Proteolipid plasmolipin: localization in polarized cells,regulated expression and lipid raft association in CNS and PNS myelin

The proteolipid plasmolipin is member of the expanding group of tetraspan (4TM) myelin proteins. Initially, plasmolipin was isolated from kidney plasma membranes, but subsequent northern blot analysis revealed highest expression in the nervous system. To gain more insight into the functional roles of plasmolipin, we have generated a plasmolipin-specific polyclonal antibody. Immunohistochemical staining confirms our previous observation of glial plasmolipin expression and proves plasmolipin localization in the compact myelin of rat peripheral nerve and myelinated tracts of the CNS. Western blot analysis indicates a strong temporal correlation of plasmolipin expression and (re-) myelination in the PNS and CNS. However, following axotomy plasmolipin expression is also recovered in non-regenerating distal nerve stumps. In addition, we detected plasmolipin expression in distinct neuronal subpopulations of the CNS. The observed asymmetric distribution of plasmolipin in compact myelin, as well as in epithelial cells of kidney and stomach, indicates a polarized cellular localization. Therefore, we purified myelin from the CNS and PNS and demonstrated an enrichement of phosphorylated plasmolipin protein in detergent-insoluble lipid raft fractions, suggesting selective targeting of plasmolipin to the myelin membranes. The present data indicate that the proteolipid plasmolipin is a structural component of apical membranes of polarized cells and provides the basis for further functional analysis.     

Identification of a novel Bardet-Biedl syndrome protein,BBS7, that shares structural features with BBS1 and BBS2

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous disorder, the primary features of which include obesity, retinal dystrophy, polydactyly, hypogenitalism, learning difficulties, and renal malformations. Conventional linkage and positional cloning have led to the mapping of six BBS loci in the human genome, four of which (BBS1, BBS2, BBS4, and BBS6) have been cloned. Despite these advances, the protein sequences of the known BBS genes have provided little or no insight into their function. To delineate functionally important regions in BBS2, we performed phylogenetic and genomic studies in which we used the human and zebrafish BBS2 peptide sequences to search dbEST and the translation of the draft human genome. We identified two novel genes that we initially named "BBS2L1" and "BBS2L2" and that exhibit modest similarity with two discrete, overlapping regions of BBS2. In the present study, we demonstrate that BBS2L1 mutations cause BBS, thereby defining a novel locus for this syndrome, BBS7, whereas BBS2L2 has been shown independently to be BBS1. The motif-based identification of a novel BBS locus has enabled us to define a potential functional domain that is present in three of the five known BBS proteins and, therefore, is likely to be important in the pathogenesis of this complex syndrome.     

Presence of the Plasma Membrane Proteolipid (Plasmolipin) in Myelin

Plasma membrane proteolipid (plasmolipin), which was originally isolated from kidney membranes, has also been shown to be present in brain. In this study, we examined the distribution of plasmolipin in brain regions, myelin, and oligodendroglial membranes. Immunoblot analysis of different brain regions revealed that plasmolipin levels were higher in regions rich in white matter. Plasmolipin was also detected in myelin, myelin subfractions, and oligodendroglial membranes. Immunocytochemical analysis of the cerebellum revealed that plasmolipin was localized in the myelinated tracts. Plasmolipin levels in myelin were enriched during five successive cycles of myelin purification, similar to the enrichment of myelin proteolipid apoprotein (PLP) and myelin basic protein (MBP). In contrast, levels of Na+,K(+)-ATPase and a 70-kDa protein were decreased. When myelin or white matter was extracted with chloroform/methanol, it contained, in addition to PLP, a significant amount of plasmolipin. Quantitative immunoblot analysis suggested that plasmolipin constitutes in the range of 2.2-4.8% of total myelin protein. Plasmolipin, purified from kidney membranes, was detected by silver stain on gels at 18 kDa and did not show immunological cross-reactivity with either PLP or MBP. Thus, it is concluded that plasmolipin is present in myelin, possibly as a component of the oligodendroglial plasma membrane, but is structurally and immunologically different from the previously characterized myelin proteolipids.     

