Functional sites of glia-derived nexin (GDN): importance of the site reacting with the protease

Glia-derived nexin (GDN) is a 43-kDa serine protease inhibitor with neurite promoting activity in mouse neuroblastoma cells (Guenther et al., 1985). In chick sympathetic neurons, GDN but not hirudin and synthetic peptide inhibitors promoted neurite outgrowth (Zurn et al., 1988). Thus, it was considered that the protease inhibitory activity cannot account for the total biological activity of GDN. We show here that synthetic peptide inhibitors with thrombin specificity mimic GDN at similar concentrations in neuroblastoma cells. Limited proteolysis of GDN with elastase causes a cleavage between sites P1 and P2, corresponding to residues Ala-344-Arg-345 of the molecule. The resulting fragments still copurify on heparin-Sepharose, but the protease inhibitor activity of GDN and the GDN neurite promoting activity are lost. The results confirm the necessity of an intact reactive site for the biological activity of GDN.     

Detection of glia-derived nexin in the olfactory system of the rat

Glia-derived nexin (GDN) is a 43 kd cell-secreted protease inhibitor with neurite promoting activity. We have raised specific polyclonal antisera to rat GDN. These antibodies stain a single band at 43 kd on immunoblots of concentrated C6 glioma-conditioned medium and have been used to demonstrate that GDN is present in the olfactory system of the rat. One band at 43 kd is recognized by the GDN antibodies on immunoblots of olfactory bulb homogenate. Immunohistochemistry shows that GDN occurs predominantly in the olfactory nerve layer of the olfactory bulb and in the olfactory submucosa. Comparative studies with antibodies against vimentin, GFAP, and fibronectin suggest that anti-GDN recognizes cells associated with the olfactory system, but not exclusively the olfactory neurons themselves. Data from the immunohistochemical studies were confirmed by RNA blots and GDN mRNA expression throughout development of the olfactory bulb. The high levels of GDN in the rat olfactory system may be related to the continuous degeneration and regeneration phenomena taking place in these structures.     

Specific inhibitors prevent proteolytic degradation of recombinant proteins expressed in High Five cells

The insect cell line BTI-TN-5B1-4 (High Five) is frequently used to express recombinant proteins in large amounts using the baculovirus expression system. However, extensive proteolytic degradation of recombinant proteins is often encountered. Furthermore, we have observed that recombinant proteins migrate in SDS-PAGE in agreement with poly-ubiquitinated forms of the protein, suggesting a ubiquitin/proteasome degradation pathway. Here, we describe a systematic study unraveling the effect of adding proteasome inhibitors or specific protease inhibitors to the growth medium of High Five insect cells infected with recombinant baculovirus. Furthermore, protease inhibitors were added to the lysis buffer to establish the most efficient way to inhibit proteolytic activity after lysis of baculovirus-infected cells expressing recombinant proteins. We conclude that a combination of adding protease inhibitors to the growth medium and to the lysis buffer minimizes the proteolytic activity in High Five cells. The most efficient protease inhibitors were E-64 in the growth medium together with Leupeptin in the lysis buffer at concentrations higher than with available cocktails of inhibitors. The optimal treatment of High Five cells is different from the optimal treatment of Sf9 cells. For proteins susceptible to ubiquitinylation, a treatment of insect cell cultures with the proteasome inhibitor MG132 (LLL) leads to a considerable reduction of the yield of production of recombinant protein.     

Neoplastic transformation by the human gene N-myc.

Amplification and abundant expression of a gene known as N-myc are found frequently in advanced stages of human neuroblastoma and may play a role in the genesis of several malignant human tumors. Previous studies have shown that N-myc can cooperate with a mutant allele of the proto-oncogene c-Ha-ras to transform embryonic rat cells in culture. Here we show that N-myc can also act alone to elicit neoplastic growth of an established line of rat fibroblasts (Rat-1). We used recombinant DNA vectors to express either N-myc or its kindred gene c-myc in transfected cells. Both genes caused morphological transformation, anchorage-independent growth, and tumorigenicity. We noticed two variables that appeared to influence the ability to isolate cells transformed by N-myc and c-myc: the abundance in which the genes were expressed and biological selection to eliminate untransformed cells from the cultures. Our findings sustain the belief that N-myc is an authentic proto-oncogene, lend further credibility to the role of N-myc in the genesis of human tumors, and establish a convenient assay that can be used to explore further the properties of both N-myc and c-myc.     

