Cyclophilin C-associated Protein Research Articles

90K, an interferon-stimulated gene product, reduces the infectivity of HIV-1

BackgroundIn response to viral infections, interferons induce the transcription of several hundred genes in mammalian cells. Specific antiviral functions, however, have only been attributed to a few of them. 90K/LGALS3BP has been reported to be an interferon-stimulated gene that is upregulated in individuals with cancer or HIV-1 infection.ResultsHere, we show that 90K expression dose-dependently decreased the particle infectivity of HIV-1 progeny. The lower infectivity of released particles correlated with reduced virion incorporation of mature envelope glycoproteins gp120 and gp41. Further, proteolytic processing of the gp160 precursor and surface expression of gp120 in the producer cell were impaired in the presence of 90K expression. In contrast, expression of Gag, Nef and Vpu, and virus release were not grossly affected by 90K expression. 90K-imposed restriction occurred in the absence of direct interaction of 90K with HIV-1 Env or entrapment of Env in the ER. The cell-associated, but not the secreted species of 90K, mediated the antiviral effect. A truncated version of human 90K, solely consisting of the two intermediate domains, displayed a similar antiviral activity as the full-length wildtype 90K, indicating that the N-terminal SRCR-like domain and the C-terminal domain are dispensable for 90K’s antiviral activity. The murine homolog of 90K, CypCAP (Cyclophilin C-associated protein), neither modulated particle infectivity of HIV-1 nor lowered the virion incorporation of mature gp120, suggesting a species-specific mode of action. 90K was expressed at basal levels in TZM-bl cells and in primary macrophages, and at low levels in CD4+ T-cells and PBMCs. 90K’s susceptibility to IFN-mediated stimulation of expression was cell type-specific. siRNA-mediated knockdown of 90K in TZM-bl cells and primary macrophages enhanced the incorporation of Env glycoproteins into progeny virions, boosted the particle infectivity of released HIV-1, and accelerated HIV-1 spread. Conversely, treatment of HIV-1 infected macrophages with IFN-α induced 90K expression and lowered the particle infectivity of HIV-1.ConclusionsThus, 90K constitutes a novel antiviral factor that reduces the particle infectivity of HIV-1, involving interference with the maturation and incorporation of HIV-1 Env molecules into virions.

Cyclophilin C-associated protein regulation of phagocytic functions via NFAT activation in macrophages

Experimental cerebral ischemia has been reportedly alleviated by the immunosuppressive agent cyclosporin A (CsA). Cyclophilin C-associated protein (CyCAP) was proposed to be an endogenous equivalent of CsA; CsA- and CyCAP-targeting protein cyclophilin C have attracted extensive attention regarding their ischemia-alleviating mechanisms. In this study we have introduced the specific CyCAP antibody for evaluating its distribution in the rat ischemic brain after middle cerebral artery occlusion. During the recovery of cerebral ischemia in rats, CyCAP was highly expressed in the activated microglia/macrophages in the ischemic lesion. However, it remains unknown what roles CyCAP plays in the activation of macrophage phagocytosis. Thus, we studied CyCAP function using a RAW264.7 macrophage cell line. When we expressed CyCAP-GFP and cyclophilin C-FLAG in RAW264.7 cells, we found that CyCAP and cyclophilin C make a complex, which is competitively inhibited by CsA. Consistently, in immunoprecipitates by anti-calcineurin antibody, cyclophilin C and CyCAP were detected, and CyCAP pulled down NFATc1, suggesting that both CyCAP and cyclophilin C form a complex with calcineurin and NFATc1. When CyCAP was adenovirally overexpressed in RAW cells, NFAT staining increased over the nucleus. Furthermore, calcineurin and IL-2 were increased with time. Thus, CyCAP appears to control macrophage functions by activating NFAT and the resultant IL-2 production. With a protein phosphatase inhibitor PhoSTOP, NFAT was localized more to the cytoplasm, and phagocytosis was decreased strikingly. Thus, we suggest that in a CyCAP-cyclophilin C pathway for macrophage activation, calcineurin phosphatase activity is essential for the phagocytosis activity via dephosphorylation of NFATc1.

