Structural and phylogenetic characterization of human SLURP-1, the first secreted mammalian member of the Ly-6/uPAR protein superfamily

Members of the Ly-6/uPAR protein family share one or several repeat units of the Ly-6/uPAR domain that is defined by a distinct disulfide bonding pattern between 8 or 10 cysteine residues. The Ly-6/uPAR protein family can be divided into two subfamilies. One comprises GPI-anchored glycoprotein receptors with 10 cysteine residues. The other subfamily includes the secreted single-domain snake and frog cytotoxins, and differs significantly in that its members generally possess only eight cysteines and no GPI-anchoring signal sequence. We report the purification and structural characterization of human SLURP-1 (secreted mammalian Ly-6/uPAR related protein 1) from blood and urine peptide libraries. SLURP-1 is encoded by the ARS (component B)-81/s locus, and appears to be the first mammalian member of the Ly-6/uPAR family lacking a GPI-anchoring signal sequence. A phylogenetic analysis based on the SLURP-1 primary protein structure revealed a closer relationship to the subfamily of cytotoxins. Since the SLURP-1 gene maps to the same chromosomal region as several members of the Ly-6/uPAR subfamily of glycoprotein receptors, it is suggested that both biologically distinct subfamilies might have co-evolved from local chromosomal duplication events.     

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 ¹⁵N-labeled analog were obtained. The recombinant SLURP-1 competed with ¹²⁵I-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 ¹H and ¹⁵N resonance assignment was obtained for both structural forms of SLURP-1. The secondary structure of SLURP-1 involves two antiparallel β-sheets formed from five β-strands and closely resembles those of three-finger snake neurotoxins.     

Structural Analysis and Deletion Mutagenesis Define Regions of QUIVER/SLEEPLESS that Are Responsible for Interactions with Shaker-Type Potassium Channels and Nicotinic Acetylcholine Receptors

Ly6 proteins are endogenous prototoxins found in most animals. They show striking structural and functional parallels to snake α-neurotoxins, including regulation of ion channels and cholinergic signaling. However, the structural contributions of Ly6 proteins to regulation of effector molecules is poorly understood. This question is particularly relevant to the Ly6 protein QUIVER/SLEEPLESS (QVR/SSS), which has previously been shown to suppress excitability and synaptic transmission by upregulating potassium (K) channels and downregulating nicotinic acetylcholine receptors (nAChRs) in wake-promoting neurons to facilitate sleep in Drosophila. Using deletion mutagenesis, co-immunoprecipitations, ion flux assays, surface labeling and confocal microscopy, we demonstrate that only loop 2 is required for many of the previously described properties of SSS in transfected cells, including interactions with K channels and nAChRs. Collectively our data suggest that QVR/SSS, and by extension perhaps other Ly6 proteins, target effector molecules using limited protein motifs. Mapping these motifs may be useful in rational design of drugs that mimic or suppress Ly6-effector interactions to modulate nervous system function.     

The Urokinase Receptor Homolog Haldisin Is a Novel Differentiation Marker of Stratum Granulosum in Squamous Epithelia

Several members of the Ly-6/uPAR (LU)-protein domain family are differentially expressed in human squamous epithelia. In some cases, they even play important roles in maintaining skin homeostasis, as exemplified by the secreted single domain member, SLURP-1, the deficiency of which is associated with the development of palmoplantar hyperkeratosis in the congenital skin disorder Mal de Meleda. In the present study, we have characterized a new member of the LU-protein domain family, which we find to be predominantly expressed in the stratum granulosum of human skin, thus resembling the expression of SLURP-1. In accordance with its expression pattern, we denote this protein product, which is encoded by the LYPD5 gene, as Haldisin (human antigen with LU-domains expressed in skin). Two of the five human glycolipid-anchored membrane proteins with multiple LU-domains characterized so far are predominantly confined to squamous epithelia (i.e., C4.4A), to stratum spinosum, and Haldisin to stratum granulosum under normal homeostatic conditions. Whether Haldisin is a prognostic biomarker for certain epithelial malignancies, like C4.4A and SLURP-1, remains to be explored.     

Bacterial Production and Structural Study of Human Neuromodulator Lynx2

Russian Journal of Bioorganic Chemistry - The human protein Lynx2 belongs to the Ly6/uPAR family of “three-finger” proteins and is associated with the cell membrane by the...     

