 37°C than at 25°C and appear independent of an IgG''s variable region. Our in vivo data in mouse and rat models demonstrate that both affinity and avidity influence an IgG''s serum half-life, which should be considered when choosing animals, especially transgenic systems, as surrogates.     

The Human CD8β M-4 Isoform Dominant in Effector Memory T Cells Has Distinct Cytoplasmic Motifs That Confer Unique Properties

The CD8 co-receptor influences T cell recognition and responses in both anti-tumor and anti-viral immunity. During evolution in the ancestor of humans and chimpanzees, the CD8B gene acquired two additional exons. As a result, in humans, there are four CD8β splice variants (M1 to M4) that differ in their cytoplasmic tails. The M-1 isoform which is the equivalent of murine CD8β, is predominantly expressed in naïve T cells, whereas, the M-4 isoform is predominantly expressed in effector memory T cells. The characteristics of the M-4 isoform conferred by its unique 36 amino acid cytoplasmic tail are not known. In this study, we identified a dihydrophobic leucine-based receptor internalization motif in the cytoplasmic tail of M-4 that regulated its cell surface expression and downregulation after activation. Further the M-4 cytoplasmic tail was able to associate with ubiquitinated targets in 293T cells and mutations in the amino acids NPW, a potential EH domain binding site, either enhanced or inhibited the interaction. In addition, the M-4 tail was itself mono-ubiquitinated on a lysine residue in both 293T cells and a human T cell line. When peripheral blood human T cells expressed CD8αβ M-4, the frequency of MIP-1β secreting cells responding to antigen presenting cells was two-fold higher as compared to CD8αβ M-1 expressing T cells. Thus, the cytoplasmic tail of the CD8β M-4 isoform has unique characteristics, which likely contributed to its selective expression and function in human effector memory T cells.     

Apical to basolateral transcytosis and apical recycling of immunoglobulin G in trophoblast-derived BeWo cells: effects of low temperature, nocodazole, and cytochalasin D

The murine neonatal Fc receptor, FcRn, carries out two functions: materno-fetal IgG delivery and maintenance of serum IgG homeostasis. During human pregnancy maternal IgG is transferred across placental syncytiotrophoblasts presumably by the human homolog of FcRn, hFcRn. Trophoblast-derived BeWo cells express hFcRn endogenously and can be considered as a model system to investigate IgG transport in syncytiotrophoblasts. Using a pulse-chase protocol, we here demonstrate that polarized BeWo cells exhibit not only apical to basolateral transcytosis but also apical IgG recycling. Thus, for the first time we demonstrate that epithelial cells can be involved in both materno-fetal IgG transmission and regulation of serum IgG levels. Lowering the temperature from 37 to 16 degrees C reduced, but did not block, IgG recycling and transcytosis. Microtubule-disruption by nocodazole did not influence transcytosis or apical recycling. Disassembly of filamentous actin by cytochalasin D stimulated apical endocytosis and recycling, while transcytosis remained unaffected. In summary, in BeWo cells apically internalized IgG enters both a transcytotic and recycling pathway. While the transcytotic route is temperature-sensitive but independent from microtubules and actin filaments, the apical recycling pathway is temperature-influenced and stimulated by actin disassembly, suggestive for the involvement of distinct endosome subcompartments in transcytosis and recycling.     

The Tyrosine Kinase c-Src Specifically Binds to the Active Integrin αIIbβ3 to Initiate Outside-in Signaling in Platelets