Cyclophilin C‐associated protein/Mac‐2 binding protein colocalizes with calnexin and regulates the expression of tissue transglutaminase

Cyclophilin C-associated protein (CyCAP) or Mac-2 binding protein has been identified as a binding protein for cyclophilin C in mice and for Mac-2 (galectin-3) in human, suggesting its multiple binding activity to proteins. In the present study, using specific anti-rat-CyCAP antibody, we found that CyCAP colocalizes with calnexin at the location near the nuclear envelope, however CyCAP does not have colocalization with calreticulin. In senescent fibroblasts and interferon-gamma (IFNgamma) treated fibroblasts, both calnexin and CyCAP form larger polymers and are released from the endoplasmic reticulum (ER) through the cellular membrane to the extracellular area. Immunoprecipitation studies further confirm that the release of calnexin is through binding to CyCAP. Further, we found that tissue transglutaminase (tTG) protein is decreased, however not at the RNA level, in CyCAP null fibroblasts, which suggests that CyCAP is involved in tTG post-translational modification. Our data give novel evidence that CyCAP regulates the post-translational modification of tTG through its colocalization with calnexin in ER.

Cyclophilin C-associated protein (CyCAP) knock-out mice spontaneously develop colonic mucosal hyperplasia and exaggerated tumorigenesis after treatment with carcinogen azoxymethane1

BackgroundThe discovery of a "serrated neoplasia pathway" has highlighted the role of hyperplastic lesions of the colon as the significant precursor of colorectal adenocarcinoma. In mice, hyperplasia of the colonic mucosa is a regular phenomenon after a challenge with colonic carcinogens indicating that mucosal hyperproliferation and thickening, even without cytological dysplasia, represents an early pre-malignant change. Cyclophilin C-associated protein (CyCAP) has been described to down-modulate endotoxin signaling in colorectal murine mucosa and is a murine orthologue of the tumor-associated antigen 90 K (TAA90K)/mac-2-binding protein.MethodsFemale Balb/c wild-type (WT) and CyCAP knock-out (KO) mice (6–8 weeks old) were administered 2 or 6 weekly subcutaneous injections of azoxymethane. The animals were evaluated post-injection at six weeks for aberrant crypt foci (ACF) study and at five months for colon tumor measurement. The thickness of the colon crypts was measured in microns and the number of colonocytes per crypt was also determined in well-oriented crypts. Morphometric analyses of the colon mucosa were also performed in untreated 6–8 weeks old KO and WT animals. Formalin-fixed/paraffin-embedded colon sections were also studied by immunohistochemistry to determine the Ki-67 proliferation fraction of the colon mucosa, β-catenin cellular localization, cyclin D1, c-myc, and lysozyme in Paneth cells.ResultsCyclophilin C-associated protein (CyCAP)-/- mice, spontaneously developed colonic mucosal hyperplasia early in life compared to wild-type mice (WT) (p < 0.0001, T-test) and crypts of colonic mucosa of the (CyCAP)-/- mice show higher proliferation rate (p = 0.039, Mann-Whitney Test) and larger number of cyclin D1-positive cells (p < 0.0001, Mann-Whitney Test). Proliferation fraction and cyclin D1 expression showed positive linear association (p = 0.019, Linear-by-Linear Association). The hyperplasia was even more pronounced in CyCAP-/- mice than in WT after challenge with azoxymethane (p = 0.005, T-test). The length of the crypts (r = 0.723, p = 0.018, Spearman Correlation) and the number of colonocytes per crypt (r = 0.863, p = 0.001, Spearman Correlation) in non-tumorous areas were positively associated with azoxymethane-induced number of tumors. CyCAP-/- developed larger numbers of tumors than WT animals (p = 0.003, T-Test) as well as overall larger tumor mass (p = 0.016, T-Test). Membranous β-catenin was focally overexpressed in KO mice including proliferative zone of the crypts.ConclusionCyCAP-/- represent the first described model of spontaneous colonic mucosal hyperplasia. We conclude that CyCAP-deficient mice spontaneously and after challenge with carcinogen develop significantly more colorectal mucosal hyperplasia, an early stage in murine colonic carcinogenesis.

