银鲫精巢特异的Ly-6/uPAR相关蛋白的分子克隆及其特征分析

Ly-6/uPAR基因超家族(Ly-6 SF)成员广泛地存在于后生动物中, 开展该家族相关功能基因研究具有重要的意义。研究从银鲫(Carassius auratus gibelio)中鉴定到一个该家族新成员, cDNA全长为570 bp, 其中开放阅读框长度为300 bp, 编码99个氨基酸, 生物软件预测该蛋白含有一个LU结构域, 不含GPI锚信号序列, N端含有信号肽, 表明其可能为Ly-6基因超家族中分泌型蛋白。组织表达分析显示, 该基因只在银鲫精巢中特异表达, 且又是Ly-6基因超家族中一员, 因此将其命名为银鲫精巢特异的Ly-6/uPAR相关蛋白(Carassius auratus gibelio testis-specific Ly-6/uPAR related protein, 简称CagTslurp)。原位杂交结果显示, 该基因在银鲫精巢的精原细胞, 初级精母细胞以及次级精母细胞中表达, 精子细胞中存在少量的表达, 而在体细胞中不表达。这种精巢特异的表达模式, 暗示CagTslurp在银鲫精子发生中可能发挥了作用。       

Cloning and expression of SOLD1 in ovine and caprine placenta,and their expected roles during the development of placentomes

Background  

The Ly-6 (Ly-6/uPAR) superfamily members share the Ly-6 domain defined by distinct disulfide bonding patterns between 8 or 10 cysteine residues. They comprise membrane- and secretory-type proteins. We recently reported the gene and protein characterization of the bovine secreted protein of Ly-6 domain 1 (SOLD1). Bovine SOLD1 is expressed in trophoblast mononucleate cells (TMCs) and is localized in the cotyledonary mesenchyme. Here, we compared the expression and functionality of SOLD1 among the ruminants. We examined mRNA expression by chorionic fibroblasts as a measure of one of the SOLD1 functions.     

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 nico- tinic 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.     

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.     

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.     

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]     

A comparison of the genes coding for canonical TRP channels and their M,V and P relatives

The mammalian transient receptor potential (TRP) protein gene family consists of a diverse group of cation channels that currently contain at least 26 members. The physiologic functions of many remain unknown. They are structurally similar to Drosophila TRP and have a wide tissue distribution. In the present report, we compare the chromosomal locations, the gene, and primary structures of each of these 26 human TRP family members. Based on primary amino acid analyses, these channels comprise four different subfamilies: C- (canonical or classical), V- (or vanilloid receptor related), M- (melastatin related), and P (PKD)-type. The highest homology within each subfamily and between subfamilies exists in the predicted ion channel domains. Belonging to a given subfamily, however, does not determine the activating stimuli. This is exemplified by the V- and M-subfamilies, both of which have members that respond to temperature and osmolarity. TRP genes vary in their intron-exon organization, with the greatest diversity in the P subfamily. Chromosomal organization analyses revealed that two TRP members are found as direct repeats; TRPV3 follows TRPV1 and TRPV6 follows TRPV5. Both of these duplications appear to be recent as TRPV1 and V3 are more similar to each other than to other members of the TRPV subfamily. The same holds true for TRPV5 and V6. The article presents complication of comparisons including exon-intron boundaries, the amino acid sequence alignments, and the chromosomal organization of each of the presently known TRP channels.     

Trace amine-associated receptors form structurally and functionally distinct subfamilies of novel G protein-coupled receptors

Trace amines are endogenous compounds structurally related to classical biogenic amines that have been studied for decades, triggered by their link to psychiatric conditions of high epidemiological and economical relevance. The understanding of their pharmacology on the molecular level was hampered until the recent discovery of trace-amine-specific receptors. We completed the identification of all members of this novel GPCR family in human, chimpanzee, rat, and mouse and observed remarkable interspecies differences, even between human and chimpanzee. The analysis of the chromosomal localizations, phylogenetic relationships, and ligand pocket vectors reveals three distinct receptor subfamilies. As most of these receptors do not respond to trace amines, each subfamily will presumably have a distinct pharmacological profile, which remains to be identified. We propose a uniform nomenclature describing this novel GPCR family in all mammalian species as trace-amine-associated receptors (TAARs), which resolves the ambiguities and contradictions of the previous naming.     

