Connective Tissue-activating Peptide Research Articles

Early angiogenic proteins associated with high risk for bronchopulmonary dysplasia and pulmonary hypertension in preterm infants.

Early pulmonary vascular disease in preterm infants is associated with the subsequent development of bronchopulmonary dysplasia (BPD) and pulmonary hypertension (PH); however, mechanisms that contribute to or identify infants with increased susceptibility for BPD and/or PH are incompletely understood. Therefore, we tested if changes in circulating angiogenic peptides during the first week of life are associated with the later development of BPD and/or PH. We further sought to determine alternate peptides and related signaling pathways with the risk for BPD or PH. We prospectively enrolled infants with gestational age <34 wk and collected blood samples during their first week of life. BPD and PH were assessed at 36 wk postmenstrual age. Samples were assayed for each of the 1,121 peptides included in the SOMAscan scan technology, with subsequent pathway analysis. Of 102 infants in the study, 82 had BPD, and 13 had PH. Multiple angiogenic proteins (PF-4, VEGF121, ANG-1, bone morphogenetic protein 10 [BMP10], hepatocyte growth factor (HGF), ANG-2) were associated with the subsequent diagnosis of BPD; and FGF-19, PF-4, connective tissue activating peptide (CTAP)-III, and PDGF-AA levels were associated with BPD severity. Early increases in BMP10 was strongly associated with the late risk for BPD and PH. We found that early alterations of circulating angiogenic peptides and others were associated with the subsequent development of BPD. We further identified peptides that were associated with BPD severity and BPD-associated PH, including BMP10. We speculate that proteomic biomarkers during the first week of life may identify infants at risk for BPD and/or PH to enhance care and research.

P71: Nitrite and nitric oxide supplementation during erythrocyte storage

Background Several studies have shown that aging red blood cells (RBCs) during storage reduces their ability to improve microvascular perfusion and oxygenation after transfusion. In this study we investigated the role of nitric oxide (NO) and nitrite supplementation during storage of RBCs. In order to study the roles of NO and nitrite in the various biochemical changes during RBC storage, NO-releasing agents, NO releasing nanoparticles (NO-nps) and nitrite were used. Methods Rat cells were stored for 14 days at 4 °C in CDPA-1, during storage NO-releasing agents, NO-nps and nitrite were supplemented at various doses. Biochemical and biomechanical changes were quantified and compared to fresh cells. Additionally, rats were instrumented and exchange transfused with stored cells with and without NO supplementation. Results During the storage duration 2,3-DPG concentration and pH decreased, and increased lactate. NO supplementation with NO-nps slowed down the biochemical changes including the depletion on important antioxidant factors, such as GSH, NADP and NADPH. NO supplementation with NO-nps decreased proteolytic enzymes, including connective tissue-activating peptide III, which participates in tissue remodeling and inflammatory process. Lastly, NO supplementation preserved static and dynamic red cell mechanics, which affect cell-to-cell and cell-to-wall interaction. Transfusion of stored cells without NO supplementation affected small arterioles hemodynamics, including diameter, velocity, blood flow, and oxygen delivery. While, infusion of cells stored under NO supplementation presented superior microvascular hemodynamics and oxygenation. Conclusions Although both NO and its donors and/or their decomposed products may have biological activities, most of the cellular responses to these donors have been postulated to reflect NO-dependent events. NO supplementation during storage preserved cell deformability, preserved antioxidant capacity and preserved microvascular endothelial cell mechanotransduction, and red cell hydrodynamics. Most of the storage changes were prevented or slowed down by NO, NO induced protection seems to be related to decrease oxidative stress in the storage conditions. Therefore, NO supplementation prevents ROS irreversible lesions in the early period of storage, rather then intervening a posteriori through the addition of rejuvenating solutions. Disclosure There is not financial interest. Supported by R01HL052684 P01HL11090.

