Similarity in cyst wall protein (CWP) trafficking between encysting Giardia duodenalis trophozoites and CWP-expressing human embryonic kidney-293 cells.
Cyst wall proteins 1 and 2 (CWP1 and CWP2) are major constituents of the giardial cyst wall and are expressed with similar kinetics by encysting trophozoites. In the present study, we were interested to determine if the expression of giardial CWPs as heterologous proteins in a higher eukaryotic cell would result in their trafficking across the secretory pathway, as is the case in encysting trophozoites. Recombinant (r)CWP1 and rPro-CWP2 were detected in the lysate and culture media of transfected HEK-293 cells. We then conducted intracellular localization experiments using confocal microscopy and found that the proteins were trafficked in membrane enclosed vesicles across the secretory pathway and released to the culture medium by transfected HEK-293 cells. We then dissected the rCWP1 and rPro-CWP2 molecules to identify the portion(s) responsible for their secretion and found that the putative N-terminal signal peptide was sufficient for directing the secretion of rCWP1, while both the putative N-terminal signal peptide and the 13kDa C-terminal regions were necessary for the secretion of rPro-CWP2 by transfected HEK-293 cells. Taken together, these results demonstrate the degree of conservation of signal peptide recognition between lower and higher eukaryotes.
Institute of Parasitology, McGill University, MacDonald Campus, 21,111 Lakeshore Road, Montréal, Quebec, Canada H9X 3V9.
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Abdul-Wahid
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Faubert
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D002460:Cell Line; D003470:Culture Media / Q000494:pharmacology; D017931:DNA Primers / Q000737:chemistry; D019286:Endopeptidase K / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005872:Giardia / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry; D014162:Transfection
D000911:Antibodies, Monoclonal; C094333:CWP1 protein, Giardia lamblia; C093811:CWP2 protein, S cerevisiae; D003470:Culture Media; D017931:DNA Primers; D008562:Membrane Glycoproteins; D010455:Peptides; D021382:Protein Sorting Signals; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D019286:Endopeptidase K