It is currently believed that inactive tyrosine kinase c-Src in platelets binds to the cytoplasmic tail of the β3 integrin subunit via its SH3 domain. Although a recent NMR study supports this contention, it is likely that such binding would be precluded in inactive c-Src because an auto-inhibitory linker physically occludes the β3 tail binding site. Accordingly, we have re-examined c-Src binding to β3 by immunoprecipitation as well as NMR spectroscopy. In unstimulated platelets, we detected little to no interaction between c-Src and β3. Following platelet activation, however, c-Src was co-immunoprecipitated with β3 in a time-dependent manner and underwent progressive activation as well. We then measured chemical shift perturbations in the ¹⁵N-labeled SH3 domain induced by the C-terminal β3 tail peptide NITYRGT and found that the peptide interacted with the SH3 domain RT-loop and surrounding residues. A control peptide whose last three residues where replaced with those of the β1 cytoplasmic tail induced only small chemical shift perturbations on the opposite face of the SH3 domain. Next, to mimic inactive c-Src, we found that the canonical polyproline peptide RPLPPLP prevented binding of the β3 peptide to the RT- loop. Under these conditions, the β3 peptide induced chemical shift perturbations similar to the negative control. We conclude that the primary interaction of c-Src with the β3 tail occurs in its activated state and at a site that overlaps with PPII binding site in its SH3 domain. Interactions of inactive c-Src with β3 are weak and insensitive to β3 tail mutations.     

On the Perplexingly Low Rate of Transport of IgG2 across the Human Placenta

The neonatal receptor, FcRn, mediates both serum half–life extension as well as active transport of maternal IgG to the fetus during pregnancy. Therefore, transport efficiency and half-life go hand-in-hand. However, while the half-life of the human IgG2 subclass is comparable to IgG1, the placental transport of IgG2 is not, with the neonatal IgG1 levels generally exceeding maternal levels at birth, but not for IgG2. We hypothesized that the unique short-hinged structure of IgG2, which enables its κ-, but not λ-isotype to form at least three different structural isoforms, might be a contributing factor to these differences. To investigate whether there was any preference for either light chain, we measured placental transport of IgG subclasses as well as κ/λ-light chain isotypes of IgG1 and IgG2 in 27 matched mother-child pairs. We also studied the half-life of IgG1 and IgG2 light chain isotypes in mice, as well as that of synthesized IgG2 structural isotypes κA and κB. In order to investigate serum clearance of IgG1 and IgG2 light-chain isotypes in humans, we quantified the relative proportions of IgG1 and IgG2 light chains in hypogammaglobulinemia patients four weeks after IVIg infusion and compared to the original IVIg isotype composition. None of our results indicate any light chain preference in either of the FcRn mediated mechanisms; half-life extension or maternal transport.     

An in vitro FcRn- dependent transcytosis assay as a screening tool for predictive assessment of nonspecific clearance of antibody therapeutics in humans

ABSTRACT

A cell-based assay employing Madin–Darby canine kidney cells stably expressing human neonatal Fc receptor (FcRn) heavy chain and β2-microglobulin genes was developed to measure transcytosis of monoclonal antibodies (mAbs) under conditions relevant to the FcRn-mediated immunoglobulin G (IgG) salvage pathway. The FcRn-dependent transcytosis assay is modeled to reflect combined effects of nonspecific interactions between mAbs and cells, cellular uptake via pinocytosis, pH-dependent interactions with FcRn, and dynamics of intracellular trafficking and sorting mechanisms. Evaluation of 53 mAbs, including 30 marketed mAb drugs, revealed a notable correlation between the transcytosis readouts and clearance in humans. FcRn was required to promote efficient transcytosis of mAbs and contributed directly to the observed correlation. Furthermore, the transcytosis assay correctly predicted rank order of clearance of glycosylation and Fv charge variants of Fc-containing proteins. These results strongly support the utility of this assay as a cost-effective and animal-sparing screening tool for evaluation of mAb-based drug candidates during lead selection, optimization, and process development for desired pharmacokinetic properties.     

