Expression pattern and secretion of human and chicken heparanase are determined by their signal peptide sequence

Cleavage of heparan sulfate (HS) proteoglycans affects the integrity and function of tissues and thereby fundamental phenomena, involving cell migration and response to changes in the extracellular microenvironment. The role of HS-degrading enzymes, commonly referred to as heparanases, in normal development has not been identified. The present study focuses on cloning, expression, and properties of a chicken heparanase and its distribution in the developing chicken embryo. We have identified a chicken EST, homologous to the recently cloned human heparanase, to clone and express a functional chicken heparanase, 60% homologous to the human enzyme. The full-length chicken heparanase cDNA encodes a 60-kDa proenzyme that is processed at the N terminus into a 45-kDa highly active enzyme. The most prominent difference between the chicken and human enzymes resides in the predicted signal peptide sequence, apparently accounting for the chicken heparanase being readily secreted and localized in close proximity to the cell surface. In contrast, the human enzyme is mostly intracellular, localized in perinuclear granules. Cells transfected with a chimeric construct composed of the chicken signal peptide preceding the human heparanase exhibited cell surface localization and secretion of heparanase, similar to cells transfected with the full-length chicken enzyme. We examined the distribution pattern of the heparanase enzyme in the developing chicken embryo. Both the chicken heparanase mRNA and protein were expressed, as early as 12 h post fertilization, in cells migrating from the epiblast and forming the hypoblast layer. Later on (72 h), the enzyme is preferentially expressed in cells of the developing vascular and nervous systems. Cloning and characterization of heparanase, the first and single functional vertebrate HS-degrading enzyme, may lead to identification of other glycosaminoglycan degrading enzymes, toward elucidation of their significance in normal and pathological processes.     

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 lambdagt11 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.     

Halofuginone: a potent inhibitor of critical steps in angiogenesis progression.

We have previously demonstrated that halofuginone, a low molecular weight quinazolinone alkaloid, is a potent inhibitor of collagen alpha1(I) and matrix metalloproteinase 2 (MMP-2) gene expression. Halofuginone also effectively suppresses tumor progression and metastasis in mice. These results together with the well-documented role of extracellular matrix (ECM) components and matrix degrading enzymes in formation of new blood vessels led us to investigate the effect of halofuginone on the angiogenic process. In a variety of experimental system, representing sequential events in the angiogenic cascade, halofuginone treatment resulted in profound inhibitory effect. Among these are the abrogation of endothelial cell MMP-2 expression and basement membrane invasion, capillary tube formation, and vascular sprouting, as well as deposition of subendothelial ECM. The most conclusive anti-angiogenic activity of halofuginone was demonstrated in vivo (mouse corneal micropocket assay) by showing a marked inhibition of basic fibroblast growth factor (bFGF) -induced neovascularization in response to systemic administration of halofuginone, either i.p. or in the diet. The ability of halofuginone to interfere with key events in neovascularization, together with its oral bioavailability and safe use as an anti-parasitic agent, make it a promising drug for further evaluation in the treatment of a wide range of diseases associated with pathological angiogenesis.     

Site-directed mutagenesis, proteolytic cleavage, and activation of human proheparanase

Heparanase is an endo-beta-D-glucuronidase that degrades heparan sulfate in the extracellular matrix and cell surfaces. Human proheparanase is produced as a latent 65-kDa polypeptide undergoing processing at two potential proteolytic cleavage sites, located at Glu109-Ser110 (site 1) and Gln157-Lys158 (site 2). Cleavage of proheparanase yields 8- and 50-kDa subunits that heterodimerize to form the active enzyme. The fate of the linker segment (Ser110-Gln157) residing between the two subunits, the mode of processing, and the protease(s) engaged in proheparanase processing are currently unknown. We applied multiple site-directed mutagenesis and deletions to study the nature of the potential cleavage sites and amino acids essential for processing of proheparanase in transfected human choriocarcinoma cells devoid of endogenous heparanase but possessing the enzymatic machinery for proper processing and activation of the proenzyme. Although mutagenesis at site 1 and its flanking sequences failed to identify critical residues for proteolytic cleavage, processing at site 2 required a bulky hydrophobic amino acid at position 156 (i.e. P2 of the cleavage site). Substitution of Tyr156 by Ala or Glu, but not Val, resulted in cleavage at an upstream site in the linker segment, yielding an improperly processed inactive enzyme. Processing of the latent 65-kDa proheparanase in transfected Jar cells was inhibited by a cell-permeable inhibitor of cathepsin L. Moreover, recombinant 65-kDa proheparanase was processed and activated by cathepsin L in a cell-free system. Altogether, these results suggest that proheparanase processing at site 2 is brought about by cathepsin L-like proteases. The involvement of other members of the cathepsin family with specificity to bulky hydrophobic residues cannot be excluded. Our results and a three-dimensional model of the enzyme are expected to accelerate the design of inhibitory molecules capable of suppressing heparanase-mediated enhancement of tumor angiogenesis and metastasis.     

