High-level expression of the native barley alpha-amylase/subtilisin inhibitor in Pichia pastoris.

An expression system for high-level expression of the native Hordeum vulgare alpha-amylase/subtilisin inhibitor (BASI) has been developed in Pichia pastoris, using the methanol inducible alcohol oxidase 1 (AOX1) promoter. To optimize expression, two codon-optimized coding regions have been designed and expressed alongside the wild-type coding region. To ensure secretion of the native mature protein, a truncated version of the alpha mating factor secretion signal from Saccharomyces cerevisiae was used. In order to be able to compare expression levels from different clones, single insertion transformants generated by gene replacement of the AOX1 gene was selected by PCR screening. Following methanol induction, expression levels reached 125 mgL(-1) from the wild-type coding region while expression from the two codon-optimized variants reached 65 and 125 mgL(-1), respectively. The protein was purified and characterized by Edman degradation, liquid chromatography mass spectrometry and insoluble blue starch assay, and was shown to possess the same characteristics as wild-type protein purified from barley grains.

Isolation of a natural inhibitor of human malignant glial cell invasion: inter alpha-trypsin inhibitor heavy chain 2.

Malignant central nervous system (CNS) tumors, such as glioblastoma multiforme, invade the brain and disrupt normal tissue architecture, making complete surgical removal virtually impossible. Here, we have developed and optimized a purification strategy to isolate and identify natural inhibitors of glioma cell invasion in a three-dimensional collagen type I matrix. Inter alpha-trypsin inhibitor heavy chain 2 (ITI H2) was identified from the most inhibitory fractions and its presence was confirmed both as a single protein and in a bikunin-bound form. Stable overexpression in U251 glioma cells validated ITI H2's strong inhibition of human glioma cell invasion together with significant inhibition of cell proliferation and promotion of cell-cell adhesion. Analysis of primary human brain tumors showed significantly higher levels of ITI H2 in normal brain and low-grade tumors compared with high-grade gliomas, indicating an inverse correlation with malignancy. The phosphatidylinositol 3-kinase/Akt signaling cascade seemed to be one of the pathways involved in the effect of ITI H2 on U251 cells. These findings suggest that reduction of ITI H2 expression correlates with brain tumor progression and that targeting factors responsible for its loss or restoring the ITI supply exogenously may serve as potential therapeutic strategies for a variety of CNS tumors.

Tumor suppressor activity and epigenetic inactivation of hepatocyte growth factor activator inhibitor type 2/SPINT2 in papillary and clear cell renal cell carcinoma.

Following treatment with a demethylating agent, 5 of 11 renal cell carcinoma (RCC) cell lines showed increased expression of hepatocyte growth factor (HGF) activator inhibitor type 2 (HAI-2/SPINT2/Bikunin), a Kunitz-type protease inhibitor that regulates HGF activity. As activating mutations in the MET proto-oncogene (the HGF receptor) cause familial RCC, we investigated whether HAI-2/SPINT2 might act as a RCC tumor suppressor gene. We found that transcriptional silencing of HAI-2 in RCC cell lines was associated with promoter region methylation and HAI-2/SPINT2 protein expression was down-regulated in 30% of sporadic RCC. Furthermore, methylation-specific PCR analysis revealed promoter region methylation in 30% (19 of 64) of clear cell RCC and 40% (15 of 38) of papillary RCC, whereas mutation analysis (in 39 RCC cell lines and primary tumors) revealed a missense substitution (P111S) in one RCC cell line. Restoration of HAI-2/SPINT2 expression in a RCC cell line reduced in vitro colony formation, but the P111S mutant had no significant effect. Increased cell motility associated with HAI-2/SPINT2 inactivation was abrogated by treatment with extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) and phospholipase C-gamma inhibitors, but not by an inhibitor of atypical protein kinase C. These findings are consistent with frequent epigenetic inactivation of HAI-2/SPINT2, causing loss of RCC tumor suppressor activity and implicate abnormalities of the MET pathway in clear cell and papillary sporadic RCC. This information provides opportunities to develop novel targeted approaches to the treatment of RCC.

