Protein LY6E as a candidate for mediating transport of adeno-associated virus across the human blood-brain barrier.

The blood-brain barrier (BBB) is a major obstacle for the treatment of central nervous system (CNS) disorders. Significant progress has been made in developing adeno-associated virus (AAV) variants with increased ability to cross the BBB in mice. However, these variants are not efficacious in non-human primates. Herein, we employed various bioinformatic techniques to identify lymphocyte antigen-6E (LY6E) as a candidate for mediating transport of AAV across the human BBB based on the previously determined mechanism of transport in mice. Our results provide insight into future discovery and optimization of AAV variants for CNS gene delivery in humans.

Structural Diversity and Dynamics of Human Three-Finger Proteins Acting on Nicotinic Acetylcholine Receptors.

Ly-6/uPAR or three-finger proteins (TFPs) contain a disulfide-stabilized ß-structural core and three protruding loops (fingers). In mammals, TFPs have been found in epithelium and the nervous, endocrine, reproductive, and immune systems. Here, using heteronuclear NMR, we determined the three-dimensional (3D) structure and backbone dynamics of the epithelial secreted protein SLURP-1 and soluble domains of GPI-anchored TFPs from the brain (Lynx2, Lypd6, Lypd6b) acting on nicotinic acetylcholine receptors (nAChRs). Results were compared with the data about human TFPs Lynx1 and SLURP-2 and snake α-neurotoxins WTX and NTII. Two different topologies of the ß-structure were revealed: one large antiparallel ß-sheet in Lypd6 and Lypd6b, and two ß-sheets in other proteins. α-Helical segments were found in the loops I/III of Lynx2, Lypd6, and Lypd6b. Differences in the surface distribution of charged and hydrophobic groups indicated significant differences in a mode of TFPs/nAChR interactions. TFPs showed significant conformational plasticity: the loops were highly mobile at picosecond-nanosecond timescale, while the ß-structural regions demonstrated microsecond-millisecond motions. SLURP-1 had the largest plasticity and characterized by the unordered loops II/III and cis-trans isomerization of the Tyr39-Pro40 bond. In conclusion, plasticity could be an important feature of TFPs adapting their structures for optimal interaction with the different conformational states of nAChRs.

Three-Finger Proteins from the Ly6/uPAR Family: Functional Diversity within One Structural Motif.

The discovery in higher animals of proteins from the Ly6/uPAR family, which have structural homology with snake "three-finger" neurotoxins, has generated great interest in these molecules and their role in the functioning of the organism. These proteins have been found in the nervous, immune, endocrine, and reproductive systems of mammals. There are two types of the Ly6/uPAR proteins: those associated with the cell membrane by GPI-anchor and secreted ones. For some of them (Lynx1, SLURP-1, SLURP-2, Lypd6), as well as for snake α-neurotoxins, the target of action is nicotinic acetylcholine receptors, which are widely represented in the central and peripheral nervous systems, and in many other tissues, including epithelial cells and the immune system. However, the targets of most proteins from the Ly6/uPAR family and the mechanism of their action remain unknown. This review presents data on the structural and functional properties of the Ly6/uPAR proteins, which reveal a variety of functions within a single structural motif.

A novel homozygous mutation disrupting the initiation codon in the SLURP1 gene underlies mal de Meleda in a consanguineous family.

Mal de Meleda (MDM) is a palmoplantar keratoderma (PPK), characterized by hyperkeratosis of the palms and soles, and keratotic skin lesions. Patients with MDM can develop perioral erythema, keratotic and lichenoid plaques over the joints (including the elbows and knees), nail abnormalities, joint contractures and stiffness, brachydactyly, sclerodactyly, pseudoainhum, and malodorous maceration. MDM is associated with mutations in the SLURP1 gene. We report a consanguineous family in which MDM was inherited in an autosomal recessive manner. Genotyping using microsatellite markers established linkage in the family to the SLURP1 gene, which has been mapped previously to chromosome 8q24.3. Sequence analysis revealed a homozygous missense mutation (c.2T>C, p.Met1Thr) in affected family members. Molecular docking studies using a ZDOCK server predicted disruption of binding of the mutant variant to its target α7-nAChR. This study further supports the previously reported findings that homozygous mutations in the SLURP1 gene cause MDM.

