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Human
African Trypanosomiasis
Most eukaryotes possess cell surface proteins that are anchored to the plasma membrane by a covalently attached glycosylphosphatidylinositol. The GPI anchors contain either glycerolipids (diacylglycerol, alkylacylglycerol, or lysoacylglycerol) or ceramide lipid moieties. Trypanosomes live extracellularly in evading the immune response by a process known as antigenic variation. The variant antigen is the GPI anchored VSG, which coats the entire surface of Trypanosomes. After the biosynthesis of the precursor (glycolipid A') the GPI-glycolipid undergoes fatty acid remodeling reactions (Milne, 1999). Besides its structural function as an anchor, other roles have been attributed to GPI anchors, such as the promotion of lateral mobility of proteins in the bilayer and sorting signals for the transport to the apical plasma membrane in polarized cells (Renato, 2001--> Ferguson, 1997, 1999). The surface protein of T. brucei is known as the variant surface glycoprotein (VSG) which functions in immune evasion. This protein is linked to the plasma membrane by a GPI anchor that is unique in that its lipid moiety is exclusively myristate (dimyristoyl glycerol) (Morita, 2000). GPI anchors in the bloodstream form of T. brucei are unusual because the fatty acids in their lipid moiety are both myristate, added in the final stages of GPI biosynthesis in a remodeling reaction and are also present with out protein on the cell surface (Morita, 2000). The precursor for GPI is first synthesized with fatty acids that are longer than myristate, and these are sequentially replaced by myristate in fatty acid remodeling reactions that involving deacylation and reacylation with myristoyl-CoA as a donor. The product is a dimyristoylated GPI, known as glycolipid A, which is then attached to VSG (Morita, 2000). Glucosylphosphatidylinositol (GPI) anchors attach a large array of glycoproteins to the plasma membrane of protozoan parasites. All GPI anchors share a core glycan structure
made of mannose, glucosamine, and phosphatidylinositol with some
form of fatty acid anchor (figure 1, bottom left corner) (Garg,
1998). The core structure of T. brucei is specifically ethanolamine-P-Man3-GlcN-phosphatidylinositol
and is highly conserved (Werbovetz, 1996). This lipid moiety is
crucial as it allows a protein-membrane interaction with an otherwise
insoluble plasma membrane. The unique structure and morphology brought
with the use of GPI anchors may aid in the sorting of domains that
promote both lateral and trans-bilayer protein-protein interactions
crucial to cell function. The moiety specificity provides switches
that allow only certain protein conformations (Baumann, 2002). GPI is synthesized in the endoplasmic reticulum
(ER), transferred to the carboxy-terminal GPI attachment signal
sequence, then transported to the cell surface (Kinoshita, 2000).
Below is the classic model used for the ubiquitous biosynthesis
of GPI-anchor glycolipids. Notice the MAM (mitochondira associated
membrane) portion of the process. Bloodstream form of T. brucei
do not use their mitochondria during this stage in their life cycle.
The biosynthesis of GPI is initiated by GPI-N-acetylglucosaminyl transferase on the cytoplasmic surface of the endoplasmic reticulum as N-acetylglucosamine is transferred to phosphatidylinositol releasing uracil diphosphate. Next, the release of acetate mediated by glucosaminylphosphatidylinositol de-N-acetylase in mitochondria-associated membranes results in glucosaminylphosphatidylinositol . The position 2 of the inositol ring of this molecule is then acylated with acyltransferase using a Palmitoyl-CoA donor to generate GLN-acyl-PI, which is translocated to the luminal side of the ER mediated by a flippase and the first Mannose from the Dolichol-phosphate-mannose synthase is transferred to position 4 of glucosamine. The molecule exits the mitochondria-associated membrane and receives its second mannose at position 6 of the first mannose and third mannose at position 2 of the second mannose both using *1-6 and *1-2 mannosyl transferases respectively. The addition of ethanolaminephosphate to position 6 of the third mannose by phosphatidylethanolamine, links GPI to proteins from to position 6. Finally, the second mannose of this molecule can be modified to generate precursors of for the protein-bound anchors. This slightly modified version can also be found protein free on the cell surface (Kinoshita, 1999).
The pre-assembled GPI is transferred to proteins using a GPI-attachment signal peptide at the carboxyl terminus. After being translocation across the ER membrane, the GPI is attached by replacing the GPI-attachment signal peptide that is mediated by a transamidase that consists of at least 2 ER membrane proteins that form a complex that is required for the generation of a carbonyl-intermediate between the transamidase and a precursor protein. After attachment of the GPI to the proteins, the acyl group on the inositol ring is eliminated in the ER right after GPI transfer. The GPI anchored proteins are then transported from the ER to the cell surface using the Golgi. The bloodstream-form trypanosomes replace both fatty acyl chains of GPI with myristate before the addition to VSG in a remodeling reaction. This fatty acid synthase preferentially generates myristate.
The sn-1 and sn-2 fatty acids are removed and replace with myristate forming the VSG precursor, glycolipid A. Subsequently, there are myristate exchange reactions that serve as a form of proofreading to ensure that myristate is the only fatty acid used in the VSG GPI anchor. There are 2 free GPI species present in significant quantities. They are glycolipid A and glycolipid C. These tow species are in equilibrium via inositol acylation and deacylation reactions. Trypanosomes synthesize much higher levels of GPI than they need for anchoring VSG. There may exist a GPI catabolic pathway, which would prevent excessive accumulation of these molecules. The intermediate has properties that that hint that it may serve as an intermediate in GPI breakdown. (Milne, 1999) In Trypanosomes, myristate is the sole fatty acid that is used to remodel GPI anchors. It is preferentially incorporated into GPI's. Interestingly enough, Myristate is not abundant in the hosts' bloodstream and it was though that trypanosomes were unable to synthesize fatty acids. Bloodstream trypanosomes can synthesize fatty acids with the major product being myristate. Now it is known that African Trypanosomes are able to elongate fatty acids that are shorter than myristate (14:0) into myristate this specific fatty acid using fatty acid synthesis. For example, laurate (12:0) and octanoate (8:0) are all fatty acids that are shorter than myristate (Morita, 2000). The acyltransferases involved in the GPI remodeling were thought to be specific to myristate, but it was found that the remodeling acyltransferases, although they completely excluded fatty acids that were longer than the 14C myristate, they functioned on shorter FA’s such as laurate (12:0) and octanoate (8:0) (although these rates of incorporation were slower). These small FA’s are present in small amounts, but do not compete effectively with myristate. Especially since they would be synthesized using specialized FA synthesize that would elongate them to myristate prior to incorporation into GPI’s (Morita, 2000). The FA’s palpitate (16:0) and separate (18:0) are found in the greatest amounts in the human body or most mammals for that matter. Constituting the most abundant saturated FA in the diet and are highly used. Mammals have lost the ability to synthesize unsaturated FA with double bonds beyond the 9C, they must be provided for within the diet. All said, even though a remodeling transferase is permissive in FA lengthening, the myristate specificity in GPI anchors is very high. This specificity is achieved using myristoyl-A protein GPI transamidase then catalyzes GPI addition to protein (Morita, 2000). Fatty acid exchange occurs where VSG attached
GPI undergoes a regular exchange of myristate with myristate donated
by myristoyl-CoA (Morita, 2000).
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senior seminar at Earlham College |
