Abstract
ADAMs (a disintegrin and metalloproteinases) are a recently discovered gene family of multifunctional proteins with the disintegrin-like and metalloproteinase domains. To analyze the biological functions of ADAM28, we screened binding molecules to secreted-type ADAM28 (ADAM28s) by the yeast two-hybrid system and identified P-selectin glycoprotein ligand-1 (PSGL-1). Binding between the disintegrin-like domain of ADAM28s and the extracellular portion of PSGL-1 was determined by yeast two-hybrid assays, binding assays of the domain-specific recombinant ADAM28s species using PSGL-1 stable transfectants and leukocyte cell lines expressing native PSGL-1 (HL-60 cells and Jurkat cells), and co-immunolocalization and co-immunoprecipitation of the molecules in these cells. Incubation of HL-60 cells with recombinant ADAM28s enhanced the binding to P-selectin-coated wells and P-selectin-expressing endothelial cells. In addition, intravenous injection of ADAM28s-treated HL-60 cells increased their accumulation in the pulmonary microcirculation and alveolar spaces in a mouse model of endotoxin-induced inflammation. These data suggest a novel function that ADAM28s promotes PSGL-1/P-selectin-mediated leukocyte rolling adhesion to endothelial cells and subsequent infiltration into tissue spaces through interaction with PSGL-1 on leukocytes under inflammatory conditions.
Highlights
ADAMs) are composed of propeptide, metalloproteinase, disintegrin-like, cysteine-rich, epidermal growth factor (EGF)like, transmembrane and cytoplasmic domains [4, 5]
Binding of ADAM28s to the Extracellular Decamer Repeats of P-selectin glycoprotein ligand-1 (PSGL-1)—We examined the region of PSGL-1 that interacts with ADAM28s by inhibition studies using three different antiPSGL-1 Abs, which recognize the tyrosine sulfation consensus sequence motif of the receptor-binding domain (KPL1 monoclonal antibody (mAb)), to HL-60 and Jurkat cells was significantly inhibited by incubation of the cells with H-300 Ab in a dosedependent manner, but the other two Abs (KPL1 mAb and PL1 mAb), nonimmune mouse IgG or nonimmune rabbit IgG, had no such effect (Fig. 3, B–D and data not shown for Jurkat cells)
We provided the first evidence that ADAM28s binds to PSGL-1 through interaction between the Dis domain of ADAM28s and the extracellular portion of PSGL-1, and that this interaction on cell surfaces enhances PSGL-1/P-selectinmediated leukocyte adhesion to endothelial cells and subsequent transendothelial migration in vitro and in vivo
Summary
5Ј-GGAATTCCATATGTGTGGGAACCAGTTGGTG-3Ј Reverse, 5Ј-CCGGAATTCTCATCTGAAATGATTTTCCTTCG-3Ј Forward, 5Ј-GGAATTCCATATGTGTGGGAACCAGTTGGTG-3Ј Reverse, 5Ј-CCGGAATTGAGGGAAGCCATTGACTTGG-3Ј Forward, 5Ј-GGAATTCCATATGTGCCATCACGGGAAGG-3Ј Reverse, 5Ј-CCGGAATTCTGGTCCCCACAGCTCTGTG-3Ј Forward, 5Ј-GGAATTCCATATGGGTAGGAGGACAAATCCTTTCC-3Ј Reverse, 5Ј-CCGGAATTCAGCAACCCTAAAACCAACCTC-3Ј Forward, 5Ј-CCGGAATTCGTGCCATGCCTCTGCAAC-3Ј Reverse, 5Ј-ACGCGTCGACAGGGAGGAAGCTGTGCAGG-3Ј Forward, 5Ј-GGAATTCCCCAGCATGTTGCAAGGTCTC-3Ј Reverse, 5Ј-ACGCGTCGACAATTGGAGTGGATATGATATCTGTAGG-3Ј. 5Ј-GGAATTCCCCAGCATGTTGCAAGGTCTC-3Ј Reverse, 5Ј-ACGCAGATCTAGCACTTACTGCAACAGAGAG-3Ј Forward, 5Ј-ACGCAGATCTATTTGTGGGAACCAGTTGG-3Ј Reverse, 5Ј-ACGCGTCGACTCTGAAATGATTTTCCTTCGC-3Ј cell proliferation. Little is known about the biological functions of human ADAM28 and their molecular mechanisms under pathophysiological conditions. We screened ADAM28s-interacting proteins by the yeast two-hybrid system and identified P-selectin glycoprotein ligand-1 (PSGL-1). Our data provide the evidence that the binding between the disintegrin-like domain of ADAM28s and the extracellular portion of PSGL-1 enhances PSGL-1/P-selectin-mediated cell adhesion to endothelial cells in vitro and the interaction promotes accumulation of PSGL-1-expressing cells in the lung microcirculation and alveolar spaces in a mouse model of endotoxin-induced inflammation. We propose a novel pathway by which ADAM28s is involved in the promotion of leukocyte rolling adhesion to blood vessel endothelial cells and the subsequent migration into tissue spaces under inflammatory conditions
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