Immunohistochemical expression of heparanase isoforms and syndecan-1 proteins in colorectal adenomas.

The proteoglycan syndecan-1 and the endoglucuronidases heparanase-1 and heparanase-2 are involved in molecular pathways that deregulate cell adhesion during carcinogenesis. Few studies have examined the expression of syndecan-1, heparanase-1 and mainly heparanase-2 proteins in non-neoplastic and neoplastic human colorectal adenoma tissues. The aim of this study was to analyze the correlation among the heparanase isoforms and the syndecan-1 proteins through immunohistochemical expression in the tissue of colorectal adenomas. Primary anti-human polyclonal anti-HPSE and anti-HPSE2 antibodies and primary anti-human monoclonal anti-SDC1 antibody were used in the immunohistochemical study. The expressions of heparanase-1 and heparanase-2 proteins were determined in tissue samples from 65 colorectal adenomas; the expression of syndecan-1 protein was obtained from 39 (60%) patients. The histological type of adenoma was tubular in 44 (67.7%) patients and tubular-villous in 21 (32.3%); there were no villous adenomas. The polyps were <1.0 cm in size in 54 (83.1%) patients and ≥1.0 cm in 11 (16.9%). The images were quantified by digital counter with a computer program for this purpose. The expression index represented the relationship between the intensity expression and the percentage of positively stained cells. The results showed that the average of heparanase-1, heparanase-2 and syndecan-1 expression index was 73.29 o.u./µm², 93.34 o.u./µm², and 55.29 o.u./µm², respectively. The correlation between the heparanase-1 and syndecan-1 expression index was positive (R=0.034) and significant (P=0.035). There was a negative (R= -0.384) and significant (P=0.016) correlation between the expression index of heparanase-1 and heparanase-2. A negative (R= -0.421) and significant (P=0.008) correlation between the expression index of heparanase-2 and syndecan-1 was found. We concluded that in colorectal adenomas, the heparanase-1 does not participate in syndecan-1 degradation; the heparanase-2 does not stimulate syndecan-1 degradation by the action of heparanase-1, and the heparanase-2 may be involved in the modulation of the heparanase-1 activity.

Downregulation of Heparanase Expression Results in Suppression of Invasion, Migration, and Adhesion Abilities of Hepatocellular Carcinoma Cells.

OBJECTIVE: Heparanase (HPSE) is high-expressed in most malignant tumors including hepatocellular carcinoma (HCC) and promotes cancer cell invasion and migration. The aim of the study is to explore whether HPSE enhances adhesion in metastasis of HCC cells. METHODS: HPSE expressions in human HCC cells were measured with real-time RT-PCR and Western blot analysis. Four recombinant miRNA vectors pcDNATM6.2-GW/EmGFP-miR-HPSE (pmiR-HPSE) were transfected into HCCLM3 cell. HPSE expression in transfected cell was measured. The cell invasion, migration, and adhesion abilities were detected, respectively. RESULTS: Both HPSE mRNA and protein relative expression levels were higher in HepG2, BEL-7402, and HCCLM3 cells than those in normal hepatocyte (P < 0.05). HPSE showed highest expression level in HCCLM3 cell (P < 0.05). Transfection efficiencies of four miRNA vectors were 75%-85%. The recombinant vectors significantly decreased HPSE expression in transfected HCCLM3 cells (P < 0.01), and pmiR-HPSE-1 showed best interference effect (P < 0.05). pmiR-HPSE-1 significantly decreased the penetrated and migrating cells numbers and adherence rate of HCCLM3 cells (P < 0.05). CONCLUSION: HPSE is a potentiator of cell adhesion in metastasis of HCC.

Expression of HpaI in Pichia pastoris and optimization of conditions for the heparinase I production.

