Expression of immunoglobulin constant domain genes in neurons of the mouse central nervous system.

General consensus states that immunoglobulins are exclusively expressed by B lymphocytes to form the first line of defense against common pathogens. Here, we provide compelling evidence for the expression of two heavy chain immunoglobulin genes in subpopulations of neurons in the mouse brain and spinal cord. RNA isolated from excitatory and inhibitory neurons through ribosome affinity purification revealed Ighg3 and Ighm transcripts encoding for the constant (Fc), but not the variable regions of IgG3 and IgM. Because, in the absence of the variable immunoglobulin regions, these transcripts lack the canonical transcription initiation site used in lymphocytes, we screened for alternative 5' transcription start sites and identified a novel 5' exon adjacent to a proposed promoter element. Immunohistochemical, Western blot, and in silico analyses strongly support that these neuronal transcripts are translated into proteins containing four Immunoglobulin domains. Our data thus demonstrate the expression of two Fc-encoding genes Ighg3 and Ighm in spinal and supraspinal neurons of the murine CNS and suggest a hitherto unknown function of the encoded proteins.

The reduced form of the antibody CH2 domain.

Among the immunoglobulin domains, the CH2 domain has the lowest thermal stability, which also depends on amino acid sequence and buffer conditions. To further identify factors that influence CH2 folding and stability, we characterized the domain in the reduced form using differential scanning fluorimetry and nuclear magnetic resonance. We show that the CH2 domain can fold, similarly to the disulfide-bridged form, without forming a disulfide-bridge, even though the protein contains two Cys residues. Although the reduced form exhibits thermal stability more than 15°C lower than the disulfide-bridged form, it does not undergo immediate full oxidization. To explain this phenomenon, we compared CH2 oxidization at different conditions and demonstrate a need for significant fluctuation of the folded conformation to enhance CH2 disulfide-bridge formation. We conclude that, since CH2 can be purified as a folded, semi-stable, reduced protein that can coexist with the oxidized form, verification of the level of oxidization at each step is critical in CH2 engineering studies.

Crystal Structures of Bat and Human Coronavirus ORF8 Protein Ig-Like Domain Provide Insights Into the Diversity of Immune Responses.

ORF8 is a viral immunoglobulin-like (Ig-like) domain protein encoded by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA genome. It tends to evolve rapidly and interfere with immune responses. However, the structural characteristics of various coronavirus ORF8 proteins and their subsequent effects on biological functions remain unclear. Herein, we determined the crystal structures of SARS-CoV-2 ORF8 (S84) (one of the epidemic isoforms) and the bat coronavirus RaTG13 ORF8 variant at 1.62 Å and 1.76 Å resolution, respectively. Comparison of these ORF8 proteins demonstrates that the 62-77 residues in Ig-like domain of coronavirus ORF8 adopt different conformations. Combined with mutagenesis assays, the residue Cys20 of ORF8 is responsible for forming the covalent disulfide-linked dimer in crystal packing and in vitro biochemical conditions. Furthermore, immune cell-binding assays indicate that various ORF8 (SARS-CoV-2 ORF8 (L84), ORF8 (S84), and RaTG13 ORF8) proteins have different interaction capabilities with human CD14+ monocytes in human peripheral blood. These results provide new insights into the specific characteristics of various coronavirus ORF8 and suggest that ORF8 variants may influence disease-related immune responses.

Dissecting the Interaction of FGF8 with Receptor FGFRL1.