Evaluation of complex inheritance involving the most common Bardet-Biedl syndrome locus (BBS1)

Bardet-Biedl syndrome (BBS) is a genetic disorder with the primary features of obesity, pigmentary retinopathy, polydactyly, renal malformations, mental retardation, and hypogenitalism. Patients with BBS are also at increased risk for diabetes mellitus, hypertension, and congenital heart disease. BBS is known to map to at least six loci: 11q13 (BBS1), 16q21 (BBS2), 3p13-p12 (BBS3), 15q22.3-q23 (BBS4), 2q31 (BBS5), and 20p12 (BBS6). Although these loci were all mapped on the basis of an autosomal recessive mode of inheritance, it has recently been suggested-on the basis of mutation analysis of the identified BBS2, BBS4, and BBS6 genes-that BBS displays a complex mode of inheritance in which, in some families, three mutations at two loci are necessary to manifest the disease phenotype. We recently identified BBS1, the gene most commonly involved in Bardet-Biedl syndrome. The identification of this gene allows for further evaluation of complex inheritance. In the present study we evaluate the involvement of the BBS1 gene in a cohort of 129 probands with BBS and report 10 novel BBS1 mutations. We demonstrate that a common BBS1 missense mutation accounts for approximately 80% of all BBS1 mutations and is found on a similar genetic background across populations. We show that the BBS1 gene is highly conserved between mice and humans. Finally, we demonstrate that BBS1 is inherited in an autosomal recessive manner and is rarely, if ever, involved in complex inheritance.     

The cloning and developmental expression of unconventional myosin IXA (MYO9A) a gene in the Bardet-Biedl syndrome (BBS4) region at chromosome 15q22-q23.

Bardet-Biedl Syndrome (BBS) is a heterogeneous, autosomal recessive disorder characterized by mental retardation, obesity, retinitis pigmentosa, syndactyly and/or polydactyly, short stature, and hypogenitalism and is caused by mutations at a number of distinct loci. Using a positional cloning approach for identifying the BBS4 (chromosome 15) gene, we identified and cloned an unconventional myosin gene, myosin IXA (HGMW-approved symbol MYO9A). Since mutations in unconventional myosins are known to cause several human diseases, and since mutations of unconventional myosin VIIa cause retinal degeneration, we evaluated myosin IXA as a candidate for BBS. We exploited PCR-based techniques to clone a 8473-nt cDNA for myosin IXA. A 7644-bp open reading frame predicts a protein with all the hallmarks of class IX unconventional myosins. Human Northern blot analysis and in situ hybridization of mouse embryos reveal that myosin IXA is expressed in many tissues consistent with BBS. Intron/exon boundaries were identified, and myosin IXA DNA and RNA from BBS4 patients were evaluated for mutation.     

BBS4 is a minor contributor to Bardet-Biedl syndrome and may also participate in triallelic inheritance

Bardet-Biedl syndrome (BBS) is an uncommon multisystemic disorder characterized primarily by retinal dystrophy, obesity, polydactyly, and renal dysfunction. BBS has been modeled historically as an autosomal recessive trait, under which premise six independent BBS loci (BBS1-BBS6) have been mapped in the human genome. However, extended mutational analyses of BBS2 and BBS6, the first two BBS genes cloned, suggest that BBS exhibits a more complex pattern of inheritance, in which three mutations at two loci simultaneously are necessary and sufficient in some families to manifest the phenotype. We evaluated the spectrum of mutations in the recently identified BBS4 gene with a combination of haplotype analysis and mutation screening on a multiethnic cohort of 177 families. Consistent with predictions from previous genetic analyses, our data suggest that mutations in BBS4 contribute to BBS in <3% of affected families. Furthermore, integrated mutational data from all three currently cloned BBS genes raise the possibility that BBS4 may participate in triallelic inheritance with BBS2 and BBS1, but not the other known loci. Establishment of the loci pairing in triallelism is likely to be important for the elucidation of the functional relationships among the different BBS proteins.     

Ectopic Expression of Human BBS4 Can Rescue Bardet-Biedl Syndrome Phenotypes in Bbs4 Null Mice

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous autosomal recessive disorder characterized by obesity, retinal degeneration, polydactyly, hypogenitalism and renal defects. Recent findings have associated the etiology of the disease with cilia, and BBS proteins have been implicated in trafficking various ciliary cargo proteins. To date, 17 different genes have been reported for BBS among which BBS1 is the most common cause of the disease followed by BBS10, and BBS4. A murine model of Bbs4 is known to phenocopy most of the human BBS phenotypes, and it is being used as a BBS disease model. To better understand the in vivo localization, cellular function, and interaction of BBS4 with other proteins, we generated a transgenic BBS4 mouse expressing the human BBS4 gene under control of the beta actin promoter. The transgene is expressed in various tissues including brain, eye, testis, heart, kidney, and adipose tissue. These mice were further bred to express the transgene in Bbs4 null mice, and their phenotype was characterized. Here we report that despite tissue specific variable expression of the transgene, human BBS4 was able to complement the deficiency of Bbs4 and rescue all the BBS phenotypes in the Bbs4 null mice. These results provide an encouraging prospective for gene therapy for BBS related phenotypes and potentially for other ciliopathies.     