Upregulation of bikunin in tumor-infiltrating macrophages as a factor of favorable prognosis in ovarian cancer.

OBJECTIVE: This study was carried out to clarify the localization of bikunin, a Kunitz-type protease inhibitor, and relation between expression of individual bikunin protein and ovarian cancer progression. METHODS: We performed a retrospective study on the immunohistochemical expression of bikunin, urokinase-type plasminogen activator (uPA) and macrophages (CD68) in surgical specimens derived from 89 ovarian cancer patients to investigate correlations between the expression of bikunin and the clinicopathologic features and the prognosis. Furthermore, bikunin and uPA levels were measured by immunoblot analysis. RESULTS: Immunohistochemical staining revealed that the localization of bikunin was similar to that of CD68 for macrophages. We identified high expression of bikunin in 40 (45%) of 89 ovarian cancers. The results of Western blot analysis showed a significant correlation with immunohistochemical data. There was a significant inverse correlation between bikunin levels and uPA levels in ovarian cancer tissues. High bikunin expression was an independent predictor for disease-free survival (P = 0.040) and overall survival (P = 0.042). The 5-year survival rate of the 49 patients with low bikunin expression in ovarian cancers was 39%, whereas that of the other 40 patients with high bikunin expression was 63%. In addition, macrophage-derived bikunin protein was induced by exogenous IL-6. CONCLUSION: Bikunin derived from tumor-infiltrating macrophages might be a prognostic indicator as an antiinvasive factor supplied from macrophages within and around the tumor possibly through down-regulation of tumor-associated uPA expression.

[The effects of the active constituents of Alisma orientalis on renal stone formation and bikunin expression in rat urolithiasis model].

OBJECTIVE: To evaluate the effects of the active constituents of Alisma orientalis on the expression of bikunin mRNA in rat urolithiasis model, and explore the mechanism of this traditional Chinese medicine on prevention of urinary calculi. METHODS: Modern phytochemistry and bioactivity guided isolation techniques were applied to extract the active constituents of Alisma orientalis. Hyperoxaluria and the renal oxalate calcium stone formation were induced in rats by infusion into the stomach with 1% ethylene glycol and 2% ammonium chloride. 30 adult male Wistar rats were randomized into 3 groups of 10 rats: control group, infused into the stomach with running water; stone-forming group, infused into the stomach with 1% ethylene glycol and 2% ammonium chloride so as to make into renal oxalate calcium stone model; and group of Alisma orientalis, infused into the stomach with 2% ammonium chloride and the constituents of Alisma orientalis. Four weeks after the rats were killed and their kidneys were taken out. Reverse transcription polymerase chain reaction technique was used to examine the bikunin mRNA expression levels in the rat renal tissues. The calcium oxalate deposits in the kidneys were detected by microscopy. The serum creatinnine and blood urea nitrogen levels, renal tissue calcium content, 24 h urinary calcium and oxalate excretion were also detected. RESULTS: In the group administered with the active constituents of Alisma orientalis, calcium oxalate deposits in the kidney, serum creatinnine and blood urea nitrogen levels, the bikunin mRNA expression levels, renal tissue calcium content and 24 h urinary calcium excretion were all significantly lower than those in the model group (the bikunin mRNA expression levels: 0.53 +/- 0.17 vs 0.71 +/- 0.25, P < 0.05; renal tissue calcium content: 4.70 mg/g +/- 0.08 mg/g vs 9.49 mg/g +/- 0.45 mg/g, P < 0.01; 24 h urinary calcium excretion: 37 micromol +/- 2 micromol vs 62 micromol +/- 2 micromol, P < 0.01). CONCLUSION: The active constituents of Alisma orientalis can down-regulate the bikunin mRNA expression, decrease the calcium oxalate formation in rat kidney, and inhibit the renal stone formation in rat urolithiasis model.

Inhibition of tumor invasion by genomic down-regulation of matriptase through suppression of activation of receptor-bound pro-urokinase.