In mammals, the novel protein fibroblast growth factor receptor-like 1 (FGFRL1) is involved in the development of metanephric kidneys. It appears that this receptor controls a crucial transition of the induced metanephric mesenchyme to epithelial renal vesicles, which further develop into functional nephrons. FGFRL1 knockout mice lack metanephric kidneys and do not express any fibroblast growth factor (FGF) 8 in the metanephric mesenchyme, suggesting that FGFRL1 and FGF8 play a decisive role during kidney formation. FGFRL1 consists of three extracellular immunoglobulin (Ig) domains (Ig1-Ig2-Ig3), a transmembrane domain and a short intracellular domain. We have prepared the extracellular domain (Ig123), the three individual Ig domains (Ig1, Ig2, Ig3) as well as all combinations containing two Ig domains (Ig12, Ig23, Ig13) in recombinant form in human cells. All polypeptides that contain the Ig2 domain (Ig123, Ig12, Ig23, Ig2) were found to interact with FGF8 with very high affinity, whereas all constructs that lack the Ig2 domain (Ig1, Ig3, Ig13) poorly interacted with FGF8 as shown by ELISA and surface plasmon resonance. It is therefore likely that FGFRL1 represents a physiological receptor for FGF8 in the kidney and that the ligand primarily binds to the Ig2 domain of the receptor. With Biacore experiments, we also measured the affinity of FGF8 for the different constructs. All constructs containing the Ig2 domain showed a rapid association and a slow dissociation phase, from which a KD of 2-3 × 10-9 M was calculated. Our data support the hypothesis that binding of FGF8 to FGFRL1 could play an important role in driving the formation of nephrons in the developing kidney.

Isolation of antigen-specific, disulphide-rich knob domain peptides from bovine antibodies.

As a novel alternative to established surface display or combinatorial chemistry approaches for the discovery of therapeutic peptides, we present a method for the isolation of small, cysteine-rich domains from bovine antibody ultralong complementarity-determining regions (CDRs). We show for the first time that isolated bovine antibody knob domains can function as autonomous entities by binding antigen outside the confines of the antibody scaffold. This yields antibody fragments so small as to be considered peptides, each stabilised by an intricate, bespoke arrangement of disulphide bonds. For drug discovery, cow immunisations harness the immune system to generate knob domains with affinities in the picomolar to low nanomolar range, orders of magnitude higher than unoptimized peptides from naïve library screening. Using this approach, knob domain peptides that tightly bound Complement component C5 were obtained, at scale, using conventional antibody discovery and peptide purification techniques.

Engineering of Group A Streptococcus Isopeptide Bonds into Immunoglobulin-Like Protein Domains.

Intramolecular isopeptide bonds, formed autocatalytically between Lys and Asn/Asp side chains, are widely present in the immunoglobulin-like domains of Gram-positive bacterial adhesins, including Group A Streptococcus, and confer considerable mechanical and chemical stability. These properties make them attractive for applications in biotechnology. Here, we detail the practical considerations that are involved in engineering isopeptide bonds into Ig-like proteins, including the choice of a site where bond-forming residues could be introduced and the appropriate methodology for mutagenesis. We specify how to determine whether an isopeptide bond has formed, what strategies can be adopted to overcome problems, and how to monitor the stability of the engineered protein.

Novel Immunoglobulin Domain Proteins Provide Insights into Evolution and Pathogenesis of SARS-CoV-2-Related Viruses.

A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was recently identified as the causative agent for the coronavirus disease 2019 (COVID-19) outbreak that has generated a global health crisis. We use a combination of genomic analysis and sensitive profile-based sequence and structure analysis to understand the potential pathogenesis determinants of this virus. As a result, we identify several fast-evolving genomic regions that might be at the interface of virus-host interactions, corresponding to the receptor binding domain of the Spike protein, the three tandem Macro fold domains in ORF1a, and the uncharacterized protein ORF8. Further, we show that ORF8 and several other proteins from alpha- and beta-CoVs belong to novel families of immunoglobulin (Ig) proteins. Among them, ORF8 is distinguished by being rapidly evolving, possessing a unique insert, and having a hypervariable position among SARS-CoV-2 genomes in its predicted ligand-binding groove. We also uncover numerous Ig domain proteins from several unrelated metazoan viruses, which are distinct in sequence and structure but share comparable architectures to those of the CoV Ig domain proteins. Hence, we propose that SARS-CoV-2 ORF8 and other previously unidentified CoV Ig domain proteins fall under the umbrella of a widespread strategy of deployment of Ig domain proteins in animal viruses as pathogenicity factors that modulate host immunity. The rapid evolution of the ORF8 Ig domain proteins points to a potential evolutionary arms race between viruses and hosts, likely arising from immune pressure, and suggests a role in transmission between distinct host species.IMPORTANCE The ongoing COVID-19 pandemic strongly emphasizes the need for a more complete understanding of the biology and pathogenesis of its causative agent SARS-CoV-2. Despite intense scrutiny, several proteins encoded by the genomes of SARS-CoV-2 and other SARS-like coronaviruses remain enigmatic. Moreover, the high infectivity and severity of SARS-CoV-2 in certain individuals make wet-lab studies currently challenging. In this study, we used a series of computational strategies to identify several fast-evolving regions of SARS-CoV-2 proteins which are potentially under host immune pressure. Most notably, the hitherto-uncharacterized protein encoded by ORF8 is one of them. Using sensitive sequence and structural analysis methods, we show that ORF8 and several other proteins from alpha- and beta-coronavirus comprise novel families of immunoglobulin domain proteins, which might function as potential immune modulators to delay or attenuate the host immune response against the viruses.

