Mice mutant for Ppap2c, a homolog of the germ cell migration regulator wunen, are viable and fertile

Phosphatidic acid phosphatases (PAPs) catalyze the conversion of phosphatidic acid to diacylglycerol and inorganic phosphate and have been postulated to function both in lipid biosynthesis and in cellular signal transduction. In Drosophila melanogaster, the Type 2 phosphatidic acid phosphatase protein encoded by the wunen gene, negatively regulates primordial germ cell migration. We recently described the cloning and characterization of the mouse Ppap2c gene, which encodes the Type 2 phosphatidic acid phosphatase Pap2c (Zhang et al., Genomics 63:142-144). To analyze the in vivo role of the Ppap2c gene we constructed a null mutation by gene targeting. Ppap2c(-/-) homozygous mutant mice were viable, fertile, and exhibited no obvious phenotypic defects. These data demonstrate that the Ppap2c gene is not essential for embryonic development or fertility in mice.     

Lipid phosphate phosphatase (LPP3) and vascular development

Lipid phosphate phosphatases (LPP) are integral membrane proteins with broad substrate specificity that dephosphorylate lipid substrates including phosphatidic acid, lysophosphatidic acid, ceramide 1-phosphate, sphingosine 1-phosphate, and diacylglycerol pyrophosphate. Although the three mammalian enzymes (LPP1-3) demonstrate overlapping catalytic activities and substrate preferences in vitro, the phenotypes of mice with targeted inactivation of the Ppap2 genes encoding the LPP enzymes reveal nonredundant functions. A specific role for LPP3 in vascular development has emerged from studies of mice lacking Ppap2b. A meta-analysis of multiple, large genome-wide association studies identified a single nucleotide polymorphism in PPAP2B as a novel predictor of coronary artery disease. In this review, we will discuss the evidence that links LPP3 to vascular development and disease and evaluate potential molecular mechanisms. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.     

Immunogenetic polymorphism of lipoproteins in swine. 1. Four additional serum beta-lipoprotein allotypes (Lpp2, Lpp4, Lpp5 and Lpp15) in the Lpp system.

Four additional swine serum lipoprotein allotypes are described. Specific anti-allotype reagents were obtained from alloimmune precipitating sera produced in lipoprotein-defined-type recipients immunized with normal sera and subsequently with lipoprotein fractions. Identification studies indicate that the four serologically defined low-density lipoprotein (LDL) variants, designated Lpp2, Lpp4, Lpp5 and Lpp15, are members of a previously described Lpp system. The individual specificities, Lpp2, Lpp4 and Lpp5, are determined by three co-dominant autosomal genes, Lpp2, Lpp4 and Lpp5, respectively, whereas the common specificity, Lpp15, is controlled by a complex of genetic information of the Lpp2 and Lpp4 genes, and by the two previously described alleles, Lpp1 and Lpp3; Lpp15 occurs on the same molecule with respective individual specificity. The Lpp5 and Lpp15 antigens behave as a pair of alternative allotypic specificities. The double immunodiffusion test in agar was employed to demonstrate independent phenotypic expression of each allelic gene in the Lpp heterozygous animals, for the analysis of the immune sera, and for lipoprotein testing of 3305 sera. Marked differences in gene frequencies were found between the swine breeds tested. As a result of characteristic frequencies, only nine of 15 possible Lpp genotypes were found in the breeding herds tested; the remaining six genotypes were obtained from testcross matings.     

A novel outer membrane lipoprotein, LolB (HemM), involved in the LolA (p20)-dependent localization of lipoproteins to the outer membrane of Escherichia coli.