Heparinase I has important applications in the fields of biomedicine and pharmaceuticals. The heparinase I gene (HpaI) from Flavobacterium heparinum was cloned and overexpressed in Pichia pastoris GS115, and the conditions for the heparinase I production were optimized by RSM. PCR analysis indicated that HpaI was integrated into the P. pastoris GS115 genome. The concentrations of key factors that affected the heparinase I activity were optimized, and were as follows: oleic acid, 0.07%, liquid volume in flask, 34.3 ml/L, and methanol, 0.96%. Under the optimal conditions, the activity of heparinase I was up to 323 U/L in shake flask. A maximal heparinase I activity of 398.5 U/L from the transformant 2 was achieved in a 5L fermentor. This study demonstrates the overproduction of heparinase I by recombinant P. pastoris.

Expression of the heparinase gene from Flavobacterium heparinum in Escherichia coli and its enzymatic properties.

Heparinase has an important application in the preparation of low-molecular-weight heparins by heparin enzymolysis. A heparinase gene from Flavobacterium heparinum was cloned and expressed in Escherichia coli BL21 in order to enhance its activity. The expressed heparinase was purified to homogeneity by a metal chelating affinity column and its enzymatic properties were evaluated. A maximal heparinase activity of 1061 IU/L toward the substrate heparin was achieved when the recombinant strain was induced with 0.5 mM isopropyl-ß-D-thiogalactoside at 28 °C for 9 h. The optimal temperature and pH of heparinase were 30 °C and 7.0, respectively. The recombinant heparinase was heat-unstable and had a higher stability at pHs from 7.0 to 10.0. Observed activities of heparinase were the highest in the presence of Ca(2+) and Cu(2+) and the lowest in the presence of Mn(2+) and Pb(2+). These results lay a good foundation for the preparation of LMWHs by heparin enzymolysis.

Two promoters with distinct activities in different tissues drive the expression of heparanase in Xenopus.

In Xenopus laevis embryos, heparanase, the enzyme that degrades heparan sulfate, is synthesized as a preproheparanase (XHpaL) and processed to become enzymatically active (XHpa active). A short nonenzymatic heparanase splice variant (XHpaS) is also expressed. Using immunohistochemistry, Western blot, and heparanase promoter analysis, we studied the dynamic developmental expression of the three heparanases. Our results indicate that (1) all three isoforms are maternally expressed; (2) XHpaS is a developmental variant; (3) in the early embryo, heparanase is localized to both the plasma membrane and the nucleus; (4) several tissues express heparanase, but expression in the developing nervous system is most evident; (5) two promoters with distinct activities in different tissues drive heparanase expression; (6) Oct binding transcription factors may modulate heparanase promoter activity in the early embryo. These data argue that heparanase is expressed widely during development, but localization and levels are finely regulated.

Cloning, expression and characterization of acharan sulfate-degrading heparin lyase II from Bacteroides stercoris HJ-15.

AIMS: This study focused on the cloning, expression and characterization of recombinant heparinase II (rHepII) from Bacteroides stercoris HJ-15. METHODS AND RESULTS: The heparinase II gene from Bact. stercoris HJ-15 was identified by Southern blotting and the sequence was deposited in GenBank. The gene was cloned and overexpressed in Escherichia coli, and rHepII was purified using two simple ion-exchange column chromatography steps. Enzymatic properties and substrate specificities of rHepII were assessed and its kinetic constants were calculated. Heparin-like glycosaminoglycans (HLGAGs) were digested with rHepII under optimal reaction conditions, and the products were analysed by SAX-HPLC. CONCLUSIONS: The heparinase II gene is 2322-bp long and consists of 773 amino acids. rHepII is most active in 50 mmol l(-1) sodium phosphate buffer with 75 mmol l(-1) NaCl (pH 7.4) at 32 degrees C, and the activity is stable at 4 degrees C for 15 days on storage. Acharan sulfate is the best substrate for rHepII, followed by heparan sulfate and heparin. The major degradation products were verified as highly sulfated disaccharides through SAX-HPLC analysis. It means that rHepII prefers iduronic acid over glucuronic acid on the HLGAG structure. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides easy and certain means for obtaining large amounts of pure rHepII and also provides important information regarding the tendencies of this enzyme and its digested products. rHepII digests HLGAGs in a different manner than heparinases from Flavobacterium heparinum; therefore, we anticipate that rHepII will be a powerful tool for studies of GAGs and GAGs lyases.

