Isolation and structural characterization of the Leishmania donovani kinetoplastid membrane protein-11, a major immunoreactive membrane glycoprotein.
A novel membrane molecule, previously observed to be co-isolated with lipophosphoglycan and called lipophosphoglycan-associated protein, has been detected in Leishmania donovani promastigotes and amastigotes. This kinetoplastid membrane protein (KMP-11) has been purified by preparative SDS/PAGE after organic solvent extraction of promastigote membranes. Isoelectric-focusing experiments indicated that this was an acidic protein with an isoelectric point of 4.8. Immunoblot analysis of subcellular fractions, together with 125I-labelling experiments, showed this molecule to be associated with the promastigote cell surface membrane. KMP-11 was expressed at a copy number similar to that of lipophosphoglycan (1 x 10(6)-2 x 10(6) molecules per cell), making this glycoprotein one of the major features on the parasite cell surface. The primary structure, less a blocked N-terminal region, was determined by automated Edman degradation of peptides derived from CNBr or enzymic fragmentation. Several post-translational modifications were also found during these studies, including an O-linked oligosaccharide and an NG-monomethylarginine functionality which was verified by m.s. Finally, a set of sequential synthetic peptides was made based on the established partial sequence allowing structural determination of two distinct antibody-binding sites for the monoclonal antibodies L98 and L157.
Jardim
A
A
Department of Biochemistry and Microbiology, University of Victoria, B.C. Canada.
Funk
V
V
Caprioli
R M
RM
Olafson
R W
RW
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001120:Arginine / Q000032:analysis; D018604:Epitope Mapping; D006031:Glycosylation; D007893:Leishmania donovani / Q000737:chemistry* / Q000254:growth & development / Q000276:immunology*; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D015800:Protozoan Proteins / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification*; D013347:Subcellular Fractions / Q000737:chemistry
D000911:Antibodies, Monoclonal; D008562:Membrane Glycoproteins; D015800:Protozoan Proteins; C090009:kinetoplastid membrane protein 11, Leishmania donovani; D001120:Arginine