CYST WALL ENDOPROTEASES AND GLYCANS OF PARASITES

Boston University Medical Campus, Boston MA

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. N-Glycans of Entamoeba histolytica, the protist that causes amebic dysentery and liver abscess, are of great interest for multiple reasons. E. histolytica makes an unusual truncated N-glycan precursor (Man(5)GlcNAc(2)), has few nucleotide sugar transporters, and has a surface that is capped by the lectin concanavalin A. Here, biochemical and mass spectrometric methods were used to examine N-glycan biosynthesis and the final N-glycans of E. histolytica with the following conclusions. Unprocessed Man(5)GlcNAc(2), which is the most abundant E. histolytica N-glycan, is aggregated into caps on the surface of E. histolytica by the N-glycan-specific, anti-retroviral lectin cyanovirin-N. Glc(1)Man(5)GlcNAc(2), which is made by a UDP-Glc: glycoprotein glucosyltransferase that is part of a conserved N-glycan-dependent endoplasmic reticulum quality control system for protein folding, is also present in mature N-glycans. A swainsonine-sensitive alpha-mannosidase trims some N-glycans to biantennary Man(3)GlcNAc(2). Complex N-glycans of E. histolytica are made by the addition of alpha1,2-linked Gal to both arms of small oligomannose glycans, and Gal residues are capped by one or more Glc. In summary, E. histolytica N-glycans include unprocessed Man(5)GlcNAc(2), which is a target for cyanovirin-N, as well as unique, complex N-glycans containing Gal and Glc. Entamoeba histolytica trophozoites are covered by proteophosphoglycans (PPGs), which are the target of anti-carbohydrate monoclonal antibodies. These anti-PPG antibodies confer resistance to Entamoeba infections in passive transfer experiments. The carbohydrate portion of the PPG consists of a polymer of alpha-1,6-linked glucose, which is linked by a phosphate group to hydroxyls on Ser or Thr. However, the protein back-bone of the PPGs has not been characterized. Biochemical and mass spectrometric methods were used to understand the binding of the monoclonal antibody on the surface of the parasite, to identify proteic backbones and to characterize PPgs glycan portion, with the following conclusions. By showing that anti-amebic antibody also reacts with bacterial dextran, that enzymatic incubation with dextranase inhibits binding of the anti-dextran antibody to blots of Concanavalin A-enriched glycoproteins of Entamoeba, we proved that PPG glycans behave like bacterial dextran. Thanks to a TFA treatment/muti-dimesional chromatography based method we then identified 7 putative candidates for specific anti-amebic antibody. Structural investigations on the PPGs glycan component are still in progress by means of high resolution mass spectrometry. We are also interested in studying the glycoproteins of Trichomonas vaginalis, the only medically important parasite that is sexually transmitted. Trichomonas has a surface lipophosphoglycan (LPG) that contains rhamnose, xylose, GlcNAc, and Gal. Other glycans of Trichomonas that are the focus of the studies here are Asn-linked glycans (N-glycans) and O-phosphodiester-linked glycans (O-P-glycans). We have now shown that the unmodified N-glycan of Trichomonas that contains 5 mannose rather than 9 mannose is recognized by the anti-retroviral lectin cyanovirin-N. We used mass spectrometric techniques to identify complex N-glycans of Trichomonas and the glycoproteins that contain the N-glycans. We have identified a novel O-P-glycan of Trichomonas that is a polymer of glucose with a small amount of mannose. The Trichomonas O-P-glycan does not digest with dextranase or amylase and so may represent a novel structure. Future studies will determine the structures of the Trichomonas complex N-glycans and O-P-glycans and determine whether either or both are immunogenic, and so may be vaccine candidates.