Multiple and alternative adhesive responses on defined substrata of an immortalized dorsal root neuron hybrid cell line

Attachment and neurite extension processes have been evaluated for an immortalized derivative cell of a rat dorsal root neuron after fusion with a mouse neuroblastoma cell (the clonal F11 hybrid cell line) and these processes compared with previous studies of neuroblastoma cells, since both cell types may be derived from the neural crest of the developing embryo. Biochemically defined substrata were provided by human plasma fibronectin (pFN), the heparan sulfate-binding protein platelet factor-4 (PF4), and the ganglioside GM1-binding protein cholera toxin B subunit (CTB). While some attachment of unsupplemented cells was noted on CTB substrata, GM1 supplementation permitted F11 cells to attach as well on CTB as on pFN or PF4. On PF4, very few neurite processes were observed while on pFN two morphologically distinct types of neurites could be identified: short, linear processes in a low percentage of cells resembling those of neuroblastoma cells and long, irregular and narrow processes in a higher percentage of cells resembling those of dorsal root neurons. On CTB, neurites of the latter class were even more prominent; however, cell bodies on CTB failed to spread by cytoplasmic extension as commonly observed in F11 cells on pFN and, to some extent, on PF4. The formation of both neurite classes on either pFN or CTB was completely inhibited by low concentrations of an RGDS (Arg-Gly-Asp-Ser) peptide in the medium of cultures, indicating the significance of pFN's binding to cell surface integrin or ganglioside GM1's possible interaction with integrin for mediating the differentiative process. In contrast, neurite formation of neuroblastoma cells is refractile to the soluble peptide as reported previously. Neurite extensions of F11 cells on either pFN or CTB were comparably sensitive to low concentrations of cytochalasin D, revealing the mediation of microfilament reorganization in these processes. Treatment of F11 cells with cycloheximide failed to inhibit neurite extension on pFN but did partially inhibit extension on CTB; this contrasts with the very high sensitivity of neurite formation by neuroblastoma cells on CTB substrata reported previously.(ABSTRACT TRUNCATED AT 400 WORDS)     

Nitric oxide-dependent proteasomal degradation of cytochrome P450 2B proteins

Exposure to inflammatory agents or cytokines causes the suppression of cytochrome P450 (CYP) enzyme activities and expression in liver and primary hepatocyte cultures. We showed previously that phenobarbital-induced CYP2B protein is down-regulated in primary cultures of rat hepatocytes after exposure to bacterial endotoxin (lipopolysaccharide) in a nitric oxide (NO) -dependent manner. In this study, we found that CYP2B proteins in primary rat hepatocyte cultures were suppressed >60% after 6 h of treatment with interleukin-1beta (IL-1). This effect was NO-dependent, and treatment of cells with the NO donors (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl) aminodiazen-1-ium-1,2-diolate (NOC-18), S-nitrosoglutathione, and S-nitroso-N-acetylpenicillamine also suppressed CYP2B proteins. However, the down-regulation by IL-1 was insensitive to inhibition of cGMP-dependent protein kinases. The down-regulation by IL-1 or NO donors was abolished by treatments with the proteasome inhibitors MG132 and lactacystin that did not affect NO production. The calpain inhibitor E64-d or the lysosomal protease inhibitors NH(4)Cl and chloroquine did not attenuate the down-regulation of CYP2B by IL-1. Treatment of HeLa cells expressing c-Myc-tagged CYP2B1 with NOC-18 down-regulated its expression and enhanced its ubiquitination. Treatment of rat liver microsomes with S-nitrosoglutathione caused S-nitrosylation of CYP2B protein and enhanced the ubiquitination pattern of CYP2B compared with unmodified CYP2B in an in vitro ubiquitination assay. These data are consistent with the hypothesis that NO-dependent CYP2B ubiquitination and proteasomal degradation are dependent on protein modification by reactive nitrogen species.     

Effect of protein kinase C inhibitors on the antiviral activity of human alpha interferon in herpes simplex virus-infected human neuroblastoma cells.

