Molecular cloning and expression of a new member of the nerve growth factor receptor family that is characteristic for Hodgkin's disease.

In man, Hodgkin's disease (HD) represents the most frequent lymphoma entity whose pathogenesis is still unknown. In order to contribute to the characterization of the molecular mechanisms of this disease, cDNAs coding for the HD characteristic antigen CD30 were cloned from expression libraries of the human HUT-102 cell line using the monoclonal antibodies Ki-1 and Ber-H2. The open reading frame of the cDNA that can be translated from two mRNA species of 2.6 kb, and 3.8 kb, respectively, predicts a 595 amino acid protein with leader, extracellular, single transmembrane, and intracellular domains. When expressed in COS-1 cells, the cDNA presented properties comparable to native CD30 antigen. The CD30 extracellular domain proved to be homologous to members of the nerve growth factor receptor superfamily. Six cysteine-rich motifs could be recognized within the putative ligand-binding domain.     

The T cell activation antigen CD27 is a member of the nerve growth factor/tumor necrosis factor receptor gene family.

CD27 is a dimeric membrane glycoprotein found on the surface of most human T lymphocytes. Activation of T cells by engagement of the Ag receptor increases CD27 surface expression, and anti-CD27 antibodies augment Ag receptor-mediated T cell proliferation. In this study a cDNA-encoding CD27 was isolated by expression and immunoselection in COS cells. The predicted polypeptide was found to belong to a recently characterized family of cysteine-rich receptors whose known ligands include nerve growth factor and TNF-alpha and -beta. Structural similarities suggest that CD27 belongs to a lymphocyte-specific subgroup of the family, comprised of the B cell Ag CD40, the rat T cell subset Ag OX40, and the mouse T cell activation Ag 4-1BB. Recent studies suggest some of these molecules may play a role in the survival of activated cells.     

Sequence analysis of the non-recurring C-terminal domains shows that insect lipoprotein receptors constitute a distinct group of LDL receptor family members

Lipoprotein-mediated delivery of lipids in mammals involves endocytic receptors of the low density lipoprotein (LDL) receptor (LDLR) family. In contrast, in insects, the lipoprotein, lipophorin (Lp), functions as a reusable lipid shuttle in lipid delivery, and these animals, therefore, were not supposed to use endocytic receptors. However, recent data indicate additional endocytic uptake of Lp, mediated by a Lp receptor (LpR) of the LDLR family. The two N-terminal domains of LDLR family members are involved in ligand binding and dissociation, respectively, and are composed of a mosaic of multiple repeats. The three C-terminal domains, viz., the optional O-linked glycosylation domain, the transmembrane domain, and the intracellular domain, are of a non-repetitive sequence. The present classification of newly discovered LDLR family members, including the LpRs, bears no relevance to physiological function. Therefore, as a novel approach, the C-terminal domains of LDLR family members across the entire animal kingdom were used to perform a sequence comparison analysis in combination with a phylogenetic tree analysis. The LpRs appeared to segregate into a specific group distinct from the groups encompassing the other family members, and each of the three C-terminal domains of the insect receptors is composed of unique set of sequence motifs. Based on conservation of sequence motifs and organization of these motifs in the domains, LpR resembles most the groups of the LDLRs, very low density lipoprotein (VLDL) receptors, and vitellogenin receptors. However, in sequence aspects in which LpR deviates from these three receptor groups, it most notably resembles LDLR-related protein-2, or megalin. These features might explain the functional differences disclosed between insect and mammalian lipoprotein receptors.     

