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PubMed1991

Human natural killer cells express different integrins and spread on fibronectin.

Human natural killer (NK) cells adhered and most of them also actively spread on cellular fibronectin (cFn), plasma Fn (pFn) and its Mr 120,000-140,000 or Mr 105,000 cell-binding proteolytic Fn-fragments as well as on heparin-binding Fn-fragments containing the alternative cell binding site. The cells did not spread on vitronectin, laminin or collagens. Adhesion on Mr 105,000 Fn fragment containing the cell binding site, could be prevented by the synthetic peptide GRGDS but not by an inactive peptide, whereas adhesion on heparin-binding Fn fragments was unaffected by the peptide. Spreading of the NK cells led to a distinct reorganization of F-actin. Immunoprecipitation with monoclonal antibodies (MoAb) against the beta 1 integrin subunit of radioactively surface-labelled cells revealed a broad polypeptide band of Mr 140,000 under reducing conditions and a polypeptide doublet of Mr 160,000 and Mr 110,000 under non-reducing conditions. Identical polypeptides, corresponding to the alpha- and beta-subunits of the Fn-receptor complex, were bound to the Mr 105,000 chymotryptic Fn-fragment coupled to Sepharose. Similar experiments with small lymphocytes did not reveal any polypeptides. Immunofluorescence results with McAbs suggested that among the alpha-subunits of integrins, the alpha 3, alpha 4, and alpha 5 subunits are expressed in NK cells. The present results suggest that non-activated NK cells, but not small lymphocytes, express beta 1-integrins, and that at least the Fn-receptors alpha 4 beta 1 and alpha 5 beta 1 may function in the adhesion and migration of NK cells.

Cell Adhesion↗
PubMed1993

Plasminogen-activator inhibitor type 2 (PAI-2) is a spontaneously polymerising SERPIN. Biochemical characterisation of the recombinant intracellular and extracellular forms.

Plasminogen-activator inhibitor type 2 (PAI-2) is a specific inhibitor of plasminogen activators (PA) that exists in an intracellular, low-molecular-mass form and a secreted, high-molecular-mass form that varies with respect to glycosylation. Here we have developed expression systems for both forms of PAI-2 and biochemically characterised the purified proteins. In order to obtain efficient secretion, we constructed an artificial signal sequence and fused it to the coding region of PAI-2. With this construct, more than 90% of PAI-2 was secreted as a glycosylated, 60-kDa molecular-mass form, but the level of expression was low and unstable. To obtain higher expression of secreted PAI-2, a novel expression vector based on the Semliki-forest-virus replicon was used. Secreted PAI-2 was purified to homogeneity and N-terminal sequence analysis showed that the artificial signal peptide was correctly removed. The intracellular, non-glycosylated form of PAI-2 was expressed in Escherichia coli and purified to homogeneity. Both the secreted and the intracellular forms of PAI-2 were found to inhibit plasminogen activators by forming SDS-resistant complexes and the second-order rate constants were similar for both forms, ranging over 2.4-2.7 x 10(6) M-1s-1 for urokinase-type PA, 2.5-2.7 x 10(5) M-1s-1 for two-chain tissue-type PA and 0.8-1.2 x 10(4) M-1s-1 for single-chain tissue-type PA. None of the purified PAI-2 forms bound to vitronectin. Circular-dichroism spectral analysis revealed that PAI-2 has a CD spectrum that resembles ovalbumin more than PA-inhibitor type 1, confirming the greater similarity between these two members of the serine-protease inhibitor family. Similar to what has been described for the Z-form of alpha 1-antitrypsin, purified PAI-2 was found to spontaneously form polymers during incubation at room temperature. Attempts to convert PAI-2 to a stable locked conformation resembling the conformation of latent PAI-1 by treatment with diluted guanidinium chloride were unsuccessful. Instead, this treatment enhanced the formation of PAI-2 polymers, possibly by the loop-sheet polymerisation mechanism described for alpha 1-antitrypsin.

Animals↗
PubMed1995

Integrin distribution in gastric carcinoma: association of beta 3 and beta 5 integrins with tumor invasiveness.