Proteomic Analysis of Human Saliva From Lung Cancer Patients Using Two-Dimensional Difference Gel Electrophoresis and Mass Spectrometry

Lung cancer is often asymptomatic or causes only nonspecific symptoms in its early stages. Early detection represents one of the most promising approaches to reduce the growing lung cancer burden. Human saliva is an attractive diagnostic fluid because its collection is less invasive than that of tissue or blood. Profiling of proteins in saliva over the course of disease progression could reveal potential biomarkers indicative of oral or systematic diseases, which may be used extensively in future medical diagnostics. There were 72 subjects enrolled in this study for saliva sample collection according to the approved protocol. Two-dimensional difference gel electrophoresis combined with MS was the platform for salivary proteome separation, quantification, and identification from two pooled samples. Candidate proteomic biomarkers were verified and prevalidated by using immunoassay methods. There were 16 candidate protein biomarkers discovered by two-dimensional difference gel electrophoresis and MS. Three proteins were further verified in the discovery sample set, prevalidation sample set, and lung cancer cell lines. The discriminatory power of these candidate biomarkers in lung cancer patients and healthy control subjects can reach 88.5% sensitivity and 92.3% specificity with AUC = 0.90. This preliminary data report demonstrates that proteomic biomarkers are present in human saliva when people develop lung cancer. The discriminatory power of these candidate biomarkers indicate that a simple saliva test might be established for lung cancer clinical screening and detection.

Discovery and biochemical characterisation of four novel biomarkers for osteoarthritis

ObjectiveKnee osteoarthritis (OA) is a heterogeneous, complex joint pathology of unknown aetiology. Biomarkers have been widely used to investigate OA but currently available biomarkers lack specificity and sensitivity. Therefore, novel...

Place for Biochemical Markers in Early-Stage Lung Cancer Detection?

Place for Biochemical Markers in Early-Stage Lung Cancer Detection?

Connective Tissue-Activating Peptide III: A Novel Blood Biomarker for Early Lung Cancer Detection

There are no reliable blood biomarkers to detect early lung cancer. We used a novel strategy that allows discovery of differentially present proteins against a complex and variable background. Mass spectrometry analyses of paired pulmonary venous-radial arterial blood from 16 lung cancer patients were applied to identify plasma proteins potentially derived from the tumor microenvironment. Two differentially expressed proteins were confirmed in 64 paired venous-arterial blood samples using an immunoassay. Twenty-eight pre- and postsurgical resection peripheral blood samples and two independent, blinded sets of plasma from 149 participants in a lung cancer screening study (49 lung cancers and 100 controls) and 266 participants from the National Heart Lung and Blood Institute Lung Health Study (45 lung cancer and 221 matched controls) determined the accuracy of the two protein markers to detect subclinical lung cancer. Connective tissue-activating peptide III (CTAP III)/ neutrophil activating protein-2 (NAP-2) and haptoglobin were identified to be significantly higher in venous than in arterial blood. CTAP III/NAP-2 levels decreased after tumor resection (P = .01). In two independent population cohorts, CTAP III/NAP-2 was significantly associated with lung cancer and improved the accuracy of a lung cancer risk prediction model that included age, smoking, lung function (FEV(1)), and an interaction term between FEV(1) and CTAP III/NAP-2 (area under the curve, 0.84; 95% CI, 0.77 to 0.91) compared to CAPIII/NAP-2 alone. We identified CTAP III/NAP-2 as a novel biomarker to detect preclinical lung cancer. The study underscores the importance of applying blood biomarkers as part of a multimodal lung cancer risk prediction model instead of as stand-alone tests.

Mast cells and neutrophils proteolytically activate chemokine precursor CTAP-III and are subject to counterregulation by PF-4 through inhibition of chymase and cathepsin G

AbstractThe CXC chemokines platelet factor 4 (PF-4/CXCL4) and connective tissue-activating peptide III (CTAP-III) are released by activated human platelets in micromolar concentrations. So far, neutrophils have been recognized to cleave the precursor CTAP-III to form the active chemokine neutrophil-activating peptide 2 (NAP-2/CXCL7) through limited proteolysis by membrane-associated cathepsin G. Here we show for the first time that activated human skin mast cells (MCs) convert CTAP-III into biologically active NAP-2 through proteolytic cleavage by released chymase. A direct comparison on a cell number basis revealed that unstimulated MCs exceed the CTAP-III–processing potency of neutrophils about 30-fold, whereas MCs activated by IgE cross-linking exhibit even 1000-fold higher CTAP-III–processing capacity than fMLP-stimulated neutrophils. Intriguingly, PF-4 counteracted MC- as well as neutrophil-mediated NAP-2 generation at physiologically relevant concentrations. Addressing the underlying mechanism, we obtained evidence that PF-4 acts as an inhibitor of the CTAP-III–processing enzymes cathepsin G and chymase without becoming cleaved itself as a competitive substrate. Because cleavage of the CTAP-III–unrelated substrate substance P was also affected by PF-4, our results suggest a regulatory role for PF-4 not only in NAP-2 generation but also in neutrophil- and MC-mediated processing of other physiologically relevant inflammatory mediators.