Pretreatment of human neuroblastoma cells with an inhibitor of protein kinase C (PKC), staurosporine or H-7, prior to the addition of human alpha interferon (HuIFN-alpha), recombinant HuIFN-alpha, or recombinant HuIFN-beta blocked the inhibitory effect of these IFNs on the release of infectious herpes simplex virus type 1 from treated cells. In addition, staurosporine blocked the inhibitory effect of HuIFNs on the expressions of herpes simplex type 1 glycoproteins B, C, and D in treated neuroblastoma cells. Furthermore, addition of HuIFNs resulted in an increased expression of PKC in treated neuroblastoma cells. These results suggest that inhibitors of PKC block the expression of HuIFN-induced genes in treated human neuroblastoma cells. Thus, the activation of PKC is an important step in the HuIFN-treated cells of neuronal origin.     

cDNA sequence coding for a rat glia-derived nexin and its homology to members of the serpin superfamily

Rat glial cells release a neurite-promoting factor with serine protease inhibitory activity. By using a rat glioma cDNA clone as a probe, it was possible to isolate rat cDNAs containing the entire sequence coding for this neurite-promoting factor. The largest rat cDNA (approximately 2100 bp) was characterized by DNA sequencing. It contained the entire coding region, 135 bp of the 5' nontranslated region, and about 750 bp of the 3' nontranslated region. The open reading frame coded for 397 amino acids including a putative signal peptide of 19 amino acids. The correct identity of the coding sequence was substantiated by the fact that the sequence of tryptic peptides, derived from the purified rat factor, matched exactly with the deduced amino acid sequence. The rat protein sequence had 84% homology with the corresponding protein from human glioma cells. Both amino acid sequences indicated that the proteins belong to the protease nexins [Baker, B.J., Low, D. A., Simmer, R. L., & Cunningham, D.D. (1980) Cell (Cambridge, Mass.) 21, 37-45] and therefore can be defined as glia-derived nexins (GDNs). Further analysis showed that both rat and human GDN belong to the serpin superfamily and share 41%, 32%, and 25% homology with human endothelial-cell-type plasminogen activator inhibitor, antithrombin III, and alpha-1 proteinase inhibitor, respectively.     

Bacterial expression and characterization of rat apolipoprotein E

Apolipoprotein (apo) E is a protein involved in both lipid metabolism and neuroprotection. Recently, it has been suggested that apoE may play a role in the regulation of food intake and body weight in rodents. However, rodent plasma apoE is difficult to purify in reasonable amounts due to numerous time-consuming steps. To circumvent this, we created a bacterial expression system for the efficient production of large amounts of rat apoE. We inserted rat apoE DNA into the pET30 expression vector and overexpressed the proteins in Escherichia coli strain BL21 (DE3). A histidine tag present at the N-terminus allowed for easy purification of the recombinant protein. The tag was removed with an IgA protease (Igase) from Neisseria gonorrhoeae leaving the mature form of the protein. The use of Igase was important as several more common proteases routinely cleave apolipoproteins at undesired sites. The recombinant protein was then compared both structurally and functionally to rat plasma apoE. This expression system will be highly useful for probing the ability of rat apoE to mediate food intake in rats.     

Identification of galectin-3-binding protein as a factor secreted by tumor cells that stimulates interleukin-6 expression in the bone marrow stroma

We have previously demonstrated that neuroblastoma cells increase the expression of interleukin-6 by bone marrow stromal cells and that stimulation does not require cell-cell contact. In this study we report the purification and identification of a protein secreted by neuroblastoma cells that stimulates interleukin-6 production by stromal cells. Using a series of chromatographic purification steps including heparin-affinity, ion exchange, and molecular sieve chromatography followed by trypsin digestion and liquid chromatography tandem mass spectrometry, we identified in serum-free conditioned medium of neuroblastoma cells several secreted peptides including galectin-3-binding protein, also known as 90-kDa Mac-2-binding protein. We demonstrated the presence of the galectin-3-binding protein in the conditioned medium of several neuroblastoma cell lines and in chromatographic fractions with interleukin-6 stimulatory activity. Consistently, bone marrow stromal cells express galectin-3, the receptor for galectin-3-binding protein. Supporting a role for galectin-3-binding protein in stimulating interleukin-6 expression in bone marrow stromal cells, we observed that recombinant galectin-3-binding protein stimulated interleukin-6 expression in these cells and that interleukin-6 stimulation by neuroblastoma-conditioned medium was inhibited in the presence of lactose or a neutralizing anti-galectin-3 antibody. Down-regulation of galectin-3-binding protein expression in neuroblastoma cells also decreased the interleukin-6 stimulatory activity of the conditioned medium on bone marrow stromal cells. We also provide evidence that stimulation of interleukin-6 by galectin-3-binding protein involves activation of the Erk1/2 pathway. The data, thus, identifies galectin-3-binding protein as a factor secreted by neuroblastoma cells that stimulates the expression of interleukin-6 in bone marrow stromal cells and provides a novel function for this protein in cancer progression.     