Response surface methodology for optimization of medium components in submerged culture of Aspergillus flavus for enhanced heparinase production.

AIMS: Aim of the study was to develop a medium for optimal heparinase production with a strain of Aspergillus flavus (MTCC-8654) by using a multidimensional statistical approach. METHODS AND RESULTS: Statistical optimization of intracellular heparinase production by A. flavus, a new isolate, was investigated. Plackett-Burman design was used to evaluate the affect of medium constituents on heparinase yield. The experimental results showed that the production of heparinase was dependent upon heparin, the inducer; chitin, structurally similar to heparin and NH(4)NO(3,) the nitrogen source. A central composite design was applied to derive a statistical model for optimizing the composition of the fermentation medium for the production of heparinase enzyme. The optimum fermentation medium consisted of (g l(-1)) Mannitol, 8.0; NH(4)NO(3), 2.5; K(2)HPO(4), 2.5; Na(2)HPO(4), 2.5; MgSO(4).7H(2)O, 0.5; Chitin, 17.1; Heparin, 0.6; trace salt solution (NaMoO(4).2H(2)O, CoCl(2).6H(2)O, CuSO(4).5H(2)O, FeSO(4).7H(2)O, CaCl(2)), 10(-4) mol l(-1). CONCLUSIONS: A 2.37-fold increase in heparinase production was achieved in economic and effective manner by the application of statistical designs in medium optimization. SIGNIFICANCE AND IMPACT OF THE STUDY: Heparinase production was doubled by statistical optimization in a cost-effective manner. This heparinase can find application in pharmaceutical industry and for the generation of low-molecular-weight heparins, active as antithrombotic and antitumour agents.

Purification and properties of recombinant GST-heparinase III and optimization of cultivation conditions.

Heparinase III is an enzyme that specifically cleaves certain sequences of heparan sulfate. Previous reports showed that this enzyme expressed in Escherichia coli was highly prone to aggregation in inclusion bodies and lacks detectable biological activity. In this paper, we fused a glutathione-S-transferase (GST) tag to the N-terminus of heparinase III gene and expressed the fusion protein in Escherichia coli to develop an expression system of soluble heparinase III. As a result, approximately 80% of the fusion protein was soluble. The protein was then purified to near homogeneity via one-step affinity chromatography. A 199.4-fold purification was achieved and the purified enzyme had a specific activity of 101.7 IU/mg protein. This represented 32.3% recovery of the total activity of recombinant GST-heparinase III. The maximum enzyme production was achieved when bacteria were induced with 0.5 mmol/L isopropyl-beta-D-thiogalactoside at 15 degrees C for 12 h. The enzyme showed maximum activity at 30 degrees C and pH 7.5. And the enzyme activity was stimulated by 1 mmol/L Ca2+ and 150 mmol/L NaCl.

Two heparanase splicing variants with distinct properties are necessary in early Xenopus development.

Heparanase is an endoglycosidase that cleaves heparan sulfate (HS) side chains from heparan sulfate proteoglycans (HSPGs) present in extracellular matrix and cell membranes. Although HSPGs have many functions during development, little is known of the role of the enzyme that degrades HS, heparanase. We cloned and characterized the expression of two heparanase splicing variants from Xenopus laevis and studied their function in early embryonic development. The heparanase gene (termed xHpa) spans over 15 kb and consists of at least 12 exons. The long heparanase (XHpaL) cDNA encodes a 531-amino acid protein, whereas the short splicing variant (XHpaS) results in a protein with the same open reading frame but missing 58 amino acids as a consequence of a skipped exon 4. Comparative studies of both isoforms using heterologous expression systems showed: 1) XHpaL is enzymatically active, whereas XHpaS is not; 2) XHpaL and XHpaS interact with heparin and HS; 3) both proteins traffic through the endoplasmic reticulum and Golgi apparatus, but XHpaL is secreted into the medium, whereas XHpaS remains associated with the membrane as a consequence of the loss of three glycosylation sites; 4) overexpression of XHpaS but not XHpaL increases cell adhesion of glioma cells to HS-coated surfaces; 5) XHpaL and XHpaS mRNA and protein levels vary as development progresses; 6) specific antisense knock-down of both XHpaL and XHpaS, but not XHpaL alone, results in failure of embryogenesis to proceed. Interestingly, rescue experiments suggest that the two heparanases regulate the same developmental processes, but via different mechanisms.