Prokaryotic Expression of the Immunoglobulin's Domains of CRTAM to Characterize a Monoclonal Antibody.

Class-I restricted T cell-associated molecule (CRTAM) is a member of the immunoglobulin superfamily, and it is closely related to nectin-like protein. CRTAM is expressed in activated CD8 T cells, NKT cells, NK cells and in a subpopulation CD4 T cells. In this study, we produce as recombinant proteins, the Ig-domains of CRTAM (IgV-IgC), the IgV, and the IgC. These proteins were successfully purified in the soluble fraction only if the stalk region was included. The recombinant CRTAM recognizes its ligand nectin-like 2 in a cell-free system. We also demonstrate that the IgC domain of CRTAM is recognized by the anti-hCRTAM monoclonal antibody C8 with a 0.62 nM affinity. In conclusion, the stalk region of CRTAM provides solubility for the expression of its Ig-domains as recombinant proteins.

Leptospiral Immunoglobulin-Like Domain Proteins: Roles in Virulence and Immunity.

The virulence mechanisms required for infection and evasion of immunity by pathogenic Leptospira species remain poorly understood. A number of L. interrogans surface proteins have been discovered, lying at the interface between the pathogen and host. Among these proteins, the functional properties of the Lig (leptospiral immunoglobulin-like domain) proteins have been examined most thoroughly. LigA, LigB, and LigC contain a series of, 13, 12, and 12 closely related domains, respectively, each containing a bacterial immunoglobulin (Big) -like fold. The multidomain region forms a mostly elongated structure that exposes a large surface area. Leptospires wield the Lig proteins to promote interactions with a range of specific host proteins, including those that aid evasion of innate immune mechanisms. These diverse binding events mediate adhesion of L. interrogans to the extracellular matrix, inhibit hemostasis, and inactivate key complement proteins. These interactions may help L. interrogans overcome the physical, hematological, and immunological barriers that would otherwise prevent the spirochete from establishing a systemic infection. Despite significant differences in the affinities of the LigA and LigB proteins for host targets, their functions overlap during lethal infection of hamsters; virulence is lost only when both ligA and ligB transcription is knocked down simultaneously. Lig proteins have been shown to be promising vaccine antigens through evaluation of a variety of different adjuvant strategies. This review serves to summarize current knowledge of Lig protein roles in virulence and immunity and to identify directions needed to better understand the precise functions of the Lig proteins during infection.

Expression of a novel class of bacterial Ig-like proteins is required for IncHI plasmid conjugation.

Antimicrobial resistance (AMR) is currently one of the most important challenges to the treatment of bacterial infections. A critical issue to combat AMR is to restrict its spread. In several instances, bacterial plasmids are involved in the global spread of AMR. Plasmids belonging to the incompatibility group (Inc)HI are widespread in Enterobacteriaceae and most of them express multiple antibiotic resistance determinants. They play a relevant role in the recent spread of colistin resistance. We present in this report novel findings regarding IncHI plasmid conjugation. Conjugative transfer in liquid medium of an IncHI plasmid requires expression of a plasmid-encoded, large-molecular-mass protein that contains an Ig-like domain. The protein, termed RSP, is encoded by a gene (ORF R0009) that maps in the Tra2 region of the IncHI1 R27 plasmid. The RSP protein is exported outside the cell by using the plasmid-encoded type IV secretion system that is also used for its transmission to new cells. Expression of the protein reduces cell motility and enables plasmid conjugation. Flagella are one of the cellular targets of the RSP protein. The RSP protein is required for a high rate of plasmid transfer in both flagellated and nonflagellated Salmonella cells. This effect suggests that RSP interacts with other cellular structures as well as with flagella. These unidentified interactions must facilitate mating pair formation and, hence, facilitate IncHI plasmid conjugation. Due to its location on the outer surfaces of the bacterial cell, targeting the RSP protein could be a means of controlling IncHI plasmid conjugation in natural environments or of combatting infections caused by AMR enterobacteria that harbor IncHI plasmids.

