LEISHMANIA-MAJOR - ASSOCIATION OF THE DIFFERENTIALLY EXPRESSED GENE B-PROTEIN AND THE SURFACE LIPOPHOSPHOGLYCAN AS REVEALED BY MEMBRANE CAPPING

The lipophosphoglycan (LPG) of Leishmania promastigotes forms a dense glycocalyx which effectively covers the entire surface of the cell, an d which undergoes structural modifications during the differentiation of promastigotes to the infective or metacyclic stage. Recently, the f irst protein marker for metacyclic promastigotes of Leishmania major h as been characterized. This protein, termed gene B protein, is located on the cell surface, yet it lacks any hydrophobic sequence for membra ne attachment. It does contain an unusual amino acid repeat that is re lated to the peptidoglycan binding domain of protein A from Staphyloco ccus aureus, suggesting that the protein might interact with metacycli c LPG via this domain for attachment to the cell. We have studied the distribution of LPG, gene B protein, and the major surface protease, g p63, by labeling them with immunogold or immunofluorescence prior to a nd during capping events. Thin sections of double-labeled parasites re vealed that the gene B protein-gold particles were colocalized with th e LPG-gold particles in the LPG capping structures at the extremities of the cell. Cocapping of LPG and gene B protein was also observed wit h two-color fluorescence. No similar redistribution was seen in gp63 o r with integral membrane proteins. In contrast to the gene B protein, gp63 could only be immunogold labeled on the metacyclic surface after capping and shedding of the LPG, providing further evidence that it an d other membrane-associated proteins are normally buried under the LPG coat. The unusual surface exposure of the gene B protein is consisten t with its hydrophilic and LPG binding properties, which allow it to b ecome incorporated into the cell coat and to localize to the most exte rnal aspects Of the cell. (C) 1994 Academic Press, Inc.