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

• 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
• ISSN: 0014-4894; 1090-2449
• DOI: 10.1006/expr.1996.0046

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.