Ig-like Domain in Endoglucanase Cel9A from Alicyclobacillus acidocaldarius Makes Dependent the Enzyme Stability on Calcium.

Endoglucanase Cel9A from Alicyclobacillus acidocaldarius (AaCel9A) has an Ig-like domain and the enzyme stability is dependent to calcium. In this study the effect of calcium on the structure and stability of the wild-type enzyme and the truncated form (the wild-type enzyme without Ig-like domain, AaCel9AΔN) was investigated. Fluorescence quenching results indicated that calcium increased and decreased the rigidity of the wild-type and truncated enzymes, respectively. RMSF results indicated that AaCel9A has two flexible regions (regions A and B) and deleting the Ig-like domain increased the truncated enzyme stability by decreasing the flexibility of region B probably through increasing the hydrogen bonds. Calcium contact map analysis showed that deleting the Ig-like domain decreased the calcium contacting residues and their calcium binding affinities, especially, in region B which has a role in calcium binding site in AaCel9A. Metal depletion and activity recovering as well as stability results showed that the structure and stability of the wild-type and truncated enzymes are completely dependent on and independent of calcium, respectively. Finally, one can conclude that the deletion of Ig-like domain makes AaCel9AΔN independent of calcium via decreasing the flexibility of region B through increasing the hydrogen bonds. This suggests a new role for the Ig-like domain which makes AaCel9A structure dependent on calcium.

Immunoglobulin-like domains of the cargo proteins are essential for protein stability during secretion by the type IX secretion system.

The periodontal pathogen Porphyromonas gingivalis secretes many potent virulence factors using the type IX secretion system (T9SS). T9SS cargo proteins that have been structurally determined by X-ray crystallography are composed of a signal peptide, functional domain(s), an immunoglobulin (Ig)-like domain and a C-terminal domain. Role of the Ig-like domains of cargo proteins in the T9SS has not been elucidated. Gingipain proteases, which are cargo proteins of the T9SS, were degraded when their Ig-like domains were lacking or truncated. The degradation was dependent on the activity of a quality control factor, HtrA protease. Another T9SS cargo protein, HBP35, which has a thioredoxin domain as a functional domain, was analyzed by X-ray crystallography, revealing that HBP35 has an Ig-like domain after the thioredoxin domain and that the hydrophobic regions of the thioredoxin domain and the Ig-like domain face each other. HBP35 with substitution of hydrophobic amino acids in the Ig-like domain was degraded depending on HtrA. These results suggest that the Ig-like domain mediates stability of the cargo proteins in the T9SS.

Discovery and Analysis of Invertebrate IgVJ-C2 Structure from Amphioxus Provides Insight into the Evolution of the Ig Superfamily.

The emergence of adaptive immunity in jawed vertebrates depended on the appearance of variable immune receptors, BCRs and TCRs, which exhibit variable-J-constant (VJ-C)-type Ig superfamily folds. Hitherto, however, the structures of IgV-J-IgC-type molecules had never been characterized in invertebrates, leaving the origin of BCR/TCR-type molecules unknown. Using x-ray crystallography, the structure of a VJ-C2 molecule, named AmpIgVJ-C2, was determined in amphioxus (Branchiostoma floridae). The first domain shows typical V folding, including the hydrophobic core, CDR analogs, and eight conserved residues. The second domain is a C2-type Ig superfamily domain, as defined by its short length and the absence of ß-strand D- and C1-typical motifs. AmpIgVJ-C2 molecules form homodimers, using "three-layer packing dimerization," as described for TCRs and BCRs. The AmpIgVJ-C2 V domain harbors a diglycine motif in ß-strand G and forms a ß-bulge structure participating in V-V intermolecular interaction. By immunohistochemistry, AmpIgVJ-C2 molecules were primarily found in mucosal tissues, whereas PCR and sequence analysis indicated considerable genetic variation at the single-gene level; these findings would be consistent with an immune function and a basic ability to adapt to binding different immune targets. Our results show a BCR/TCR-ancestral like molecule in amphioxus and help us to understand the evolution of the adaptive immune system.