Immunolocalization of a variety of integrins using monoclonal antibodies against beta 3, beta 5, alpha 3 beta 1 and alpha 6, and polyclonal antibodies against vitronectin receptor (alpha v beta 3) and beta 1 were investigated on PLP-fixed paraffin sections of 19 cases of advanced gastric carcinomas. The beta 5 integrin, which pairs only with the alpha v subunit, was positive in seven cases (37%), and was associated closely with scirrhous invasion (P < 0.05). beta 5 was positive chiefly in the cytoplasm of carcinoma cells and infrequently in cell membranes. beta 3, which is another subunit pairing with alpha v, was positive in six cases (32%), and tended to be associated with scirrhous invasion (P < 0.01). beta 3 was also located chiefly in the cytoplasm. Five of the seven beta 5-positive cases showed coexpression of beta 3. Polyclonal antibodies to alpha v beta 3 also showed a significant difference among the amounts of stroma (P < 0.05). Anti-beta 1 antibodies showed clear positivity in many cases (89%). Of the beta 1 integrins, alpha 3 beta 1 was positive in a few cases (26%) without any preferential pattern, and laminin receptor subunit alpha 6 stained on cell membranes of neoplastic epithelia in many cases of carcinoma (89%) except for mucinous carcinoma These distinctive patterns of integrin positivity indicate a close association of beta 5 and beta 3 expression with scirrhous invasion in gastric carcinoma.

Adult↗
PubMed1997

Interactions between the constitutive host defences of tears and Staphylococcus epidermidis.

As part of the normal microbiota of the external eye, Staphylococcus epidermidis has probably developed strategies to overcome tear defences. The interaction of this bacteria with constitutive tear proteins was examined. Isolates of S. epidermidis grown in 10% serum or artificial tear fluid containing lysozyme or lactoferrin were resistant to the action of these proteins. Using ELISA, cell wall binding of C3, vitronectin and lactoferrin differed quantitatively between strains and in closed-eye compared to open-eye conditions. No differences were observed between ocular and non-ocular strains. This suggests that ocular isolates originate from the general host microbiota and S. epidermidis isolates are resistant to individual constitutive tear proteins.

Bacterial Adhesion↗
PubMed2006

Allosteric disulfide bonds in thrombosis and thrombolysis.

Allosteric disulfide bonds control protein function by mediating conformational change when they undergo reduction or oxidation. The known allosteric disulfide bonds are characterized by a particular bond geometry, the -RHStaple. A number of thrombosis and thrombolysis proteins contain one or more disulfide bonds of this type. Tissue factor (TF) was the first hemostasis protein shown to be controlled by an allosteric disulfide bond, the Cys186-Cys209 bond in the membrane-proximal fibronectin type III domain. TF exists in three forms on the cell surface: a cryptic form that is inert, a coagulant form that rapidly binds factor VIIa to initiate coagulation, and a signaling form that binds FVIIa and cleaves protease-activated receptor 2, which functions in inflammation, tumor progression and angiogenesis. Reduction and oxidation of the Cys186-Cys209 disulfide bond is central to the transition between the three forms of TF. The redox state of the bond appears to be controlled by protein disulfide isomerase and NO. Plasmin(ogen), vitronectin, glycoprotein 1balpha, integrin beta(3) and thrombomodulin also contain -RHStaple disulfides, and there is circumstantial evidence that the function of these proteins may involve cleavage/formation of these disulfide bonds.

Allosteric Site↗
PubMed1996

Radicular cysts are involved in the recruitment of osteoclast precursors.