Connective tissue activation: I. The nature, specificity, measurement and distribution of connective tissue activating peptide

A polypeptide which induces hypermetabolism in normal synovial fibroblasts in tissue culture is described. The metabolic changes induced included a marked increase in hyaluronate formation, lactate formation and glucose uptake; formation of soluble and fibrous collagen was depressed. An assay method is presented which permits quantitative comparisons of samples for connective tissue activating peptide (CTAP) activity. The CTAP appeared to be widely distributed in human tissues, the amount of activity being a function of cell density. A role for CTAP in rheumatoid inflammation is proposed.

Decreased levels of CXC-chemokines in serum of benzene-exposed workers identified by array-based proteomics

Benzene is an important industrial chemical and environmental contaminant that causes leukemia. To obtain mechanistic insight into benzene's mechanism of action, we examined the impact of benzene on the human serum proteome in a study of exposed healthy shoe-factory workers and unexposed controls. Two sequential studies were performed, each using sera from 10 workers exposed to benzene (overall mean benzene air level >30 ppm) and 10 controls. Serum samples were subjected to anion-exchange fractionation and bound to three types of ProteinChip arrays (Ciphergen Biosystems, Fremont, CA) [hydrophobic (H50), metal affinity (IMAC3-Cu), and cation exchange (WCX2)]. Protein-expression patterns were detected by surface-enhanced laser desorption/ionization (SELDI)-TOF MS. Three proteins (4.1, 7.7, and 9.3 kDa) were consistently down-regulated in exposed compared with control subjects in both studies. All proteins were highly inversely correlated with individual estimates of benzene exposure (r > 0.75). The 7.7- and 9.3-kDa proteins were subsequently identified as platelet factor (PF)4 and connective tissue activating peptide (CTAP)-III. Initial proteomic results for PF4 and CTAP-III were subsequently confirmed in a single experiment using a ProteinChip-array-based immunoassay(Ciphergen Biosystems). The altered expression of the platelet-derived CXC-chemokines (40% and 63% for PF4 and CTAP-III, respectively) could not be explained by changes in absolute platelet counts. Thus, SELDI-TOF analysis of a limited number of exposed and unexposed subjects revealed that lowered expression of PF4 and CTAP-III proteins is a potential biomarker of benzene's early biologic effects and may play a role in the immunosuppressive effects of benzene.

Induction and antimicrobial activity of platelet basic protein derivatives in human monocytes.

The antimicrobial activity of a number of chemokines has recently come into focus of research about innate immunity. We have previously shown that platelet basic protein (PBP), which gives rise to several antimicrobial peptides of platelets, is also expressed in human monocytes. In the present studies, we show that exposure of human monocytes to bacteria or microbial components (lipopolysaccharide and zymosan) induces a several-fold greater expression of derivates of PBP. Also, activation of proteinase-activated receptors (PARs) by thrombin or the synthetic peptide ligand SFLLRN of PAR-1 significantly increased PBP expression, presumably on the transcriptional level, as evidenced by higher mRNA levels. Derivates of PBP appeared to reach phago-lysosomes, as higher concentration was found in latex phagosomes isolated by a flotation method. By the gel-overlay technique, two bactericidal derivatives of PBP could be visualized, which were immunoreactive with anti-PBP antibody in Western blots. By matrix-assisted laser desorption/ionization time of flight and surface-enhanced laser desorption and ionization techniques, it was confirmed that the bands corresponded to PBP derivates. After immunofixation with a monoclonal antibody to PBP, the major peptide in zymosan-stimulated monocytes was identified to correspond by molecular weight to connective tissue-activating peptide III, which has been reported to be a major antimicrobial PBP derivate also in platelets. Our observations indicate that PBP and its derivates are constituents of the antimicrobial arsenal of human monocytes. Their increased expression after exposure to microorganisms allows a rapid host response to pathogens.