Investigating the Interaction between the Neonatal Fc Receptor and Monoclonal Antibody Variants by Hydrogen/Deuterium Exchange Mass Spectrometry

The recycling of immunoglobulins by the neonatal Fc receptor (FcRn) is of crucial importance in the maintenance of antibody levels in plasma and is responsible for the long half-lives of endogenous and recombinant monoclonal antibodies. From a therapeutic point of view there is great interest in understanding and modulating the IgG–FcRn interaction to optimize antibody pharmacokinetics and ultimately improve efficacy and safety. Here we studied the interaction between a full-length human IgG₁ and human FcRn via hydrogen/deuterium exchange mass spectrometry and targeted electron transfer dissociation to map sites perturbed by binding on both partners of the IgG–FcRn complex. Several regions in the antibody Fc region and the FcRn were protected from exchange upon complex formation, in good agreement with previous crystallographic studies of FcRn in complex with the Fc fragment. Interestingly, we found that several regions in the IgG Fab region also showed reduced deuterium uptake. Our findings indicate the presence of hitherto unknown FcRn interaction sites in the Fab region or a possible conformational link between the IgG Fc and Fab regions upon FcRn binding. Further, we investigated the role of IgG glycosylation in the conformational response of the IgG–FcRn interaction. Removal of antibody glycans increased the flexibility of the FcRn binding site in the Fc region. Consequently, FcRn binding did not induce a similar conformational stabilization of deglycosylated IgG as observed for the wild-type glycosylated IgG. Our results provide new molecular insight into the IgG–FcRn interaction and illustrate the capability of hydrogen/deuterium exchange mass spectrometry to advance structural proteomics by providing detailed information on the conformation and dynamics of large protein complexes in solution.Antibodies and variants thereof constitute the fastest growing category of therapeutic agents, and currently more than 30 immunoglobulins (Igs)¹ have been approved for the treatment of cancer, immunological diseases, and infectious diseases (1). The success of therapeutic monoclonal antibodies (mAbs) is based on the ability to specifically target diverse antigens and activate immunological effector responses. An Ig is a “dimer of a dimer” consisting of light chains and heavy chains in which each light chain is linked to a heavy chain and the light–heavy dimers are connected by disulfide bridges to form the intact antibody. IgG is the most prevalent Ig isotype in plasma and is the most commonly used isotype for therapeutic antibodies because of its strong ability to induce antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity (2). The IgG₁ subtype is a 150 kDa Y-shaped glycoprotein. Its stem and arms are referred to as the fragment crystallizable (Fc) and fragment antigen binding (Fab) regions, respectively. The Fab region is composed of a variable (V) and constant (C) domain from both the light chain and the heavy chain (V_L, C_L, V_H, C_H1). Antigen binding is achieved through three highly variable complementary determining regions in each variable domain (V_L and V_H) of the Fab region. The Fc region is composed of additional constant domains of the heavy chain (C_H2 and C_H3); it mediates antibody-dependent cellular cytotoxicity through interaction with Fcγ receptors (3, 4) and activates complement-dependent cytotoxicity through interaction with C1q (5). The Fc region also interacts with the neonatal Fc receptor (FcRn), which regulates the maintenance of antibody levels in plasma and thus the half-life of endogenous and recombinant monoclonal antibodies (6). The interaction between IgG and FcRn displays a characteristic pH dependence that is the basis for the function of FcRn in IgG recycling (7). FcRn rescues and recycles IgG from lysosomal degradation by binding with low micromolar affinity to internalized IgG in the slightly acidic late endosome of, for example, vascular endothelial cells (pH < 6.5). The IgG is rescued from intracellular degradation as the IgG–FcRn complex returns to the cell surface, where the IgG is released into circulation as FcRn binding is abolished in the neutral pH of plasma (6). FcRn-mediated IgG recycling contributes to the long catabolic half-life of endogenous and therapeutic antibodies of ∼22 days (8).The FcRn is a heterodimer of an MHC-class-I-like heavy chain and a β₂-microglobulin (β₂m) light chain. The FcRn heavy chain (α-chain) is composed of three structural domains, α₁, α₂, and α₃, followed by a transmembrane region and a cytoplasmic domain. The three-dimensional structure of FcRn is similar to that of MHC class I molecules in which domains α₁ and α₂ are stacked against domain α₃ and β₂m (9, 10). The pH dependence of the IgG–FcRn interaction is attributed to highly conserved residues in both FcRn and IgG (10). The first crystal structures of rat FcRn and rat Fc revealed that FcRn binds to the C_H2 and C_H3 domains of the IgG Fc region—specifically, C_H2 residues 252–254 and 309–311, as well as C_H3 residues 434–436 (11, 12). Several