Identification of a Ly-6 superfamily gene expressed in lateral line neuromasts in zebrafish.

Lymphocyte antigen-6 (Ly-6) superfamily members have been identified in zebrafish, but the expression and function of these Ly-6 genes remain largely unknown. Posterior lateral line (pLL) system is produced by migrating pLL primordium (pLLp). Chemokine signaling, Notch, Wnt, and fibroblast growth factor (FGF) signaling regulate migration of pLLp cells and formation of neuromasts. However, the mechanism of neuromast deposition remains to be explored. Identification of novel genes expressed in pLLp will certainly help the study of such a process. Here we identified a Ly-6 gene called neuromast-expressed gpi-anchored lymphocyte antigen-6 (negaly6), which was specifically expressed in neuromast. Quantitative real-time PCR (qRT-PCR) analysis showed that negaly6 started to be expressed at 24 hpf, and whole-mount in situ hybridization analysis indicated that negaly6 was highly expressed in the trailing zone of pLLp and mature neuromast. Furthermore, negaly6 expression was inhibited by FGF signaling antagonist but not by Wnt signaling agonist or antagonist. Collectively, these data indicate that negaly6 may be associated with the regulation of neuromast deposition via FGF signaling pathway.

SOLD1 is expressed in bovine trophoblast cell lines and regulates cell invasiveness.

BACKGROUND: Secreted protein of Ly-6 domain 1 (SOLD1), a secretory-type member of the Ly-6 superfamily, is expressed in both fetal and maternal tissues throughout gestation. SOLD1 mRNA is expressed in the endometrium and in trophoblast mononucleate and binucleate cells, suggesting it plays an important role not only in placental architecture at early gestation, but also in remodeling the endometrium at late gestation. Here, we investigate the expression of SOLD1 mRNA and protein in trophoblast cell lines. In addition, we examine the effect of SOLD1 on the invasive ability of trophoblast cells. METHODS: We measured SOLD1 gene expression in thirteen bovine trophoblast (BT) cell lines by using quantitative reverse transcription PCR (qRT-PCR). SOLD1 protein levels were examined in two cell lines, BT-C and BT-K, by using Western blotting and immunocytochemistry. In addition, we measured the invasive activity of BT cells in the presence or absence of anti-bovine SOLD1 antibodies. RESULTS: At variable levels, SOLD1 was expressed in all thirteen cell lines; however, expression remained below that of proximal fetal membrane tissue. SOLD1 protein, which was approximately 28 kDa in size, was detected in perinuclear area of the cytoplasm in BT cells. Treatment with anti-bovine SOLD1 antibody had a dose-dependent suppressive effect on the invasiveness of BT-K cell lines. CONCLUSIONS: The present study is the first to investigate SOLD1 expression in vitro, in trophoblastic cell lines. Our data suggested that SOLD1 is involved in the regulation of the trophoblast invasiveness. Therefore, SOLD1 may play an active and crucial role in mediating communication at the fetomaternal interface.

Characterization of Ly108 in the thymus: evidence for distinct properties of a novel form of Ly108.

Ly108 (CD352) is a member of the signaling lymphocyte activation molecule (SLAM) family of receptors that signals through SLAM-associated protein (SAP), an SH2 domain protein that can function by the recruitment of Src family kinases or by competition with phosphatases. Ly108 is expressed on a variety of hematopoietic cells, with especially high levels on developing thymocytes. We find that Ly108 is constitutively tyrosine phosphorylated in murine thymi in a SAP- and Fyn kinase-dependent manner. Phosphorylation of Ly108 is rapidly lost after thymocyte disaggregation, suggesting dynamic contact-mediated regulation of Ly108. Similar to recent reports, we find at least three isoforms of Ly108 mRNA and protein in the thymus, which are differentially expressed in the thymi of C57BL/6 and 129S6 mice that express the lupus-resistant and lupus-prone haplotypes of Ly108, respectively. Notably, the recently described novel isoform Ly108-H1 is not expressed in mice having the lupus-prone haplotype of Ly108, but is expressed in C57BL/6 mice. We further provide evidence for differential phosphorylation of these isoforms; the novel Ly108-H1does not undergo tyrosine phosphorylation, suggesting that it functions as a decoy isoform that contributes to the reduced overall phosphorylation of Ly108 seen in C57BL/6 mice. Our study suggests that Ly108 is dynamically regulated in the thymus, shedding light on Ly108 isoform expression and phosphorylation.