Comparative analysis of nonaspanin protein sequences and expression studies in zebrafish

Nonaspanins constitute a family of proteins, also called TM9SF, characterized by a large non-cytoplasmic domain and nine putative transmembrane domains. This family is highly conserved through evolution and comprises three members in Saccharomyces cerevisiae, Dictyostelium discoideum, and Drosophila melanogaster, and four members are reported in mammals (TM9SF1–TM9SF4). Genetic studies in Dictyostelium and Drosophila have shown that TM9SF members are required for adhesion and phagocytosis in innate immune response, furthermore, human TM9SF1 plays a role in the regulation of autophagy and human TM9SF4 in tumor cannibalism. Here we report that the zebrafish genome encodes five members of this family, TM9SF1–TM9SF5, which show high level of sequence conservation with the previously reported members. Expression analysis in zebrafish showed that all members are maternally expressed and continue to be present throughout embryogenesis to adults. Gene expression could not be regulated by pathogen-associated molecular patterns such as LPS, CpG, or Poly I:C. By bioinformatic analyses of 80 TM9SF protein sequences from yeast, plants, and animals, we confirmed a very conserved protein structure. An evolutionary conserved immunoreceptor tyrosine-based inhibition motif has been detected in the cytoplasmic domain between transmembrane domain (TM) 7 and TM8 in TM9SF1, TM9SF2, TM9SF4 and TM9SF5, and at the extreme C-terminal end of TM9SF4. Finally, a conserved TRAF2 binding domain could also be predicted in the cytoplasmic regions of TM9SF2, TM9SF3, TM9SF4, and TM9SF5. This confirms the hypothesis that TM9SF proteins may play a regulatory role in a specific and ancient cellular mechanism that is involved in innate immunity.     

Comparative genomics and gene expression analysis identifies BBS9, a new Bardet-Biedl syndrome gene

Bardet-Biedl syndrome (BBS) is an autosomal recessive, genetically heterogeneous, pleiotropic human disorder characterized by obesity, retinopathy, polydactyly, renal and cardiac malformations, learning disabilities, and hypogenitalism. Eight BBS genes representing all known mapped loci have been identified. Mutation analysis of the known BBS genes in BBS patients indicate that additional BBS genes exist and/or that unidentified mutations exist in the known genes. To identify new BBS genes, we performed homozygosity mapping of small, consanguineous BBS pedigrees, using moderately dense SNP arrays. A bioinformatics approach combining comparative genomic analysis and gene expression studies of a BBS-knockout mouse model was used to prioritize BBS candidate genes within the newly identified loci for mutation screening. By use of this strategy, parathyroid hormone-responsive gene B1 (B1) was found to be a novel BBS gene (BBS9), supported by the identification of homozygous mutations in BBS patients. The identification of BBS9 illustrates the power of using a combination of comparative genomic analysis, gene expression studies, and homozygosity mapping with SNP arrays in small, consanguineous families for the identification of rare autosomal recessive disorders. We also demonstrate that small, consanguineous families are useful in identifying intragenic deletions. This type of mutation is likely to be underreported because of the difficulty of deletion detection in the heterozygous state by the mutation screening methods that are used in many studies.     

Cloning and characterization of a splice variant of human Bardet-Biedl syndrome 4 gene (BBS4).

Bardet-Biedl syndrome (BBS) is a heterogeneous multisystemic disorder characterized primarily by five cardinal features of retinal degeneration, obesity, polydactyly, hypogenitalism and mental retardation. To date, six distinct BBS loci that have been identified on different chromosomes. BBS4 gene is mapped to 15q22.2-23, which when mutated can cause BBS4. Its protein shows strong homology to O-linked N-acetylglucosamine (O-GlcNAc) transferase. Here we report a splice variant of BBS4, which is 2556 bp in length and has an open reading frame coding a predicted 527 amino-acids protein. RT-PCR shows that the cDNA is widely expressed while it has higher expression levels in pancreas, liver and prostate.     