The chicken leukocyte receptor complex: a highly diverse multigene family encoding at least six structurally distinct receptor types

The chicken Ig-like receptors (CHIR) have been described as two Ig domain molecules with long cytoplasmic tails containing inhibitory motifs. In this study, we demonstrate that CHIR form a large family, with multiple members showing great sequence variability among members as well as a great diversity in domain organization and properties of the transmembrane and cytoplasmic segments. We characterize various novel receptor types with motifs indicative of inhibitory, activating, or both functions. In addition to the inhibitory receptors with two ITIM, receptors with a single immunoreceptor tyrosine-based switch motif or receptors lacking a cytoplasmic domain were isolated. Activating receptors with a short cytoplasmic domain and a transmembrane arginine assembled with the newly identified chicken FcepsilonRIgamma chain. Three bifunctional receptor types were characterized composed of one or two C2-type Ig-like domains, a transmembrane region with a positively charged residue and combinations of cytoplasmic motifs such as ITIM, immunoreceptor tyrosine-based switch motif, and YXXM. RT-PCR revealed distinct expression patterns of individual CHIR. All receptor types shared a conserved genomic architecture, and in single Ig domain receptors a pseudoexon replaced the second Ig exon. Southern blot analyses with probes specific for the Ig1 domain were indicative of a large multigene family. Of 103 sequences from the Ig1 domain of a single animal, 41 unique sequences were obtained that displayed extensive variability within restricted Ig regions. Fluorescence in situ hybridization localized the CHIR gene cluster to microchromosome 31 and identified this region as orthologous to the human leukocyte receptor complex.     

Characterization of the MRC OX40 antigen of activated CD4 positive T lymphocytes--a molecule related to nerve growth factor receptor.

The antigen recognized by the monoclonal antibody (mAb) MRC OX40 is present on activated rat CD4 positive T lymphocytes but not other cells. cDNA clones were isolated from an expression library using the MRC OX40 mAb and the protein sequence for the OX40 antigen deduced. It contains a typical signal sequence and a single putative transmembrane sequence of 25 predominantly hydrophobic amino acids giving an extracellular domain of 191 amino acids and a cytoplasmic domain of 36 amino acids. The sequence of the extracellular domain includes a cysteine-rich region with sequence similarities with the low affinity nerve growth factor receptor (NGFR) of neurons and the CD40 antigen present on human B cells. Within this region three cysteine-rich motifs can be recognized in OX40 compared with four similar motifs in both NGFR and CD40. OX40, CD40 and NGFR constitute a new superfamily of molecules with expression including lymphoid cells (OX40, CD40) and neuronal cells (NGFR). This is reminiscent of the immunoglobulin superfamily whose molecules are variously found at the surface of lymphoid or brain cells or both.     

Structural model for the mannose receptor family uncovered by electron microscopy of Endo180 and the mannose receptor

The mannose receptor family comprises four members in mammals, Endo180 (CD280), DEC-205 (CD205), phospholipase A(2) receptor (PLA(2)R) and the mannose receptor (MR, CD206), whose extracellular portion contains a similar domain arrangement: an N-terminal cysteine-rich domain (CysR) followed by a single fibronectin type II domain (FNII) and 8-10 C-type lectin-like domains (CTLDs). These proteins mediate diverse functions ranging from extracellular matrix turnover through collagen uptake to homeostasis and immunity based on sugar recognition. Endo180 and the MR are multivalent transmembrane receptors capable of interacting with multiple ligands; in both receptors FNII recognizes collagens, and a single CTLD retains lectin activity (CTLD2 in Endo180 and CTLD4 in MR). It is expected that the overall conformation of these multivalent molecules would deeply influence their function as the availability of their binding sites could be altered under different conditions. However, conflicting reports have been published on the three-dimensional arrangement of these receptors. Here, we have used single particle electron microscopy to elucidate the three-dimensional organization of the MR and Endo180. Strikingly, we have found that both receptors display distinct three-dimensional structures, which are, however, conceptually very similar: a bent and compact conformation built upon interactions of the CysR domain and the lone functional CTLD. Biochemical and electron microscopy experiments indicate that, under a low pH mimicking the endosomal environment, both MR and Endo180 experience large conformational changes. We propose a structural model for the mannose receptor family where at least two conformations exist that may serve to regulate differences in ligand selectivity.     

