KOJO MENSA-WILMOT
Professor
Ph.D., Johns Hopkins University
RESEARCH
The protozoans Trypanosoma brucei and Leishmania major are parasites of humans. T. brucei causes trypanosomiasis in Africa, and Leishmania infections manifest as a range of symptoms from cutaneous lesions to visceral disease in tropical to subtropical regions of the world. Our laboratory is interested in three aspects of the biochemistry and molecular genetics of these parasites.
GLYCOSYLATED PHOSPHATIDYLINOSITOLS (GPIS): FUNCTIONS AND REGULATED CLEAVAGE BY A GPI-PHOSPHOLIPASE C IN TRYPANOSOMATIDS SPECIES-SPECIFICITY IN SIGNAL PEPTIDE SELECTION AT THE ENDOPLASMIC RETICULUM Signal sequences are believed to function universally across species, from prokaryotes to vertebrates. To our surprise, signal sequences from T. brucei and Leishmania hardly function at the endoplasmic reticulum of vertebrate microsomes. We are studying the biochemical mechanism(s) for rejection of kinetoplastid signal peptides by the mammalian translocation machinery. The difference in signal peptide design/function between vertebrates and animals represents a new lead for possible rationale drug design.
To address possible mechanisms of this species-specificity in signal sequence recognition, a variant surface glycoprotein (VSG117) from T. brucei has been studied. VSG 117 ribosome-nascent-chain complexes (RNC) are targeted to the canine ER membrane. However, docked VSG117-RNC displayed post-targeting defects in interactions with the canine translocon. Other studies reveal that VSGsp is "kinetically excluded" from import into the canine ER during co-translational translocation.
Other trypanosome signal peptides are being tested in order to examine the possibility that kinetic exclusion is a general phenomenon for conferring species-specificity in signal peptide selection. We are also cloning trypanosome homologs of the mammalian protein that displayed defective interactions with the kinetoplastid signal sequences.
We are exploring the mechanisms by which these LTEs function. We also are analyzing the 5' untranslated regions (UTRs) in the Leishmania genome, in order to learn about nucleotide usage patterns. From these, testable hypotheses have been formulated with regard to possible function of these cis-elements. The physiological significance, if any of the sequences will be tested experimentally. The knowledge gained from these experiments will be used to develop tools that will facilitate molecular genetic studies in Leishmania and possibly in other trypanosomatids.
Along these lines we have discovered and patented short sequences that promote protein synthesis in both eukaryotic and prokaryotes.
In the mammalian bloodstream, T. brucei is protected from complement-mediated lysis by a surface coat composed of about 10 million molecules of a variant surface glycoprotein (VSG). All VSGs are attached to the parasite plasma membrane by a GPI anchor. Through switching of the VSG coat (antigenic variation) the parasite population avoids total elimination, triggered by production of anti-VSG antibody, by the host immune system. Frequent switching of VSG possess a major obstacle for development of a vaccine against T. brucei. The polypeptide segment of each VSG is different and therefore not recognized by antibody made by the host against a previous VSG. However, the GPI anchor of VSG remains the same. Without a GPI anchor, VSG cannot be attached to the plasma membrane, and the parasites are susceptible to complement-mediated lysis. GPIs seem to be a reasonable target where antigenic variation of T. brucei could be interfered with.
One of our goals is to understand how T. brucei controls the biosynthesis and degradation of GPIs. Specifically, the parasite contains a GPI-phospholipase C (GPI-PLC), which releases most of the VSG from the parasite plasma membrane under appropriate circumstances. Not surprisingly, GPI-PLC is "quiescent" in T. brucei. Using related (kinetoplastid) protozoan parasites, Leishmania major and Trypanosoma cruzi, as models, we have demonstrated that heterologous expression of GPI-PLC can lead to a GPI deficiency, resulting in replication defects in both Leishmania and T. cruzi. GPIs are essential in amastigotes (intracellular mammalian stage) of both Leishmania and T. cruzi. We hypothesize that constitutive activation of the GPI-PLC in T. brucei will cause a GPI deficiency and produce a phenotype similar to that observed in Leishmania. We are interested in learning how T. bucei controls activity of GPI-PLC in vivo. Our recent work indicates that modification of GPI-PLC with lipid, and tetramerization of the protein in vivo might be major pathways for regulation of the enzyme activity in T. brucei. We are also testing a hypothesis that localization of GPI-PLC away from intracellular regions where GPIs are synthesized contributes to prevention of GPI cleavage in T. brucei.