Multiple Heparanases Are Expressed in Polymorphonuclear Cells

Background. Heparan sulfate proteoglycans are complex cell surface molecules containing polysaccharides called heparan sulfate. Lysosomes, platelet granules, and neutrophils (polymorphonuclear cells) contain heparanases that degrade heparan sulfate. There are at least two groups of heparanases: connective tissue–activating-peptide (CTAP-III) and mammalian heparanase (hpa). The purpose of this study was to quantify the expression of both CTAP-III and hpa in neutrophils and their heparanase activity.Materials and methods. Neutrophils were isolated from whole blood, total RNA collected, and reverse transcriptase–polymerase chain reaction (RT-PCR) performed. Primers were designed for CTAP-III and hpa-1 sequences from GenBank. Neutrophil lysate underwent Western blot analysis (and quantification) with antibodies to the C-terminus of CTAP-III and the 50-kDa subunit of hpa1. Chromatography separated these components of lysate, which were then tested for heparanase activity.Results. Both CTAP-III (281 bp) and hpa-1 (485 bp) messenger RNA (mRNA) were expressed equally by neutrophils with use of quantitative RT-PCR. By Western blot analysis, a CTAP-III-like protein was detected at 80 kDa, and hpa-1 was detected as a 50-kDa protein, with expression not significantly different (P > 0.05). Heparanase activity was significantly different (P < 0.0001) for the 50-kDa hpa-1 protein (1.51 × 10−6 μmol/min) and the 80-kDa CTAP-III-like protein (0.85 × 10−6 μmol/min).Conclusions. Human neutrophils express mRNA and protein for both a CTAP-III-like protein and hpa-1. Although expressed in similar quantity for mRNA and protein, Hpa-1 was more active as heparanase than the CTAP-III-like protein. With more than one class of heparanase in their granules, neutrophils may be able to modify different kinds of heparan sulfate chains.

Megakaryocyte maturation is associated with expression of the CXC chemokine connective tissue-activating peptide CTAP III

Connective tissue-activating peptide III (CTAP III), a CXC chemokine derived from the chemokine precursor platelet basic protein (PBP) by proteolytic cleavage, has been identified in platelets, activated macrophages, neutrophils and T lymphocytes. CTAP III can support stem cell-derived haematopoiesis yet inhibits the proliferation of committed megakaryocyte (MK) progenitors. This investigation was aimed at characterizing CTAP III expression in human MKs and determining it's role in MK differentiation. We report high expression of CTAP III in mature human bone marrow (BM) MKs and megakaryoblast cell lines following differentiation induction with phorbol ester 12-myristate 13-acetate (PMA). Immunostaining with anti-CTAP III antibodies demonstrated its prominent presence in platelet producing zones in the cytoplasm and intense staining around the periphery of the large BM MKs. In cultures of logarithmically growing megakaryoblast cell lines DAMI, CHRF-288 or MEG01, which contain primarily 2N cells, only 15% of the cells expressed CTAP III. The addition of PMA stimulated high levels of CTAP III after 24 h in more than 75% of the cells, being expressed in both the 2N and large polyploid MKs. Reverse transcription polymerase chain reaction (RT-PCR) revealed upregulation of CTAP III mRNA after only 1 h of exposure to PMA that was sustained for 24 h. In the bone marrow of idiopathic thrombocytopenic pupura (ITP) patients undergoing accelerated MK maturation and thrombopoiesis, 99% of large MKs and 95% of small MKs expressed high levels of CTAP III. While the biological function of this chemokine in MKs is not known, these studies demonstrate that molecular upregulation of CTAP III in MKs is associated with maturation and, as with other chemokines, may be involved in proliferation arrest and cellular interactions with extracellular matrix and platelet production.

Megakaryocyte maturation is associated with expression of the CXC chemokine connective tissue-activating peptide CTAP III.

