Mutations in DDHD2, Encoding an Intracellular Phospholipase A1, Cause a Recessive Form of Complex Hereditary Spastic Paraplegia

We report on four families affected by a clinical presentation of complex hereditary spastic paraplegia (HSP) due to recessive mutations in DDHD2, encoding one of the three mammalian intracellular phospholipases A₁ (iPLA₁). The core phenotype of this HSP syndrome consists of very early-onset (<2 years) spastic paraplegia, intellectual disability, and a specific pattern of brain abnormalities on cerebral imaging. An essential role for DDHD2 in the human CNS, and perhaps more specifically in synaptic functioning, is supported by a reduced number of active zones at synaptic terminals in Ddhd-knockdown Drosophila models. All identified mutations affect the protein’s DDHD domain, which is vital for its phospholipase activity. In line with the function of DDHD2 in lipid metabolism and its role in the CNS, an abnormal lipid peak indicating accumulation of lipids was detected with cerebral magnetic resonance spectroscopy, which provides an applicable diagnostic biomarker that can distinguish the DDHD2 phenotype from other complex HSP phenotypes. We show that mutations in DDHD2 cause a specific complex HSP subtype (SPG54), thereby linking a member of the PLA₁ family to human neurologic disease.     

Comparative Proteomic Profiling of Dystroglycan-Associated Proteins in Wild Type, mdx, and Galgt2 Transgenic Mouse Skeletal Muscle

Dystroglycan is a major cell surface glycoprotein receptor for the extracellular matrix in skeletal muscle. Defects in dystroglycan glycosylation cause muscular dystrophy and alterations in dystroglycan glycosylation can impact extracellular matrix binding. Here we describe an immunoprecipitation technique that allows isolation of beta dystroglycan with members of the dystrophin-associated protein complex (DAPC) from detergent-solubilized skeletal muscle. Immunoprecipitation, coupled with shotgun proteomics, has allowed us to identify new dystroglycan-associated proteins and define changed associations that occur within the DAPC in dystrophic skeletal muscles. In addition, we describe changes that result from overexpression of Galgt2, a normally synaptic muscle glycosyltransferase that can modify alpha dystroglycan and inhibit the development of muscular dystrophy when it is overexpressed. These studies identify new dystroglycan-associated proteins that may participate in dystroglycan's roles, both positive and negative, in muscular dystrophy.     

The Mouse p (pink‐eyed dilution) and Human P Genes,Oculocutaneous Albinism Type 2 (OCA2), and Melanosomal pH

Recessive mutations of the mouse p (pink‐eyed dilution) gene lead to hypopigmentation of the eyes, skin, and fur. Mice lacking a functional p protein have pink eyes and light gray fur (if non‐agouti) or cream‐colored fur (if agouti). The human orthologue is the P protein. Humans lacking a functional P protein have oculocutaneous albinism type 2 (OCA2). Melanocytes from p‐deficient mice or OCA2 individuals contain small, minimally pigmented melanosomes. The mouse and human proteins are predicted to have 12 membrane spanning domains and possess significant sequence homology to a number of membrane transport proteins, some of which are involved in the transport of anions. The p protein has been localized to the melanosome membrane. Recently, it has been shown that melanosomes from p protein‐deficient melanocytes have an abnormal pH. Melanosomes in cultured melanocytes derived from wild‐type mice are typically acidic, whereas melanosomes from p protein‐deficient mice are non‐acidic. Melanosomes and related endosome‐derived organelles (i.e., lysosomes) are thought to have an adenosine triphosphate (ATP)‐driven proton pump that helps to generate an acidic lumen. To compensate for the charge of these protons, anions must also be transported to the lumen of the melanosome. In light of these observations, a model of p protein function is presented in which the p protein, together with the ATP‐driven proton pump, regulates the pH of the melanosome.     

Differentiation of the Gene Clusters Encoding Botulinum Neurotoxin Type A Complexes in Clostridium botulinum Type A, Ab, and A(B) Strains

We describe a strategy to identify the clusters of genes encoding components of the botulinum toxin type A (boNT/A) complexes in 57 strains of Clostridium botulinum types A, Ab, and A(B) isolated in Italy and in the United States from different sources. Specifically, we combined the results of PCR for detecting the ha33 and/or p47 genes with those of boNT/A PCR-restriction fragment length polymorphism analysis. Three different type A toxin gene clusters were revealed; type A1 was predominant among the strains from the United States, whereas type A2 predominated among the Italian strains, suggesting a geographic distinction between strains. By contrast, no relationship between the toxin gene clusters and the clinical or food source of strains was evident. In two C. botulinum type A isolates from the United States, we recognized a third type A toxin gene cluster (designated type A3) which was similar to that previously described only for C. botulinum type A(B) and Ab strains. Total genomic DNA from the strains was subjected to pulsed-filed gel electrophoresis and randomly amplified polymorphic DNA analyses, and the results were consistent with the boNT/A gene clusters obtained.     

Mouse proton pump ATPase C subunit isoforms (C2-a and C2-b) specifically expressed in kidney and lung

The vacuolar-type H+-ATPases (V-ATPases) are multimeric proton pumps involved in a wide variety of physiological processes. We have identified two alternative splicing variants of C2 subunit isoforms: C2-a, a lung-specific isoform containing a 46-amino acid insertion, and C2-b, a kidney-specific isoform without the insert. Immunohistochemistry with isoform-specific antibodies revealed that V-ATPase with C2-a is localized specifically in lamellar bodies of type II alveolar cells, whereas the C2-b isoform is found in the plasma membranes of renal alpha and beta intercalated cells. Immunoprecipitation combined with immunohistological analysis revealed that C2-b together with other kidney-specific isoforms was selectively assembled to form a unique proton pump in intercalated cells. Furthermore, a chimeric yeast V-ATPase with mouse the C2-a or C2-b isoform showed a lower Km(ATP) and lower proton transport activity than that with C1 or Vma5p (yeast C subunit). These results suggest that V-ATPases with the C2-a and C2-b isoform are involved in luminal acidification of lamellar bodies and regulation of the renal acid-base balance, respectively.     