positively charged histidines in the IgG C_H2 and C_H3 domains (H310, H433, H435, and H436; the latter is not found in humans) interact with acidic residues E117, E132, W133, E135, and D137 in the FcRn α₂ domain, accounting for the pH-sensitive nature of the IgG–FcRn interaction. The interface is also composed of a hydrophobic core around Fc I253 that interacts with FcRn W133 and the N-terminal I1 residue of the β₂m, which has been proposed to contact Fc residues 309–311. The interaction of FcRn and IgG occurs in a 2:1 stoichiometry, where two FcRn molecules bind to one IgG through binding sites on each heavy chain (12). Two distinct binding modes have been suggested in which the FcRn molecules bind in a symmetric or asymmetric fashion to the Fc. In symmetric models FcRns bind to opposite sites on the Fc, whereas in the asymmetric models two FcRn molecules form a homodimer with only one FcRn molecule binding the Fc directly (6, 11). The extracellular domains of rat and human FcRn have 68% sequence identity and are structurally similar (9, 10). The first crystal structure of human FcRn in complex with an engineered human Fc fragment (Fc-YTE) as well as human serum albumin was published recently (13) and showed a binding mode similar to that of rodent IgG–FcRn variants, with the exception of the additional interaction sites caused by substitutions in the Fc domain. To the best of our knowledge, no crystal structures of full-length human IgG and human FcRn are currently available.From a therapeutic point of view there is great interest in understanding and modulating the IgG–FcRn interaction to optimize the pharmacokinetics and thus ultimately the efficacy of therapeutic monoclonal antibodies. The goal of FcRn modulation is typically prolongation of the in vivo half-life in order to reduce dosing frequency and ultimately the cost of treatment. However, a shorter half-life can also be desirable, for example, for antibody–toxin conjugates or antibodies used in bioimaging (6). Several engineered therapeutic mAb variants with improved in vitro FcRn binding affinity and extended in vivo half-life have been generated via mutation of residues in the Fc domain (14–19). For example, the engineered variants of palivizumab (M252Y/S254T/T256E) (15, 16) and bevacizumab (M428L/N434S) (17) show 10- and 11-fold increases in relative FcRn affinity that result in increases of the in vivo half-life in cynomolgus monkeys of 4- and 3-fold, respectively. Mutation can also impact half-life negatively: mAb engineering can improve FcRn affinity at both pH 6 and 7.5 such that the pH-dependent release of IgGs is prohibited, leading to increased IgG clearance (16). Interestingly, post-translational modifications such as oxidation of conserved methionines in the C_H2 and C_H3 domains of IgG₁ and IgG₂ have been shown to affect FcRn affinity negatively. Antibody oxidation that can occur during production or storage significantly reduces FcRn binding in vitro (20, 21), which also translates to a reduced in vivo half-life in human FcRn transgenic mice models (22). The molecular origins of the effect of post-translational modifications on the IgG–FcRn interaction are, however, unclear. Further, the impact of FcRn binding on the conformational properties and dynamics of IgG in solution is currently not well understood.In this study we investigated the interaction between human FcRn and two variants of a full-length IgG₁ by means of hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS). HDX-MS has become a popular approach for studying protein dynamics and interactions (23–27), as the technique provides access to proteins at native solution conditions with modest sample requirements. Amide HDX rates in native proteins are highly influenced by higher order structure: fully solvated (non-hydrogen-bonded) amides exchange rapidly, whereas structurally protected (hydrogen-bonded) amides exchange up to 7 orders of magnitude slower (28, 29). Protein interactions can be studied and mapped via HDX-MS, as binding events can perturb HDX rates as solvation and hydrogen bonding changes directly in the binding interface or indirectly in conformationally linked regions. The structural resolution of a classic peptide-level HDX-MS experiment is dependent on the generation of overlapping peptides by acid-stable proteases, such as pepsin, typically used in HDX-MS workflows. More recently, the use of gas-phase fragmentation of deuterated peptides with ETD (30–33) has become a viable option for sublocalizing deuterium uptake to short peptide stretches or even individual amino acids, thus increasing the spatial resolution of the classical bottom-up HDX-MS method.Here, we used HDX-MS to probe the solution-phase interactions of human FcRn with a full-length recombinant human IgG₁ and its deglycosylated variant. Our results allowed us to map antibody and FcRn regions that displayed changes in HDX upon complex formation and examine the impact of antibody glycosylation on FcRn binding. Additionally, by coupling ETD to the HDX-MS workflow in a targeted manner, we obtained high-resolution information on the HDX of individual sites that became protected upon IgG₁–FcRn complex formation.     

