The outer surface of the Giardia lamblia trophozoite is covered with one of a group of extraordinarily (approximately 12%)cysteine-rich variable surface proteins (VSPs). We have cloned, expressed, and begun to characterize the first complete VSP gene, TSA 417. Questions of how nascent proteins fold and form the proper S-S bonds are at the forefront of modern cell biology. With up to 41 S-S bonds buried within a single chain, TSA 417 presents a formidable topological challenge. It is an ideal substrate for studies of basic mechanisms of protein folding and S-S bond formation and their relationship to transport and function of a major surface protein. Delineating the pathway of TSA 417 folding, its quaternary structure, and its transport to the cell surface may yield valuable insights into its protease-resistant structure and its function.
The Specific Aims are:
Aim A: To characterize the kinetics and basic cell biologic pathways of the synthesis, S-S bond formation, and transport of TSA 417 to the cell surface to probe the relationship between these key functions.
Aim B: To evaluate the quaternary structure and functions of TSA 417 by asking: [1] Is native TSA 417 non-covalently bound to itself or other proteins on the cell surface and is correct folding necessary for oligomerization? [2] What is the native shape and overall structure of TSA 417 and its relationship to the lipid membrane? [3] Does TSA 417 have specific post-translational modifications? [4] Does it bind calcium or other metal ions? Aim C: To probe the native structure of TSA 417 by mapping sites of proteolytic cleavage by N-terminal sequencing. Defining the sites of protease susceptibility will give key insights into the secondary and tertiary structure of TSA 417.
Aim D: To determine the effects of blocking transport of TSA 417 or of removing defined segments of it on trophozoite: [1] survival or growth; [2] adherence; [3] osmotic sensitivity; [4] uncovering other proteins; [5] flow of lipids to the plasma membrane, [6] ultrastructure. This will test the hypothesis that a major function of TSA 417 is to protect the cell surface and will give us valuable insights into its importance.
Aim E: To localize two putative protein disulfide isomerases within the giardial cell and evaluate their roles in folding of TSA 417.
Aim F: To identify the vesicular carriers that transport TSA 417 to the plasmalemma and determine if it is normally released from the surface or internalized. These studies will yield important new information on conserved features of the structure and functions of the VSPs that cover the G. lamblia trophozoite. They may give valuable clues to the evolution of folding and transport of surface proteins with cysteine-rich domains. They will have profound implications for understanding the basic biology of Giardia and its interactions with the environment.
