Dirofilaria immitis:Ultrastructural Localization, Molecular Characterization, and Analysis of the Expression of p27, a Small Heat Shock Protein Homolog of Nematodes

A cDNA fragment encoding the complete coding region of a 27-kDa protein (p27) ofDirofilaria immitiswas cloned. Antibody to the recombinant p27 bound to hypodermal tissues of third (L3) and fourth stage larvae (L4) ofD. immitisand to both the hypodermis and the cuticle of L3s ofOnchocerca volvulus,as...

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Bibliographic Details
Published in:	Experimental parasitology Vol. 83; no. 1; pp. 30 - 45
Main Authors:	Lillibridge, C.David, Rudin, Werner, Philipp, Mario T.
Format:	Journal Article
Language:	English
Published:	San Diego, CA Elsevier Inc 01-06-1996 Elsevier
Subjects:	Amino Acid Sequence Animals Antigens, Helminth - analysis Antigens, Helminth - chemistry Antigens, Helminth - genetics Antigens, Surface - analysis Antigens, Surface - chemistry Antigens, Surface - genetics Base Sequence Biochemistry. Physiology. Immunology. Molecular biology Biological and medical sciences Blotting, Northern Blotting, Southern Blotting, Western Brugia malayi Caenorhabditis elegans chaperone chemical composition composicion quimica composition chimique cuticle Dirofilaria immitis Dirofilaria immitis - genetics Dirofilaria immitis - immunology Dirofilaria immitis - metabolism Dirofilaria immitis - ultrastructure DNA, Helminth - chemistry dNTP, deoxynucleoside triphosphate EDTA, ethylenediaminetetraacetic acid estres termico Female filariae Fundamental and applied biological sciences. Psychology gDNA, genomic DNA heat shock heat stress Heat-Shock Proteins - analysis Heat-Shock Proteins - chemistry Heat-Shock Proteins - genetics Helminth Proteins - analysis Helminth Proteins - chemistry Helminth Proteins - genetics horseradish peroxidase Hot Temperature HRP1 Invertebrates L3, third stage larva L4, fourth stage larva Male MBP, maltose binding protein Microscopy, Immunoelectron Molecular Sequence Data Molting Nemathelminthia. Plathelmintha Nematoda - genetics Nematoda - immunology Nematoda - metabolism Nematode nucleotide sequence Onchocerca volvulus Onchocerca volvulus - genetics Onchocerca volvulus - metabolism Onchocerca volvulus - ultrastructure ORF, open reading frame PBS, phosphate-buffered saline PCR, polymerase chain reaction Physiology. Development PNGase F, peptideN-glycosidase F proteinas proteine proteins RNA, Helminth - analysis SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis secuencia nucleotidica Sequence Homology, Amino Acid sequence nucleotidique sHsp, small heat shock protein SSC, sodium chloride, sodium citrate SSPE, sodium chloride, sodium phosphate stress thermique TCA, trichloroacetic acid ultraestructura ultrastructure α-crystallin gDNA, genomic DNA EDTA, ethylenediaminetetraacetic acid SSC, sodium chloride, sodium citrate HRP1 Dirofilaria immitis SSPE, sodium chloride, sodium phosphate PBS, phosphate-buffered saline chaperone L3, third stage larva sHsp, small heat shock protein cuticle dNTP, deoxynucleoside triphosphate horseradish peroxidase Nematode TCA, trichloroacetic acid α-crystallin ORF, open reading frame Onchocerca volvulus heat shock filariae L4, fourth stage larva PNGase F, peptideN-glycosidase F Caenorhabditis elegans SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis Brugia malayi PCR, polymerase chain reaction MBP, maltose binding protein Chaperone Parasite Cuticle Gene expression Larva Heat shock protein Helmintha Immunoelectron microscopy Nemathelminthia Invertebrata Localization Nematoda
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Summary:	A cDNA fragment encoding the complete coding region of a 27-kDa protein (p27) ofDirofilaria immitiswas cloned. Antibody to the recombinant p27 bound to hypodermal tissues of third (L3) and fourth stage larvae (L4) ofD. immitisand to both the hypodermis and the cuticle of L3s ofOnchocerca volvulus,as visualized by immunoelectronmicroscopy. The deduced amino acid sequence of the central and C-terminal regions of p27 (amino acids S83to H222) is 18–36% identical to members of the sHsp/α-crystallin family of proteins. The homologous region is thought to be responsible for the molecular chaperone activity of members of this family. The p27 cDNA does not encode a hydrophobic signal peptide. At least two homologous yet distinct p27 genes were identified in theD. immitisgenome by Southern hybridization using the p27 cDNA as a probe. The p27 transcript was 0.9 kb in length on Northern blots. The expression of p27 in L3s ofD. immitiswas neither upregulated by heat shock (43°C) nor by incubation at the physiologic temperature of 37°C. Pulse-labeling experiments of bothD. immitisandBrugia malayiL3s during the L3–L4 moltin vitroshowed that synthesis of p27 is also not upregulated during this developmental phase. However, p27 is expressed constitutively throughout theD. immitisL3–L4 molt and therefore by both larval stages. In addition, both female and male adult worms of this species express p27 constitutively. P27, or an allomorph thereof, was detected in each of nine species representing four nematode superfamilies, thus indicating that this molecule is ubiquitous within the phylum Nematoda. In view of the hypodermal localization of p27, its constitutive expression, and its retention among nematodes, the function of this protein in essential housekeeping roles such as that of molecular chaperone during the molting process is discussed.
Bibliography:	L L72 ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 ObjectType-Article-1 ObjectType-Feature-2
ISSN:	0014-4894 1090-2449
DOI:	10.1006/expr.1996.0046