In consideration of the close relationship between radicular cysts and alveolar bone, it is important to evaluate the potential involvement of the neighbouring bone tissue in such lesions. In the present study, using cytochemical, immunocytochemical and morphological analyses, presumptive osteoclast precursors were revealed in the connective tissue of radicular cyst capsules. The osteoclastic nature of these cells was postulated by their positive staining for the enzyme tartrate-resistant acid phosphatase (TRAP) and by the expression of vitronectin receptor (VnR) on their cell surface. However, these cells did not express the vacuolar-type proton pump, suggesting that they may represent early osteoclast precursors infiltrating the cyst capsule. Cysts also contained activated small blood vessels whose endothelial cells expressed the VnR. This integrin receptor is important in the adhesion of preosteoclasts to the endothelial lining, a necessary step for their emigration out of the vasculature. Therefore, the intracystic vessels could represent a substrate for preosteoclast recruitment. These precursor cells may then reach the perialveolar bone surface and contribute to bone demolition together with those recruited by the resorbing surfaces. The bone-destroying potential of radicular cysts was confirmed by the presence of numerous osteoclasts with large resorption areas on the perialveolar bone surfaces exposed to the cyst capsules. The resorbed surfaces were usually located around the vascular canals of the Haversian systems.

Acid Phosphatase↗
PubMed1995

Osteopontin adhesion receptors on gingival fibroblasts.

Osteopontin (OPN) promotes attachment and spreading of cells in an RGD dependent fashion, suggesting that OPN interacts with integrins on cell surfaces. Here in, we show that LM-609, a monoclonal antibody to the alpha v beta 3 integrin (a vitronectin receptor), inhibited OPN-mediated attachment of gingival fibroblasts. To characterize the cell surface receptors responsible for this interaction, we performed OPN-sepharose affinity chromatography using detergent extracts of 35S-methionine or 125I-surface labeled gingival fibroblasts. Proteins bound to the OPN-matrix were eluted with EDTA and subjected to SDS-PAGE under reducing conditions. EDTA eluates from both 125I-surface labeled and 35S-methionine labeled extracts demonstrated prominent bands in the 90kDa and 50kDa regions, by both autoradiography and fluorography, respectively. These studies suggest that OPN is associated with other cell surface molecules in addition to alpha v beta 3. Furthermore, these as yet to be characterized proteins, may prove to have a stronger affinity for OPN than alpha v beta 3.

Cell Adhesion↗
PubMed1999

Regulation of cell adhesion by PAI-1.

Type I plasminogen activator inhibitor (PAI-1) is the primary inhibitor of tissue- and urokinase-type plasminogen activators. It circulates in plasma complexed with vitronectin (VN), the primary PAI-1 binding protein. The somatomedin B (SMB) domain of VN contains both the high affinity PAI-1 binding site and the specific site for urokinase plasminogen activator receptor (uPAR). PAI-1 is able to regulate uPAR-mediated cell adhesion by competing with uPAR for VN binding. Binding of PAI-1 to SMD may also affect integrin-mediated cell adhesion to VN by hindering integrin binding to the RGD sequence adjacent to the uPAR binding site.

Animals↗
PubMed1999

Urokinase receptor antagonists: discovery and application to in vivo models of tumor growth.

Urokinase receptor antagonists based on the growth factor domains of both human and murine urokinase which show sub-nanomolar affinities for their homologous receptors have been expressed as recombinant proteins. Further modification of these molecules by preparing fusions with the constant region of human IgG has led to molecules with high affinities and long in vivo half-lives. Smaller peptidic inhibitors have been obtained by a combination of bacteriophage display and peptide analog synthesis. All of these molecules inhibit the binding of the growth factor domain of uPA to the uPA receptor and enhance binding of the uPA receptor to vitronectin. Protein uPA receptor antagonists were tested in an in vivo tumor model using the human breast carcinoma MDAmb231 in immunodeficient mice. Both human and murine receptor antagonists showed significant inhibition of primary tumor growth, demonstrating that in vivo, both tumor and stromal cell uPA receptor dependent plasminogen activation can modulate tumor growth.

Amino Acid Sequence↗
PubMed1991

Immunoregulatory role of gamma delta T cells.