Recombinant production and structural studies of the human Lypd6 and Lypd6b proteins

Lypd6 and Lypd6b are human three-finger proteins expressed in various tissues and sharing a high degree of sequence homology (~54%). Unlike other members of the Ly6/uPAR family, both proteins additionally bear extended N- and С-terminal sequences flanking the LU-domain. The role of these sequences is unclear. It is known that Lypd6 can increase the amplitude of nicotine-induced calcium currents in mouse trigeminal ganglion neurons. The Danio rerio fish Lypd6 is involved in the regulation of the Wnt/β-cathenin signaling pathway and the knockdown of the lypd6 expression leads to the impairment of embryonic development. The Lypd6b expression in X. laevis oocytes increased the sensitivity of nicotinic acetylcholine receptors to acetylcholine and increased their desensitization rate. Molecular mechanisms of Lypd6 and Lypd6b action as well as their spatial structures remain unknown. In this work, the genes encoding water-soluble variants of human Lypd6 and Lypd6b lacking N-terminal sequences (rLypd6 and rLypd6b) and Lypd6 bearing the N-terminal sequence (N-rLypd6) were obtained and expressed. The proteins were expressed in cytoplasmic inclusion bodies followed by solubilization under denaturing conditions and renaturation. The renaturation conditions were screened to optimize recombinant protein yields. The analysis of NMR spectra of recombinant N-rLypd6, rLypd6, and rLypd6b demonstrated that N-rLypd6 may not have a structured form. The production of milligram quantities of isotope-labeled rLypd6 and rLypd6b analogues allowed characterization of the secondary structure of these proteins and study of their intramolecular dynamics. It was found that rLypd6 and rLypd6b have structural elements typical for the Ly6/uPAR family, although with some unique features, particularly, an additional disulfide bond in the third loop and helical regions in the first and third loops.     

Mal de Meleda (MDM) caused by mutations in the gene for SLURP-1 in patients from Germany,Turkey, Palestine,and the United Arab Emirates

Mal de Meleda (MDM) or keratosis palmoplantaris transgrediens of Siemens is an autosomal recessive skin disorder characterized by diffuse palmoplantar keratoderma (PPK) and transgressive keratosis with an onset in early infancy. There is no associated involvement of other organs; however, a spectrum of clinical presentations with optional and variable features has been described. Mutations in the ARS (component B)-81/s gene ( LY6LS) on chromosome 8q24-qter, which encodes SLURP-1, have recently been identified in patients with MDM. Here, we have analyzed four MDM families for mutations in SLURP-1. In a large Palestinian pedigree with multiple consanguinity, patients are homozygous for a new mutation that substitutes an arginine for a conserved glycine residue at position 86. A different mutation in Turkish patients results in the same amino acid exchange. Some remarkable similarities are seen in the clinical picture of patients from both families. Patients of an Emirati Bedouin family have a homozygous alteration of the translation initiation codon. In a German family with no known consanguinity, we have shown pseudodominant inheritance. Three affected children and their affected mother are homozygous for the missense mutation W15R. Our findings indicate that the MDM type of transgressive PPK is caused by SLURP-1 mutations in patients from various origins and demonstrate allelic heterogeneity for mutations in SLURP-1.     

Characterization of the five novel Ly-6 superfamily members encoded in the MHC, and detection of cells expressing their potential ligands

Lymphocyte Antigen 6 (Ly-6) superfamily members are cysteine-rich, generally GPI-anchored cell surface proteins, which have definite or putative immune related roles. There are 27 members of this family described so far in the human genome and 37 in the mouse. Five of them are clustered in the class III region of the human and mouse MHCs. Following computational analyses, we functionally characterized the encoded proteins by creating epitope-tagged fusion constructs to determine molecular weight, complex formation, subcellular localization, post-translational modifications and ligand binding. We found that all human and mouse proteins were glycosylated, and most could form part of larger complexes. Human and mouse Ly6G6c and Ly6G6d, and mouse Ly6g6e were found to be GPI-anchored cell surface proteins, highly expressed at the leading edges of cells, on filopodia, which are normally involved in cell adhesion and migration. However, analysis of Ly6G5c and Ly6G5b indicated that they are potentially secreted proteins. Our results indicate that there are two subclusters of related Ly-6 proteins in this region of the MHC, with Ly6G6c, Ly6G6d, and Ly6G6e forming one and Ly6G5c and Ly6G5b forming another. In addition, by FACS analysis we have found that the potential ligands for human LY6G6C, LY6G6D, and LY6G5C are expressed on K562 cells, an undifferentiated megakaryocyte cell line, indicating a potential role in hematopoietic cell differentiation. This characterization of the five MHC class III region Ly-6 family members is of great relevance, as they represent 18% of the human Ly-6 protein family and 50% of the secreted ones.     