TrkA amino acids controlling specificity for nerve growth factor

Neurotrophins are important for the development and maintenance of the vertebrate nervous system, mediating their signal into the cell by specific interaction with tyrosine kinase receptors of the Trk family. The extracellular portion of the Trk receptors has been previously proposed to consist of a cysteine-rich motif, a leucine-rich motif, a second cysteine-rich motif followed by two immunoglobulin-like domains. Earlier studies have shown that a major neurotrophin-binding site in the Trk receptors resides in the second immunoglobulin-like domain. Although the individual amino acids in TrkA involved in binding to nerve growth factor (NGF) and those in TrkC involved in binding to neurotrophin-3 have been mapped in this domain, the Trk amino acids that provide specificity remained unclear. In this study, a minimum set of residues in the human TrkC second immunoglobulin-like domain, which does not bind nerve growth factor (NGF), were substituted with those from human TrkA. The resulting Trk variant recruited binding of NGF equivalent to TrkA, maintained neurotrophin-3 binding equivalent to TrkC, and also bound brain-derived neurotrophin, although with lower affinity compared with TrkB. This implies that the amino acids in the second immunoglobulin-like domain that determine Trk specificity are distinct for each Trk.     

An extended conformation of the macrophage mannose receptor

The macrophage mannose receptor mediates phagocytosis of pathogenic microorganisms and endocytosis of potentially harmful soluble glycoproteins by recognition of their defining carbohydrate structures. The mannose receptor is the prototype for a family of receptors each having an extracellular region consisting of 8-10 domains related to C-type carbohydrate recognition domains (CRDs), a fibronectin type II repeat and an N-terminal cysteine-rich domain. Hydrodynamic analysis and proteolysis experiments performed on fragments of the extracellular region of the receptor have been used to investigate its conformation. Size and shape parameters derived from sedimentation and diffusion coefficients indicate that the receptor is a monomeric, elongated and asymmetric molecule. Proteolysis experiments indicate the presence of close contacts between several pairs of domains and exposed linker regions separating CRDs 3 and 6 from their neighboring domains. Hydrodynamic coefficients predicted for modeled receptor conformations are consistent with an extended conformation with close contacts between three pairs of CRDs. The N-terminal cysteine-rich domain and the fibronectin type II repeat appear to increase the rigidity of the molecule. The rigid, extended conformation of the receptor places domains with different functions at distinct positions with respect to the membrane.     

Molecular evolution of lysin motif-type receptor-like kinases in plants

The lysin motif (LysM) domain is an ancient and ubiquitous protein module that binds peptidoglycan and structurally related molecules. A genomic survey in a large number of species spanning all kingdoms reveals that the combination of LysM and receptor kinase domains is present exclusively in plants. However, the particular biological functions and molecular evolution of this gene family remain largely unknown. We show that LysM domains in plant LysM proteins are highly diversified and that a minimum of six distinct types of LysM motifs exist in plant LysM kinase proteins and five additional types of LysM motifs exist in nonkinase plant LysM proteins. Further, motif similarities suggest that plant LysM motifs are ancient. Although phylogenetic signals are not sufficient to resolve the earliest relationships, plant LysM motifs may have arisen through common ancestry with LysM motifs in other kingdoms. Within plants, the gene family has evolved through local and segmental duplications. The family has undergone further duplication and diversification in legumes, where some LysM kinase genes function as receptors for bacterial nodulation factor. Two pairs of homeologous regions were identified in soybean (Glycine max) based on microsynteny and fluorescence in situ hybridization. Expression data show that most plant LysM kinase genes are expressed predominantly in the root and that orthologous LysM kinase genes share similar tissue expression patterns. We also examined synteny around plant LysM kinase genes to help reconstruct scenarios for the evolution of this important gene family.     

Structure of the Hodgkin's lymphoma-associated human CD30 gene and the influence of a microsatellite region on its expression in CD30(+) cell lines.