Identification of the myelin protein plasmolipin as the cell entry receptor for Mus caroli endogenous retrovirus

The Asian wild mouse species Mus caroli harbors an endogenous retrovirus (McERV) that is closely related to but distinct from the endogenous retrovirus family defined by the Mus dunni endogenous virus and the Mus musculus endogenous retrovirus. McERV could infect some cell types from humans, dogs, and rats, but not all, and did not infect any mouse cell line tested. Because of its interesting host range and proposed ancestral relationship to primate retroviruses and because none of the entry receptors for this family of retroviruses had been identified, we began a search for the McERV receptor. We determined the chromosomal location of the receptor gene in the human genome by phenotypic screening of the G3 human-hamster radiation hybrid cell line panel and confirmed the localization by assaying for receptor activity conferred by bacterial artificial chromosome (BAC) clones spanning the region. We next localized the gene more precisely in one positive BAC by assaying for receptor activity following BAC digestion with several restriction enzymes that cleaved different sets of genes, and we confirmed that the final candidate gene, plasmolipin (PLLP; TM4SF11), is the novel receptor by showing that the expression of the human PLLP cDNA renders hamster and mouse cells susceptible to McERV infection. PLLP functions as a voltage-dependent potassium ion channel and is expressed primarily in kidney and brain, helping to explain the limited range of cell types that McERV can infect. Interestingly, mouse PLLP also functioned well as a receptor for McERV but was simply not expressed in the mouse cell types that we originally tested.     

Myelin and lymphocyte protein (MAL/MVP17/VIP17) and plasmolipin are members of an extended gene family

An increasing number of four-transmembrane proteins has been found to be associated with CNS and PNS myelin. Some of these proteins play crucial roles in the development and maintenance of the nervous system. In the CNS, proteolipid protein (PLP) is mutated in the myelin disorder Pelizaeus-Merzbacher disease and in spastic paraplegia, while in the PNS, peripheral myelin protein 22 (PMP22) and connexin32 (C×32) are culprit genes in the most frequent forms of hereditary peripheral neuropathies. Myelin and lymphocyte protein (MAL; also called MVP17 or VIP17) and plasmolipin are additional tetraspan proteins that are highly expressed by myelinating glial cells. However, little is known about the role of these proteins in the nervous system. As a prerequisite for functional genetic approaches in the mouse, we have isolated and characterized a mouse MAL cDNA and the corresponding structural MAL gene. Computer-aided analysis and database searches revealed that MAL belongs to a larger gene family which also includes plasmolipin, BENE and the expressed sequence tag (EST) H09290. While the overall amino acid sequence identities between mouse MAL and the related proteins are relatively low (29–37%), the conserved motif -[Q/Y-G-W-V-M-F/Y-V]- which is found at the junction of the first extracellular loop and the second membrane-associated domain serves as a fingerprint for the MAL protein family. Expression analysis of the members of the MAL gene family indicates widespread expression in various tissues, suggesting a common role of these proteins in cell biology.     

The effect of ST2 gene product on anchorage-independent growth of a glioblastoma cell line, T98G.

The ST2 gene, which is specifically induced by growth stimulation in fibroblasts, encodes interleukin-1 receptor-related proteins and is widely expressed in hematopoietic, helper T, and various cancer cells. However, the physiological as well as pathological functions of the ST2 gene products are not yet fully understood. In this study, we analyzed the expression of the ST2 gene in human glioma cell lines and human brain tumor samples with real-time polymerase chain reaction method, the results of which revealed that the expression level of the ST2 gene in glioma cell lines and glioblastoma samples is significantly lower than that in a fibroblastic cell line, TM12, and benign brain tumors, suggesting the reverse relationship between malignancy and ST2 expression. As we could not detect the soluble ST2 protein in the culture fluid of the T98G glioblastic cell line by ELISA, we established stable transformants of T98G that continuously produce and secrete the ST2 protein, in order to study the effect of the ST2 protein on malignancy. Although we could not detect a remarkable difference in proliferation between transformants and control cells in conventional tissue culture dishes, the efficiency of colony formation in soft agar was significantly decreased in the case of cells that continuously produce the ST2 protein. Furthermore, inhibition of colony formation in soft agar was observed in wild-type T98G cells when purified soluble ST2 protein was added to the culture, in a dose-dependent manner. Taken together, the results suggest that the expression of ST2 suppressed the anchorage-independent growth and malignancy.     