Characterization and function of human Ly-6/uPAR molecules

Human Ly-6/uPAR molecules are a superfamily composed of two subfamilies; one is the membrane bound proteins with a GPI-anchor and the other are secreted proteins without the GPI-anchor. Ly-6/uPAR molecules have remarkable amino acid homology through a distinctive 8-10 cysteine-rich domain that is associated predominantly with O-linked glycans. These molecules are encoded by multiple tightly linked genes located on Chr. 8q23, and have a conserved genomic organization. Ly-6/uPAR molecules have an interesting expression pattern during hematopoiesis and on specific tumors indicating that Ly-6/uPAR molecules are associated with development of the immune system and carcinogenesis. Thus, Ly-6/uPAR molecules are useful antigens for diagnostic and therapeutic targets. This review summarizes our understanding of human Ly-6/uPAR molecules with regard to molecular structure as well as what is known about their function in normal and malignant tissues and suggest Ly-6/uPAR molecules as target antigens for cancer immunotherapy. [BMB Reports 2012; 45(11): 595-603]     

NMR structure and action on nicotinic acetylcholine receptors of water-soluble domain of human LYNX1

Discovery of proteins expressed in the central nervous system sharing the three-finger structure with snake α-neurotoxins provoked much interest to their role in brain functions. Prototoxin LYNX1, having homology both to Ly6 proteins and three-finger neurotoxins, is the first identified member of this family membrane-tethered by a GPI anchor, which considerably complicates in vitro studies. We report for the first time the NMR spatial structure for the water-soluble domain of human LYNX1 lacking a GPI anchor (ws-LYNX1) and its concentration-dependent activity on nicotinic acetylcholine receptors (nAChRs). At 5-30 μM, ws-LYNX1 competed with (125)I-α-bungarotoxin for binding to the acetylcholine-binding proteins (AChBPs) and to Torpedo nAChR. Exposure of Xenopus oocytes expressing α7 nAChRs to 1 μM ws-LYNX1 enhanced the response to acetylcholine, but no effect was detected on α4β2 and α3β2 nAChRs. Increasing ws-LYNX1 concentration to 10 μM caused a modest inhibition of these three nAChR subtypes. A common feature for ws-LYNX1 and LYNX1 is a decrease of nAChR sensitivity to high concentrations of acetylcholine. NMR and functional analysis both demonstrate that ws-LYNX1 is an appropriate model to shed light on the mechanism of LYNX1 action. Computer modeling, based on ws-LYNX1 NMR structure and AChBP x-ray structure, revealed a possible mode of ws-LYNX1 binding.     

Isolation and Characterization of a New Member of the HumanLy6Gene Family(LY6H)

TheLy6family of genes encodes glycosylphosphatidylinositol-anchored cell surface glycoproteins expressed on various types of cells. Intriguing patterns of expression ofLy6genes on specific subpopulations of lymphoid and myeloid cells suggest that Ly6 molecules may be involved in the development and homeostasis of hematopoietic cells. We have isolated a new member of the humanLy6gene family,LY6H,from a human fetal brain cDNA library. Fluorescencein situhybridization and radiation hybrid analyses assignedLY6Hto chromosome 8, where other members of theLy6gene family are also located. Northern analysis revealed thatLY6His highly expressed in particular subdivisions of human brain and also in MOLT-3 and -4 acute lymphoblastic leukemia cells. These data suggest that LY6H may play a role(s) in both the central nervous system and the immune system.     