The Escherichia coli major outer membrane lipoprotein (Lpp) is released from the inner membrane into the periplasm as a complex with a carrier protein, LolA (p20), and is then specifically incorporated into the outer membrane. An outer membrane protein playing a critical role in Lpp incorporation was identified, and partial amino acid sequences of the protein, named LolB, were identical to those of HemM, which has been suggested to play a role in 5-aminolevulinic acid synthesis in the cytosol. In contrast to this suggested role, the deduced amino acid sequence of HemM implied that the gene encodes a novel outer membrane lipoprotein. Indeed, an antibody raised against highly purified LolB revealed its outer membrane localization, and inhibited in vitro Lpp incorporation into the outer membrane. Furthermore, LolB was found to be synthesized as a precursor with a signal sequence and then processed to a lipid-modified mature form. An E.coli strain possessing chromosomal hemM under the control of the lac promoter-operator required IPTG for growth, indicating that hemM (lolB) is an essential gene. Outer membrane prepared from LolB-depleted cells did not incorporate Lpp. When the Lpp-LolA complex was incubated with a water-soluble LolB derivative, Lpp was transferred from LolA to LolB. Based on these results, the outer membrane localization pathway for E.coli lipoprotein is discussed with respect to the functions of LolA and LolB.     

Lethality of the covalent linkage between mislocalized major outer membrane lipoprotein and the peptidoglycan of Escherichia coli.

The major outer membrane lipoprotein (Lpp) of Escherichia coli possesses serine at position 2, which is thought to function as the outer membrane sorting signal, and lysine at the C terminus, through which Lpp covalently associates with peptidoglycan. Arginine (R) is present before the C-terminal lysine in the wild-type Lpp (LppSK). By replacing serine (S) at position 2 with aspartate (D), the putative inner membrane sorting signal, and by deleting lysine (K) at the C terminus, Lpp mutants with a different residue at either position 2 (LppDK) or the C terminus (LppSR) or both (LppDR) were constructed. Expression of LppSR and LppDR little affected the growth of E. coli. In contrast, the number of viable cells immediately decreased when LppDK was expressed. Prolonged expression of LppDK inhibited separation of the inner and outer membranes by sucrose density gradient centrifugation, whereas short-term expression did not. Pulse-labeled LppDK and LppDR were localized in the inner membrane, indicating that the amino acid residue at position 2 functions as a sorting signal for the membrane localization of Lpp. LppDK accumulated in the inner membrane covalently associated with the peptidoglycan and thus prevented the separation of the two membranes. Globomycin, an inhibitor of lipoprotein-specific signal peptidase II, was lethal for E. coli only when Lpp possessed the C-terminal lysine. Taken together, these results indicate that the inner membrane accumulation of Lpp per se is not lethal for E. coli. Instead, a covalent linkage between the inner membrane Lpp having the C-terminal lysine and the peptidoglycan is lethal for E. coli, presumably due to the disruption of the cell surface integrity.     

Immunogenetic polymorphism of lipoproteins in swine

Five new antigenic markers (allotypes) of swine serum lipoproteins are described. Specific antiallotype reagents were obtained from alloimmune precipitating sera. Identification studies and genetic analysis indicate that the five serum alloantigens—designated Lpp6, Lpp11, Lpp12, Lpp13, and Lpp14—are markers of low-density lipoproteins (LDL),d 1.002–1.075 g/ml, and are members of a previously described Lpp system. The Lpp6 allotype belongs to the group of individual markers and is determined by a new codominant allelic gene,Lpp ⁶, whereas the remaining four antigens—Lpp11, Lpp12, Lpp13, and Lpp 14—named common specificities, behave as alternative variants to Lpp1, Lpp2, Lpp3, and Lpp4, respectively, forming pairs of mutually exclusive alloantigens. Each Lpp gene in a heterozygous animal expresses itself independently on separate molecules and each haplotype carries one individual and at least four common specificities. The relationship between common and individual specificities, together with their number in the complex haplotypes, seems to shed some light on evolution of Lpp genes. It is proposed in this concept that the original gene for low-density lipoproteins in swine, and also in rhesus monkeys and the human, consisted of genetic information for common specificities only, the individual specificities evolving later as a result of point mutations.     