BBS8蛋白参与衣藻鞭毛膜蛋白的运输

真核细胞的纤毛(也称鞭毛)是一种突出于细胞表面的极性细胞器,纤毛不仅参与细胞运动,还参与信号传导等过程,其结构或功能异常引发的一系列人类疾病称为"纤毛相关性疾病"。纤毛相关性疾病巴德-毕德氏综合征(Bardet-Biedl syndrome,简称BBS)由BBS相关基因缺陷导致,为了研究致病基因BBS8的生理作用和功能,构建模式生物莱茵衣藻BBS8基因缺陷突变体,利用性状观测和生化分析检测突变体的表现型和生理功能。免疫荧光表明BBS8蛋白是一种鞭毛蛋白且在基体有特异性定位;bbs8突变体感光极性运动消失,并在解聚诱导实验中鞭毛解聚缓慢;鞭毛的银染和质谱结果表明突变体的鞭毛膜蛋白在鞭毛内异常积累。文中通过实验证据说明BBS8蛋白在参与鞭毛内膜蛋白运输中起到重要作用,并极可能通过介导膜蛋白反向运输发挥生理功能。     

Functional analysis by minigene assay of putative splicing variants found in Bardet–Biedl syndrome patients

Bardet–Biedl syndrome (BBS) and Alström syndrome (ALMS) are rare diseases belonging to the group of ciliopathies. Although mutational screening studies of BBS/ALMS cohorts have been extensively reported, little is known about the functional effect of those changes. Thus, splicing variants are estimated to represent 15% of disease‐causing mutations, and there is growing evidence that many exonic changes are really splicing variants misclassified. In this study, we aimed to analyse for the first time several variants in BBS2, ARL6/BBS3, BBS4 and ALMS1 genes predicted to produce aberrant splicing by minigene assay. We found discordance between bioinformatics analysis and experimental data when comparing wild‐type and mutant constructs. Remarkably, we identified nonsense variants presumably resistant to nonsense‐mediated decay, even when a premature termination codon would be introduced in the second amino acid (p.(G2*) mutation in ARL6/BBS3 gene). As a whole, we report one of the first functional studies of BBS/ALMS1 variants using minigene assay, trying to elucidate their role in disease. Functional studies of variants identified in BBS and ALMS patients are essential for their proper classification and subsequent genetic counselling and could also be the start point for new therapeutic approaches, currently based only on symptomatic treatment.     

Intrinsic protein-protein interaction-mediated and chaperonin-assisted sequential assembly of stable bardet-biedl syndrome protein complex, the BBSome

The pleiotropic features of obesity, retinal degeneration, polydactyly, kidney abnormalities, cognitive impairment, hypertension, and diabetes found in Bardet-Biedl syndrome (BBS) make this disorder an important model disorder for identifying molecular mechanisms involved in common human diseases. To date, 16 BBS genes have been reported, seven of which (BBS1, 2, 4, 5, 7, 8, and 9) code for proteins that form a complex known as the BBSome. The function of the BBSome involves ciliary membrane biogenesis. Three additional BBS genes (BBS6, BBS10, and BBS12) have homology to type II chaperonins and interact with CCT/TRiC proteins and BBS7 to form a complex termed the BBS-chaperonin complex. This complex is required for BBSome assembly. Little is known about the process and the regulation of BBSome formation. We utilized point mutations and null alleles of BBS proteins to disrupt assembly of the BBSome leading to the accumulation of BBSome assembly intermediates. By characterizing BBSome assembly intermediates, we show that the BBS-chaperonin complex plays a role in BBS7 stability. BBS7 interacts with BBS2 and becomes part of a BBS7-BBS2-BBS9 assembly intermediate referred to as the BBSome core complex because it forms the core of the BBSome. BBS1, BBS5, BBS8, and finally BBS4 are added to the BBSome core to form the complete BBSome.     

Comparative genomic analysis identifies an ADP-ribosylation factor-like gene as the cause of Bardet-Biedl syndrome (BBS3)

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous, pleiotropic human disorder characterized by obesity, retinopathy, polydactyly, renal and cardiac malformations, learning disabilities, and hypogenitalism. Eight BBS loci have been mapped, and seven genes have been identified. BBS3 was previously mapped to chromosome 3 by linkage analysis in a large Israeli Bedouin kindred. The rarity of other families mapping to the BBS3 locus has made it difficult to narrow the disease interval sufficiently to identify the gene by positional cloning. We hypothesized that the genomes of model organisms that contained the orthologues to known BBS genes would also likely contain a BBS3 orthologue. Therefore, comparative genomic analysis was performed to prioritize BBS candidate genes for mutation screening. Known BBS proteins were compared with the translated genomes of model organisms to identify a subset of organisms in which these proteins were conserved. By including multiple organisms that have relatively small genome sizes in the analysis, the number of candidate genes was reduced, and a few genes mapping to the BBS3 interval emerged as the best candidates for this disorder. One of these genes, ADP-ribosylation factor-like 6 (ARL6), contains a homozygous stop mutation that segregates completely with the disease in the Bedouin kindred originally used to map the BBS3 locus, identifying this gene as the BBS3 gene. These data illustrate the power of comparative genomic analysis for the study of human disease and identifies a novel BBS gene.