PTD-NPY融合基因的克隆及其在毕赤酵母中的分泌表达

应用重叠延伸PCR方法扩增HIV-1 TAT蛋白转导结构域(PTD)与鼠源神经肽Y(NPY)的融合基因,克隆目的片段并插入酵母表达载体pPICZαA,构建成重组表达质粒pPICZα-PTD-NPY.PCR和酶切鉴定及测序正确后,经限制性内切酶Sac Ⅰ线性化重组表达质粒并通过电转化整合到巴斯德毕赤酵母菌GS115的染色体基因组中.阳性重组酵母菌用含1%甲醇的培养基诱导其分泌表达.经过120 h的诱导,取上清浓缩除盐后进行SDS-PAGE电泳,表明该系统成功表达了PTD-NPY融合蛋白,Western blotting实验证实表达产物具有特异性.获得真核表达的PTD-NPY融合蛋白,为下一步的应用研究提供了物质基础.     

DIFFERENTIATION OF NEUROBLASTOMA, GLIOMA, AND HYBRID CELLS IN CULTURE AS MEASURED BY THE SYNTHESIS OF SPECIFIC PROTEIN SPECIES: EVIDENCE FOR NEUROBLAST-GLIOBLAST RECIPROCAL GENETIC REGULATION

Abstract— Protein species from differentiating neuroblastoma, glioma, and hybrid neuroblastoma-glioma cell lines in cell culture were separated and identified initially in the first dimension by the use of isoelectric focusing gels and were further separated in the second dimension by SDS-acrylamide gels. There were two main classes of proteins identified: proteins which were dominantly expressed in neuroblastoma and also in hybrid cell cultures, and proteins which were expressed in glioma and also hybrid cell cultures. In general, proteins were identified which were significantly expressed in neuroblastoma cells and much reduced in glioma cultures, and also conversely so. The hybrid cell line expressed many of the neuroblastoma-type proteins and relatively fewer of the glioma type proteins. A specific protein species (2) was identified in hybrid cells and was not present in either parental neuroblastoma or glioma cultures. Protein z was expressed however by the co-culturing of neuroblastoma and glioma cells suggesting its induction is dependent on a soluble factor. Protein z in hybrid cells was demonstrated in both stained gels and by autoradiography. Chromosome analysis of hybrid cells confirmed the presence of both rat and mouse chromosomes. It is suggested that similar neuronal-glial interaction may be functional in the intact brain, and that similar reciprocal modulation between neurons and glia may be a central mechanism of differentiation in the nervous system.     

HtrA1 inhibits mineral deposition by osteoblasts: requirement for the protease and PDZ domains

HtrA1 is a secreted multidomain protein with serine protease activity. In light of increasing evidence implicating this protein in the regulation of skeletal development and pathology, we investigated the role of HtrA1 in osteoblast mineralization and identified domains essential for this activity. We demonstrate increased HtrA1 expression in differentiating 2T3 osteoblasts prior to the appearance of mineralization. HtrA1 is subsequently down-regulated in fully mineralized cultures. The functional role of HtrA1 in matrix calcification was investigated using three complementary approaches. First, we transfected a full-length HtrA1 expression plasmid into 2T3 cells and showed that overexpression of HtrA1 delayed mineralization, reduced expression of Cbfa1 and collagen type I mRNA, and prevented BMP-2-induced mineralization. Second, knocking down HtrA1 expression using short interfering RNA induced mineral deposition by 2T3 cells. Third, by expressing a series of recombinant HtrA1 proteins, we demonstrated that the protease domain and the PDZ domain are essential for the inhibitory effect of HtrA1 on osteoblast mineralization. Finally, we tested whether HtrA1 cleaves specific matrix proteins that are known to regulate osteoblast differentiation, mineralization, and/or BMP-2 activity. Full-length recombinant HtrA1 cleaved recombinant decorin, fibronectin, and matrix Gla protein. Both the protease domain and the PDZ domain were necessary for the cleavage of matrix Gla protein, whereas the PDZ domain was not required for the cleavage of decorin or fibronectin. Type I collagen was not cleaved by recombinant HtrA1. These results suggest that HtrA1 may regulate matrix calcification via the inhibition of BMP-2 signaling, modulating osteoblast gene expression, and/or via the degradation of specific matrix proteins.     