044 Up-Regulated p38, PCNA, and Decreased Smooth Muscle Actin in Cyclophilin C-Associated Protein Null Mice During Skin Wound Healing

Introduction: Cyclophilin C-associated protein (CyC-AP) expression is up-regulated during wound healing. CyC-AP deletion causes greater expression of TNFα, MMP-3, MMP-13, and MMP-14 during cutaneous repair. Furthermore, CyC-AP is a mediator for fibronectin-fragment induced MMP-13 expression. In order to understand the effects of the deletion of CyC-AP at the cellular level, we investigated the intracellular signal transduction pathways of CyC-AP null and wild-type mice during skin wound healing. Methods: Two-month-old CyC-AP null or wild-type mice were anesthetized and two 0.6 cm diameter excisional skin wounds were made on the dorsum of each mouse. Wounds were collected up to 10 days after injury. Total protein was purified from each wound and the protein expression of TNFα converting enzyme (TACE), integrin beta 1, ERK 1, ERK 2, p38, PCNA, and smooth muscle actin (sm-actin) was analyzed by immunoblot. Results: TNFα expression was higher in CyC-AP null mice in unwounded skin. However, the elevated TNFα in CyC-AP null wounds was lower compared to the wild-type mice. To further understand the relationship of CyC-AP and TNFα, TACE expression was examined. TACE expression was less in the skin of CyC-AP null mice compared to wild-type mice. Expression of p38 and PCNA increased while sm-actin decreased in both unwounded and wounded CyC-AP null skin compared to wild type. PCNA expression in CyC-AP null fibroblasts was also higher compared to wild type, supporting our in vivo observations. In addition, the expression of integrin beta 1 was lower during wound healing. Conclusion: The increased PCNA and decreased sm-actin expression suggests that CyC-AP null fibroblasts have higher proliferation and lower differentiation rates compared to wild types. These effects might be regulated through the p38 signaling pathway, which was increased in CyC-AP null mice. The decreased TACE expression in CyC-AP null skin suggests CyC-AP may also regulate TACE via the altered expression of integrin beta 1. Our data suggest CyC-AP is involved in TNFα activation.

Cyclophilin C‐associated protein is up‐regulated during wound healing

Cyclophilin C-associated protein (CyCAP) is identified from macrophages. It locates in intracellular, membrane bound and extracellular, suggesting it has an important role, however both of its regulation and function have not been elucidated. The expression of CyCAP in skin and during wound healing is also unknown. We demonstrate that CyCAP is expressed in both dermal fibroblasts and keratinocytes. In the dermis, the majority of CyCAP protein is located intracellular in a filamentous protein form while a lesser amount is in the extracellular matrix (ECM). CyCAP gene and protein expression is increased 1 day after skin wound healing in both fetal and adult rats and remains elevated level up to 1 week in adult rats. Immunohistochemistry studies demonstrate that the increased CyCAP expression locates mainly to inflammatory cells, including macrophages, monocytes and lymphocytes during wound healing. Interferon-gamma increases CyCAP gene and protein expression in cultured rat fibroblasts. We also found that wound healing is slower and less collagen is expressed in skin of CyCAP null mice. These data are the first observations of CyCAP expression in skin and during wound repair. Our data indicates that CyCAP is regulated by IFNgamma and may function on immune defense in macrophages, lymphocytes, dermal fibroblasts and keratinocytes during wound healing.

