Isolation and structural characterization of the Leishmania donovani kinetoplastid membrane protein-11, a major immunoreactive membrane glycoprotein

Abstract

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.

The kinetoplastid membrane protein 11 of Leishmania donovani and African trypanosomes is a potent stimulator of T-lymphocyte proliferation

“…2, lanes 6 to 11). Amastigotes were shown previously to contain KMP-11 (20). A similar band was also detected in the African trypanosomes (Fig.…”

“…The isolation of the KMP-11 molecule from the LPG was achieved by reverse-phase chromatography as described elsewhere (22). The leishmania KMP-11 molecule is a 92-amino-acid surface membrane glycoprotein that is present at approximately 1 million copies per L. donovani LD3 promastigote (20,21).…”

A critical role for lipophosphoglycan in proinflammatory responses of dendritic cells to Leishmania mexicana

“…Tight association of LPG with other molecules on intact parasites, for example the geneB product [40] or KMP-11 [41], has been documented. Based on our results for L. mexicana, we favor the idea that LPG is a PAMP, because purified LPG induced DC maturation and, more relevant, LPG expression was necessary for parasite-induced maturation.…”

Kinetoplastid Membrane Protein-11 DNA Vaccination Induces Complete Protection against Both Pentavalent Antimonial-Sensitive and -Resistant Strains of Leishmania donovani That Correlates with Inducible Nitric Oxide Synthase Activity and IL-4 Generation: Evidence for Mixed Th1- and Th2-Like Responses in Visceral Leishmaniasis

“…KMP-11, a highly conserved surface membrane protein present in all members of the family Kinetoplastidae, is differentially expressed both in amastigote and promastigote forms of Leishmania (13)(14)(15). In a previous report, we have implicated an association of KMP-11 expression with the vaccine potential of an attenuated, avirulent Leishmania strain UR6 (16).…”