Ultrastructural localization of giardins to the edges of disk microribbons of Giarida lamblia and the nucleotide and deduced protein sequence of alpha giardin

The giardins are a group of 29-38-kD proteins in the ventral disk of the protozoan parasite Giardia lamblia. The disk attaches the parasite to the host's intestinal epithelium and is composed of parallel, coiled microtubules that are adjacent to the ventral plasma membrane and from which processes called microribbons extend into the cytoplasm; the microribbons are connected by crossbridges. G. lamblia cytoskeletons, consisting of disks and attached flagella, were isolated and used to show that the 29-38-kD proteins separate into five bands by one- dimensional electrophoresis and into 23 species by two-dimensional analysis. Rabbit antibodies raised against a 33-kD protein band, purified by one-dimensional gel electrophoresis and shown to contain three proteins by two-dimensional electrophoresis, recognized 17 proteins by two-dimensional immunoblot analysis. By immunofluorescence these antibodies reacted with the ventral disk but not with the flagella in isolated cytoskeletons. Electron microscopy revealed that the anti-giardin antibodies bound to the edges of the microribbons but not to the microtubules, crossbridges, or other, nondisk structures. Antibodies to tubulin reacted with both the disk and flagella in isolated cytoskeletons but bound only to the microtubules in these structures. The amino-terminal sequence of the 33-kD immunogen was determined and used to construct a DNA oligomer, and the oligomer was used to isolate the alpha giardin gene. The gene was used to hybrid select RNA, and the in vitro translation product from this RNA was precipitated by the antibodies against the 33-kD immunogen. The gene sequence was a single open reading frame of 885 nucleotides that predicted a protein of 33.8 kD. The protein sequence is unique, having no significant homology to two other giardin sequences or to any sequences within the Protein Identification Resource. It is predicted to be 82% alpha helical. The downstream sequence of the gene indicates that the sequence AGT-PuAA is located six to nine nucleotides beyond the stop codon in all protein-encoding genes of G. lamblia that have been sequenced and reported to date.