Urokinase-type plasminogen activator (uPA) degrades the extracellular matrix and plays critical roles in tumor invasion and metastasis. Matriptase, a membrane-bound serine protease, was shown to activate uPA in a uPA receptor-free, solution-based study. We now investigate whether matriptase affects activation of receptor-bound uPA and contributes to the invasiveness of HRA human ovarian cancer cells in vitro and tumor behavior in nude mice. Here we show the following. 1) uPA expression was effectively stimulated by TGF-beta1 in HRA cells. 2) Antisense (AS)-matriptase transfection achieved a marked inhibition of receptor-bound pro-uPA activation without altering expression of uPA and uPA receptor mRNA and proteins, irrespective of whether cells were stimulated with TGF-beta1. 3) Tumor cell receptor-bound pro-uPA could be efficiently cleaved by matriptase to generate enzymatically active two-chain uPA. Thus, matriptase can substitute for plasmin in the proteolytic activation of pro-uPA to enzymatically active uPA. 4) The AS-matriptase-treated cells had a decreased ability to invade an extracellular matrix layer, as compared with control cells. 5) When the AS-matriptase-treated cells were injected intraperitoneally into nude mice, the mice developed smaller tumors. Our data identify a novel role for matriptase for activation of receptor-bound uPA.

Hepatocyte growth factor activator inhibitor types 1 and 2 are expressed by tubular epithelium in kidney and down-regulated in renal cell carcinoma.

PURPOSE: Hepatocyte growth factor (HGF) and its receptor MET have been implicated in kidney development and renal cell carcinoma (RCC) progression. HGF is secreted as an inactive proform and it must be activated to initiate MET signaling. HGF activator (HGFA) activates pro-HGF in injured tissue. We evaluated the expression of HGFA and its endogenous inhibitors HAI-1 and HAI-2 in normal kidney and RCC. MATERIALS AND METHODS: We examined the gene expression of HGFA, HAI-1, HAI-2, HGF and MET in a normal kidney by laser captured microdissection, followed by reverse transcriptase-polymerase chain reaction. We also quantified the mRNA levels of these proteins in 14 RCC cases by real-time reverse transcriptase-polymerase chain reaction. RESULTS: HAI-1 and HAI-2 were abundant in the normal kidney. The uriniferous tubules showed the highest levels of HAI-1 and HAI-2 mRNA. HGFA was hardly detectable in the normal kidney. However, in the kidney with RCC a low but distinct level of HGFA mRNA became detectable in the tumor and adjacent renal tissue. The HAI-1 mRNA level was significantly and consistently down-regulated in RCC relative to normal tissue. HAI-2 mRNA was also significantly low in the advanced stage of RCC. MET was up-regulated in most cases of RCC. CONCLUSIONS: HAI-1 and HAI-2 were expressed in renal tubular epithelial cells. The expression of the 2 HAIs was significantly down-regulated in RCC, whereas HGFA expression was enhanced in the diseased kidney, suggesting an imbalance between HAI and its target proteinases, including HGFA, in favor of proteinase activities in RCC.

Purification and characterization of the beta-trefoil fold protein barley alpha-amylase/subtilisin inhibitor overexpressed in Escherichia coli.