PATE gene clusters code for multiple, secreted TFP/Ly-6/uPAR proteins that are expressed in reproductive and neuron-rich tissues and possess neuromodulatory activity

We report here syntenic loci in humans and mice incorporating gene clusters coding for secreted proteins each comprising 10 cysteine residues. These conform to three-fingered protein/Ly-6/urokinase-type plasminogen activator receptor (uPAR) domains that shape three-fingered proteins (TFPs). The founding gene is PATE, expressed primarily in prostate and less in testis. We have identified additional human PATE-like genes (PATE-M, PATE-DJ, and PATE-B) that co-localize with the PATE locus, code for novel secreted PATE-like proteins, and show selective expression in prostate and/or testis. Anti-PATE-B-specific antibodies demonstrated the presence of PATE-B in the region of the sperm acrosome and at high levels on malignant prostatic epithelial cells. The syntenic mouse Pate-like locus encompasses 14 active genes coding for secreted proteins, which are all, except for Pate-P and Pate-Q, expressed primarily in prostate and/or testis. Pate-P and Pate-Q are expressed solely in placental tissue. Castration up-regulates prostate expression of mouse Pate-B and Pate-E, whereas testosterone ablates this induced expression. The sequence similarity between TFP/Ly-6/uPAR proteins that modulate activity of nicotinic acetylcholine receptors and the PATE (Pate)-like proteins stimulated us to see whether these proteins possess analogous activity. Pharmacological studies showed significant modulation of the nicotinic acetylcholines by the PATE-B, Pate-C, and Pate-P proteins. In concert with these findings, certain PATE (Pate)-like genes were extensively expressed in neuron-rich tissues. Taken together, our findings indicate that in addition to participation of the PATE (Pate)-like genes in functions related to fertility and reproduction, some of them likely act as important modulators of neural transmission.     

Random expression of main and vomeronasal olfactory receptor genes in immature and mature olfactory epithelia of Fugu rubripes

Main olfactory receptor genes were isolated from a seawater fish, Fugu rubripes (pufferfish), and characterized. Two subfamilies of genes encoding seven transmembrane receptors were identified; one consists of five or more members, termed FOR1-1 to 5 of FOR1 subfamily, and the other appears to be a single copy gene, termed the FOR2 subfamily. FOR1 members show extremely high amino acid sequence similarities of about 95% to one another, and are distantly related to catfish-1 with the highest similarity of 37%. FOR2 shows 43% similarity to goldfish-A28. Phylogenically, both FOR members are categorized among pedigrees of the fish main olfactory receptor family outside the mammalian receptor family, although similarities between Fugu receptors and those of fresh-water fishes are lower than those among fresh-water fishes. In situ hybridization shows that both subfamilies of receptor genes are expressed randomly over the olfactory epithelium throughout all developmental stages, and no segregation of the signals was found. On the other hand, when three members of a vomeronasal olfactory receptor gene family, related to the Ca(2+)-sensing receptor, were used as probes, they were also randomly expressed over the same epithelium as the main olfactory receptors. This is in contrast to the expression profiles observed for zebrafish and goldfish, where the main or vomeronasal olfactory receptors are expressed in segregated patterns. It is thus suggested that the expression pattern of fish olfactory receptors varies depending on the species, although fish olfactory receptors are highly related to one another in their primary structures, and are phylogenically distinct from those of mammals.     

Evolution of the βGRP/GNBP/β-1,3-glucanase family of insects

The βGRP/GNBP/β-1,3-glucanase protein family of insects includes several proteins involved in innate immune recognition, such as the β-glucan recognition proteins of Lepidoptera and the Gram-negative bacteria-binding proteins of Drosophila. A phylogenetic analysis supported the existence of two distinct subfamilies, designated the pattern recognition receptor (PRR) and glucanase subfamilies, which originated by gene duplication prior to the origin of the Holometabola. In the C-terminal region (CTR) shared by both subfamilies, the PRR subfamily has evolved significantly more rapidly at the amino acid sequence level than has the glucanase subfamily, implying a relative lack of constraint on the amino acid sequence of this region in the PRR subfamily. PRR subfamily members also include an N-terminal region (NTR), involved in carbohydrate recognition, which is not shared by glucanase subfamily members. In comparisons between paralogous PRR subfamily members, there were no conserved amino acid residues in the NTR. However, when pairs of putatively orthologous PRR subfamily members were compared, the NTR was most often as conserved as the CTR or more so. This pattern suggests that the NTR may be important in functions specific to the different paralogs, while amino acid sequence changes in the NTR may have been important in functional differentiation among paralogs, specifically with regard to the types of carbohydrates that they recognize.     