Cooperative interactions between VEGFR2 extracellular Ig-like subdomains ensure VEGFR2 dimerization.

BACKGROUND: Prior studies have suggested that the interactions occurring between VEGFR2 extracellular domains in the absence of ligand are complex. Here we seek novel insights into these interactions, and into the role of the different Ig-like domains (D1 through D7) in VEGFR2 dimerization. METHODS: We study the dimerization of a single amino acid mutant and of three deletion mutants in the plasma membrane using two photon microscopy and fully quantified spectral imaging. RESULTS: We demonstrate that a set of cooperative interactions between the different Ig-like domains ensure that VEGFR2 dimerizes with a specific affinity instead of forming oligomers. CONCLUSIONS: The contributions of subunits D7 and D4 seem to be the most critical, as they appear essential for strong lateral interactions and for the formation of dimers, respectively. GENERAL SIGNIFICANCE: This study provides new insights into the mechanism of VEGFR2 dimerization and activation.

Conformational plasticity and evolutionary analysis of the myotilin tandem Ig domains.

Myotilin is a component of the sarcomere where it plays an important role in organisation and maintenance of Z-disk integrity. This involves direct binding to F-actin and filamin C, a function mediated by its Ig domain pair. While the structures of these two individual domains are known, information about their relative orientation and flexibility remains limited. We set on to characterise the Ig domain pair of myotilin with emphasis on its molecular structure, dynamics and phylogeny. First, sequence conservation analysis of myotilin shed light on the molecular basis of myotilinopathies and revealed several motifs in Ig domains found also in I-band proteins. In particular, a highly conserved Glu344 mapping to Ig domain linker, was identified as a critical component of the inter-domain hinge mechanism. Next, SAXS and molecular dynamics revealed that Ig domain pair exists as a multi-conformation species with dynamic exchange between extended and compact orientations. Mutation of AKE motif to AAA further confirmed its impact on inter-domain flexibility. We hypothesise that the conformational plasticity of the Ig domain pair in its unbound form is part of the binding partner recognition mechanism.

Morphological Diversity of C. elegans Sensory Cilia Instructed by the Differential Expression of an Immunoglobulin Domain Protein.

Cilia on dendritic endings of sensory neurons organize distinct types of sensory machinery [1]. Ciliated endings display neuron-type-specific patterns of membrane elaborations [1-3], but it is not well understood how such neuron-type-specific morphologies are generated and whether they are coupled to the specification of other identity aspects of a terminally differentiated sensory neuron. In the course of a genome-wide analysis of members of a small family of immunoglobulin domain proteins, we found that OIG-8, a previously uncharacterized transmembrane protein with a single immunoglobulin (Ig) domain, instructs the distinct, neuron-type-specific elaboration of ciliated endings of different olfactory neuron types in the nematode C. elegans. OIG-8 protein localizes to ciliated endings of these sensory neurons, and is transcribed at different levels in distinct olfactory neuron types. oig-8 expression levels correlate with the extent of sensory cilia growth and branching patterns. Loss of oig-8 leads to a reduction in the branching patterns of cilia, whereas raising the levels of oig-8 results in an increase in elaborations. Levels of OIG-8 expression are controlled by the specific combination of a terminal selector type of transcription factors that also specify other identity features of distinct olfactory neuron types.

Immunoglobulin-like domain 4-mediated ligand-independent dimerization triggers VEGFR-2 activation in HUVECs and VEGFR2-positive breast cancer cells.

