This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Many parasites exhibit cell-surface glycans that interact with the host, helping to facilitate the entry of the parasite, e.g., by masquerading as host-derived species, contributing to adhesion, and causing cytotoxicity. We are currently studying such interactions of Giardia lamblia which infiltrates the intestine and causes diarrhea, Entamobea, which is harbored by up to 50% of the population worldwide (1-3% in US), is the main cause of amebic dysentery and liver abcesses in the developing world. Trichomonas vaginalis infection initiates a sexually transmitted disease that can cause sterility and cancer. Structural determinations of unique glycans represent opportunities for development of therapeutic drugs that will interfere with the parasite without producing side effects in the host. The infectious and diagnostic stage of Giardia lamblia (also known as G. intestinalis or G. duodenalis) is the cyst. The Giardia cyst wall contains fibrils of a unique ¿1,3-linked N-acetylgalactosamine (GalNAc) homopolymer and at least three cyst wall proteins (CWPs) composed of Leu-rich repeats (CWPLRR) and a C-terminal conserved Cys-rich region (CWPCRR). Our goals were to dissect the structure of the cyst wall and determine how it is disrupted during excystation. Our results showed that the Leu-rich repeat domain of CWP1 is a lectin that binds to curled fibrils of the GalNAc homopolymer. During excystation, host and Giardia proteases appear to degrade bound CWPs, exposing fibrils of the GalNAc homopolymer that are digested by a stage-specific glycohydrolase. Entamoeba histolytica, the protist that causes amebic dysentery and liver abscess, has a truncated Asn-linked glycan (N-glycan) precursor composed of seven sugars (Man5GlcNAc2). We showed that glycoproteins with unmodified N-glycans are aggregated and capped on the surfaceof E. histolytica trophozoites by the antiretroviral lectin cyanovirin-N and then replenished from large intracellular pools. Cyanovirin-N cocaps the Gal/GalNAc adherence lectin, as well as glycoproteins containing O-phosphodiester-linked glycans recognized by an anti-proteophosphoglycan monoclonal antibody. Cyanovirin-N inhibits phagocytosis by E. histolytica trophozoites of mucin-coated beads, a surrogate assay for amebic virulence. We enriched the secreted and membrane proteins for mass spectrometric identification, finding E. histolytica glycoproteins with occupied N-glycan sites including Gal/GalNAc lectins, proteases, and 17 previously hypothetical proteins. These and 50 previously hypothetical proteins may be vaccine targets as they are abundant and unique. Trichomonas vaginalis has on its surface a unique lipophosphoglycan (LPG) that plays a role in adherence and cytotoxicity to host cells. We are investigating the Asn-linked (N-Glycans) and O-phosphodiester-linked (O-P-glycans) glycans that modify Trichomonas proteins. Trichomonas N-glycans are built on a truncated precursor containing 5 mannose residues that are recognized by the anti-retroviral lectin cyanovirin-N. O-P-glycans form a linear polymer predominantly composed of glucose that is not digested with either amylase or dextranase. The plant lectin Concanavalin A dramatically enriches Trichomonas secreted and membrane glycoproteins that were identified by mass spectrometry after trypsin digestion. The "Con Aome" of Trichomonas contains ER proteins involved in N-glycan-dependent quality control of protein folding (calreticulin) and N-glycan-independent quality control of protein folding (BiP). The ConAome also contains lysosomal proteins including acid phosphatase and cysteine proteinase. Of great interest, the ConAome is dominated by unique transmembrane proteins and unique secreted proteins that may be novel targets for vaccination against this protist. In summary, these results show that Trichomonas glycoproteins have novel N-glycans and O-P-glycans and include many previously hypothetical proteins.