In order to study the glycans in the extracellular sporozoites and merozoites, it is crucial that they are of high purity, free from mosquito and human rbc debris respectively. The collection and purification of Pb sporozoites from infected mosquitoes were performed at the partner organization during the 6-month period. After dissecting hundreds of infected mosquitoes, we successfully obtained over 10 million Pb sporozoites with purity greater than 97% using state-of-the-art fluorescence-activated cell sorting (FACS) flow cytometry. However, due to the minute size of sporozoites, only less than 100 μg protein could be extracted and were insufficient for protein glycosylation study. Obtaining larger quantities proved to be challenging due to variations in mosquito infection rates, which were difficult to control and took months to stabilize. The alternate source from commercially available sporozoites was isolated using methods that did not produce sporozoites of high purity for glycan analysis. Instead we acquired Pf-infected and non-Pf-infected mosquito salivary glands for further proteomics analysis. Nevertheless, we have established the parameters using FACS to sort fluorescently-labelled sporozoites as previously known method (density gradient) used to separate sporozoites from mosquito debris did not result in such high purity. As for merozoites, using existing optimized methods in our laboratory, we successfully obtained highly purified and large quantities of tightly synchronized Pf merozoites. We also isolated sufficient quantities of other blood-stage parasites including rbc-infected rings, trophozoites, schizonts and gametocytes. As these samples inevitably contained rbc debris, uninfected rbcs were also collected simultaneously as controls. Using lectins, anti-α-gal (m86) and blood stage-specific antibodies, we investigated the protein glycosylation in the extracted protein samples. The results showed specific α-gal expression patterns, which appeared to relate to their growth cycle during the blood-stage development. They were not detected in the mature parasites (merozoites, schizonts and gametocytes) but was repeatedly observed in the young parasites that exist as rings and trophozoites in the rbcs. As it was not detected in the uninfected rbcs, we concluded that the α-gal epitopes were from the parasites and may be under specific developmental regulation. We further validated the results using α-galactosidase enzyme capable of removing α-gal structure from the proteins. The type of protein glycosylation was then determined using enzymatic or chemical reactions to study the glycan-protein linkage. The results indicated that these glycans were not likely to be O-linked or GPI-anchor protein glycosylation, while signals seemed to be slightly reduced with N-linked deglycosylation. In the late phase of the project, isolation of the glycan-bound protein(s) from the extracted protein mixture was performed using immunoprecipitation and m86 antibody and analysed using LC-MS/MS analysis. In addition to blood-stage parasites, comparative analysis of proteins obtained from Pf-infected and non-infected salivary glands of Anopheles mosquito was performed. Preliminary analysis identified a total of 1053 non-redundant proteins with 193 and 3 of those uniquely present in the infected and non-infected samples, respectively. Given the reported significance of α-gal in malaria, we are currently working with collaborators to gain further insights into the data obtained.