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.     

Analysis of hydrophobicity in the alpha and beta chemokine families and its relevance to dimerization.

The chemokine family of chemotactic cytokines plays a key role in orchestrating the immune response. The family has been divided into 2 subfamilies, alpha and beta, based on the spacing of the first 2 cysteine residues, function, and chromosomal location. Members within each subfamily have 25-70% sequence identity, whereas the amino acid identity between members of the 2 subfamilies ranges from 20 to 40%. A quantitative analysis of the hydrophobic properties of 11 alpha and 9 beta chemokine sequences, based on the coordinates of the prototypic alpha and beta chemokines, interleukin-8 (IL-8), and human macrophage inflammatory protein-1 beta (hMIP-1 beta), respectively, is presented. The monomers of the alpha and beta chemokines have their strongest core hydrophobic cluster at equivalent positions, consistent with their similar tertiary structures. In contrast, the pattern of monomer surface hydrophobicity between the alpha and beta chemokines differs in a manner that is fully consistent with the observed differences in quaternary structure. The most hydrophobic surface clusters on the monomer subunits are located in very different regions of the alpha and beta chemokines and comprise in each case the amino acids that are buried at the interface of their respective dimers. The theoretical analysis of hydrophobicity strongly supports the hypothesis that the distinct dimers observed for IL-8 and hMIP-1 beta are preserved for all the alpha and beta chemokines, respectively. This provides a rational explanation for the lack of receptor crossbinding and reactivity between the alpha and beta chemokine subfamilies.     

The GRK4 subfamily of G protein-coupled receptor kinases. Alternative splicing, gene organization, and sequence conservation.

G protein-coupled receptor kinases (GRKs) desensitize G protein-coupled receptors by phosphorylating activated receptors. The six known GRKs have been classified into three subfamilies based on sequence and functional similarities. Examination of the mouse GRK4 subfamily (GRKs 4, 5, and 6) suggests that mouse GRK4 is not alternatively spliced in a manner analogous to human or rat GRK4, whereas GRK6 undergoes extensive alternative splicing to generate three variants with distinct carboxyl termini. Characterization of the mouse GRK 5 and 6 genes reveals that all members of the GRK4 subfamily share an identical gene structure, in which 15 introns interrupt the coding sequence at equivalent positions in all three genes. Surprisingly, none of the three GRK subgroups (GRK1, GRK2/3, and GRK4/5/6) shares even a single intron in common, indicating that these three subfamilies are distinct gene lineages that have been maintained since their divergence over 1 billion years ago. Comparison of the amino acid sequences of GRKs from various mammalian species indicates that GRK2, GRK5, and GRK6 exhibit a remarkably high degree of sequence conservation, whereas GRK1 and particularly GRK4 have accumulated amino acid changes at extremely rapid rates over the past 100 million years. The divergence of individual GRKs at vastly different rates reveals that strikingly different evolutionary pressures apply to the function of the individual GRKs.     

Autophosphorylation activity of the Arabidopsis ethylene receptor multigene family

