Interaction of fibrin(ogen) with leukocyte receptor alpha M beta 2 (Mac-1): further characterization and identification of a novel binding region within the central domain of the fibrinogen gamma-module.
The fibrinogen gamma-module sequences, gamma190-202 or P1, and gamma377-395 or P2, were implicated in interaction with the alpha(M)I-domain of the leukocyte receptor alpha(M)beta(2). P1 is an integral part of the gamma-module central domain, while P2 is inserted into this domain forming an antiparallel beta-strand with P1. We hypothesized earlier that separation of P2 from P1 may regulate interaction of fibrin(ogen) with leukocytes during the inflammatory response. To test the relative contributions of these sequences to the interaction and the effect of their separation, we prepared the recombinant gamma-module (gamma148-411) and its halves, gamma148-286 and gamma287-411 fragments containing P1 and P2, respectively, and evaluated their affinities for the recombinant alpha(M)I-domain. In a solid-phase binding assay, the immobilized gamma-module exhibited high affinity for alpha(M)I (K(d) = 22 nM), while the affinities of the isolated gamma148-286 and gamma287-411 halves were much lower (K(d)'s = 521 and 194 nM, respectively), indicating that both halves contribute to the interaction in a synergistic manner. This is consistent with the above hypothesis. Further, we prepared the recombinant gamma148-191 and gamma192-286 fragments corresponding to the NH(2)-terminal and central domains, respectively, as well as gamma148-226 containing P1, and tested their interaction with alpha(M)I. The immobilized gamma192-286 fragment bound to alpha(M)I with K(d) = 559 nM, while both gamma148-191 and gamma148-226 failed to bind suggesting that P1 does not contribute substantially to the binding and that the binding occurs mainly through the gamma227-286 region. To further localize a putative binding sequence, we cleaved gamma192-286 and analyzed the resulting peptides. The only alpha(M)I-binding activity was associated with the gamma228-253 peptide, indicating that this region of the central domain contains a novel alpha(M)beta(2)-binding sequence.
Yakovlev
Sergiy
S
University of Maryland School of Medicine, Rockville, Maryland 20855, USA.
Zhang
Li
L
Ugarova
Tatiana
T
Medved
Leonid
L
D020816:Amino Acid Motifs; D015153:Blotting, Western; D002448:Cell Adhesion; D002460:Cell Line; D005340:Fibrinogen / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014644:Genetic Variation; D006801:Humans; D008024:Ligands; D016177:Macrophage-1 Antigen / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011869:Radioligand Assay; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D020349:Surface Plasmon Resonance
D008024:Ligands; D016177:Macrophage-1 Antigen; D010446:Peptide Fragments; D021122:Protein Subunits; D011994:Recombinant Proteins; D005340:Fibrinogen