Integrin αIIbβ3 Transmembrane Domain Separation Mediates Bi-Directional Signaling across the Plasma Membrane

Integrins play an essential role in hemostasis, thrombosis, and cell migration, and they transmit bidirectional signals. Transmembrane/cytoplasmic domains are hypothesized to associate in the resting integrins; whereas, ligand binding and intracellular activating signals induce transmembrane domain separation. However, how this conformational change affects integrin outside-in signaling and whether the α subunit cytoplasmic domain is important for this signaling remain elusive. Using Chinese Hamster Ovary (CHO) cells that stably expressed different integrin α_IIbβ₃ constructs, we discovered that an α_IIb cytoplasmic domain truncation led to integrin activation but not defective outside-in signaling. In contrast, preventing transmembrane domain separation abolished both inside-out and outside-in signaling regardless of removing the α_IIb cytoplasmic tail. Truncation of the α_IIb cytoplasmic tail did not obviously affect adhesion-induced outside-in signaling. Our research revealed that transmembrane domain separation is a downstream conformational change after the cytoplasmic domain dissociation in inside-out activation and indispensable for ligand-induced outside-in signaling. The result implicates that the β TM helix rearrangement after dissociation is essential for integrin transmembrane signaling. Furthermore, we discovered that the PI3K/Akt pathway is not essential for cell spreading but spreading-induced Erk1/2 activation is PI3K dependent implicating requirement of the kinase for cell survival in outside-in signaling.     

Localization of the human neonatal Fc receptor (FcRn) in human nasal epithelium

The airway epithelium is a central player in the defense against pathogens including efficient mucociliary clearance and secretion of immunoglobulins, mainly polymeric IgA, but also IgG. Pulmonary administration of therapeutic antibodies on one hand, and intranasal immunization on the other, are powerful tools to treat airway infections. In either case, the airway epithelium is the primary site of antibody transfer. In various epithelia, bi-polar transcytosis of IgG and IgG immune complexes is mediated by the human neonatal Fc receptor, FcRn, but FcRn expression in the nasal epithelium had not been demonstrated, so far. We prepared affinity-purified antibodies against FcRn α-chain and confirmed their specificity by Western blotting and immunofluorescence microscopy. These antibodies were used to study the localization of FcRn α-chain in fixed nasal tissue. We here demonstrate for the first time that ciliated epithelial cells, basal cells, gland cells, and endothelial cells in the underlying connective tissue express the receptor. A predominant basolateral steady state distribution of the receptor was observed in ciliated epithelial as well as in gland cells. Co-localization of FcRn α-chain with IgG or with early sorting endosomes (EEA1-positive) but not with late endosomes/lysosomes (LAMP-2-positive) in ciliated cells was observed. This is indicative for the presence of the receptor in the recycling/transcytotic pathway but not in compartments involved in lysosomal degradation supporting the role of FcRn in IgG transcytosis in the nasal epithelium.