Isolation of a laminin-binding protein from the protozoan parasite Leishmania donovani that may mediate cell adhesion.
Extracellular matrix (ECM)-binding proteins on the surface of Leishmania are thought to play a crucial role in the onset of leishmaniasis, as these parasites invade mononuclear phagocytes in various organs after migrating through the ECM. In a previous report, we presented several lines of evidence suggesting that Leishmania has a specific receptor for laminin, a major ECM protein, with a Kd in the nanomolar range. Here we describe the identification, purification and biochemical characterization of the Leishmania laminin receptor. When the outer membrane proteins of L. donovani were blotted on to nitrocellulose paper and probed with laminin, a prominent laminin-binding protein of 67 kDa was identified. The purified protein was isolated by a three-step process involving DEAE-cellulose, Con A (concanavalin A)-Sepharose and laminin-Sepharose affinity chromatography and was used to raise a monospecific antibody. The same protein was obtained when parasite membrane extracts were adsorbed to antibody affinity matrix and eluted with glycine. The affinity-purified protein bound to laminin in a detergent-solubilized form as well as after integration into artificial bilayers, and was subsequently characterized as an integral membrane protein. Metaperiodate oxidation and metabolic inhibition of glycosylation studies indicate the binding protein to be glycoprotein in nature and that N-linked oligosaccharides play a part in the interaction of laminin with the binding protein. Surface-labelled parasites attached to microtitre wells coated with laminin and the 67 kDa protein blocked the adhesion to laminin substrate. We propose that the 67 kDa protein is an adhesin involved in the attachment of Leishmania to host tissues.
Ghosh
A
A
Molecular Cell Biology Laboratory, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Jadavpur, Calcutta 700 032, India.
Bandyopadhyay
K
K
Kole
L
L
Das
P K
PK
D000818:Animals; D002448:Cell Adhesion; D007797:Laminin / Q000378:metabolism / Q000502:physiology; D007893:Leishmania donovani / Q000737:chemistry* / Q000502:physiology; D008081:Liposomes; D011233:Precipitin Tests; D017474:Receptors, Laminin / Q000302:isolation & purification* / Q000378:metabolism / Q000502:physiology
D007797:Laminin; D008081:Liposomes; D017474:Receptors, Laminin