Biosynthesis of Entamoeba histolytica proteophosphoglycan in vitro.
A complex glycoconjugate proteophosphoglycan (PPG) is present on the surface of the pathogenic protozoan parasite Entamoeba histolytica but not in the non-pathogenic Entamoeba dispar. It is thought to be an important molecule involved in pathogenesis. In order to study its biosynthesis, an in vitro cell-free system was developed. The specificity of the system was demonstrated by various criteria including immunoprecipitation by a specific monoclonal antibody. The in vitro synthesized molecule was found to be susceptible to mild acid hydrolysis, digestion by phosphoinositol-specific phospholipase C and nitrous acid deamination, the salient features for a PPG-like molecule. The in vitro product was not synthesized when heat-treated cellular-extract was used in the assay or when the cell extract was prepared from Entamoeba invadens, a species that lacks these glycoconjugates. Analysis of the glycan side chains of the in vitro synthesized product by thin layer chromatography revealed side chains of variable sizes including a fraction greater than six glycan units. The crude membranes used in the cell-free system were further fractionated by sucrose density gradient centrifugation. The fraction containing the PPG synthesizing activity when used in the assay resulted in a 10-fold increase in specific activity. Development of this cell-free system will facilitate further studies on the nature of intracellular organelles and the pathways that are involved in PPG biosynthesis.
Arya
Ranjana
R
School of Life Sciences, Jawaharlal Nehru University, New Mehrauli Road, New Delhi 110067, India.
Mehra
Alka
A
Bhattacharya
Sudha
S
Vishwakarma
Ram A
RA
Bhattacharya
Alok
A
D000818:Animals; D002458:Cell Fractionation; D004748:Entamoeba histolytica / Q000378:metabolism* / Q000472:pathogenicity; D004749:Entamoebiasis / Q000378:metabolism; D005690:Galactose / Q000378:metabolism; D006020:Glycopeptides / Q000096:biosynthesis*; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D007202:Indicators and Reagents; D008562:Membrane Glycoproteins / Q000096:biosynthesis; D010710:Phosphates / Q000378:metabolism; D011509:Proteoglycans / Q000096:biosynthesis*
D006020:Glycopeptides; D017261:Glycosylphosphatidylinositols; D007202:Indicators and Reagents; D008562:Membrane Glycoproteins; D010710:Phosphates; D011509:Proteoglycans; D005690:Galactose