Leukocyte migration to sites of inflammation is regulated by several endothelial adhesion molecules. Vascular adhesion protein-1 (VAP-1) is unique among the homing-associated molecules as it is both an enzyme that oxidizes primary amines and an adhesin. Although granulocytes can bind to endothelium via a VAP-1-dependent manner, the counter-receptor(s) on this leukocyte population is(are) not known. Here we used a phage display approach and identified Siglec-9 as a candidate ligand on granulocytes. The binding between Siglec-9 and VAP-1 was confirmed by in vitro and ex vivo adhesion assays. The interaction sites between VAP-1 and Siglec-9 were identified by molecular modeling and confirmed by further binding assays with mutated proteins. Although the binding takes place in the enzymatic groove of VAP-1, it is only partially dependent on the enzymatic activity of VAP-1. In positron emission tomography, the 68 Gallium-labeled peptide of Siglec-9 specifically detected VAP-1 in vasculature at sites of inflammation and cancer. Thus, the peptide binding to the enzymatic groove of VAP-1 can be used for imaging conditions, such as inflammation and cancer. (Blood. 2011;118(13):3725-3733) IntroductionLeukocyte migration from the blood into the nonlymphoid tissues is a hallmark of inflammation. Several molecules on the endothelial cell surface and their counter-receptors on leukocytes mediate a multistep adhesion cascade featuring tethering, rolling, activation, adhesion, crawling, and transmigration phases. 1,2 Vascular adhesion protein-1 (VAP-1/AOC3) is an endothelial cell molecule that is rapidly translocated from the intracellular storage granules to the endothelial cell surface on inflammation. It contributes to several steps in the extravasation cascade and controls trafficking of lymphocytes, granulocytes, and monocytes to sites of inflammation. VAP-1 has unique features distinct from other conventional adhesion molecules because, besides being an adhesin, it is also an enzyme. It catalyzes oxidative deamination of primary amines and produces hydrogen peroxide, aldehyde, and ammonium. 3 The end products of the enzymatic activity are highly potent inflammatory mediators and can up-regulate other adhesion molecules, such as E-and P-selectin, ICAM-1, and VCAM-1. 4,5 We recently found the first lymphocyte ligand for VAP-1, Siglec-10. 6 It is expressed on B cells, monocytes, and eosinophils but is absent from granulocytes. 7 However, VAP-1 is also involved in granulocyte migration to sites of inflammation. This has been demonstrated in studies with acute inflammation models (peritonitis, lung, and air pouch inflammation) in mouse. In these studies, significant reduction in granulocyte migration to sites of inflammation was obtained with a function blocking anti-VAP-1 antibody and a small molecular inhibitor against VAP-1. [8][9][10] Contribution of VAP-1 both at the rolling and transmigration steps during leukocyte extravasation has been demonstrated, and the enzymatic activity of VAP-1 seems to be important in these proc...

Semicarbazide-sensitive amine oxidases (SSAOs) catalyze oxidative deamination of primary amines, but the true physiological function of these enzymes is still poorly understood. Here, we have studied the functional and structural characteristics of a human cell-surface SSAO, AOC2, which is homologous to the better characterized family member, AOC3. The preferred in vitro substrates of AOC2 were found to be 2-phenylethylamine, tryptamine and p-tyramine instead of methylamine and benzylamine, the favored substrates of AOC3. Molecular modeling suggested structural differences between AOC2 and AOC3, which provide AOC2 with the capability to use the larger monoamines as substrates. Even though AOC2 mRNA was expressed in many tissues, the only tissues with detectable AOC2-like enzyme activity were found in the eye. Characterization of AOC2 will help in evaluating the contribution of this enzyme to the pathological processes attributed to the SSAO activity and in designing specific inhibitors for the individual members of the SSAO family.