Cyclophilin C‐associated protein is an intracellular mediator between tissue transglutaminase to TNFa

Introduction. Previous study reveals that cyclophilin C-associated protein (CyCAP) null cells do not release MMP-13 after treatment with 45 kd fibronectin fragment (FN45). Since FN45 has a tissue transglutaminase (tTG) binding site but not integrin binding motif, therefore, the effects of FN45 may through tTG binding. In the present study, the connection of CyCAP with tTG, TACE and TNFa release was tested. Methods. The expression of tTG in CyCAP null fibroblasts was tested with Western blot. The release of TNFa by FN45, FN70, IL-1b, tTG and heparin in CyCAP null and wild-type fibroblasts was examined with ELISA. The gene expression of TACE, tTG and TNFa in CyCAP null and wild-type dermal fibroblasts was tested by real-time quantitative PCR. Results. Western blots indicate that tTG expression in CyCAP null cell is lower than wild-type cells. FN45 induces TNFa gene expression more than 13 folds at eight-hour time point only in wild-type cells but not in CyCAP null cells. The released TNFa is 57 fold higher by FN45 and 43 folds higher by tTG in wild-type comparing to CyCAP null cells. The tTG inhibitory antibody showed slight inhibitory effects on MMP-13 expression in wild-type cells. Conclusion. Our data indicate that deletion of CyCAP reduces tTG activity, which in turn damages the function of TACE, i.e. decrease the cleavage of TNFa into extracellular matrix. The release of MMP-13 by FN45 is abolished in CyCAP null cells, suggesting FN45 induced MMP-13 is through the effect of TNFa. Our data give first evidence that CyCAP has intracellular connection with tTG. Deletion of CyCAP abolished TACE effects on cleaving TNFa, therefore, CyCAP may play important role in anti-inflammatory diseases.

Expression of cyclophilin C-associated protein and cyclophilin C mRNAs in focal cerebral ischemia

Expression of cyclophilin C-associated protein and cyclophilin C mRNAs in focal cerebral ischemia

Cyclophilin C-Associated Protein and Cyclophilin C mRNA are Upregulated in Penumbral Neurons and Microglia after Focal Cerebral Ischemia

Immunophilin ligands, such as cyclosporin A and FK506, have neuroprotective effects in experimental stroke models, although the precise mechanism is unclear. Cyclophilin C-associated protein (CyCAP) is a natural cellular ligand for the immunophilin, cyclophilin C, and has a protective effect against endotoxins by downmodulating the proinflammatory response. Expressions of CyCAP and cyclophilin C mRNA in a rat middle cerebral artery (MCA) occlusion ischemia model were investigated by Northern blotting and in situ hybridization. Both CyCAP and cyclophilin C mRNAs were ubiquitously distributed in the neurons of the normal brain. Expression increased in neurons of the periinfarct zone up to 7 days after MCA occlusion. The neuronal distribution was confirmed by counterimmunostaining of NeuN. Both mRNAs were predominantly expressed in microglia of the ischemic core at 7 days, confirmed by immunostaining with the microglial marker, ED1. The quantification of CyCAP and cyclophilin C mRNAs at 7 days by Northern blot analysis showed the 8.5-fold increase (P<0.005, n=6) and 6.8-fold increase (P<0.005, n=6), respectively, in ischemic core compared with control. The coincidence of CyCAP and cyclophilin C expression in neurons and microglia suggests distinct roles in each cellular population. In particular, the early increase in penumbral neurons might be related to protection in periinfarct neurons.

Cyclophilin C-associated Protein Is a Mediator for Fibronectin Fragment-induced Matrix Metalloproteinase-13 Expression

The function of cyclophilin C-associated protein (CyC-AP) on expression of extracellular matrix and matrix metalloproteinases (MMPs) was studied in CyC-AP-null mice. Fibronectin showed increased expression of the 53- and 29-kDa fragments in skin and wounds from CyC-AP-null mice. Type I collagen had an initial degraded pattern in the skin of CyC-AP-null mice, which did not occur in wild-type mice. MMP-3, MMP-13, MMP-14, and tumor necrosis factor-alpha (TNFalpha) had a higher expression in CyC-AP-null skin. During wound healing, MMP-13 and TNFalpha were stimulated to an even higher level, suggesting they are regulated by multiple factors. To understand the regulatory mechanisms of the up-regulated MMPs, the direct effects of TNFalpha, IL-1beta, 45-kDa fibronectin fragment (FN-45), and the 70-kDa fibronectin fragments (FN-70) on the expression of MMPs were studied. MMP-13 expression increased significantly in both CyC-AP-null and wild-type dermal fibroblasts after treatment with IL-1beta or with TNFalpha. However, MMP-13 expression did not increase in CyC-AP-null fibroblasts but did increase only in wild-type fibroblasts after FN-45 and FN-70 treatment. MMP-3 activation was induced by FN-45 and did not show a difference between CyC-AP-null and wild-type fibroblasts, suggesting different regulatory pathways for FN-45 on MMP-13 and MMP-3 expression. Our data are the first to demonstrate that deletion of CyC-AP can abolish fibronectin fragment-induced MMP-13 expression through an unknown mechanism. CyC-AP is an important factor for the regulation of MMP-13 expression.

