Membrane orientation of laminin binding protein.
Earlier we presented several lines of evidence that a 67-kDa laminin binding protein (LBP) in Leishmania donovani, that is different from the putative mammalian 67-kDa laminin receptor, may play an important role in the onset of leishmaniasis, as these parasites invade macrophages in various organs after migrating through the extracellular matrix. Here we describe the membrane orientation of this Leishmania laminin receptor. Flow cytometric analysis using anti-LBP Ig revealed its surface localization, which was further confirmed by enzymatic radiolabeling of Leishmania surface proteins, autoradiography and Western blotting. Efficient incorporation of LBP into artificial lipid bilayer, as well as its presence in the detergent phase after Triton X-114 membrane extraction, suggests that it may be an integral membrane protein. Limited trypsinization of intact parasite and subsequent immunoblotting of trypsin released material using laminin as primary probe revealed that a major part of this protein harbouring the laminin binding site is oriented extracellularly. Carboxypeptidase Y treatment of the whole cell, as well as the membrane preparation, revealed that a small part of the C-terminal is located in the cytosol. A 34-kDa transmembrane part of LBP could be identified using the photoactive probe, 3-(trifluoromethyl)-3-(m-iodophenyl)diazirine (TID). Partial sequence comparison of the intact protein to that with the trypsin-released fragment indicated that N-terminal may be located extracellularly. Together, these results suggest that LBP may be an integral membrane protein, having significant portion of N-terminal end as well as the laminin binding site oriented extracellularly, a membrane spanning domain and a C-terminal cytosolic end.
Bandyopadhyay
Keya
K
Molecular Cell Biology Laboratory, Indian Institute of Chemical Biology, Calcutta, India.
Karmakar
Sudipan
S
Biswas
Aruna
A
Das
Pijush K
PK
D000818:Animals; D001389:Azirines / Q000737:chemistry; D001665:Binding Sites; D002268:Carboxypeptidases / Q000378:metabolism; D005109:Extracellular Matrix / Q000737:chemistry* / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D007457:Iodine Radioisotopes; D007797:Laminin / Q000737:chemistry / Q000378:metabolism; D007893:Leishmania donovani / Q000737:chemistry* / Q000378:metabolism; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry; D019920:Photoaffinity Labels / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017474:Receptors, Laminin / Q000737:chemistry* / Q000378:metabolism*
D001389:Azirines; D007457:Iodine Radioisotopes; D007797:Laminin; D008081:Liposomes; D008565:Membrane Proteins; D010446:Peptide Fragments; D019920:Photoaffinity Labels; D017474:Receptors, Laminin; D011092:Polyethylene Glycols; C033174:3-(trifluoromethyl)-3-(3-iodophenyl)diazirine; D002268:Carboxypeptidases