Cloning And Subcellular Localization Of Human Heparanase Alternatively Spliced Variant

Heparan sulfate proteoglycans (HSPG) are macromolecules associated with the cell surface and extracellular matrix (ECM) of a wide range of cells. The basic HSPG structure consists of a protein core to which several linear heparan sulfate (HS) are connected. Heparan sulfate binds to and assembles ECM proteins, including growth factor and cytokine, is playing important roles in pathology and physiological processes. Heparanase (HPSE) is a mammalian endo-β-glucuronidase that can cleave heparin sulfate side chain of HSPG, facilitating structural alterations of the extracellular matrix and basement membrane underlying epithelial and endothelial cells, which makes it more susceptible to cellular invasion, and liberation of a multitude of biological mediators. Heparanase activity has been traditionally correlated with cancer metastasis, angiogenesis, inflammatory process and autoimmune disease.Almost all eukaryotic genes are interrupted gene which consists of exons and introns. RNA splicing is the process of excising the sequences in RNA that corresponds to introns, so that the sequence corresponding to exons are connected into a continuous messenger RNA. About half of the gene exons correlate with protein domains and motifs. Through recognizing different splicing sites, alternative splicing (AS) of precursor messenger RNA generates different isoforms from a single gene, which plays an important role in the generation of functional diversity of protein and the complexity of gene expression. Alternative splicing is also important for regulation of the levels and tissue specificity of gene expression and, if disrupted, can lead to disease. Along with profound study on this field will reveal the pathogenesis of hereditary disease which mechanism are not clear yet and find novel target of tumor therapy.The purpose of this research is to clone heparanase alternatively spliced variants from human peripheral blood leukocytes and tumor cells, then study their subcellular localization and biological function.The main contents of this research include three aspects as follows: 1). Screening and identification of heparanase alternatively spliced variants from human peripheral blood leukocytes and tumor cells. 2). Construction, expression of recombinant plasmids of heparanase alternatively spliced variants and studying their subcellular localization. 3). Construction eukaryotic expression plasmid of heparanase alternatively spliced variants and expression in mammalian cells for the purpose of enzymatic activity studying and tumorigenesis.First, the human heparanase alternatively spliced variants were amplified from peripheral blood leukocytes, SGC-7901, SMMC-7721 and Du145 by RT-PCR using specific primers based on the sequence of heparanase cDNA in GenBank, then cloned into pGEM-T Easy vector. The recombinant vector were further transformed into Escherichia coli strain DH5αand positive clones were screened and sequenced. Second, the human heparanase alternatively spliced variants were cloned into pEGFP-N3 vector. Transient transfection of HEK293FT and CHO-K1 were performed and heparanase alternatively spliced variants subcellular location were observed using laser scanning confocal fluorescence microscope. Third, the human heparanase alternatively spliced variants were inserted into the expression vector pcDNA3.1(+)and transferred into U87 and MCF-7. Stable transfected cells were selected and identified.Results: 1). We obtained five alternatively spliced variants of human heparanase, lacked exon 5, exon 6, exon 5 and exon 6, exon 9 and exon 10, exon 10 respectively, named splice5,splice6,slice5&6,splice9&10,splice10. The latter four were novel spliced form of human heparanase and were registered (GenBank :FJ517659,FJ517660,GQ337901,GQ337902). We obtainned all of the five heparanase alternatively spliced variants in peripheral blood leukocytes, however, only splice5 and splice9&10 in SMMC-7721 ,splice5 in SMMC-7721 and splice10 in Du145. 2). Plasmids pEGFP- HPSE/s5/s9&10/s10 were constructed and expressed in HEK293FT, B16F10 and CHO-K1. Results showed that wild-type heparanase exhibited predominantly granular pattern in cytoplasm, in contrast, splice5,splice9&10,splice10 all exhibited diffused pattern in cytoplasm. 3). Plasmid pcDNA- HPSE/s5/s9&10/s10 were constructed and transferred into U87 and MCF-7, but positive candidate cell line have not got yet.In this study, we cloned four novel splice variants of human heparanase splice6,splice5&6,splice9&10,splice10. It was noting that splice10, lack of exon 10, results in a premature termination codon (PTC), but it was not degradation by nonsense mediated decay (NMD). Furthermore, different from other heparanase alternatively spliced variants, splic10 have heparin sulfate binding sites and active sites. This means that splice10 may have potential heparanase enzymatic activity and its biological function may be different.Pre-pro-heparanase is first targeted to the endoplasmic reticulum via its own signal peptide. The 65 kD pro-heparanase is then shuttled to the Golgi apparatus, and is subsequently secreted via vesicles that bud from Golgi. Once secreted, heparanase rapidly interacts with cell membrane HSPGs, followed by a rapid endocytosis of heparanase-HSPG complex that appears to accumulated in endosomes. Conversion of endosomes to lysosome results in heparanase processing and activation. In this study, we found that wild-type heparanase exhibited predominantly granular pattern in cytoplasm. In contrast, splice5,splice9&10,splice10 exhibited diffused pattern in cytoplasm. These results suggested two possible explanations: On one hand, these alternatively spliced variants were not trafficked to lysosome and lack of heparanase enzymatic activity because they were not processed and activated. On the other hand, these alternatively spliced variants were trafficked through lysosome and then became heterodimer, an active form of heparanase, hereby localization in other place. The difference of subcellular localization implies different functions thus renders further study on these alternatively spliced variants necessary.