The Yersinia adhesin YadA mediates the adhesion of the human enteropathogen Yersinia enterocolitica to collagens and other components of the extracellular matrix. Though YadA has been proposed to bind to a specific site in collagens, the exact binding determinants for YadA in native collagen have not previously been elucidated. We investigated the binding of YadA to collagen Toolkits, which are libraries of triple-helical peptides spanning the sequences of type II and III human collagens. YadA bound to many of them, in particular to peptides rich in hydroxyproline but with few charged residues. We were able to block the binding of YadA to collagen type IV with the triple-helical peptide (Pro-Hyp-Gly) 10 , suggesting that the same site in YadA binds to triple-helical regions in network-forming collagens as well. We showed that a single Gly-Pro-Hyp triplet in a triple-helical peptide was sufficient to support YadA binding, but more than six triplets were required to form a tight YadA binding site. This is significantly longer than the case for eukaryotic collagenbinding proteins. YadA-expressing bacteria bound promiscuously to Toolkit peptides. Promiscuous binding could be advantageous for pathogenicity in Y. enterocolitica and, indeed, for other pathogenic bacteria. Many of the tightly binding peptides are also targets for eukaryotic collagen-binding proteins, and YadA was able to inhibit the interaction between selected Toolkit peptides and platelets. This leads to the intriguing possibility that YadA may interfere in vivo with host processes mediated by endogenous collagen-binding proteins.

Human membrane primary amine oxidase (hAOC3; also known as vascular adhesion protein-1, VAP-1) is expressed upon inflammation in most tissues, where its enzymatic activity plays a crucial role in leukocyte trafficking. We have determined two new structures of a soluble, proteolytically cleaved form of hAOC3 (sAOC3), which was extracted from human plasma. In the 2.6 Å sAOC3 structure, an imidazole molecule is hydrogen bonded to the topaquinone (TPQ) cofactor, which is in an inactive on-copper conformation, while in the 2.95 Å structure, an imidazole molecule is covalently bound to the active off-copper conformation of TPQ. A second imidazole bound by Tyr394 and Thr212 was identified in the substrate channel. We furthermore demonstrated that imidazole has an inhibitory role at high concentrations used in crystallization. A triple mutant (Met211Val/Tyr394Asn/Leu469Gly) of hAOC3 was previously reported to change substrate preferences toward those of hAOC2, another human copper-containing monoamine oxidase. We now mutated these three residues and Thr212 individually to study their distinct role in the substrate specificity of hAOC3. Using enzyme activity assays, the effect of the four single mutations was tested with four different substrates (methylamine, benzylamine, 2-phenylethylamine, and p-tyramine), and their binding modes were predicted by docking studies. As a result, Met211 and Leu469 were shown to be key residues for substrate specificity. The native structures of sAOC3 and the mutational data presented in this study will aid the design of hAOC3 specific inhibitors.

The Yersinia adhesin A (YadA) is a collagen-binding trimeric autotransporter of Yersinia enterocolitica, an enteropathogen that causes a range of gastroenteric and systemic diseases, and YadA is essential for Y. enterocolitica virulence. Although previous studies suggest a specific binding site in collagen for YadA, we found that recombinant YadA binds to both major cyanogen bromide fragments of collagen type II and the collagen-like model peptide (Pro-Hyp-Gly)(10) [(POG)(10)]. To further characterise the YadA-collagen interaction, we investigated the binding of YadA to (POG)(10) and three other model peptides, (Pro-Pro-Gly)(10) which lacks the hydroxyl groups of (POG)(10), T3-785 which contains a stretch of the collagen type III sequence and Gly(-) which is similar to (POG)(10) but lacks the central glycine. All the peptides except Gly(-) adopt a collagen-like triple-helical conformation at room temperature. All three triple-helical peptides bound to YadA, with (POG)(10) being the tightest, whereas binding of Gly(-) was hardly detectable. The affinity of (POG)(10) for YadA was 0.28 microM by isothermal titration calorimetry and 0.17 microM by surface plasmon resonance (SPR), similar to that of collagen type I. Our results show that a collagen-like triple-helical conformation, strengthened by the presence of hydroxyproline residues, is both necessary and sufficient for YadA binding.