Defects in cell spreading and ERK1/2 activation in fibroblasts with lamin A/C mutations

In-frame mutations in nuclear lamin A/C lead to a multitude of tissue-specific degenerative diseases known as the ‘laminopathies’. Previous studies have demonstrated that lamin A/C-null mouse fibroblasts have defects in cell polarisation, suggesting a role for lamin A/C in nucleo-cytoskeletal-cell surface cross-talk. However, this has not been examined in patient fibroblasts expressing modified forms of lamin A/C. Here, we analysed skin fibroblasts from 3 patients with Emery–Dreifuss muscular dystrophy and from 1 with dilated cardiomyopathy. The emerin–lamin A/C interaction was impaired in each mutant cell line. Mutant cells exhibited enhanced cell proliferation, collagen-dependent adhesion, larger numbers of filopodia and smaller cell spread size, compared with control cells. Furthermore, cell migration, speed and polarization were elevated. Mutant cells also showed an enhanced ability to contract collagen gels at early time points, compared with control cells. Phosphotyrosine measurements during cell spreading indicated an initial temporal lag in ERK1/2 activation in our mutant cells, followed by hyper-activation of ERK1/2 at 2 h post cell attachment. Deregulated ERK1/2 activation is linked with cardiomyopathy, cell spreading and proliferation defects. We conclude that a functional emerin–lamin A/C complex is required for cell spreading and proliferation, possibly acting through ERK1/2 signalling.     

Cloning of cDNA and Genomic DNA Encoding Human Type XVIII Collagen and Localization of the α1(XVIII) Collagen Gene to Mouse Chromosome 10 and Human Chromosome 21

Types XV and XVIII collagen belong to a unique and novel subclass of the collagen superfamily for which we have proposed the name the MULTIPLEXIN family. Members of this class contain polypeptides with multiple triple-helical domains separated and flanked by non-triple-helical regions. In this paper, we report the isolation of human cDNAs and genomic DNAs encoding the α1(XVIII) collagen chain. Utilizing a genomic clone as probe, we have mapped the COL18A1 gene to chromosome 21q22.3 by fluorescence in situ hybridization. In addition, using an interspecific backcross panel, we have shown that the murine Col18a1 locus is on chromosome 10, close to the loci for Col6a1 and Col6a2.     

一类新的编码PRPs基因的分离及其在棉花纤维等组织细胞中的表达

富含脯氨酸的细胞壁蛋白(proline-rich proteins,PRPs)在植物中广泛分布,它们在建造围绕特定细胞类型的细胞壁结构上起着很重要的作用.从棉花的cDNA文库中分离了5个编码富含脯氨酸的细胞壁蛋白质基因,这5个基因推断的氨基酸序列最普遍的特点就是脯氨酸含量非常高.根据氨基酸组成、富含脯氨酸的重复单元和结构域组织的特点,将这5个蛋白质分成2个亚类:一类(包括GhPRP3-6)与典型的PRPs结构相似,由N端疏水区(或信号肽)与不同富含脯氨酸的重复序列组成;另一类(GhPRPL)与典型的PRPs结构不同,这个蛋白质的N端为亲水序列,GhPRPL在靠近C端有8个5肽(类似PPKKE)的重复基序,与典型PRPs所含有的重复序列PPVYK非常相似.实时RT-PCR(Real-time RT-PCR)分析表明,GhPRP3和GhPRP5在10dpa纤维中特异表达,而GhPRPL在子叶中优势表达.GhPRP4和GhPRP6在所分析的组织中都有表达,GhPRP4mRNA在下胚轴中最丰富,在花药中次之,而GhPRP6在10dpa纤维中表达最强,在10dpa胚珠中次之.此外,GhPRP3,GhPRP5基因表达受纤维发育调节,表明它们可能在棉纤维发育中起重要作用.     

Human C-type lectin domain family 4, member C (CLEC4C/BDCA-2/CD303) is a receptor for asialo-galactosyl-oligosaccharides

Plasmacytoid dendritic cells are specialized in the production of type I interferon (type I IFN), which promotes antiviral and antitumor responses, as well as autoimmune disorders. Activation of type I IFN secretion depends on the pattern recognition receptors TLR7 and TLR9, which sense microbial RNA and DNA, respectively. Type I IFN production is modulated by several receptors, including the type II C-type lectin domain family 4, member C (CLEC4C). The natural ligand of CLEC4C is unknown. To identify it, here we probed a glycan array with a soluble form of the CLEC4C ectodomain. We found that CLEC4C recognizes complex type sugars with terminal galactose. Importantly, soluble CLEC4C bound peripheral blood leukocytes and tumor cells that express glycans with galactose residues at the non-reducing ends. The positive and negative modulation of galactose residues on cell membranes was paralleled by the regulation of type I IFN secretion by plasmacytoid dendritic cells in co-culture experiments in vitro. These results suggest that the modulation in the expression of non-sialylated oligosaccharides by invading pathogens or transformed cells may affect type I IFN response and immune surveillance.