Heterogeneity in the Biosynthesis of Mucin O-Glycans from Trypanosoma cruzi Tulahuen Strain with the Expression of Novel Galactofuranosyl-Containing Oligosaccharides † Christopher Jones, ‡,§ Adriane R. Todeschini, ‡,| Orlando A. Agrellos, | Jose ´ O. Previato, | and Lucia Mendonc ¸ a-Previato* ,| National Institute for Biological Standards and Control, Potters Bar, Hertfordshire EN6 3QG, U.K., and Instituto de Biofisica Carlos Chagas Filho, UniVersidade Federal do Rio de Janeiro, 21949-900, Cidade UniVersita ´ ria, Rio de Janeiro, RJ, Brazil ReceiVed May 24, 2004; ReVised Manuscript ReceiVed July 22, 2004 ABSTRACT: Sialoglycoprotein from Trypanosoma cruzi strains participates in important biological functions in which the O-linked glycans play a pivotal role, and their structural diversity may be related to the parasite’s virulence pattern. To provide supporting evidence for this idea, we have determined the structure of novel linear and branched R-O-GlcNAc-linked oligosaccharides present on the mucins of the T. cruzi Tulahuen strain. The O-glycans were isolated as oligosaccharide alditols by reductive -elimination, purified, and characterized by nuclear magnetic resonance spectroscopy and methylation analysis. Two core families were synthesized by the parasite: the Galf1f4GlcNAc and Galp1f4GlcNAc. The Galf1f4GlcNAc core yields three series of O-chain structures. In the first, the Galf residue is nonsubstituted, while in the other series it is elongated by the activity of galactopyranosyl or galactofuranosyl transferases giving rise to Galp--(1f2)-Galf--(1f4) or Galf--(1f2)-Galf--(1f4) substructures not previously observed. The three series can arise by further galactopyranosylation of the GlcNAc O-6 arm. Sialylation was the only observed elaboration of the Galp1f4GlcNAc core family. Thus the determination of the structures of the O-glycans from T. cruzi Tulahuen mucins confirms the strain specificity of the glycosylation and predicts a relationship between it and parasite pathogenicity and the epidemiology of Chagas’ disease. Trypanosoma cruzi, the parasite responsible for Chagas’ disease, infects 18-20 million people in South and Central America (1). T. cruzi is a heterogeneous group of strains that establish infection in a wide range of mammalian hosts, exhibiting tropism for different tissue types (2), varying in the pathology and clinical manifestation of infection, and leading to death or serious damage to the heart or digestive tract during its chronic phase (3). The causes of this wide variability are not known. However, recently a correlation between the clinical variations and the genetic diversities of T. cruzi was proposed (4, 5). Several grouping schemes for T. cruzi strains have been developed in order to understand the role of parasite diversity in the pathogenesis of the disease (4). On the basis of biochemical and molecular studies, it has been observed that T. cruzi strains can be divided into two major groups (6-8), which have been recently standard- ized as T. cruzi I and T. cruzi II (9). Current biological and epidemiological studies provide evidence for an association of T. cruzi II with the domestic cycle, mainly involved in human infection, whereas T. cruzi I is associated with the sylvatic cycle, affecting marsupials and edentates (10), and rarely and asymptomatically infects humans (11). Presumably variability observed during infection by dif- ferent T. cruzi strains is a result of diversity in parasite/host interactions resulting from variability of the macromolecules expressed on both the parasite and host cell surface. T. cruzi is an intracellular parasite and must invade cells of the vertebrate host in order to replicate and liberate infective forms (trypomastigotes) to complete its life cycle. Specific T. cruzi surface sialoglycoproteins, known as mucin-like molecules, are implicated in the interaction of the parasite with host cells and modulation of the host immune system (12, 13). The protein expressed by T. cruzi mucin genes contains a short hypervariable N-terminal region, a threonine- (Thr-) 1 rich central domain where O-glycosylation occurs, and a C-terminus containing the GPI-anchor sequence. Recent data suggest that the T. cruzi mucins are stage- † This work was supported by grants from Conselho Nacional de Cie ˆncia e Tecnologia (CNPq), Programa Nu ´cleo de Excele ˆncia (PRONEX), Fundac ¸ a ˜o Carlos Chagas Filho de Amparo a ` Pesquisa do Estado do Rio de Janeiro (FAPERJ), and TWAS. The research of J.O.P. was supported in part by a fellowship from the John Simon Guggenheim Memorial Foundation. * To whom correspondence should be addressed. Telephone: 55 21 2562 6646. Fax: 55 21 2280 8193. E-mail: luciamp@biof.ufrj.br. ‡ C.J. and A.R.T. contributed equally to this work. § National Institute for Biological Standards and Control. | Universidade Federal do Rio de Janeiro. 1 Abbreviations: Thr, threonine; GPI, glycosylphosphatidylinositol; GalNAc, N-acetylgalactosamine; Ser, serine; GlcNAc, N-acetylglu- cosamine; Galf, galactofuranose; Galp, galactopyranose; Neu5Ac, N-acetylneuraminic acid; SDS-PAGE, sodium dodecyl sulfate- polyacrylamide gel electrophoresis; TLC, thin-layer chromatography; HPLC, high-pressure liquid chromatography; PGC, porous graphitic carbon; GC, gas-liquid chromatography; NMR, nuclear magnetic resonance spectroscopy; Man, mannose; Glc, glucose; Ins, inositol; NOE, nuclear Overhauser enhancement; GlcNAc-ol, N-acetylglu- cosaminitol; ManNAc-ol, N-acetylmannosaminitol; HexNAc-ol, N- acetylhexosaminitol; ROESY, rotating frame NOE spectroscopy; TOCSY, total correlation spectroscopy. 11889 Biochemistry 2004, 43, 11889-11897 10.1021/bi048942u CCC: $27.50 © 2004 American Chemical Society Published on Web 08/26/2004