Membrane orientation of laminin binding protein

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Abstract
Earlier we presented several lines of evidence that a 67-kDa\ud laminin binding protein (LBP) in Leishmania donovani, that\ud is di.erent from the putative mammalian 67-kDa laminin\ud receptor, may play an important role in the onset of leishmaniasis,\ud as these parasites invade macrophages in various\ud organs after migrating through the extracellular matrix.\ud Here we describe the membrane orientation of this Leishmania\ud laminin receptor. Flow cytometric analysis using anti-\ud LBP Ig revealed its surface localization, which was further\ud con.rmed by enzymatic radiolabeling of Leishmania surface\ud proteins, autoradiography and Western blotting. E.cient\ud incorporation of LBP into arti.cial lipid bilayer, as well as its\ud presence in the detergent phase after Triton X-114 membrane\ud extraction, suggests that it may be an integral membrane\ud protein. Limited trypsinization of intact parasite and\ud subsequent immunoblotting of trypsin released material\ud using laminin as primary probe revealed that amajor part of\ud this protein harbouring the laminin binding site is oriented\ud extracellularly. Carboxypeptidase Y treatment of the whole\ud cell, as well as the membrane preparation, revealed that a\ud small part of the C-terminal is located in the cytosol. A\ud 34-kDa transmembrane part of LBP could be identi.ed\ud using the photoactive probe, 3-(tri.uoromethyl)-3-(m-iodophenyl)\ud diazirine (TID). Partial sequence comparison of the\ud intact protein to that with the trypsin-released fragment\ud indicated that N-terminal may be located extracellularly.\ud Together, these results suggest that LBP may be an integral\ud membrane protein, having signi.cant portion of N-terminal\ud end as well as the laminin binding site oriented extracellularly,\ud a membrane spanning domain and a C-terminal\ud cytosolic end.\ud \u