While the major population of T lymphocytes express T cell receptor (TCR) alpha beta-chains and recognize peptide antigens in association with either Major Histocompatibility Complex class I or class II molecules, a consensus view does not exist concerning either the nature of the antigen recognized or the nature of the restriction element utilized by the minor population of T cells which express TCR gamma delta-chains. We have identified a unique subpopulation of gamma delta T cells which uniformly express the C gamma 4, V delta 6 TCR and which produce a number of cytokines in the absence of exogenous stimulation. Adaption of these cell lines to serum-free culture conditions resulted in a cessation of cytokine production which could then be induced by the addition of extracellular matrix (ECM)-proteins to the culture. The response to the ECM-proteins could be completely inhibited by an antibody to the murine vitronectin receptor (VNR). However, engagement of the VNR by its ligand was not sufficient for the induction of cytokine production as anti-TCR antibodies inhibited the response to ECM-proteins and gamma delta TCR loss mutants failed to respond. Collectively, these data demonstrate that not only is coexpression of the VNR and the gamma delta TCR required for the induction of cytokine production by this subpopulation of T cells, but that the TCR must also be engaged by its ligand, most likely a cell surface autoantigen expressed by the T cells themselves.

Animals↗
PubMed2006

Novel alpha1-adrenergic receptor signaling pathways: secreted factors and interactions with the extracellular matrix.

alpha1-Adrenergic receptor (alpha1-ARs) subtypes (alpha1A, alpha1B, and alpha1D) regulate multiple signal pathways, such as phospholipase C, protein kinase C (PKC), and mitogen-activated protein kinases. We employed oligonucleotide microarray technology to explore the effects of both short- (1 h) and long-term (18 h) activation of the alpha1A-AR to enable RNA changes to occur downstream of earlier well characterized signaling pathways, promoting novel couplings. Polymerase chain reaction (PCR) studies confirmed that PKC was a critical regulator of alpha1A-AR-mediated gene expression, and secreted interleukin (IL)-6 also contributed to gene expression alterations. We next focused on two novel signaling pathways that might be mediated through alpha1A-AR stimulation because of the clustering of gene expression changes for cell adhesion/motility (syndecan-4 and tenascin-C) and hyaluronan (HA) signaling. We confirmed that alpha1-ARs induced adhesion in three cell types to vitronectin, an interaction that was also integrin-, FGF7-, and PKC-dependent. alpha1-AR activation also inhibited cell migration, which was integrin- and PKC-independent but still required secretion of FGF7. alpha1-AR activation also increased the expression and deposition of HA, a glycosaminoglycan, which displayed two distinct structures: pericellular coats and long cable structures, as well as increasing expression of the HA receptor, CD44. Long cable structures of HA can bind leukocytes, which this suggests that alpha1-ARs may be involved in proinflammatory responses. Our results indicate alpha1-ARs induce the secretion of factors that interact with the extracellular matrix to regulate cell adhesion, motility and proinflammatory responses through novel signaling pathways.

Adrenergic alpha-1 Receptor Antagonists↗
PubMed1996

Regulation of integrin function by the urokinase receptor.

Integrin function is central to inflammation, immunity, and tumor progression. The urokinase-type plasminogen activator receptor (uPAR) and integrins formed stable complexes that both inhibited native integrin adhesive function and promoted adhesion to vitronectin via a ligand binding site on uPAR. Interaction of soluble uPAR with the active conformer of integrins mimicked the inhibitory effects of membrane uPAR. Both uPAR-mediated adhesion and altered integrin function were blocked by a peptide that bound to uPAR and disrupted complexes. These data provide a paradigm for regulation of integrins in which a nonintegrin membrane receptor interacts with and modifies the function of activated integrins.

Amino Acid Sequence↗
PubMed1998

Integrins alpha(v)beta3 and alpha5beta1 mediate attachment of lyme disease spirochetes to human cells.

Borrelia burgdorferi (sensu lato), the agent of Lyme disease, is able to cause chronic, multisystemic infections in human and animal hosts. Attachment of the spirochete to host cells is likely to be important for the colonization of diverse tissues. The platelet-specific integrin alpha(IIb)beta3 was previously identified as a receptor for all three species of Lyme disease spirochetes (B. burgdorferi sensu stricto, B. garinii, and B. afzelii). Here we show that B. burgdorferi also recognizes the widely expressed integrins alpha(v)beta3 and alpha5beta1, known as the vitronectin and fibronectin receptors, respectively. Three representatives of each species of Lyme disease spirochete were tested for the ability to bind to purified alpha(v)beta3 and alpha5beta1. All of the strains tested bound to at least one integrin. Binding to one integrin was not always predictive of binding to other integrins, and several different integrin preference profiles were identified. Attachment of the infectious B. burgdorferi strain N40 to purified alpha(v)beta3 and alpha5beta1 was inhibited by RGD peptides and the appropriate receptor-specific antibodies. Binding to alpha(v)beta3 was also shown by using a transfected cell line that expresses this receptor but not alpha(IIb)beta3. Attachment of B. burgdorferi N40 to human erythroleukemia cells and to human saphenous vein endothelial cells was mediated by both alpha5beta1 and alpha(v)beta3. Our results show that multiple integrins mediate attachment of Lyme disease spirochetes to host cells.