Structural model of lymphocyte receptor NKR-P1C revealed by mass spectrometry and molecular modeling.

NKR-P1C is an activating immune receptor expressed on the surface of mouse natural killer cells. It has been widely used as a marker for NK cell identification in different mice strains. Recently we solved a crystal structure of the C-type lectin-like domain of a homologous protein, NKR-P1A, using X-ray crystallography and also described the strategy for rapid characterization of the protein conformation in solution. This procedure utilized chemical cross-linking, hydrogen/deuterium exchange, and molecular modeling. It was found that the solution structure differs from the crystal structure in the conformation of the loop region. The loop, detached from the protein compact core in the crystal structure, is closely attached to the core of the protein in solution. Here we present and interpret the solution structure of the C-type lectin-like domain of NKR-P1C using chemical cross-linking and molecular modeling. The validation of the model and conformation of the loop region in NKR-P1C were addressed using ion-mobility mass spectrometry.

Recognition of class I MHC by a rat Ly49 NK cell receptor is dependent on the identity of the P2 anchor amino acid of bound peptide.

Members of the rodent Ly49 receptor family control NK cell responsiveness and demonstrate allele specificity for MHC class I (MHC-I) ligands. For example, the rat Ly49i2 inhibitory NK cell receptor binds RT1-A1(c) but not other rat MHC class Ia or Ib molecules. RT1-A1(c) preferentially binds peptides with proline at the second, or P2, position, which defines it as an HLA-B7 supertype MHC-I molecule. Previously, our laboratory showed that mutations within the MHC-I supertype-defining B-pocket of RT1-A1(c) could lead to alterations in P2 anchor residues of the peptide repertoire bound by RT1-A1(c) and loss of recognition by Ly49i2. Although suggestive of peptide involvement, it was unclear whether the peptide P2 anchor residue or alteration of the RT1-A1(c) primary sequence influenced Ly49i2 recognition. Therefore, we directly investigated the role of the P2 anchor residue of RT1-A1(c)-bound peptides in Ly49i2 recognition. First, fluorescent multimers generated by refolding soluble recombinant RT1-A1(c) with individual synthetic peptides differing only at the P2 anchor residue were examined for binding to Ly49i2 NK cell transfectants. Second, cytotoxicity by Ly49i2-expressing NK cells toward RMA-S target cells expressing RT1-A1(c) bound with peptides that only differ at the P2 anchor residue was evaluated. Our results demonstrate that Ly49i2 recognizes RT1-A1(c) bound with peptides that have Pro or Val at P2, whereas little or no recognition is observed when RT1-A1(c) is complexed with peptide bearing Gln at P2. Thus, the identity of the P2 peptide anchor residue is an integral component of MHC-I recognition by Ly49i2.

Human neuromodulator SLURP-1: bacterial expression, binding to muscle-type nicotinic acetylcholine receptor, secondary structure, and conformational heterogeneity in solution.

