Structural and antigenic characterization of a species- and promastigote-specific Leishmania mexicana amazonensis membrane protein.
A Leishmania mexicana amazonensis promastigote membrane glycoprotein (Mr 46,000) expressing the species-specific and promastigote-specific epitope of monoclonal antibody IX 2H7-E10(M-2) has been purified to homogeneity, and studies have been made to determine the minimum peptide fragment that retained antigenic activity. Peptide mapping experiments performed with the metabolically labeled or surface radioiodinated protein illustrated its highly folded nature and marked resistance to proteolytic digestion. The M-2 epitope was readily destroyed by limited proteolysis and/or reduction and alkylation, indicating disulfide bond involvement in its formation by at least the secondary protein structure. The stability of approximately half of the molecular mass of the protein (46kDa/M-2) was also dependent on disulfide bonding. Enzymic digests under various conditions generated a glycopolypeptide (Mr 22,000 to 27,000), extremely resistant to further enzymic digestion, that was the dominant immunogenic portion of the purified protein recognized by a specific rabbit heteroserum. No smaller or larger fragments were antigenic. Data obtained by using the radioiodinated hydrophobic probe 3-(trifluoromethyl)-3-([m-125]iodophenyl)-diazirine ([125I]TID) indicate that 46kDa/M-2 is an integral membrane protein with a component polypeptide (Mr 23,000 to 27,000), highly resistant to further enzymic cleavage and containing sequences within the external promastigote membrane. Data indicate that the [125I]TID-labeled fragment is identical to the immunodominant fragment. We suggest that hydrophobic interactions maintain the integrity of this fragment as amino acids within it fold through the parasite membrane.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000953:Antigens, Protozoan / Q000032:analysis*; D000954:Antigens, Surface / Q000276:immunology; D002462:Cell Membrane / Q000276:immunology / Q000648:ultrastructure; D006023:Glycoproteins / Q000276:immunology; D007163:Immunosorbent Techniques; D007894:Leishmania mexicana / Q000032:analysis* / Q000276:immunology; D008565:Membrane Proteins / Q000276:immunology*; D008970:Molecular Weight; D010449:Peptide Mapping
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D000953:Antigens, Protozoan; D000954:Antigens, Surface; D006023:Glycoproteins; D008565:Membrane Proteins