Giardia lamblia: post-translational processing and status of exposed cysteine residues in TSA 417, a variable surface antigen.
Giardia lamblia develops and thrives within the harsh and variable environment of the human small intestine. To survive this environment, Giardia has evolved a unique family of antigenically variable, extremely cysteine-rich surface proteins. We have characterized the expression of one of these antigens, TSA 417, at the gene and protein levels. TSA 417 mRNA and protein were expressed at constant levels during both logarithmic growth and encystation. On the trophozoite surface, TSA 417 exists in two forms, an 85-kDa protein and a derived 66-kDa form. Both species have identical amino-terminal sequences that match the translated sequence of TSA 417 after removal of a predicted 17 amino acid signal peptide. Since TSA 417 is highly cysteine rich (ca. 12 mole%), we asked whether it was complexed with itself or with other molecules. Although we found no interchain disulfide bonds, there was substantial intrachain bonding that helped retain the gross structure of isolated TSA 417 after partial trypsin digestion. Because the hydropathy profile of TSA 417 suggested that most of the cysteine and all 29 of its conserved CXXC motifs are on the cell surface, we investigated whether TSA 417 might contain some of the free thiols previously shown to be on the trophozoite surface (Gillin et al., Molecular and Biochemical Parasitology, 13, 1-12, 1984). However, no free thiols were detected, either exposed on native TSA 417 or in the unfolded protein. The absence of free thiols and resistance to proteolytic digestion suggest that most of the cysteine residues are in intrachain bridges, probably either disulfide bonds or cysteine-metal complexes. This internal crosslinking may help explain the resistance of Giardia to proteases and other degradative enzymes in the intestinal fluid.
Aley
S B
SB
Department of Pathology, University of California San Diego Medical Center 92103-8416.
Gillin
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FD
D000595:Amino Acid Sequence; D000818:Animals; D000953:Antigens, Protozoan / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000954:Antigens, Surface / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015152:Blotting, Northern; D015139:Blotting, Southern; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D016054:DNA, Protozoan / Q000032:analysis; D015870:Gene Expression; D016829:Giardia lamblia / Q000235:genetics / Q000276:immunology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011499:Protein Processing, Post-Translational; D015800:Protozoan Proteins; D012333:RNA, Messenger / Q000096:biosynthesis; D016053:RNA, Protozoan / Q000096:biosynthesis; D014357:Trypsin / Q000378:metabolism
D000953:Antigens, Protozoan; D000954:Antigens, Surface; D016054:DNA, Protozoan; D015800:Protozoan Proteins; D012333:RNA, Messenger; D016053:RNA, Protozoan; C081832:tsa417 protein, Giardia lamblia; D014357:Trypsin; D003545:Cysteine