Connective tissue-activating peptide III (CTAP III), a CXC chemokine derived from the chemokine precursor platelet basic protein (PBP) by proteolytic cleavage, has been identified in platelets, activated macrophages, neutrophils and T lymphocytes. CTAP III can support stem cell-derived haematopoiesis yet inhibits the proliferation of committed megakaryocyte (MK) progenitors. This investigation was aimed at characterizing CTAP III expression in human MKs and determining it's role in MK differentiation. We report high expression of CTAP III in mature human bone marrow (BM) MKs and megakaryoblast cell lines following differentiation induction with phorbol ester 12-myristate 13-acetate (PMA). Immunostaining with anti-CTAP III antibodies demonstrated its prominent presence in platelet producing zones in the cytoplasm and intense staining around the periphery of the large BM MKs. In cultures of logarithmically growing megakaryoblast cell lines DAMI, CHRF-288 or MEG01, which contain primarily 2N cells, only 15% of the cells expressed CTAP III. The addition of PMA stimulated high levels of CTAP III after 24 h in more than 75% of the cells, being expressed in both the 2N and large polyploid MKs. Reverse transcription polymerase chain reaction (RT-PCR) revealed upregulation of CTAP III mRNA after only 1 h of exposure to PMA that was sustained for 24 h. In the bone marrow of idiopathic thrombocytopenic pupura (ITP) patients undergoing accelerated MK maturation and thrombopoiesis, 99% of large MKs and 95% of small MKs expressed high levels of CTAP III. While the biological function of this chemokine in MKs is not known, these studies demonstrate that molecular upregulation of CTAP III in MKs is associated with maturation and, as with other chemokines, may be involved in proliferation arrest and cellular interactions with extracellular matrix and platelet production.

Stimulation of Cellular Sphingomyelin Import by the Chemokine Connective Tissue-activating Peptide III

The selective import of phospholipids into cells could be mediated by proteins secreted from the cells into the extracellular compartment. We observed that the supernatants obtained from suspensions of thrombin-activated platelets stimulated the exchange of pyrene (py)-labeled sphingomyelin between lipid vesicles in vitro. The proteins with sphingomyelin transfer activity were purified and identified as the chemokine connective tissue-activating peptide III (CTAP-III) and platelet basic protein. Isolated CTAP-III stimulated the exchange of py-sphingomyelin between lipid vesicles but did not affect the translocations of py-labeled phosphatidylcholine and phosphatidylethanolamine. CTAP-III rapidly increased the transfer of py-sphingomyelin from low density lipoproteins into peripheral blood lymphocytes, other immune cells, and fibroblasts. In the presence of heparin, CTAP-III was unable to insert sphingomyelin into the peripheral blood lymphocytes. The activation energy of the py-sphingomyelin transfer suggested that the translocation proceeded entirely in a hydrophobic environment. [(3)H]Sphingomyelin transferred to the cells by CTAP-III was hydrolyzed to [(3)H]ceramide and [(3)H]sphingosine after activation with tumor necrosis factor alpha. The generation of the [(3)H]sphingolipid messengers was catalyzed by acid sphingomyelinase. Our results identify CTAP-III as the first mediator of the selective (endocytosis-independent) cellular import of sphingomyelin allowing the paracrine modulation of the sphingolipid signaling.

Down-regulation of neutrophil functions by the ELR+CXC chemokine platelet basic protein

AbstractThe platelet-derived neutrophil-activating peptide 2 (NAP-2, 70 amino acids) belongs to the ELR+ CXC subfamily of chemokines. Similar to other members of this group, such as IL-8, NAP-2 activates chemotaxis and degranulation in neutrophils (polymorphonuclear [PMN]) through chemokine receptors CXCR-1 and CXCR-2. However, platelets do not secrete NAP-2 as an active chemokine but as the C-terminal part of several precursors that lack PMN-stimulating capacity. As we have previously shown, PMN themselves may liberate NAP-2 from the precursor connective tissue-activating peptide III (CTAP-III, 85 amino acids) by proteolysis. Instead of inducing cell activation, continuous accumulation of the chemokine in the surroundings of the processing cells results in the down-regulation of specific surface-expressed NAP-2 binding sites and in the desensitization of chemokine-induced PMN degranulation. Thus, NAP-2 precursors may be regarded as indirect mediators of functional desensitization in neutrophils. In the current study we investigated the biologic impact of another major NAP-2 precursor, the platelet basic protein (PBP, 94 amino acids). We show that PBP is considerably more potent than CTAP-III to desensitize degranulation and chemotaxis in neutrophils. We present data suggesting that the high desensitizing capacity of PBP is based on its enhanced proteolytic cleavage into NAP-2 by neutrophil-expressed cathepsin G and that it involves efficient down-regulation of surface-expressed CXCR-2 while CXCR-1 is hardly affected. Correspondingly, we found PBP and, less potently, CTAP-III to inhibit CXCR-2– but not CXCR-1– dependent chemotaxis of neutrophils toward NAP-2. Altogether our findings demonstrate that the anti-inflammatory capacity of NAP-2 is governed by the species of its precursors.