Bacterial Adhesion↗
PubMed1998

Isolation and characterization of two proteins from Moraxella catarrhalis that bear a common epitope.

The UspA1 and UspA2 proteins of Moraxella catarrhalis are potential vaccine candidates for preventing disease caused by this organism. We have characterized both proteins and evaluated their vaccine potential using both in vitro and in vivo assays. Both proteins were purified from the O35E isolate by Triton X-100 extraction, followed by ion-exchange and hydroxyapatite chromatography. Analysis of the sequences of internal peptides, prepared by enzymatic and chemical cleavage of the proteins, revealed that UspA1 and UspA2 exhibited distinct structural differences but shared a common sequence including an epitope recognized by the monoclonal antibody 17C7. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), purified UspA1 exhibited a molecular weight of approximately 350, 000 when unheated and a molecular weight of 100,000 after being heated for 10 min at 100 degreesC. In contrast, purified UspA2 exhibited an apparent molecular weight of 240,000 by SDS-PAGE that did not change with the length of time of heating. Their sizes as determined by gel filtration were 1,150,000 and 830,000 for UspA1 and UspA2, respectively. Preliminary results indicate the proteins have separate functions in bacterial pathogenesis. Purified UspA1 was found to bind HEp-2 cells, and sera against UspA1, but not against UspA2, blocked binding of the O35E isolate to the HEp-2 cells. UspA1 also bound fibronectin and appears to have a role in bacterial attachment. Purified UspA2, however, did not bind fibronectin but had an affinity for vitronectin. Both proteins elicited bactericidal antibodies in mice to homologous and heterologous disease isolates. Finally, mice immunized with each of the proteins, followed by pulmonary challenge with either the homologous or a heterologous isolate, cleared the bacteria more rapidly than mock-immunized mice. These results suggest that UspA1 and UspA2 serve different virulence functions and that both are promising vaccine candidates.

Amino Acid Sequence↗
PubMed1999

Sulfated polysaccharide-directed recruitment of mammalian host proteins: a novel strategy in microbial pathogenesis.

Fundamental to the virulence of microbial pathogens is their capacity for adaptation and survival within variable, and often hostile, environments encountered in the host. We describe a novel, extragenomic mechanism of surface modulation which may amplify the adaptive and pathogenic potential of numerous bacterial species, including Staphylococcus, Yersinia, and pathogenic Neisseria species, as well as Helicobacter pylori and Streptococcus pyogenes. The mechanism involves specific bacterial recruitment of heparin, glycosaminoglycans, or related sulfated polysaccharides, which in turn serve as universal binding sites for a diverse array of mammalian heparin binding proteins, including adhesive glycoproteins (vitronectin and fibronectin), inflammatory (MCP-3, PF-4, and MIP-1alpha) and immunomodulatory (gamma interferon) intermediates, and fibroblast growth factor. This strategy impacts key aspects of microbial pathogenicity as exemplified by increased bacterial invasion of epithelial cells and inhibition of chemokine-induced chemotaxis. Our findings illustrate a previously unrecognized form of parasitism that complements classical virulence strategies encoded within the microbial genome.

Animals↗
PubMed2005

Enhanced killing of Candida albicans by human macrophages adherent to type 1 collagen matrices via induction of phagolysosomal fusion.