Barley alpha-amylase/subtilisin inhibitor (BASI) is a beta-trefoil fold protein related to soybean trypsin inhibitor (Kunitz) and inhibits barley alpha-amylase isozyme 2 (AMY2), which is de novo synthesized in the seed during germination. Recombinant BASI was produced in Escherichia coli in an untagged form (untagged rBASI), in two His(6)-tag forms (His(6)-rBASI and His(6)-Xa-rBASI), and in an intein-CBD-tagged form (rBASI (intein)). The yields per liter culture after purification were (i) 25 mgl(-1) His(6)-rBASI; (ii) 6 mgl(-1) rBASI purified after cleavage of His(6)-Xa-rBASI by Factor Xa; (iii) 3 mgl(-1) untagged rBASI; and (iv) 0.2 mgl(-1) rBASI after a chitin-column and autohydrolysis of the rBASI-intein-CBD. In Pichia pastoris, rBASI was secreted at 0.1 mgl(-1). The recombinant BASI forms and natural seed BASI (sBASI) all had an identical isoelectric point of 7.2 and a mass of 19,879 Da, as determined by mass spectrometry. The fold of rBASI from the different preparations was confirmed by circular dichroism spectroscopy and rBASI (intein), His(6)-rBASI, and sBASI inhibited AMY2 catalyzed starch hydrolysis with K(i) of 0.10, 0.06, and 0.09 nM, respectively. Surface plasmon resonance analysis of the formation of AMY2/rBASI (intein) gave k(on)=1.3x10(5)M(-1)s(-1), k(off)=1.4x10(-4)s(-1), and K(D)=1.1 nM, and of the savinase-His(6)-rBASI complex k(on)=21.0x10(4)M(-1)s(-1), k(off)=53.0x10(-4)s(-1), and K(D)=25.0 nM, in agreement with sBASI values. K(i) was 77 and 65 nM for inhibition of savinase activity by His(6)-rBASI and sBASI, respectively.

A family of potato genes that encode Kunitz-type proteinase inhibitors: structural comparisons and differential expression.

Potato tubers contain a complex group of proteins of 20 to 24 kDa that exhibit homology to Kunitz-type proteinase inhibitors. We isolated three cDNAs and two genomic clones that encode members of the potato Kunitz-type proteinase inhibitor (PKPI) family. Comparison of the structures of these and other cloned genes indicated that genes of the PKPI family can be classified into three major homology groups, namely, A, B and C. The PKPI-A and -B genes exhibit higher homology to one another than to the PKPI-C genes. Determination of the N-terminal amino acid sequences of 18 polypeptides from the complex group of 20- to 24-kDa proteins that had been separated by column chromatography and subsequently gel electrophoresis revealed three different sequences that corresponded to PKPI-A, -B, and -C. PKPI-A genes include those coding for a cathepsin D inhibitor, while PKPI-B and -C genes include those coding for trypsin and/or chymotrypsin inhibitors and a subtilisin inhibitor. Precursors to PKPIs are synthesized with an N-terminal extra peptide that appears to contain, in addition to the signal peptide, a short propeptide with a highly conserved Asn-Pro-Ile-Xxx-Leu-Pro motif that is identical to the potential vacuolar-sorting determinant in the N-terminal propeptide of a precursor to sporamin of sweet potato. Expression of the PKPI-A and -B genes is differentially regulated: PKPI-A mRNA but not PKPI-B mRNA were induced in leaves after wounding or upon treatment with methyl jasmonate. Nuclear genes for PKPI-A and -B do not contain introns, and the homology between the two types of gene extends only 72 bp upstream from the site of initiation of transcription.

A Brassica napus transcript encoding a protein related to the Künitz protease inhibitor family accumulates upon water stress in leaves, not in seeds.

A cDNA clone encoding a Brassica napus drought-induced 22 kDa (BnD22) protein has been isolated and characterized. The BnD22 transcript accumulated in response to drought reversibly, and to other conditions of leaf water deficit such as rapid water stress or salt acclimation, but not to cold acclimation or heat shock. Exogenously applied abscisic acid induced both changes in leaf morphology similar to the drought-adaptive response and a pronounced accumulation of the BnD22 mRNA. In control and drought-adapted plants, the BnD22 transcript was expressed in an organ-specific manner: the mRNA level was highest in leaves, low in hypocotyls and undetectable in roots. Sequence analysis indicates that the BnD22 protein is related to the Künitz family of protease inhibitors. In contrast to most members of this family, and also to most polypeptides expressed in vegetative tissues upon drought, the BnD22 mRNA was absent in seeds, before or during the seed desiccation phase. The BnD22 gene represents a new class of genes which are strictly induced in vegetative tissues upon environmental stress, and its pattern of expression shows that the responses to water deficit differ, at least partially, in seeds and in leaves.