Heparin, heparan sulfate and heparanase in cancer: remedy for metastasis?

Malignant tumor cells invade normal tissues in the vicinity of cancer through devastating the extracelluar matrix and blood vessel wall of the tissues. An important step in this process is degradation of heparan sulfate proteoglycan, a carbohydrate-protein complex. Heparan sulfate proteoglycan is a major component of the extracellular matrix, and is essential for the self-assembly, insolubility and barrier properties of basement membranes. Heparanase is an endoglucuronidase that cleaves heparan sulfate and expression level of this enzyme correlates with metastatic potential of tumor cells. Treatment with heparanase inhibitors markedly reduces the incidence of metastasis in experimental animals. Heparin, a widely used anticoagulant, is structurally related to heparan sulfate and a natural substrate of heparanase. Long-term treatment of cancer patients having venous thromboembolism with low molecular weight heparin showed improved survival rate. Understanding the functional roles and the corresponding molecular mechanisms of heparin, heparan sulfate and heparanase in cancer development may pave the way for exploring remedies against tumor metastasis.

Heparanase induces tissue factor expression in vascular endothelial and cancer cells.

BACKGROUND: Over-expression of tissue factor (TF) and activation of the coagulation system are common in cancer patients. Heparanase is an endo-beta-D-glucuronidase that cleaves heparan sulfate chains on cell surfaces and in the extracellular matrix, activity that closely correlates with cell invasion, angiogenesis and tumor metastasis. The study was undertaken to investigate the involvement of heparanase in TF expression. METHODS: Tumor-derived cell lines were transfected with heparanase cDNA and TF expression was examined. The effect of exogenous addition of active and inactive heparanase on TF expression and activity was studied in tumor cell lines and primary human umbilical vein endothelial cells. TF expression was also explored in heparanase over-expressing transgenic (Tg) mice. Blast cells were collected from acute leukemia patients and TF and heparanase expression levels were analyzed. RESULTS: Over-expression of heparanase in tumor-derived cell lines resulted in a 2-fold increase in TF expression levels, and a similar trend was observed in heparanase Tg mice in vivo. Likewise, exogenous addition of heparanase to endothelial or tumor-derived cells resulted in enhanced TF expression and activity. Interestingly, TF expression was also induced in response to enzymatically inactive heparanase, suggesting that this effect was independent of heparanase enzymatic activity. The regulatory effect of heparanase on TF expression involved activation of the p38 signaling pathway. A positive correlation between TF expression levels and heparanase activity was found in blasts collected from 22 acute leukemia patients. CONCLUSIONS: Our results indicate that in addition to its well-known function as an enzyme paving a way for invading cells, heparanase also participates in the regulation of TF gene expression and its related coagulation pathways.

[Strain screening and fermentation conditions of a novel heparinase-producing strain].

The novel heparinase-producing bacterial strain Corynebacterium sp. was screened and isolated from soil. The optimum medium composition is (g/L): Trypticase 20, NaCl 1, K2HPO4 2.5, MgSO4 0.5, Heparin 2, maltose 20, pH 6.5. The optimum growth temperature was 27 degrees C while, maximum enzyme production was achieved at temperature 31 degrees C. When cultured at a rotating shaker at 30 degrees C for 24 hours, 200 r/min, 40 mL medium in 500 mL flask, the Production of heparinase reached 1700 u/L.