Ly49A allelic variation and MHC class I specificity

The Ly49 family of natural killer (NK) cell receptors is encoded by a polygenic genetic locus. Allelic forms have been described and their expression appears to be regulated. The best-characterized Ly49 molecule, the C57BL/6 form of Ly49A, is an NK cell inhibitory receptor that binds H2Dd. To determine whether differences between Ly49a alleles may have functional consequences, allelic variants of Ly49a were cloned from several inbred mouse strains. Stable transfectants expressing each Ly49a allelic variant were generated and tested for reactivity with a panel of monoclonal antibodies (mAbs A1, JR9.318, YE1/32, and YE1/48) that recognize the C57BL/6 form of Ly49A. Binding to H2Dd was also assessed using fluorescently labeled H2Dd tetramers. Furthermore, cytotoxicity assays were performed using anti-Ly49A mAb-separated interleukin-2-activated NK cells. We show that despite binding to fluorescently labeled H2Dd tetramers, the Ly49A+ NK cells from representative mouse strains displayed significantly different degrees of inhibition with H2Dd targets. These results can be interpreted in the light of recent structural data on the Ly49A-H2Dd complex. Thus, the Ly49 family displays functionally significant allelic polymorphism which adds to the repertoire of NK cell receptors.     

Localization of acetylcholine-related molecules in the retina: implication of the communication from photoreceptor to retinal pigment epithelium

It has been long speculated that specific signals are transmitted from photoreceptors to the retinal pigment epithelium (RPE). However, such signals have not been identified. In this study, we examined the retinal expression and localization of acetylcholine-related molecules as putative candidates for these signals. Previous reports revealed that α7 nicotinic acetylcholine receptors (nAChRs) are present in the microvilli of RPE cells that envelope the tips of photoreceptor outer segments (OS). Secreted mammalian leukocyte antigen 6/urokinase-type plasminogen activator receptor-related protein-1 (SLURP-1) is a positive allosteric modulator of the α7 nAChR. Therefore, we first focused on the expression of SLURP-1. SLURP-1 mRNA was expressed in the outer nuclear layer, which is comprised of photoreceptor cell bodies. SLURP-1 immunoreactivity co-localized with rhodopsin and S-opsin in photoreceptor OS, while choline acetyltransferase (ChAT) and high affinity choline transporter (CHT-1) were also expressed in photoreceptor OS. Immunoelectron microscopy identified that the majority of SLURP-1 was localized to the plasma membranes of photoreceptor OS. These results provide evidence that SLURP-1 is synthesized in photoreceptor cell bodies and transported to photoreceptor OS, where SLURP-1 may also be secreted. Our findings suggest that photoreceptor OS communicate via neurotransmitters such as ACh and SLURP-1, while RPE cells might receive these signals through α7 nAChRs in their microvilli.     

Conserved structural determinants in three-fingered protein domains

The three-dimensional structures of some components of snake venoms forming so-called 'three-fingered protein' domains (TFPDs) are similar to those of the ectodomains of activin, bone morphogenetic protein and transforming growth factor-beta receptors, and to a variety of proteins encoded by the Ly6 and Plaur genes. The analysis of sequences of diverse snake toxins, various ectodomains of the receptors that bind activin and other cytokines, and numerous gene products encoded by the Ly6 and Plaur families of genes has revealed that they differ considerably from each other. The sequences of TFPDs may consist of up to six disulfide bonds, three of which have the same highly conserved topology. These three disulfide bridges and an asparagine residue in the C-terminal part of TFPDs are essential for the TFPD-like fold. Analyses of the three-dimensional structures of diverse TFPDs have revealed that the three highly conserved disulfides impose a major stabilizing contribution to the TFPD-like fold, in both TFPDs contained in some snake venoms and ectodomains of several cellular receptors, whereas the three remaining disulfide bonds impose specific geometrical constraints in the three fingers of some TFPDs.     

Assessing the role of the glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1) three-finger domain in binding lipoprotein lipase

Glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1) is an endothelial cell protein that transports lipoprotein lipase (LPL) from the subendothelial spaces to the capillary lumen. GPIHBP1 contains two main structural motifs, an amino-terminal acidic domain enriched in aspartates and glutamates and a lymphocyte antigen 6 (Ly6) motif containing 10 cysteines. All of the cysteines in the Ly6 domain are disulfide-bonded, causing the protein to assume a three-fingered structure. The acidic domain of GPIHBP1 is known to be important for LPL binding, but the involvement of the Ly6 domain in LPL binding requires further study. To assess the importance of the Ly6 domain, we created a series of GPIHBP1 mutants in which each residue of the Ly6 domain was changed to alanine. The mutant proteins were expressed in Chinese hamster ovary (CHO) cells, and their expression level on the cell surface and their ability to bind LPL were assessed with an immunofluorescence microscopy assay and a Western blot assay. We identified 12 amino acids within GPIHBP1, aside from the conserved cysteines, that are important for LPL binding; nine of those were clustered in finger 2 of the GPIHBP1 three-fingered motif. The defective GPIHBP1 proteins also lacked the ability to transport LPL from the basolateral to the apical surface of endothelial cells. Our studies demonstrate that the Ly6 domain of GPIHBP1 is important for the ability of GPIHBP1 to bind and transport LPL.     