Escherichia coli amine oxidase (ECAO), encoded by the tynA gene, catalyzes the oxidative deamination of aromatic amines into aldehydes through a well-established mechanism, but its exact biological role is unknown. We investigated the role of ECAO by screening environmental and human isolates for tynA and characterizing a tynA-deletion strain using microarray analysis and biochemical studies. The presence of tynA did not correlate with pathogenicity. In tynA+ Escherichia coli strains, ECAO enabled bacterial growth in phenylethylamine, and the resultant H2O2 was released into the growth medium. Some aminoglycoside antibiotics inhibited the enzymatic activity of ECAO, which could affect the growth of tynA+ bacteria. Our results suggest that tynA is a reserve gene used under stringent environmental conditions in which ECAO may, due to its production of H2O2, provide a growth advantage over other bacteria that are unable to manage high levels of this oxidant. In addition, ECAO, which resembles the human homolog hAOC3, is able to process an unknown substrate on human leukocytes.

Naturally competent bacteria acquire DNA from their surroundings to survive in nutrient-poor environments and incorporate DNA into their genomes as new genes for improved survival. The secretin HofQ from the oral pathogen Aggregatibacter actinomycetemcomitans has been associated with DNA uptake. Cytokine sequestering is a potential virulence mechanism in various bacteria and may modulate both host defense and bacterial physiology. The objective of this study was to elucidate a possible connection between natural competence and cytokine uptake in A. actinomycetemcomitans. The extramembranous domain of HofQ (emHofQ) was shown to interact with various cytokines, of which IL-8 exhibited the strongest interaction. The dissociation constant between emHofQ and IL-8 was 43 nM in static settings and 2.4 μM in dynamic settings. The moderate binding affinity is consistent with the hypothesis that emHofQ recognizes cytokines before transporting them into the cells. The interaction site was identified via crosslinking and mutational analysis. By structural comparison, relateda type I KH domain with a similar interaction site was detected in the Neisseria meningitidis secretin PilQ, which has been shown to participate in IL-8 uptake. Deletion of hofQ from the A. actinomycetemcomitans genome decreased the overall biofilm formation of this organism, abolished the response to cytokines, i.e., decreased eDNA levels in the presence of cytokines, and increased the susceptibility of the biofilm to tested β-lactams. Moreover, we showed that recombinant IL-8 interacted with DNA. These results can be used in further studies on the specific role of cytokine uptake in bacterial virulence without interfering with natural-competence-related DNA uptake.

Sialic acid-binding immunoglobulin-like lectin-9 (Siglec-9) on leukocyte surface is a counter-receptor for endothelial cell surface adhesin, human primary amine oxidase (hAOC3), a target protein for anti-inflammatory agents. This interaction can be used to detect inflammation and cancer in vivo, since the labeled peptides derived from the second C2 domain (C22) of Siglec-9 specifically bind to the inflammation-inducible hAOC3. As limited knowledge on the interaction between Siglec-9 and hAOC3 has hampered both hAOC3-targeted drug design and in vivo imaging applications, we have now produced and purified the extracellular region of Siglec-9 (Siglec-9-EC) consisting of the V, C21 and C22 domains, modeled its 3D structure and characterized the hAOC3–Siglec-9 interactions using biophysical methods and activity/inhibition assays. Our results assign individual, previously unknown roles for the V and C22 domains. The V domain is responsible for the unusually tight Siglec-9–hAOC3 interactions whereas the intact C22 domain of Siglec-9 is required for modulating the enzymatic activity of hAOC3, crucial for the hAOC3-mediated leukocyte trafficking. By characterizing the Siglec-9-EC mutants, we could conclude that R120 in the V domain likely interacts with the terminal sialic acids of hAOC3 attached glycans whereas residues R284 and R290 in C22 are involved in the interactions with the active site channel of hAOC3. Furthermore, the C22 domain binding enhances the enzymatic activity of hAOC3 although the sialic acid-binding capacity of the V domain of Siglec-9 is abolished by the R120S mutation. To conclude, our results prove that the V and C22 domains of Siglec-9-EC interact with hAOC3 in a multifaceted and unique way, forming both glycan-mediated and direct protein-protein interactions, respectively. The reported results on the mechanism of the Siglec-9–hAOC3 interaction are valuable for the development of hAOC3-targeted therapeutics and diagnostic tools.