Human protein SLURP-1 is an endogenous neuromodulator belonging to the Ly-6/uPAR family and acting on nicotinic acetylcholine receptors. In the present work, the gene of SLURP-1 was expressed in E. coli. The bacterial systems engineered for SLURP-1 expression as fused with thioredoxin and secretion with leader peptide STII failed in the production of milligram quantities of the protein. The SLURP-1 was produced with high-yield in the form of inclusion bodies, and different methods of the protein refolding were tested. Milligram quantities of recombinant SLURP-1 and its (15)N-labeled analog were obtained. The recombinant SLURP-1 competed with (125)I-α-bungarotoxin for binding to muscle-type Torpedo californica nAChR at micromolar concentrations, indicating a partial overlap in the binding sites for SLURP-1 and α-neurotoxins on the receptor surface. NMR study revealed conformational heterogeneity of SLURP-1 in aqueous solution, which was associated with cis-trans isomerization of the Tyr39-Pro40 peptide bond. The two structural forms of the protein have almost equal population in aqueous solution, and exchange process between them takes place with characteristic time of about 4 ms. Almost complete (1)H and (15)N resonance assignment was obtained for both structural forms of SLURP-1. The secondary structure of SLURP-1 involves two antiparallel ß-sheets formed from five ß-strands and closely resembles those of three-finger snake neurotoxins.

Ly6C supports preferential homing of central memory CD8+ T cells into lymph nodes.

Ly6C is a murine cell-surface antigen expressed by plasma cells, subsets of myeloid cells and many T cells, including memory T cells. We previously documented that Ly6C crosslinking induces LFA-1 clustering on naïve CD8(+) T cells. Here, we show that in vitro and in vivo differentiation of naïve CD8(+) T cells into central (Tcm) but not effector (Tem) memory T cells enhances Ly6C expression, and its crosslinking induces strong LFA-1 clustering on Tcm. Blocking Ly6C function inhibits in vivo Tcm homing to LNs as efficiently as blocking L-selectin but it does not potentiate the inhibition provided by blocking either L-selectin or LFA-1 function. Thus, Ly6C, L-selectin and LFA-1 all appear to be part of a common homing pathway. In vitro, Ly6C crosslinking enhances Tcm adherence to ICAM-1 in the presence of CCL21. In summary, Tcm homing involves Ly6C, in addition to L-selectin and LFA-1, and appears to potentiate firm adhesion of Tcm to ICAM-1 in synergy with a chemokine. We propose that Ly6C augments Tcm compartmentalization into LNs during their homing.

Polymorphisms in Ly6 genes in Msq1 encoding susceptibility to mouse adenovirus type 1.

Strain-specific differences in susceptibility to mouse adenovirus type 1 (MAV-1) are linked to the quantitative trait locus Msq1 on mouse chromosome 15. This region contains 14 Ly6 or Ly6-related genes, many of which are known to be expressed on the surface of immune cells, suggesting a possible role in host defense. We analyzed these genes for polymorphisms between MAV-1-susceptible and MAV-1-resistant inbred mouse strains. Sequencing of cDNAs identified 12 coding-region polymorphisms in 2010109I03Rik, Ly6e, Ly6a, Ly6c1, and Ly6c2, six of which were nonsynonymous and five of which were previously unlisted in dbSNP Build 132. We also clarified sequence discrepancies in GenBank for the coding regions of I830127L07Rik and Ly6g. Additionally, Southern blotting revealed size polymorphisms within the DNA regions of Ly6e, Ly6a, and Ly6g. Collectively, these genetic variations have implications for the structure, function, and/or expression of Ly6 and Ly6-related genes that may contribute to the observed strain-specific differences in susceptibility to MAV-1.

Ligand-dependent inhibition of CD1d-restricted NKT cell development in mice transgenic for the activating receptor Ly49D.

In addition to their CD1d-restricted T cell receptor (TCR), natural killer T (NKT) cells express various receptors normally associated with NK cells thought to act, in part, as modulators of TCR signaling. Immunoreceptor-tyrosine activation (ITAM) and inhibition (ITIM) motifs associated with NK receptors may augment or attenuate perceived TCR signals respectively, potentially influencing NKT cell development and function. ITIM-containing Ly49 family receptors expressed by NKT cells are proposed to play a role in their development and function. We have produced mice transgenic for the ITAM-associated Ly49D and ITIM-containing Ly49A receptors and their common ligand H2-Dd to determine the importance of these signaling interplays in NKT cell development. Ly49D/H2-Dd transgenic mice had selectively and severely reduced numbers of thymic and peripheral NKT cells, whereas both ligand and Ly49D transgenics had normal numbers of NKT cells. CD1d tetramer staining revealed a blockade of NKT cell development at an early precursor stage. Coexpression of a Ly49A transgene partially rescued NKT cell development in Ly49D/H2-Dd transgenics, presumably due to attenuation of ITAM signaling. Thus, Ly49D-induced ITAM signaling is incompatible with the early development of cells expressing semi-invariant CD1d-restricted TCRs and appropriately harmonized ITIM-ITAM signaling is likely to play an important role in the developmental program of NKT cells.

