title: Trypanosoma cruzi GP63 proteins undergo stage-specific differential posttranslational modification and are important for host cell infection.
abstract: The protozoan Trypanosoma cruzi expresses multiple isoforms of the GP63 family of metalloproteases. Polyclonal antiserum against recombinant GP63 of T. cruzi (TcGP63) was used to study TcGP63 expression and localization in this organism. Western blot analysis revealed that TcGP63 is 61 kDa in epimastigotes, amastigotes, and tissue culture-derived trypomastigotes but 55 kDa in metacyclic trypomastigotes. Antiserum specific for Leishmania amazonensis GP63 specifically reacted with a 55-kDa TcGP63 form in metacyclic trypomastigotes, suggesting stage-specific expression of different isoforms. Surface biotinylation and endoglycosidase digestion experiments showed that TcGP63 is an ecto-glycoprotein in epimastigotes but is intracellular and lacking in N-linked glycans in metacyclic trypomastigotes. Immunofluorescence microscopy showed that TcGP63 is localized on the surfaces of epimastigotes but distributed intracellularly in metacyclic trypomastigotes. TcGP63 is soluble in cold Triton X-100, in contrast to Leishmania GP63, which is detergent resistant in this medium, suggesting that GP63 is not raft associated in T. cruzi. Western blot comparison of our antiserum to a previously described anti-peptide TcGP63 antiserum indicates that each antiserum recognizes distinct TcGP63 proteins. Preincubation of trypomastigotes with either TcGP63 antiserum or a purified TcGP63 C-terminal subfragment reduced infection of host myoblasts. These results show that TcGP63 is expressed at all life stages and that individual isoforms play a role in host cell infection.
authors.affiliation: Division of Infectious Diseases and Center for Microbial Interface Biology, The Ohio State University, Biomedical Research Tower, Rm. 1012, 460 W. 12th Ave., Columbus, OH 43210-1240, USA.
authors.forename: Manjusha M
authors.initials: MM
authors.lastname: Kulkarni
authors.forename: Cheryl L
authors.initials: CL
authors.lastname: Olson
authors.forename: David M
authors.initials: DM
authors.lastname: Engman
authors.forename: Bradford S
authors.initials: BS
authors.lastname: McGwire
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000737:chemistry; D003600:Cytosol / Q000737:chemistry; D045726:Metalloproteases / Q000737:chemistry / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008970:Molecular Weight; D011499:Protein Processing, Post-Translational; D015800:Protozoan Proteins / Q000737:chemistry / Q000378:metabolism*; D014349:Trypanosoma cruzi / Q000737:chemistry / Q000502:physiology*; D037521:Virulence Factors / Q000737:chemistry / Q000378:metabolism*
chemical_list: D015800:Protozoan Proteins; D037521:Virulence Factors; D045726:Metalloproteases