Mammalian heparanase: gene cloning, expression and function in tumor progression and metastasis.

Heparan sulfate proteoglycans interact with many extracellular matrix constituents, growth factors and enzymes. Degradation of heparan sulfate by endoglycosidic heparanase cleavage affects a variety of biological processes. We have purified a 50-kDa heparanase from human hepatoma and placenta, and now report cloning of the cDNA and gene encoding this enzyme. Expression of the cloned cDNA in insect and mammalian cells yielded 65-kDa and 50-kDa recombinant heparanase proteins. The 50-kDa enzyme represents an N-terminally processed enzyme, at least 100-fold more active than the 65-kDa form. The heparanase mRNA and protein are preferentially expressed in metastatic cell lines and specimens of human breast, colon and liver carcinomas. Low metastatic murine T-lymphoma and melanoma cells transfected with the heparanase cDNA acquired a highly metastatic phenotype in vivo, reflected by a massive liver and lung colonization. This represents the first cloned mammalian heparanase, to our knowledge, and provides direct evidence for its role in tumor metastasis. Cloning of the heparanase gene enables the development of specific molecular probes for early detection and treatment of cancer metastasis and autoimmune disorders.     

Human heparanase is localized within lysosomes in a stable form

Heparanase is an endo-beta-D-glucuronidase involved in degradation of heparan sulfate (HS) and extracellular matrix (ECM) of a wide range of cells of vertebrate and invertebrate tissues. The enzymatic activity of heparanase is characterized by specific intrachain cleavage of glycosidic bonds with a hydrolase mechanism. This enzyme facilitates cell invasion and hence plays a role in tumor metastasis, angiogenesis, inflammation, and autoimmunity. Although the expression pattern and molecular properties of heparanase have been characterized, its subcellular localization has not been unequivocally determined. We have previously suggested that heparanase subcellular localization is a major determinant in regulating the enzyme's biological functions. In the present study we examined heparanase localization in three different cell types, utilizing immunofluorescent staining and electron microscopy. Our results indicate that heparanase is localized primarily within lysosomes and the Golgi apparatus. A construct composed of heparanase cDNA fused to green fluorescent protein, utilized in order to visualize the enzyme within living cells, confirmed its localization in acidic vesicles. We suggest that following synthesis, heparanase is transported into the Golgi apparatus and subsequently accumulates in a stable form within the lysosomes, where it functions in HS turnover. The lysosomal compartment may also serve as a site for heparanase confinement within the cells, limiting its secretion and uncontrolled extracellular activities associated with tumor metastasis and angiogenesis.     

Mapping Risk of Nipah Virus Transmission from Bats to Humans in Thailand

Nipah virus (NiV) is a zoonotic virus that can pose a serious threat to human and livestock health. Old-world fruit bats (Pteropus spp.) are the natural reservoir hosts for NiV, and Pteropus lylei, Lyle’s flying fox, is an important host of NiV in mainland Southeast Asia. NiV can be transmitted from bats to humans directly via bat-contaminated foods (i.e., date palm sap or fruit) or indirectly via livestock or other intermediate animal hosts. Here we construct risk maps for NiV spillover and transmission by combining ecological niche models for the P. lylei bat reservoir with other spatial data related to direct or indirect NiV transmission (livestock density, foodborne sources including fruit production, and human population). We predict the current and future (2050 and 2070) distribution of P. lylei across Thailand, Cambodia, and Vietnam. Our best-fit model predicted that central and western regions of Thailand and small areas in Cambodia are currently the most suitable habitats for P. lylei. However, due to climate change, the species range is predicted to expand to include lower northern, northeastern, eastern, and upper southern Thailand and almost all of Cambodia and lower southern Vietnam. This expansion will create additional risk areas for human infection from P. lylei in Thailand. Our combined predictive risk maps showed that central Thailand, inhabited by 2.3 million people, is considered highly suitable for the zoonotic transmission of NiV from P. lylei. These current and future NiV transmission risk maps can be used to prioritize sites for active virus surveillance and developing awareness and prevention programs to reduce the risk of NiV spillover and spread in Thailand.