Human Secreted Ly-6/uPAR Related Protein-1 (SLURP-1) Is a Selective Allosteric Antagonist of α7 Nicotinic Acetylcholine Receptor

SLURP-1 is a secreted toxin-like Ly-6/uPAR protein found in epithelium, sensory neurons and immune cells. Point mutations in the slurp-1 gene cause the autosomal inflammation skin disease Mal de Meleda. SLURP-1 is considered an autocrine/paracrine hormone that regulates growth and differentiation of keratinocytes and controls inflammation and malignant cell transformation. The majority of previous studies of SLURP-1 have been made using fusion constructs containing, in addition to the native protein, extra polypeptide sequences. Here we describe the activity and pharmacological profile of a recombinant analogue of human SLURP-1 (rSLURP-1) differing from the native protein only by one additional N-terminal Met residue. rSLURP-1 significantly inhibited proliferation (up to ~ 40%, EC₅₀ ~ 4 nM) of human oral keratinocytes (Het-1A cells). Application of mecamylamine and atropine,—non-selective inhibitors of nicotinic acetylcholine receptors (nAChRs) and muscarinic acetylcholine receptors, respectively, and anti-α7-nAChRs antibodies revealed α7 type nAChRs as an rSLURP-1 target in keratinocytes. Using affinity purification from human cortical extracts, we confirmed that rSLURP-1 binds selectively to the α7-nAChRs. Exposure of Xenopus oocytes expressing α7-nAChRs to rSLURP-1 caused a significant non-competitive inhibition of the response to acetylcholine (up to ~ 70%, IC₅₀ ~ 1 μM). It was shown that rSLURP-1 binds to α7-nAChRs overexpressed in GH₄C_l cells, but does not compete with ¹²⁵I-α-bungarotoxin for binding to the receptor. These findings imply an allosteric antagonist-like mode of SLURP-1 interaction with α7-nAChRs outside the classical ligand-binding site. Contrary to rSLURP-1, other inhibitors of α7-nAChRs (mecamylamine, α-bungarotoxin and Lynx1) did not suppress the proliferation of keratinocytes. Moreover, the co-application of α-bungarotoxin with rSLURP-1 did not influence antiproliferative activity of the latter. This supports the hypothesis that the antiproliferative activity of SLURP-1 is related to ‘metabotropic’ signaling pathway through α7-nAChR, that activates intracellular signaling cascades without opening the receptor channel.     

Crystal structure of the Ly49I natural killer cell receptor reveals variability in dimerization mode within the Ly49 family

Natural killer (NK) cells play a crucial role in the detection and destruction of virally infected and tumor cells during innate immune responses. The cytolytic activity of NK cells is regulated through a balance of inhibitory and stimulatory signals delivered by NK receptors that recognize classical major histocompatabilty complex class I (MHC-I) molecules, or MHC-I homologs such as MICA, on target cells. The Ly49 family of NK receptors (Ly49A through W), which includes both inhibitory and activating receptors, are homodimeric type II transmembrane glycoproteins, with each subunit composed of a C-type lectin-like domain tethered to the membrane by a stalk region. We have determined the crystal structure, at 3.0 A resolution, of the murine inhibitory NK receptor Ly49I. The Ly49I monomer adopts a fold similar to that of other C-type lectin-like NK receptors, including Ly49A, NKG2D and CD69. However, the Ly49I monomers associate in a manner distinct from that of these other NK receptors, forming a more open dimer. As a result, the putative MHC-binding surfaces of the Ly49I dimer are spatially more distant than the corresponding surfaces of Ly49A or NKG2D. These structural differences probably reflect the fundamentally different ways in which Ly49 and NKG2D receptors recognize their respective ligands: whereas the single MICA binding site of NKG2D is formed by the precise juxtaposition of two monomers, each Ly49 monomer contains an independent binding site for MHC-I. Hence, the structural constraints on dimerization geometry may be relatively relaxed within the Ly49 family. Such variability may enable certain Ly49 receptors, like Ly49I, to bind MHC-I molecules bivalently, thereby stabilizing receptor-ligand interactions and enhancing signal transmission to the NK cell.