Immunogenetic polymorphism of lipoproteins in swine. 1. Four additional serum β-lipoprotein allotypes (Lpp2, Lpp4, Lpp5 and Lpp15) in the Lpp system1

Four additional swine serum lipoprotein allotypes are described. Specific anti-allotype reagents were obtained from alloimmune precipitating sera produced in lipoprotein-defined-type recipients immunized with normal sera and subsequently with lipoprotein fractions. Identification studies indicate that the four serologically defined low-density lipoprotein (LDL) variants, designated Lpp2, Lpp4, Lpp5 and Lpp15, are members of a previously described Lpp system. The individual specificities, Lpp2, Lpp4 and Lpp5, are determined by three co-dominant autosomal genes, Lpp², Lpp⁴ and Lpp⁵, respectively, whereas the common specificity, Lpp 15, is controlled by a complex of genetic information of the Lpp- and Lpp* genes, and by the two previously described alleles, Lpp¹ and Lpp³; Lpp15 occurs on the same molecule with respective individual specificity. The Lpp5 and Lpp15 antigens behave as a pair of alternative allotypic specificities. The double immunodiffusion test in agar was employed to demonstrate independent phenotypic expression of each allelic gene in the Lpp heterozygous animals, for the analysis of the immune sera, and for lipoprotein testing of 3305 sera. Marked differences in gene frequencies were found between the swine breeds tested. As a result of characteristic frequencies, only nine of 15 possible Lpp genotypes were found in the breeding herds tested; the remaining six genotypes were obtained from testcross matings.     

The free and bound forms of Lpp occupy distinct subcellular locations in Escherichia coli

The lipoprotein Lpp is the most numerically abundant protein in Escherichia coli, has been investigated for over 40 years, and has served as the paradigmatic bacterial lipoprotein since its initial discovery. It exists in two distinct forms: a 'bound-form', which is covalently bound to the cell's peptidoglycan layer, and a 'free-form', which is not. Although it is known that the carboxyl-terminus of bound-form Lpp is located in the periplasm, the precise location of free-form Lpp has never been determined. For decades, it has been widely assumed that free-form Lpp is associated with bound-form. In this work, we show that the free and bound forms of Lpp are not largely associated with each other, but are found in distinct subcellular locations. Our results indicate that free-form Lpp spans the outer membrane and is surface-exposed, whereas bound-form Lpp resides in the periplasm. Thus, Lpp represents a novel example of a single lipoprotein that is able to occupy distinct subcellular locations, and challenges models in which the free and bound forms of Lpp are assumed to be associated with each other.     

A novel periplasmic carrier protein involved in the sorting and transport of Escherichia coli lipoproteins destined for the outer membrane.

Lipoproteins are localized in the outer or inner membrane of Escherichia coli, depending on the species of amino acid located next to the N-terminal fatty acylated Cys. The major outer membrane lipoprotein (Lpp) expressed in spheroplasts was, however, retained in the inner membrane as a mature form. A novel protein that is essential for the release of Lpp from the inner membrane was discovered in the periplasm and purified. The partial amino acid sequence of this 20 kDa protein (p20) was determined and used to clone a gene for p20. Sequencing of the gene revealed that p20 is synthesized as a precursor with a signal sequence. p20 formed a soluble complex only with outer membrane-directed lipoproteins such as Lpp, indicating that p20 plays a critical role in the sorting of lipoproteins. Lpp released from the inner membrane in the presence of p20 was specifically assembled into the outer membrane in vitro. These results indicate that p20 is a periplasmic carrier protein involved in the translocation of lipoproteins from the inner to the outer membrane.     