Receptors for the gaseous phytohormone ethylene show sequence similarity to bacterial two-component histidine kinases. These receptors are encoded by a multigene family that can be divided into subfamilies 1 and 2. It has been previously shown that a subfamily 1 Arabidopsis thaliana ethylene receptor, ETR1, autophosphorylates in vitro on a conserved histidine residue (1). However, sequence comparisons between the five ethylene receptor family members suggest that subfamily 2 members do not have all the motifs necessary for histidine kinase activity. Further, a tobacco subfamily 2 receptor, NTHK1, autophosphorylates on serines and threonines in vitro (2). Here we show that all five Arabidopsis ethylene receptor proteins autophosphorylate in vitro. We analyzed the nature of the phosphorylated amino acids by acid/base stability and bi-dimensional thin layer electrophoresis and demonstrated that unlike ETR1 all other ethylene receptors autophosphorylate predominantly on serine residues. ERS1, the only other subfamily 1 receptor, is able to phosphorylate on both histidine and serine residues in the presence of Mn2+. However, histidine autophosphorylation is lost when ERS1 is assayed in the presence of both Mg2+ and Mn2+, suggesting that this activity may not occur in vivo. Furthermore, mutation of the histidine residue conserved in two-component systems does not abolish serine autophosphorylation, eliminating the possibility of a histidine to serine phosphotransfer. Our biochemical observations complement the recently published genetic data that histidine kinase activity is not necessary for ethylene receptor function in plants and suggest that ethylene signal transduction does not occur through a phosphorelay mechanism.     

Functional analysis of a mammalian odorant receptor subfamily

Phylogenetic analysis groups mammalian odorant receptors into two broad classes and numerous subfamilies. These subfamilies are proposed to reflect functional organization. Testing this idea requires an assay allowing detailed functional characterization of odorant receptors. Here we show that a variety of Class I and Class II mouse odorant receptors can be functionally expressed in Xenopus laevis oocytes. Receptor constructs included the N-terminal 20 residues of human rhodopsin and were co-expressed with Galphaolf and the cystic fibrosis transmembrane regulator to allow electrophysiological measurement of receptor responses. For most mouse odorant receptors tested, these conditions were sufficient for functional expression. Co-expression of accessory proteins was required to allow functional surface expression of some mouse odorant receptors. We used this assay to examine the receptive ranges of all members of the mouse odorant receptor 42 (MOR42) subfamily. MOR42-1 responded to dicarboxylic acids, preferring a 10-12 carbon chain length. MOR42-2 responded to monocarboxylic acids (7-10 carbons). MOR42-3 responded to dicarboxylic acids (8-10 carbons) and monocarboxylic acids (10-12 carbons). Thus, the receptive range of each receptor was unique. However, overlap between the individual receptive ranges suggests that the members of this subfamily form one contiguous subfamily receptive range, suggesting that odorant receptor subfamilies do constitute functional units.     

SAMP14, a novel,acrosomal membrane-associated,glycosylphosphatidylinositol-anchored member of the Ly-6/urokinase-type plasminogen activator receptor superfamily with a role in sperm-egg interaction

We report a new member of the Ly-6/urokinase-type plasminogen activator receptor (uPAR) superfamily of receptors, SAMP14, which is retained on the inner acrosomal membrane of the human spermatozoan following the acrosome reaction and may play a role in fertilization. The SAMP14 sequence predicted a glycosylphosphatidylinositol (GPI)-anchored protein with a signal peptide, a transmembrane domain near the carboxyl terminus, and a putative transamidase cleavage site in the proprotein. Attachment of SAMP14 to the membrane by a lipid anchor was confirmed by its sensitivity to phosphatidylinositol phospholipase C. SAMP14 has a single functional domain similar to the Ly-6 and urokinase plasminogen activator receptor superfamily of proteins, and the gene mapped to 19q13.33, near the PLAUR locus for uPAR at 19q13.2. Northern and dot blotting showed that SAMP14 expression was testis-specific. Indirect immunofluorescence and immunoelectron microscopy with antisera to purified recombinant SAMP14 localized the protein to outer and inner acrosomal membranes as well as the acrosomal matrix of ejaculated human sperm. Acrosome-reacted sperm demonstrated SAMP14 immunofluorescence, indicating its retention on the inner acrosomal membrane following the acrosome reaction. However, SAMP14 localized to the entire sperm when unwashed swim-up sperm from the ejaculate were stained, indicating that some SAMP14 is loosely associated with the plasma membrane. Antibodies against recombinant SAMP14 inhibited both the binding and the fusion of human sperm to zona free hamster eggs, suggesting that SAMP14 may have a role in sperm-egg interaction. SAMP14 represents a GPI-anchored putative receptor in the Ly-6/uPAR family that is exposed on the inner acrosomal membrane after the acrosome reaction.