The CD30 antigen is a member of the tumor necrosis factor receptor (TNFR) family which is overexpressed on the surface of the tumor cells of Hodgkin's lymphoma, anaplastic large cell lymphoma (ALCL), and embryonal carcinoma of the testis. In this study the entire cd30 gene which is more than 24000 bp long and organized in eight exons was characterized by analyzing cosmid and phage lambda clones from human placental libraries with long-range polymerase chain reaction (PCR) and sequencing. Differences to other genes of the TNFR family were detected in the region encoding the extracellular domain of the cd30 gene. In nearly all other TNFR genes, the coding region of each cysteine-rich repeat is interrupted by one intron, i.e., the 3-4 cysteine-rich repeats of these receptors are encoded by at least 4-5 exons, whereas the six cysteine-rich repeats of the cd30 gene are encoded by two exons, i.e., each of these exons encode three cysteine-rich repeats. In addition, we also found a genetic polymorphism of tetranucleotide ATCC-repeats in the 5' part of the CD30 promoter. This region was amplified by PCR from seven CD30 overexpressing human lymphoid cell lines and five human tissues with an absent or very low CD30 expression. The amplification products showed length differences of more than 550 bp. The number of the ATCC-repeats was higher in CD30(+) cell lines than in normal tissues. Comparison of the individual PCR products in reporter gene assays revealed that the CD30 promoter activity increased with the length of this polymorphic region up to eightfold. The data suggest that the number of ATCC-repeats in the 5' region of the CD30 promoter modulates the regulation of CD30 expression.     

Characterization of sugar binding by the mannose receptor family member,Endo180

Members of the mannose receptor family, the mannose receptor, the phospholipase A(2) receptor, DEC-205, and Endo180, contain multiple C-type lectin-like domains (CTLDs) within a single polypeptide. In addition, at their N termini, all four family members contain a cysteine-rich domain similar to the R-type carbohydrate recognition domains of ricin. However, despite the common presence of multiple lectin-like domains, these four endocytic receptors have divergent ligand binding activities, and it is clear that the majority of these domains do not bind sugars. Here the functions of the lectin-like domains of the most recently discovered family member, Endo180, have been investigated. Endo180 is shown to bind in a Ca(2+)-dependent manner to mannose, fucose, and N-acetylglucosamine but not to galactose. This activity is mediated by one of the eight CTLDs, CTLD2. Competition assays indicate that the monosaccharide binding specificity of Endo180 CTLD2 is similar to that of mannose receptor CTLD4. However, additional experiments indicate that, unlike the cysteine-rich domain of the mannose receptor, the cysteine-rich domain of Endo180 does not bind sulfated sugars. Thus, although Endo180 and the mannose receptor are now both known to be mannose binding lectins, each receptor is likely to have a distinct set of glycoprotein ligands in vivo.     

真核生物FANCJ-like蛋白的结构与进化

FANCJ-like蛋白是一类ATP依赖的5′-3′DNA解旋酶,参与DNA损伤修复、同源重组及G4-DNA拆解,在基因组稳定性维持过程中发挥重要功能。文章系统分析了47种真核生物的FANCJ-like蛋白,对其序列结构特征及起源进化进行了深入探讨。真核生物FANCJ-like蛋白包含4类成员——XPD、CHL1、RTEL1和FANCJ,但在真菌的一些世系及昆虫中存在严重缺失现象,如接合菌门(Zygomycota)缺失了RTEL1,担子菌门(Basidiomycota)和子囊菌门(Ascomycota)缺失了RTEL1和FANCJ,双翅目昆虫缺失了FANCJ。FANCJ-like蛋白不仅包含经典解旋酶共有HD1和HD2结构域,而且在HD1结构域中插入了自身特有的Fe-S、Arch和Extra-D结构域。Fe-S和Arch结构域在4类成员中较保守,Extra-D结构域在XPD中不存在,在其他3类成员中也各不相同。在FANCJ-like蛋白的Fe-S、Arch和Extra-D结构域中分别发现了7个、10个和2个特有模体;除了已报道的保守模体外,HD1和HD2中分别发现了5个和12个特有模体。从这些特有模体的组成和排布来看,RTEL1和FANCJ最为相近,它们在HD2区包含两个独有模体Vb2和Vc,可能与其G4-DNA解旋活性相关。进化方面的证据表明,FANCJ-like蛋白起源于一种HD1区插入了Fe-S和Arch结构域的DNA解旋酶,在多细胞真核生物出现之前,该蛋白通过3次复制事件和随后的特异化过程,依次形成了目前真核生物所包含的4类FANCJ-like蛋白。     