Down-regulation of neutrophil functions by the ELR+CXC chemokine platelet basic protein

The platelet-derived neutrophil-activating peptide 2 (NAP-2, 70 amino acids) belongs to the ELR+ CXC subfamily of chemokines. Similar to other members of this group, such as IL-8, NAP-2 activates chemotaxis and degranulation in neutrophils (polymorphonuclear [PMN]) through chemokine receptors CXCR-1 and CXCR-2. However, platelets do not secrete NAP-2 as an active chemokine but as the C-terminal part of several precursors that lack PMN-stimulating capacity. As we have previously shown, PMN themselves may liberate NAP-2 from the precursor connective tissue-activating peptide III (CTAP-III, 85 amino acids) by proteolysis. Instead of inducing cell activation, continuous accumulation of the chemokine in the surroundings of the processing cells results in the down-regulation of specific surface-expressed NAP-2 binding sites and in the desensitization of chemokine-induced PMN degranulation. Thus, NAP-2 precursors may be regarded as indirect mediators of functional desensitization in neutrophils. In the current study we investigated the biologic impact of another major NAP-2 precursor, the platelet basic protein (PBP, 94 amino acids). We show that PBP is considerably more potent than CTAP-III to desensitize degranulation and chemotaxis in neutrophils. We present data suggesting that the high desensitizing capacity of PBP is based on its enhanced proteolytic cleavage into NAP-2 by neutrophil-expressed cathepsin G and that it involves efficient down-regulation of surface-expressed CXCR-2 while CXCR-1 is hardly affected. Correspondingly, we found PBP and, less potently, CTAP-III to inhibit CXCR-2– but not CXCR-1– dependent chemotaxis of neutrophils toward NAP-2. Altogether our findings demonstrate that the anti-inflammatory capacity of NAP-2 is governed by the species of its precursors.

The beta-thromboglobulins and platelet factor 4: blood platelet-derived CXC chemokines with divergent roles in early neutrophil regulation.

The recruitment of neutrophil granulocytes to sites of tissue injury is one of the earliest events during host defense. Several chemotactic cytokines belonging to the CXC subfamily of chemokines are thought to be implicated in this kind of response. Especially those CXC chemokines that are stored in blood platelets and become immediately released upon activation are likely to dominate neutrophil-dependent host defense at the onset of inflammation. The major platelet-derived CXC chemokines are the beta-thromboglobulins and platelet factor 4 (PF-4), which are both released into the blood at micromolar concentrations. The availability as well as the functional activity of these mediators appear to be subject to tight control by diverse regulatory mechanisms. These include proteolytic processing of chemokine precursors, oligomer formation, and the differential usage of neutrophil-expressed receptors. Herein we review our work on early neutrophil regulation by PF-4, the beta-thromboglobulin neutrophil-activating peptide 2 (NAP-2) and its major precursor connective tissue-activating peptide III (CTAP-III). We moreover propose a model to assess the contribution by either of these chemokines to coordinated recruitment and activation of neutrophils in response to acute tissue injury.

Production of chemokines CTAPIII and NAP/2 by digestion of recombinant ubiquitin-CTAPIII with yeast ubiquitin C-terminal hydrolase and human immunodeficiency virus protease.