Distinct ligand binding properties of Mac-2-binding protein and mouse cyclophilin C-associated protein*

Human Mac-2-binding protein (Mac-2-BP) is a secreted glycoprotein that is widely expressed. It binds to the human macrophage-associated lectin Mac-2 and has been suggested to have a role in host defence. Mouse cyclophilin C-associated protein (mCyCAP) is also a secreted glycoprotein that binds with high affinity to cyclophilin C in the absence of the immunosuppresive drug cyclosporin A. The two proteins share a similar domain structure and considerable sequence identity, including a highly conserved scavenger receptor cysteine-rich domain, and both of them exert their function within the immune system. To elucidate whether these molecules are also functional homologues, we compared their ligand binding properties using cell lines which express Mac-2-BP or mCyCAP as well as transfected cell lines stably expressing mCyCAP or a mutant version lacking the scavenger domain. These experiments show that Mac-2-BP is unable to bind to either human or mouse cyclophilin C and thatmCyCAP cannot bind to Mac-2. The scavenger domain is not required for the interaction between mCyCAP and cyclophilin C. We conclude that these proteins may be part of a larger family of proteins of immunological importance in which closer functional homologues might exists.

Distinct ligand binding properties of Mac-2-binding protein and mouse cyclophilin [correction of mousephilin] C-associated protein.

Human Mac-2-binding protein (Mac-2-BP) is a secreted glycoprotein that is widely expressed. It binds to the human macrophage-associated lectin Mac-2 and has been suggested to have a role in host defence. Mouse cyclophilin C-associated protein (mCyCAP) is also a secreted glycoprotein that binds with high affinity to cyclophilin C in the absence of the immunosuppressive drug cyclosporin A. The two proteins share a similar domain structure and considerable sequence identity, including a highly conserved scavenger receptor cysteine-rich domain, and both of them exert their function within the immune system. To elucidate whether these molecules are also functional homologues, we compared their ligand binding properties using cell lines which express Mac-2-BP or mCyCAP as well as transfected cell lines stably expressing mCyCAP or a mutant version lacking the scavenger domain. These experiments show that Mac-2-BP is unable to bind to either human or mouse cyclophilin C and thatmCyCAP cannot bind to Mac-2. The scavenger domain is not required for the interaction between mCyCAP and cyclophilin C. We conclude that these proteins may be part of a larger family of proteins of immunological importance in which closer functional homologues might exists.

Monoclonal antibodies specific for human tumor-associated antigen 90K/Mac-2 binding protein: tools to examine protein conformation and function.