Trypanosoma brucei (top) and mutant Trypanosoma brucei (bottom)
The endoplasmic reticulum (ER) of eukaryotes is the entry site into the exocytic pathway for secretory and most membrane proteins. Translocation therein normally requires an N-terminal signal peptide, signal recognition particle (SRP), SRP receptor (SR), and a translocon composed principally of the Sec61p complex.
LEISHMANIA GENE EXPRESSION: ROLE OF PROXIMAL 5' UNTRANSLATED SEQUENCES
Regulation of gene expression in Leishmania is predominantly a post-transcriptional phenomenon. Consequently, control of protein synthesis from mature mRNAs could be a major contributor to the gene expression program in these parasites. Cis-elements in mature mRNAs can influence protein synthesis in many biological families: No such sequences been identified in trypanosomatids. Using brute-force approaches we have found several short sequences, termed Leishmania Translation Enhancers (LTEs), that increase, by up to a thousand-fold, protein synthesis in the parasite. The critical regions for LTE activity appear to be nucleotides close to the initiation codon for translation of reporter genes.
| CONTACT INFORMATION |
| (706) 542-3355, mensawil@cb.uga.edu |
| SEE ALSO |
| A World of Sorrow and Hope," Franklin Chronicle Columns profile of Dr. Mensa-Wilmot Biomedical & Health Sciences Institute |
OF NOTE
SUPPORT STAFF
|
|
| Lane |
| NAME |
|
POSITION | |
E-MAIL |
|
||||
| Josh Duffy | |
Grad Student | |
duffy1@uga.edu | |
Frank Hardin | |
Grad Student | |
chardin@uga.edu |
| Ariel Lane | |
Grad Student | |
alane@uga.edu |
|
||||
REPRESENTATIVE PUBLICATIONS
Armah, D.A. and K. Mensa-Wilmot. 1999. "S-Myristoylation of a GPI—Phospholipase C in Trypanosoma brucei." J. Biol. Chem. 274: 5931-5959.Rashid, M. B., M. Russell, and K. Mensa-Wilmot. 1999. "Roles of Gln81 and Cys80 in catalysis by glycosylphosphatidylinositol-phospholipase C from Trypanosoma brucei." Eur. J. Biochem. 264: 914-920.
Teilhet, M., M.B. Rashid, A. Hwak, A. Al-Qahtani, K. and Mensa-Wilmot. 1998. "Effect of short 5' UTRs on protein synthesis in two biological kingdoms." Gene 222: 91-7.
Al-Qahtani, A., M. Teilhet, and K. Mensa-Wilmot. 1998. "Species-specificity in endoplasmic reticulum signal peptide utilization revealed by proteins from Trypanosoma brucei and Leishmania." Biochem. J. 331: 521-529.
Morris, J.C. and K. Mensa-Wilmot. 1997. "Role of 2,6-dideoxy-2,6-diaminoglucose in activation of a eukaryotic phospholipase C by aminoglycoside antibiotics." J. Biol. Chem. 272: 29554-29559.
Garg, N., R.L. Tarleton, and K. Mensa-Wilmot. 1997. "Proteins with glycosylphosphatidylinositol (GPI) signal sequences have divergent fates during a GPI deficiency: GPIs are essential for nuclear division in Trypanosoma cruzi." J. Biol. Chem. 272: 2482-12491.
Mensa-Wilmot, K., J.H. LeBowitz, K.P. Chang, A. Al-Qahtani, B.S. McGwire, S. Tucker, and J.C. Morris. 1994. "A glycosylphosphatidylinositol (GPI)-negative phenotype produced in Leishmania major by GPI phospholipase C from Trypanosoma brucei: Topography of two GPI pathways." J. Cell Biol. 124:935-947.