富含半胱氨酸的分泌蛋白（CRISPs）在哺乳动物授精过程中的作用

雌雄配子间的结合与融合是哺乳动物授精成功的关键步骤,哺乳动物富含半胱氨酸的分泌蛋白CRISPs家族是一个进化上高度保守的蛋白家族,参与了精卵结合与融合过程,并在其中扮演了多种角色。目前从雄性小鼠生殖道中分离出4个CRISPs家族成员：附睾的CRISP1、睾丸的CRISP2、分布广泛的CRISP3以及与人CRISP1同源的CRISP4,对CRISPs家族蛋白成员的晶体结构分析揭示出CRISP蛋白含有两个功能域,一个是位于N末端的结构保守的CAP结构域,另一个是位于C末端的CRISP蛋白家族特有的CRISP功能域。CAP功能域中含有CAP基序,CRISP功能域由一个短的铰链区和一个离子通道调节区组成,并通过铰链区与CAP结构域相连接。简要回顾了各种CRISP蛋白的发现和特性鉴别过程,希望能从CRISPs的角度对哺乳动物精卵识别、结合与融合的分子机制有更好的了解。     

The mannose receptor family

The mannose receptor family comprises four glycoproteins each of which is a type I transmembrane receptor with an N-terminal cysteine-rich domain, a single fibronectin type II (FNII) domain and eight to ten C-type lectin-like domains (CTLDs). Characteristically, these proteins are able to recycle between the plasma membrane and the endosomal apparatus due to discrete motifs present within their cytoplasmic domains. This review discusses the structure and function of these four proteins-the mannose receptor (MR), the M-type receptor for secretory phospholipases A(2) (PLA(2)R), DEC-205/gp200-MR6 and Endo180/uPARAP. Despite their overall structural similarity, these four receptors have evolved to use different domains to interact with discrete ligands. In addition, they differ in their ability to mediate endocytic and phagocytic events and in their intracellular destinations. Together, they represent a unique group of multidomain, multifunctional receptors.     

Human epidermal growth factor (EGF) module-containing mucin-like hormone receptor 3 is a new member of the EGF-TM7 family that recognizes a ligand on human macrophages and activated neutrophils

The epidermal growth factor (EGF)-TM7 subgroup of G-protein-coupled receptors is composed predominantly of leukocyte-restricted glycoproteins defined by their unique hybrid structure, in which extracellular EGF-like domains are coupled to a seven-span transmembrane moiety via a mucin-like stalk. The EGF-TM7 group comprises mouse F4/80, human EGF module-containing mucin-like hormone receptor (EMR) 1, human EMR2, and human and mouse CD97, the genes for which map to human chromosome 19p13 and the syntenic regions of the mouse genome. In this study we describe the cloning and characterization of EMR3, a novel human EGF-TM7 molecule, and show the existence of its cellular ligand. The EMR3 gene maps closely to the existing members of the EGF-TM7 family on human chromosome 19p13.1 and, in common with other EGF-TM7 genes, is capable of generating different protein isoforms through alternative splicing. Two alternative splice forms have been isolated: one encoding a 652-amino acid cell surface protein consisting of two EGF-like domains, a mucin stalk, and a putative G-protein-coupled receptor domain and the other encoding a truncated soluble form containing only two EGF-like domains. As with other members of the EGF-TM7 family, EMR3 mRNA displays a predominantly leukocyte-restricted expression pattern, with highest levels in neutrophils, monocytes, and macrophages. Through the use of soluble EMR3 multivalent probes we have shown the presence of a ligand at the surface of monocyte-derived macrophages and activated human neutrophils. These interactions suggest a potential role for EMR3 in myeloid-myeloid interactions during immune and inflammatory responses.