As part of our effort to identify glycoproteins that contribute to colon cancer progression, we have previously described a family of structurally related glycoproteins expressing beta1-6 branched asparagine(Asn)-linked oligosaccharides defined by monoclonal antibody (MAb 1H9), which are differentially expressed, processed, and glycosylated by human colon carcinoma cell lines (Laferté and Loh [1992]; Biochem J; 283:193-201). MAb 1H9 immunoprecipitates three glycoproteins having apparent sizes of 92-100, 66-70, and 25 kDa, the size heterogeneity attributable to cell-type specific glycosylation differences. We report on the basis of partial protein and cDNA sequence information, that the 100-kDa glycoprotein detected by MAb 1H9 is identical to the 90-kDa glycoprotein variably known as tumor-associated antigen 90K (TAA90K), Mac-2 binding protein, and cyclophilin C-associated protein. Using a PCR-based cloning strategy, the complete cDNA encoding TAA90K was cloned into the eukaryotic expression vector pCDNA-3 (pCD-TAA90K(wt)) and the protein expressed in COS-1 cells. A [(35)S]methionine-labeled 60-kDa polypeptide, processed to an endoglycosidase H-sensitive 74-kDa glycoprotein in the presence of dog pancreas microsomes, was detected in a coupled transcription/translation in vitro reaction. The in vitro-translated 60-kDa polypeptide and N-glycanase-treated TAA90K (60-kDa species) immunoprecipitated from HT29 cells were shown to be structurally identical by limited proteolytic peptide mapping. Using a new panel of 11 TAA90K-specific monoclonal antibodies, including five specific for human TAA90K and six cross-reactive with a 90-kDa species expressed by COS-1 cells, we have detected conformational differences between recombinant wild-type TAA90K, in vitro-synthesized TAA90K, and mutant forms of TAA90K containing point mutations at residues 189, 223, and 259. Furthermore, we have shown that these mutant forms of TAA90K, as well as a truncated form of TAA90K containing amino acid residues 1-383, are defective in secretion. These studies demonstrate the potential usefulness of TAA90K-specific monoclonal antibodies for examining the structure and function of TAA90K, and highlight the contribution of specific amino acid residues to its normal processing and secretion.

Cyclophilin C-associated protein: a normal secreted glycoprotein that down-modulates endotoxin and proinflammatory responses in vivo.

Mouse cyclophilin C-associated protein (CyCAP) is a member of the scavenger-receptor cysteine-rich domain superfamily and is 69% identical to the human Mac-2 binding protein. Here, we show that CyCAP is a widely expressed secreted glycoprotein that modulates the host response to endotoxin. Gene-targeted CyCAP-deficient mice are more sensitive to the lethal effects of endotoxin. In response to endotoxin, CyCAP-deficient mice overproduced interleukin 12 and interferon-gamma systemically and tumor necrosis factor alpha locally; these are proinflammatory molecules that also promote T helper 1 responses. Furthermore, macrophages stimulated in vitro with endotoxin in serum deficient in CyCAP secreted more tumor necrosis factor alpha, supporting the proposal that CyCAP specifically down-modulates endotoxin signaling.

Cloning and expression of a mouse macrophage cDNA coding for a membrane glycoprotein of the scavenger receptor cysteine-rich domain family.

We have cloned from murine macrophages a cDNA coding for a new protein of the scavenger receptor family whose mRNA is increased very strongly by adherence and moderately by exposure to tumor necrosis factor and interferon-gamma. The nucleotidic sequence extends for 2168 bases and encodes a protein of 559 amino acids with six potential glycosylation sites. The first 100 NH2-terminal amino acids represent a single scavenger receptor cysteine-rich domain, whereas the COOH-terminal end of the molecule is compatible either with a transmembrane hydrophobic peptide followed by a very short intracytoplasmic sequence or a signal sequence for an anchoring via a glycophosphatidylinositol. The protein is highly homologous to most of the very recently identified human MAC-2-binding protein and murine cyclophilin C-associated protein.

Cloning and characterization of cyclophilin C-associated protein: a candidate natural cellular ligand for cyclophilin C.

We report the protein purification and the cloning and characterization of a cDNA encoding the proteins that bind with high affinity to cyclophilin C (Cyp-C) in the absence of cyclosporin A. Transfection of this cDNA into COS cells directs the production of a glycoprotein of 77 kDa that binds to Cyp-C in the absence, but not the presence, of cyclosporin A. Homology comparisons reveal that this protein and gene, termed CyCAP for Cyp-C-associated protein, possess a cysteine-rich domain (scavenger receptor cysteine-rich domain) found in a variety of cell-surface molecules; the rest of the sequence is apparently specific. This result raises the possibility that Cyp-C serves as a mediator or regulator of an as-yet-unidentified signal or cellular process initiated via the Cyp-C-associated protein.