Biosynthesis and glycosylation of serine/threonine-rich secreted proteins from Toxocara canis larvae.
Toxocara canis infective stage larvae continually produce excretory-secretory (TES) glycoproteins in long-term in vitro culture. The kinetics of synthesis and secretion were studied by metabolic labelling with radioactive [35S]methionine, [14C]serine and [14C]threonine. Maximal incorporation rates required overnight pre-incubation of parasites in medium depleted of the appropriate amino acid. Larvae rapidly incorporated isotope into their somatic tissues, but there was a minimum delay of 10 h before secretion of labelled antigens. Labelling with [14C]serine and [14C]threonine demonstrated a relative abundance of these amino acids in the major surface/secreted glycoproteins of this nematode (TES-32 and 120). Pulse-chase experiments suggested that TES-120 may be derived from a 58 kDa precursor, reflecting extensive posttranslational glycosylation. Inhibition of N-glycosylation with tunicamycin and digestion with N-glycanase provided evidence of N-glycosylation in the lower molecular weight ES components (TES-32, 55 and 70). These agents had no effect on the higher molecular weight components (TES-120 and 400) implying that for these molecules glycosylation is predominantly O-linked. The largest ES component (TES-400) was unusual, in incorporating serine and threonine but not methionine, and by exhibiting increased apparent molecular weight following pronase digestion; it is suggested that this molecule is a proteoglycan.
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Department of Biology, Imperial College of Science, Technology and Medicine, London.
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D000818:Animals; D000947:Antigens, Helminth / Q000096:biosynthesis / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000096:biosynthesis*; D006031:Glycosylation; D015801:Helminth Proteins / Q000096:biosynthesis* / Q000378:metabolism; D011134:Polysaccharides / Q000096:biosynthesis / Q000378:metabolism; D011233:Precipitin Tests; D012694:Serine / Q000378:metabolism*; D013912:Threonine / Q000378:metabolism*; D017166:Toxocara canis / Q000276:immunology / Q000378:metabolism*
D000947:Antigens, Helminth; D006023:Glycoproteins; D015801:Helminth Proteins; D011134:Polysaccharides; D013912:Threonine; D012694:Serine