Identification and characterization of human LYPD6, a new member of the Ly-6 superfamily.

The Ly-6 protein superfamily is usually identified as a group of proteins with a LU protein domain. LU domain is about 80 amino acids long and characterized by a conserved pattern of 10 cysteine residues. Here we report the cloning and characterization of a novel human LU domain containing gene, LYPD6, isolated from human testis cDNA library, and mapped to 2q23.1-23.2 by searching the UCSC genomic database. The LYPD6 cDNA sequence of 3,501 base pairs contains an open reading frame encoding 171 amino acids. Subcellular localization of LYPD6 demonstrated that the protein was localized in the cytoplasm when overexpressed in COS-7 cells. RT-PCR analysis showed that LYPD6 was widely expressed in human tissues and the expression levels in brain and heart were relatively high. Furthermore, the subsequent analysis based on reporter gene assays suggested that overexpression of LYPD6 in HEK 293T cells was able to suppress the transcriptional activities of AP1.

The human E48 antigen, highly homologous to the murine Ly-6 antigen ThB, is a GPI-anchored molecule apparently involved in keratinocyte cell-cell adhesion.

The E48 antigen, a putative human homologue of the 20-kD protein present in desmosomal preparations of bovine muzzle, and formerly called desmoglein III (dg4), is a promising target antigen for antibody-based therapy of squamous cell carcinoma in man. To anticipate the effect of high antibody dose treatment, and to evaluate the possible biological involvement of the antigen in carcinogenesis, we set out to molecularly characterize the antigen. A cDNA clone encoding the E48 antigen was isolated by expression cloning in COS cells. Sequence analysis revealed that the clone contained an open reading frame of 128 amino acids, encoding a core protein of 13,286 kD. Database searching showed that the E48 antigen has a high level of sequence similarity with the mouse ThB antigen, a member of the Ly-6 antigen family. Phosphatidylinositol-specific (PI-specific) phospholipase-C treatment indicated that the E48 antigen is glycosylphosphatidylinositol-anchored (GPI-anchored) to the plasma membrane. The gene encoding the E48 antigen is a single copy gene, located on human chromosome 8 in the 8q24-qter region. The expression of the gene is confined to keratinocytes and squamous tumor cells. The putative mouse homologue, the ThB antigen, originally identified as an antigen on cells of the lymphocyte lineage, was shown to be highly expressed in squamous mouse epithelia. Moreover, the ThB expression level is in keratinocytes, in contrast to that in lymphocytes, not mouse strain related. Transfection of mouse SV40-polyoma transformed mouse NIH/3T3 cells with the E48 cDNA confirmed that the antigen is likely to be involved in cell-cell adhesion.

Identification of Ly-6K as a novel marker for mouse plasma cells.

Plasma cells are the main producers of antibody and key effector cells of the immune system. Despite their importance, analytics of plasma cells still suffers from the limited availability of specific markers. Currently, plasma cell identification relies on the expression of a single marker, CD138/syndecan-1. However, syndecan-1 is widely expressed on various cell types outside the hematopoietic compartment, and furthermore, not expressed on all subsets of plasma cells. To discover novel surface markers, a differential screening followed by signal sequence trap cloning was developed, leading to the identification of mouse Ly-6K (mLy-6K). Expression profiling confirmed that mLy-6K is expressed by plasma cells but not B cells or tissues not containing plasma cells. Expression at the surface of plasma cells isolated from spleen, lymph node, bone marrow, and lamina propria of the small intestine was demonstrated at the protein level using a polyclonal rabbit antibody. This novel plasma cell marker shows promise to help broaden our understanding of plasma cell differentiation and function.