PURPOSE: The extracellular region (EC) of the vascular endothelial growth factor (VEGF) receptor-2 (VEGFR-2) contains seven immunoglobulin-like (Ig-like) domains that are required for specific ligand binding and receptor dimerization. Studies of domain 4-7 deletions and substitutions provided insights into the interaction between receptors in the absence of VEGF. In this study, we investigated the effect of domain 4 in ligand-independent VEGFR-2 dimerization and activation in human vascular endothelial cells and human breast cancer cells. METHODS: To confirm the role of domain 4 in ligand-independent receptor dimerization and activation, two VEGFR-2 fragments with and without domain 4, KFP1 and KFP2, were generated by recombinant DNA technology. We measured the affinity of KFP1 and KFP2 with VEGFR-2, and the roles of KFP1 and FKP2 in dimerization and phosphorylation of VEGFR-2. We also evaluated the effect of KFP1 and FKP2 on cell proliferation and migration in HUVECs and in human breast cancer cells. RESULTS: We showed that KFP1 did not affect the interaction of VEGFR-2 and VEGF but bound VEGFR-2 in the absence of VEGF. Furthermore, cross-linking and cross-linking immunoblotting demonstrated that KFP1 could form a complex with VEGFR-2, which resulted in VEGFR-2 dimerization in the absence of VEGF. Importantly, we found that the KDR fragment with domain 4 induced phosphorylation of VEGFR-2, as well as phosphorylation of downstream receptor kinases in HUVECs and VEGFR-2-positive breast cancer cells. Consistent with these results, this ligand-independent activation of VEGFR-2 also promoted downstream signaling and cell proliferation and migration. CONCLUSIONS: The domain 4 of VEGFR-2 plays an important role in the interaction between VEGFR receptors in the absence of VEGF.

L1 syndrome diagnosis complemented with functional analysis of L1CAM variants located to the two N-terminal Ig-like domains.

L1CAM gene mutations cause neurodevelopmental disorders collectively termed L1 syndrome. Insufficient information about L1CAM variants complicates clinical prognosis, genetic diagnosis and genetic counseling. We combined clinical data, in silico effect predictions and functional analysis of four L1CAM variants, p.I37N, p.T38M, p.M172I and p.D202Y, located to the two N-terminal Ig-like domains present in five families with symptoms of L1 syndrome. Software tools predicted destabilizing effects of p.I37N and p.D202Y but results for p.T38M and p.M172I were inconsistent. Cell surface expression of mutant proteins L1-T38M, L1-M172I and L1-D202Y was normal. Conversely, L1-I37N accumulated in the endoplasmic reticulum (ER) and showed temperature-sensitive protein maturation suggesting that p.I37N induces protein misfolding. L1CAM-mediated cell-cell aggregation was severely impaired by L1CAM variants p.I37N, p.M172I and p.D202Y but was preserved by the variant p.T38M. Our experimental data indicate that protein misfolding and accumulation in the ER affect function of the L1CAM variant p.I37N whereas the variants p.M172I and p.D202Y impair homophilic interaction at the cell surface.

Identification of an Activating Chicken Ig-like Receptor Recognizing Avian Influenza Viruses.

Chicken Ig-like receptors (CHIRs) represent a multigene family encoded by the leukocyte receptor complex that encodes a variety of receptors that are subdivided into activating CHIR-A, inhibitory CHIR-B, and bifunctional CHIR-AB. Apart from CHIR-AB, which functions as an Fc receptor, CHIR ligands are unknown. In the current study, we used a panel of different BWZ.36 CHIR reporter cells to identify an interaction between specific CHIRs and avian influenza virus (AIV). The specificity of the CHIR-AIV interaction was further demonstrated using CHIR fusion proteins that bound to AIV-coated plates and were able to reduce the interaction of reporter cells with AIV. There was no difference in binding of CHIR to different AIV strains. Furthermore, CHIR fusion proteins reduced AIV-induced in vitro activation of NK cells obtained from lungs of AIV-infected animals, as judged by the lower frequency of CD107+ cells. Because the original CHIR reporter lines were generated based on sequence information about extracellular CHIR domains, we next identified a full-length CHIR that displayed similar binding to AIV. The sequence analysis identified this CHIR as a CHIR-A. Neuraminidase treatment of coated CHIR-human Ig proteins reduced binding of trimeric H5 proteins to CHIR. This suggests that the interaction is dependent on sialic acid moieties on the receptor. In conclusion, this article identifies AIV as a ligand of CHIR-A and describes the functional consequences of this interaction.