幽门螺杆菌Lpp20-IL2核酸疫苗免疫活性

【目的】观察pcDNA3.1(+)/Lpp20-IL2免疫C57BL/6小鼠后所产生的体液免疫和细胞免疫应答水平,为研制高效、新型的幽门螺杆菌核酸疫苗提供实验依据。【方法】构建pcDNA3.1(+)/Lpp20-IL2重组载体,并转染HeLa细胞,用Western-blot观察鉴定其在真核细胞得到表达后免疫C57BL/6小鼠,ELISA间接法测定小鼠血清中抗Lpp20IgG抗体水平,ELISA双抗体夹心法检测脾淋巴细胞培养上清中IFN-γ、IL4水平,MTT比色法检测脾淋巴细胞增殖反应,免疫荧光组化法检测Lpp20蛋白在小鼠肌肉组织中的表达情况。【结果】成功构建了pcDNA3.1(+)/Lpp20-IL2真核表达载体,且重组质粒能在HeLa细胞内有效表达目的蛋白;小鼠接种pcDNA3.1(+)/Lpp20-IL2核酸疫苗后能产生特异性IgG抗体,8w后ELISA测定血清抗体A450值明显升高。核酸疫苗pcDNA3.1(+)/Lpp20-IL2免疫组小鼠脾淋巴细胞经特异性抗原刺激后,培养上清中IFN-γ、IL4含量明显升高。pcDNA3.1(+)/Lpp20-IL2和pcDNA3.1(+)/Lpp20核酸疫苗组小鼠脾淋巴细胞经特异性抗原刺激后,刺激指数明显高于空质粒组和PBS组。Lpp20蛋白在小鼠肌肉组织中能够有效表达。【结论】幽门螺杆菌Lpp20-IL2融合基因核酸疫苗和Lpp20单基因核酸疫苗均能刺激机体产生较强细胞免疫应答和体液免疫应答,且前者能诱导更强的细胞免疫应答。     

Lpp is involved in Wnt/PCP signaling and acts together with Scrib to mediate convergence and extension movements during zebrafish gastrulation

The zyxin-related LPP protein is localized at focal adhesions and cell–cell contacts and is involved in the regulation of smooth muscle cell migration. A known interaction partner of LPP in human is the tumor suppressor protein SCRIB. Knocking down scrib expression during zebrafish embryonic development results in defects of convergence and extension (C&E) movements, which occur during gastrulation and mediate elongation of the anterior–posterior body axis. Mediolateral cell polarization underlying C&E is regulated by a noncanonical Wnt signaling pathway constituting the vertebrate planar cell polarity (PCP) pathway. Here, we investigated the role of Lpp during early zebrafish development. We show that morpholino knockdown of lpp results in defects of C&E, phenocopying noncanonical Wnt signaling mutants. Time-lapse analysis associates the defective dorsal convergence movements with a reduced ability to migrate along straight paths. In addition, expression of Lpp is significantly reduced in Wnt11 morphants and in embryos overexpressing Wnt11 or a dominant-negative form of Rho kinase 2, which is a downstream effector of Wnt11, suggesting that Lpp expression is dependent on noncanonical Wnt signaling. Finally, we demonstrate that Lpp interacts with the PCP protein Scrib in zebrafish, and that Lpp and Scrib cooperate for the mediation of C&E.     

Apolipoprotein E Gene Polymorphisms Are Associated with Primary Hyperuricemia in a Chinese Population

Objective

Primary hyperuricemia, an excess of uric acid in the blood, is a major public health problem. In addition to the morbidity that is attributable to gout, hyperuricemia is also associated with metabolic syndrome, hypertension, and cardiovascular disease. This study aims to assess the genetic associations between Apolipoprotein E (APOE) polymorphisms and hyperuricemia in a Chinese population.

Methods

A total of 770 subjects (356 hyperuricemic cases and 414 normouricemic controls) were recruited from the Ningxia Hui Autonomous Region, China. A physical examination was performed and fasting blood was collected for biochemical tests, including determination of the levels of serum lipid, creatinine, and uric acid. Multi-ARMS PCR was applied to determine the APOE genotypes, followed by an investigation of the distribution of APOE genotypes and alleles frequencies in the controls and cases.

Results

The frequencies of the APOE-ε2ε3 genotype (17.70% vs. 10.39%, P = 0.003) and the APOE-ε2 allele (10.53% vs. 5.80%, P = 0.001) were significantly higher in the hyperuricemic group than in the normouricemic group. Furthermore, male cases were more likely to have the APOE-ε2ε3 genotype and APOE-ε2 allele, compared with male controls. In both Han and Hui subjects, cases were more likely to have the APOE-ε2ε3 genotype and the APOE-ε2 allele compared with controls. Furthermore, multivariate logistic regression showed that carriers of the APOE-ε2ε3 genotype (P = 0.001, OR = 2.194) and the ε2 allele (P = 0.001, OR = 2.099) were significantly more likely to experience hyperuricemia than carriers of the ε3/ε3 genotype and the ε3 allele after adjustment for sex, body mass index (BMI), diastolic blood pressure (DBP), triglyceride (TG), low density lipoprotein cholesterol (LDL-C), creatinine (Cr) and fasting blood glucose(FBG).