Recombinant yeast ubiquitin C-terminal hydrolase (YUH1), which has an N-terminal (His)(6) tag, and an autolysis-resistant mutant of the human immunodeficiency virus-1 protease (HIV-1 Pr) have been used as specific proteases to yield peptides from a ubiquitin conjugate. In the present example, connective tissue-activating peptide (CTAPIII) and neutrophil-activating peptide 2 (NAP/2) were generated by digestion of a ubiquitin-CTAPIII conjugate with YUH1 and HIV Pr, respectively, as indicated below: [see text] YUH1 cleaved at the peptide bond formed by the C-terminal Gly(76) of ubiquitin (Ub) and the N-terminal Asn(1) of the 85-residue peptide CTAPIII. The HIV-1 Pr cleaved between Tyr(15) and Ala(16), the N-terminal Ala of the 70-residue peptide NAP/2. Both enzymes produced authentic peptides from the Ub fusion protein, with a nearly 100% yield. The liberated CTAPIII and NAP/2 were separated from (His)(6)-Ub, the trace amounts of unreacted (His)(6)-Ub-CTAPIII, HIV-1 Pr, and the (His)(6)-YUH1 by passage over a nickel-chelate column; the final yield was about 10 mg of peptide/liter of cell culture. (His)(6)-YUH1, the HIV Pr mutant, and the (His)(6)-Ub-CTAPIII substrate were all expressed individually in Escherichia coli. (His)(6)-YUH1 and (His)(6)-Ub-CTAPIII were highly expressed in a soluble form, but about 75% of the total (His)(6)-YUH1 was also found in inclusion bodies. Both proteins from the soluble fractions were easily purified in a single step by immobilized metal ion affinity chromatography with a yield of about 27 mg of (His)(6)-Ub-CTAPIII and 13.6 mg of (His)(6)-YUH1 protein/liter of cell culture. Chemotactic factor activity, as assessed by the neutrophil shape change assay, was observed for NAP/2, but not for CTAPIII. This strategy, which employs YUH1 and the HIV-1 Pr as tools for the highly selective cleavage of the chimeric substrate, should be applicable to the large-scale production of a variety of peptides.

A Cellulose-Binding Domain-Fused Recombinant Human T Cell Connective Tissue-Activating Peptide-III Manifests Heparanase Activity

The chemokine connective tissue-activating peptide (CTAP)-III, which belongs to the leukocyte-derived growth factor family of mediators, was previously shown to be mitogenic for fibroblasts. However, it has recently been shown that CTAP-III, released from platelets, can act like a heparanase enzyme and degrade heparan sulfate. This suggests that CTAP-III may also function as a proinflammatory mediator. We have successfully cloned CTAP-III from a λgt11 cDNA library of PHA-activated human CD4+T cells and produced recombinant CTAP-III as a fusion protein with a cellulose-binding domain moiety. This recombinant CTAP-III exhibited heparanase activity and released degradation products from metabolically labeled, naturally produced extracellular matrix. We have also developed polyclonal and monoclonal antibodies, and these antibodies against the recombinant CTAP-III detected the CTAP-III molecule in human T cells, polymorphonuclear leukocytes, and placental extracts. Thus, our study provides tools to examine further immune cell behavior in inflamed sites rich with extracellular moieties and proinflammatory mediators.

Regulation of neutrophil activation by proteolytic processing of platelet-derived alpha-chemokines.

In recent years evidence has been accumulated that platelets besides their function in coagulation play an important role in inflammation and wound repair. Upon activation platelets release a variety of mediators, among which members of the α-chemokine subfamily of proinflammatory cytokines have been identified. These platelet-derived polypeptides do not only comprise members of the so-called ß-thromboglobulin family, such as platelet basic protein (PBP), connective tissue-activating peptide III (CTAP-III), and neutrophil-activating peptide 2 (NAP-2)1, but also platelet factor 4 (PF4)2,3 and the ß-chemokine RANTES (Regulated upon activation normal T cell expressed and probably secreted)4. While ß-chemokines have been shown to activate monocytes, T lymphocytes and eosinophils, α-chemokines such as IL-8, NAP-2 and melanoma growth-stimulating activity (MGSA/gro-α) appear to represent rather selective activators of polymorphonuclear leukocytes (PMN)5. Importantly, their biological activity, such as chemotaxis and degranulation-inducing capacity, has been demonstrated to be closely connected with the presence of an N-terminal glutamic acid-leucine-arginine (ELR) motif. Only recently, attention has been paid to the regulatory properties of α-chemokines. In the present article we will focus on two aspects of regulation of PMN functions, namely 1. the proteolytic processing of platelet-derived α-chemokines as a regulatory event in the induction and modulation of PMN activation, and 2. the phenotypic and functional consequences for the PMN under the constraints of such regulatory events.KeywordsProteolytic ProcessingNeutrophil ActivationPlatelet FactorInactive PrecursorElastase ReleaseThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.