Project Grant R01AI179950

PROTEIN GLYCOSYLATION AND TRAFFICKING IN PLASMODIUM FALCIPARUM - PROJECT SUMMARY IN ALL DOMAINS OF LIFE PROTEINS ARE MODIFIED VIA GLYCOSYLATION. THIS ADDITION OF SUGAR MOLECULES TO PROTEINS IS CRITICAL FOR THEIR FOLDING, FUNCTION, AND RECOGNITION BY THEIR PARTNERS. RIGOROUS STUDIES HAVE SHOWN THAT IN WELL-STUDIED EUKARYOTIC ORGANISMS, PROTEINS SYNTHESIZED IN THE ENDOPLASMIC RETICULUM (ER) ARE GLYCOSYLATED ON ASPARAGINE RESIDUES AS WELL AS ON SERINE OR THREONINE RESIDUES. THESE MODIFICATIONS ARE CRITICAL FOR THE TRAFFICKING OF PROTEINS IN THE SECRETORY PATHWAY. PROTEIN GLYCOSYLATION ACTS AS A MARKER FOR PROTEIN FOLDING TO ENSURE ONLY FOLDED AND FUNCTIONAL PROTEINS ARE SENT FROM THE ER. ONCE OUTSIDE THE ER, GLYCANS ON PROTEINS ALSO ACT AS CRITICAL RECOGNITION MOTIFS TO ROUTE PROTEINS ACCURATELY TO THEIR CORRECT SUBCELLULAR LOCATION. THESE GLYCAN-CENTRIC MECHANISMS ARE FOUND IN ALMOST ALL WELL STUDIED EUKARYOTES. SURPRISINGLY, THE INTRACELLULAR EUKARYOTIC PARASITE, PLASMODIUM FALCIPARUM, DOES NOT APPEAR TO UTILIZE ANY OF THESE CANONICAL PROTEIN GLYCOSYLATION-BASED MECHANISMS TO TRAFFIC PROTEINS IN ITS SECRETORY PATHWAY. THIS PARASITE CAUSES MALARIA, AND THE CLINICAL SYMPTOMS OF MALARIA ARE A DIRECT RESULT OF ITS GROWTH AND EXPANSION WITH HUMAN RED BLOOD CELLS (RBC). THE PARASITE DEPENDS UPON PROTEIN SECRETION TO ENSURE ITS SURVIVAL WITHIN THE RBC. EVEN THOUGH GLYCAN MODIFICATIONS ON PROTEINS ARE A MAJOR DRIVER OF IMMUNE RESPONSES, WE DO NOT KNOW IF AND HOW P. FALCIPARUM GLYCOSYLATES PROTEINS IN ITS SECRETORY PATHWAY DURING THE CLINICALLY RELEVANT ASEXUAL BLOOD STAGES. IN FACT, THE P. FALCIPARUM GENOME LACKS SEVERAL GENES IN THIS PATHWAY THAT ARE WELL CONSERVED IN OTHER EUKARYOTIC ORGANISMS, INCLUDING OTHER CLOSELY RELATED PARASITES. TO FILL THIS MAJOR GAP IN OUR KNOWLEDGE, WE WILL COMBINE GENETIC AND CELLULAR ASSAYS TO STUDY IF CONDITIONAL KNOCKDOWN OF THE TARGETED GLYCOSYLTRANSFERASES INHIBITS PROTEIN TRAFFICKING WITHIN THE HIGHLY BRANCHED AND UNCONVENTIONAL SECRETORY PATHWAY OF P. FALCIPARUM. OUR PRELIMINARY DATA SHOW THAT THE PARASITE PROTEIN GLYCOSYLATION PATHWAY GENES ARE ESSENTIAL FOR PARASITE GROWTH WITHIN THE RBC. THE EXPERIMENTAL APPROACH WILL TEST IF PROTEIN GLYCOSYLATION ALSO FUNCTIONS IN AN UNCONVENTIONAL PROTEIN QUALITY CONTROL PATHWAY IN THE PARASITE ER. WE WILL UTILIZE GLYCOPROTEOMIC APPROACHES TO DETERMINE THE LANDSCAPE OF PROTEIN GLYCOSYLATION DURING THE INTRAERYTHROCYTIC GROWTH OF P. FALCIPARUM. THE COLLABORATIVE RESEARCH TEAM WILL COMBINE THESE PROTEOMIC APPROACHES WITH GENETIC TOOLS TO DETERMINE WHICH GLYCOSYLTRANSFERASES ARE RESPONSIBLE FOR WHICH THE GLYCAN MODIFICATIONS. THE SPECIFIC GLYCOSYLTRANSFERASE ACTIVITIES WILL BE VALIDATED USING COMPLEMENTATION ASSAYS AS WELL AS IN VITRO ASSAYS USING RECOMBINANTLY EXPRESSED GLYCOSYLTRANSFERASE. THE TOOLS AND REAGENTS DEVELOPED FOR THIS PROJECT MAY FIND WIDER APPLICABILITY IN GLYCOBIOLOGY AND PARASITOLOGY. GIVEN THE IMPORTANCE OF GLYCANS IN DRIVING THE HOST IMMUNE SYSTEM, THE PROPOSED STUDIES MAY HAVE IMPORTANT IMPLICATIONS FOR OUR UNDERSTANDING OF THE ANTIMALARIAL IMMUNE RESPONSE.