Conclusions

The APOE-ε2ε3 genotype and the APOE-ε2 allele are associated with serum uric acid levels in Chinese subjects, indicating that individuals carrying the APOE-ε2 allele have a higher risk of hyperuricemia than non-carriers.     

幽门螺杆菌Lpp20融合蛋白的表达、标签切除及鉴定

目的:利用GST融合基因表达系统表达Lpp20-GST融合蛋白,并利用凝血酶切除GST标签.方法:IPTG诱导重组质粒Lpp20/pGEX-4T -1在大肠杆菌BL21 (DE3)中表达,菌体经反复冻融、溶菌酶裂解及超声破菌后,发现Lpp20-GST融合蛋白以部分可溶性的形式表达.采用GST蛋白纯化系统对其纯化,得到Lpp20- GST融合蛋白,再用凝血酶进行GST标签的切除,所得产物进行Western Blot鉴定.结果:高效表达出Lpp20-GST融合蛋白的相对分子质量约4.5kDa,凝血酶成功切除了GST标签,Western Blot证实Lpp20蛋白能被鼠抗Lpp20单克隆抗体识别.结论:成功表达和纯化了重组Lpp20蛋白,为深入研究Lpp20的功能奠定了基础.     

幽门螺杆菌Lpp20蛋白的生物信息学分析

目的：分析幽门螺杆菌Lpp20蛋白的主要化学和免疫学分子特征,为基因工程疫苗和诊断抗原的研究奠定基础。方法：根据Lpp20蛋白的氨基酸序列,应用生物信息学工具分析其蛋白序列,预测其信号肽、跨膜区、疏水性、二级结构、三级结构等性质。结果：Lpp20蛋白具有一段信号肽、脂蛋白信号肽酶切位点及脂盒模体,没有跨膜区,可能是一个外周膜蛋白;Lpp20蛋白的二级结构以α螺旋为主,其三级结构为一个致密的球状。结论：为基于幽门螺杆菌Lpp20蛋白的疫苗开发打下了基础。     

Overexpression of LolCDE allows deletion of the Escherichia coli gene encoding apolipoprotein N-acyltransferase

Bacterial lipoproteins represent a subset of membrane-associated proteins that are covalently modified with lipids at the N-terminal cysteine. The final step of lipoprotein modification, N-acylation of apolipoproteins, is mediated by apolipoprotein N-acyltransferase (Lnt). Examinations with reconstituted proteoliposomes and a conditional mutant previously indicated that N-acylation of lipoproteins is required for their efficient release from the inner membrane catalyzed by LolA and LolCDE, the lipoprotein-specific chaperone and ABC transporter, respectively. Because Lnt is essential for Escherichia coli, a mutant lacking Lnt activity has not been isolated. However, we report here that lnt-null strains can be constructed when LolCDE is overproduced in strains lacking either the major outer membrane lipoprotein Lpp or transpeptidases that cross-link Lpp with peptidoglycan. Lipoproteins purified from the lnt-null strain exhibited increased mobility on SDS-PAGE compared to those from wild-type cells and could be sequenced by Edman degradation, indicating that lipoproteins in this mutant exist as apolipoproteins that lack N-acylation. Overexpression of Lpp in the lnt-null strain resulted in the accumulation of apoLpp in the inner membrane and caused growth arrest. In contrast to the release of mature Lpp in the presence of LolA and LolCDE, that of apoLpp from the inner membrane was significantly retarded. Furthermore, the amount of lipoproteins copurified with LolCDE was significantly reduced in the lnt-null strain. These results indicate that the affinity of LolCDE for apolipoprotein is very low, and therefore, overexpression of LolCDE is required for its release and sorting to the outer membrane.     