Candida albicans, a component of the normal flora of the alimentary tract and mucocutaneous membranes, is the leading cause of invasive fungal disease in premature infants, diabetics, and surgical patients and of oropharyngeal disease in AIDS patients. As little is known about the regulation of monocyte/macrophage anti-Candida activity, we sought to determine if fungicidal activity might be regulated by extracellular matrix proteins to which monocytes/macrophages are adherent in vivo. Compared to monocyte/macrophages that adhered to plastic, human monocytes and monocyte-derived macrophages that adhered to type 1 collagen matrices, but not to fibronectin, vitronectin, or laminin, demonstrated a significant increase in candidacidal activity. The enhancement of monocyte fungicidal activity was maintained over a 4-h period, whereas macrophage fungicidal activity was maximum at 1 h. Although adherence of monocytes and macrophages to collagen matrices concomitantly enhanced the production of superoxide anion, only the fungicidal activity of collagen-adherent monocytes was partially blocked by superoxide dismutase and catalase. Remarkably, we found that only 10% of the phagosomes in C. albicans-infected macrophages that adhered to plastic fused with lysosomes. In contrast, 80% of yeast-containing phagosomes of collagen-adherent macrophages fused with lysosomes. These data suggest that nonoxidative mechanisms are critical for human macrophage anti-Candida activity and that C. albicans pathogenicity is mediated, in part, by its ability to inhibit phagolysosomal fusion in macrophages.

Candida albicans↗
PubMed2006

A conserved tetranucleotide repeat is necessary for wild-type expression of the Moraxella catarrhalis UspA2 protein.

The UspA2 protein has been shown to be directly involved in the serum-resistant phenotype of Moraxella catarrhalis. The predicted 5'-untranslated regions (UTR) of the uspA2 genes in several different M. catarrhalis strains were shown to contain various numbers (i.e., 6 to 23) of a heteropolymeric tetranucleotide (AGAT) repeat. Deletion of the AGAT repeats from the uspA2 genes in the serum-resistant M. catarrhalis strains O35E and O12E resulted in a drastic reduction in UspA2 protein expression and serum resistance. PCR and transformation were used to construct a series of M. catarrhalis O12E strains that differed only in the number of AGAT repeats in their uspA2 genes. Expression of UspA2 was maximal in the presence of 18 AGAT repeats, although serum resistance attained wild-type levels in the presence of as few as nine AGAT repeats. Increased UspA2 expression was correlated with both increased binding of vitronectin and decreased binding of polymerized C9. Real-time reverse transcription-PCR analysis showed that changes in the number of AGAT repeats affected the levels of uspA2 mRNA, with 15 to 18 AGAT repeats yielding maximal levels. Primer extension analysis indicated that these AGAT repeats were contained in the 5'-UTR of the uspA2 gene. The mRNA transcribed from a uspA2 gene containing 18 AGAT repeats was found to have a longer half-life than that transcribed from a uspA2 gene lacking AGAT repeats. These data confirm that the presence of the AGAT repeats in the 5'-UTR of the uspA2 gene is necessary for both normal expression of the UspA2 protein and serum resistance.

5' Untranslated Regions↗
PubMed1991

Binding of S protein by Neisseria gonorrhoeae and potential role in invasion.

An agglutination assay was used to examine the binding of purified human S protein (vitronectin, serum spreading factor) to 201 clinical isolates of Neisseria gonorrhoeae. Strains belonging to the protein IA serovars were significantly (P less than 0.001) more reactive in agglutination tests with human S protein and were more serum resistant than strains belonging to the protein IB serovars. The strains from patients with disseminated infections belonged predominantly to the IA serovar (19 of 23) and, with the exception of IA-4 and certain IB serovars, avidly agglutinated with S protein. The serovar IA-4 and IB strains isolated from joint or cerebrospinal fluid failed to agglutinate with S protein and appeared to be less serum resistant than most other IA isolates. Cysteine hydrochloride or 2-mercaptoethanol inhibited agglutination of S protein and a more than twofold increase in resistance to killing by fresh human serum following preincubation with S protein; the serum-sensitive parent strain did not agglutinate S protein, and serum resistance was not increased following preincubation with this protein. Binding of S protein by gonococci may represent a novel pathogenic mechanism that can contribute to serum resistance.

Agglutination↗