Purification and characterization of recombinant rat mast cell protease 7 expressed in Pichia pastoris

Rat mast cell protease 7 (rMCP7) is a neutral serine protease and a component of mast cells, where it is stored in secretory granules. Mast cells express numerous proteases so in order to characterize rMCP7, it was cloned and expressed as a recombinant protein in Pichia pastoris. During expression, rMCP7 protein was cleaved from the alpha-mating factor signal at the engineered KEX2 cleavage site to produce active rMCP7. The protein produced was stable at pH 5.5 and active in the absence of heparin. The rMCP7 was glycosylated and treatment with N-glycosidase F resulted in a protein of the predicted molecular mass of 30 kDa. The rMCP7 was purified via an ammonium sulfate precipitation, using casein as a carrier protein, followed by cation exchange chromatography. The purified protein was assayed using a range of substrates and where possible, k(m) and k(cat) values were determined. The substrate profile displayed by the recombinant rMCP7 was consistent with that of tryptase isolated from rat skin. The expression and purification of recombinant rMCP7 offer an efficient, low-cost method of producing large amounts of protein. It also offers the opportunity of easy manipulation and mutagenesis of rMCP7 for further biochemical, structural, and physiological studies.     

Expression of a hepatocyte growth-factor activator protein in turkey (Meleagris gallopavo) deferent duct epithelial cells

In previous research, we discovered that turkey deferent duct epithelial cells express a serine protease. Our experimental objective was to identify the gene that encodes this protein. A lambda phage cDNA library from duct cell mRNA was constructed. The library was screened using monoclonal antibodies previously produced against the turkey deferent-duct serine protease. Phage containing the protease cDNA was excised and re-circularized into plasmids. E. coli were transformed with plasmids containing protease cDNA, which was then isolated for sequencing. NCBI searches within the GenBank™ database returned 63.5 and 61.7% identity with murine and human hepatocyte growth-factor activator (HGFA) precursor, respectively. The turkey protease cDNA was then cloned into the pQE-32 expression vector and transformed into M15 cells for HIS-tagged expression of the recombinant protein, which was then purified using nickel-chelated Sepharose spin columns. Afterwards, Western blot analysis of the purified recombinant turkey protein revealed recognition by a monoclonal antibody specific to the proteolytic subunit of the turkey deferent duct protease. Therefore, these findings indicate that the recombinant HGFA precursor isolated from the deferent duct is the turkey seminal plasma protease that is secreted from the deferent duct. HGFA, a member of the Kringle-serine proteinase superfamily, can initiate diverse mitogenic, morphogenic and motogenic effects through its substrate hepatocyte growth factor. Although the presence of hepatocyte growth factor and its c-MET receptor have been reported in male mammalian reproductive tracts, our novel findings on the secretion of HGFA precursor from turkeys may help to elucidate the regulation of activated hepatocyte growth factor.     

Inhibition of protease activity by antisense RNA improves recombinant protein production in Nicotiana tabacum cv. Bright Yellow 2 (BY‐2) suspension cells

Recombinant proteins produced in plant suspension cultures are often degraded by endogenous plant proteases when secreted into the medium, resulting in low yields. To generate protease‐deficient tobacco BY‐2 cell lines and to retrieve the sequence information, we cloned four different protease cDNAs from tobacco BY‐2 cells (NtAP, NtCP, NtMMP1, and NtSP), which represent the major catalytic classes. The simultaneous expression of antisense RNAs against these endogenous proteases led to the establishment of cell lines with reduced levels of endogenous protease expression and activity at late stages of the cultivation cycle. One of the cell lines showing reduced proteolytic activity in the culture medium was selected for the expression of the recombinant full‐length IgG1(κ) antibody 2F5, recognizing the gp41 surface protein of HIV‐1. This cell line showed significantly reduced degradation of the 2F5 heavy chain, resulting in four‐fold higher accumulation of the intact antibody heavy chain when compared to transformed wild type cells expressing the same antibody. N‐terminal sequencing data revealed that the antibody has two cleavage sites within the CDR‐H3 and one site at the end of the H4‐framework region. These cleavage sites are found to be vulnerable to serine proteases. The data provide a basis for further improvement of plant cells for the production of recombinant proteins in plant cell suspension cultures.