In-silico Analyses of Disease Causing Mutations in SLURP1 Gene.

The SLURP1 (secreted LY6/urokinase type plasminogen activator receptor related protein-1) belongs to the gene family of urokinase, a type of plasminogen activator receptor (uPAR). Mutations in the SLURP1 have been reported to cause serious genetic problems of skin, Mal De Meleda, and malignancies. With the advancement of computational tools, it became possible to predict the potential impact of gene variants on the structure and function of protein. Therefore, in present study, we aimed to perform in-silico analyses of the disease causing SLURP1 mutations using online tools. In-total, 21 variants occurring in coding and non-coding regions of SLURP1 were found from public databases. In curated data, we have found 57.14% (12/21) missense, 23.81% (5/21) splice site, 9.52% (2/21) nonsense, 4.76% (1/21) deletion, and 4.76% (1/21) frameshift mutations. Moreover, heterogeneity in genotypes and phenotypes, along with 7 hotspot points in SLURP1 has been noted. In-silico analyses of the subjected variants have depicted a range of pathogenicity by combinatorial predictions of different tools from being lowly to highly pathogenic. Thus, the present study paves a platform to link computational analyses of mutations for important regulatory genes that can be undertaken for their phenotypes and their correlation with the disease status in case control studies.

Delivering genes across the blood-brain barrier: LY6A, a novel cellular receptor for AAV-PHP.B capsids.

The engineered AAV-PHP.B family of adeno-associated virus efficiently delivers genes throughout the mouse central nervous system. To guide their application across disease models, and to inspire the development of translational gene therapy vectors for targeting neurological diseases in humans, we sought to elucidate the host factors responsible for the CNS tropism of the AAV-PHP.B vectors. Leveraging CNS tropism differences across 13 mouse strains, we systematically determined a set of genetic variants that segregate with the permissivity phenotype, and rapidly identified LY6A as an essential receptor for the AAV-PHP.B vectors. Interfering with LY6A by CRISPR/Cas9-mediated Ly6a disruption or with blocking antibodies reduced transduction of mouse brain endothelial cells by AAV-PHP.eB, while ectopic expression of Ly6a increased AAV-PHP.eB transduction of HEK293T and CHO cells by 30-fold or more. Importantly, we demonstrate that this newly discovered mode of AAV binding and transduction can occur independently of other known AAV receptors. These findings illuminate the previously reported species- and strain-specific tropism characteristics of the AAV-PHP.B vectors and inform ongoing efforts to develop next-generation AAV vehicles for human CNS gene therapy.

Ly49 cluster sequence analysis in a mouse model of diabetes: an expanded repertoire of activating receptors in the NOD genome.

The mouse Ly49 and human killer cell immunoglobulin-like receptors (KIR) gene clusters encode activating and inhibitory class I MHC receptors on natural killer (NK) cells. A direct correlation between the presence of multiple activating KIR and various human autoimmune diseases including diabetes has been shown. Previous studies have implicated NK cell receptors in the development of diabetes in the non-obese diabetic (NOD) inbred mouse strain. To assess the contribution of Ly49 to NOD disease acceleration the Ly49 gene cluster of these mice was sequenced. Remarkably, the NOD Ly49 haplotype encodes the largest haplotype and the most functional activating Ly49 of any known mouse strain. These activating Ly49 include three Ly49p-related and two Ly49h-related genes. The NOD cluster contains large regions highly homologous to both C57BL/6 and 129 haplotypes, suggesting unequal crossing over as a mechanism of Ly49 haplotype evolution. Interestingly, the 129-like region has duplicated in the NOD genome. Thus, the NOD Ly49 cluster is a unique mix of elements seen in previously characterized Ly49 haplotypes resulting in a disproportionately large number of functional activating Ly49 genes. Finally, the functionality of activating Ly49 in NOD mice was confirmed in cytotoxicity assays.