The Legionella pneumophila F‐box protein Lpp2082 (AnkB) modulates ubiquitination of the host protein parvin B and promotes intracellular replication

The environmental pathogen Legionella pneumophila encodes three proteins containing F‐box domains and additional protein–protein interaction domains, reminiscent of eukaryotic SCF ubiquitin–protein ligases. Here we show that the F‐box proteins of L. pneumophila strain Paris are Dot/Icm effectors involved in the accumulation of ubiquitinated proteins associated with the Legionella‐containing vacuole. Single, double and triple mutants of the F‐box protein encoding genes were impaired in infection of Acanthamoeba castellanii, THP‐1 macrophages and human lung epithelial cells. Lpp2082/AnkB was essential for infection of the lungs of A/J mice in vivo , and bound Skp1, the interaction partner of the SCF complex in mammalian cells, similar to AnkB from strain AA100/130b. Using a yeast two‐hybrid screen and co‐immunoprecipitation analysis we identified ParvB a protein present in focal adhesions and in lamellipodia, as a target. Immunofluorescence analysis confirmed that ectopically expressed Lpp2082/AnkB colocalized with ParvB at the periphery of lamellipodia. Unexpectedly, ubiquitination tests revealed that Lpp2082/AnkB diminishes endogenous ubiquitination of ParvB. Based on these results we propose that L. pneumophila modulates ubiquitination of ParvB by competing with eukaryotic E3 ligases for the specific protein–protein interaction site of ParvB, thereby revealing a new mechanism by which L. pneumophila may employ translocated effector proteins to promote bacterial survival.     

ApoE −491A/T Promoter Polymorphism is not an Independent Risk Factor,but Associated with the ε4 Allele in Hungarian Alzheimer’s Dementia Population

Apolipoprotein E gene (Apoε) has three common alleles (ε2, ε3, and ε4), of which ε4 has been shown to be associated with an increased risk for Alzheimer’s disease (AD). Possible additional genetic factors, like the −491A variant of ApoE promoter may modify the development of AD, independently of the ApoE allele status. The objective of this study was to investigate whether A/T allelic polymorphism at site−491 of the ApoE promoter is associated with AD in a Hungarian population. The genomic DNA isolated from peripheral blood lymphocytes of 52 late-onset AD and 53 control individuals was used as a template for the two examined polymorphisms and PCR assay was applied. The ε4 allele was significantly over-represented in the AD group (28%) as compared with the control population (7%). No significant differences have been found between the control and the AD populations regarding the occurrence of the promoter A allele frequencies (control: 77%, AD: 70%). However, the AA genotype was more frequent in the AD group (48%) than in the control (10%) when the presence of ε4 allele was also considered. It is unlikely therefore that the −491A variant of the ApoE promoter gene is an independent risk factor in the Hungarian AD population, but a linkage disequilibrium exists between the two examined mutations.     

Identification and developmental expression pattern of van gogh-like 1, a second zebrafish strabismus homologue

Cell movement plays a central role in both normal embryogenesis and the development of diseases such as cancer. Therefore, identification and analysis of proteins controlling cell movement is of special importance. The zebrafish trilobite locus encodes a Van Gogh/Strabismus homologue, which regulates diverse cell migratory behaviors during embryogenesis. Trilobite is most similar to human Van Gogh-like 2 (VANGL2)/Strabismus 1 and mouse Loop-tail associated protein/Lpp1. Both human and mouse genomes encode a second Strabismus homologue referred to as VANGL1/Strabismus 2 and Lpp2, respectively. This prompted us to ask whether another van gogh/strabismus gene, one more closely related to human VANGL1, exists in the zebrafish genome. This paper describes the identification of zebrafish vangl1 and provides the first spatiotemporal expression and functional analysis of a vertebrate vangl1 homologue. Our data indicate that vangl1 and trilobite/vangl2 are expressed in largely non-overlapping domains during embryogenesis. Injection of synthetic vangl1 RNA partially suppressed the gastrulation defect in trilobite mutant embryos, suggesting that Vangl1 and Trilobite/Vangl2 have similar biochemical activities.