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Supplementary Fig from Identification and characterization of NVP-BEZ235, a new orally available dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor with potent in vivo antitumor activity

Supplementary Material from Identification and characterization of NVP-BEZ235, a new orally available dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor with potent in vivo antitumor activity

The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin inhibitor (mTOR) pathway is often constitutively activated in human tumor cells, providing unique opportunities for anticancer therapeutic intervention. NVP-BEZ235 is an imidazo[4,5-c]quinoline derivative that inhibits PI3K and mTOR kinase activity by binding to the ATP-binding cleft of these enzymes. In cellular settings using human tumor cell lines, this molecule is able to effectively and specifically block the dysfunctional activation of the PI3K pathway, inducing G1 arrest. The cellular activity of NVP-BEZ235 translates well in in vivo models of human cancer. Thus, the compound was well tolerated, displayed disease stasis when administered orally, and enhanced the efficacy of other anticancer agents when used in in vivo combination studies. Ex vivo pharmacokinetic/pharmacodynamic analyses of tumor tissues showed a time-dependent correlation between compound concentration and PI3K/Akt pathway inhibition. Collectively, the preclinical data show that NVP-BEZ235 is a potent dual PI3K/mTOR modulator with favorable pharmaceutical properties. NVP-BEZ235 is currently in phase I clinical trials. [Mol Cancer Ther 2008;7(7):1–13 [Mol Cancer Ther 2008;7(7):1851–13]

The mevalonate pathway leads to synthesis of cholesterol and isoprenoid lipids. Prenyltransferases attach the isoprenoid lipids to the C-terminus of several small guanosine triphosphate–binding proteins. The prenyl groups are essential for the biological activity of these proteins. The prenyltransferases and other components of the mevalonate pathway are either present or potential drug targets for cancer, osteoporosis, restenosis, or high serum cholesterol level. Until recently, cellular assays to study protein prenylation have been tedious, low-throughput assays. The authors have developed a high-content imagingbased assay to study protein prenylation. The assay is based on a green fluorescent protein (GFP) reporter, which is tagged with the prenylation motif of human H-Ras. The C-terminus of H-Ras targets GFP to the plasma membrane. When protein prenylation is inhibited, the tagged GFP cannot be localized to plasma membrane but is soluble in the cells. The localization of the GFP reporter can be analyzed in the 96- or 384-well format using automated microscopy and automated image analysis. Information about cell number and nuclear intensity can be obtained from the same images. In compound screening, these readouts provide valuable information about the toxicity of the compounds. The authors have validated their assay using several inhibitors of the mevalonate pathway as well as siRNA against farnesyl pyrophosphate synthase, a critical enzyme in the synthesis of the isoprenoid lipids. (Journal of Biomolecular Screening XXXX:xx-xx)

The function of alpha globin in the context of oxygen transport in erythroid cells is well described. Recently the expression of alpha globin was shown to be upregulated upon specific apoptotic stimuli like cytokine deprivation or cisplatin treatment in the hematopoietic pro-B cell line, FL5.12. In contrast to alpha globin, beta globin or globin-like genes were expressed at a very low level or were not expressed at all. Further, we found that alpha globin was not associated with heme. Apoptotic cells neither produced hemoglobin nor displayed a phenotype of cells differentiating down the erythroid lineage. Also other cell lines of variable differentiation status (NIH3T3, HeLa, K562) upregulated alpha globin during treatment with apoptosis-inducing agents. Under IL-3-deprived conditions GFP-alpha globin accelerated the progression of apoptosis comparable to GFP-Bax. GFP-alpha globin was expressed at a low level and enrichment of FL5.12 cells expressing GFP-alpha globin was difficult even in the presence of IL-3. Caspase-8, -9 and -3 as well as the proapoptotic factor Bax and cytochrome c were activated. Antisense alpha globin downregulated the expression of endogenous alpha globin und reduced caspase activity. Taken together these data indicate that alpha globin is a new and crucial factor in apoptosis especially supporting the mitochondrial pathway.

Nogo-A is a potent neurite growth inhibitorin vitroand plays a role both in the restriction of axonal regeneration after injury and in structural plasticity in the CNS of higher vertebrates. The regions that mediate inhibition and the topology of the molecule in the plasma membrane have to be defined. Here we demonstrate the presence of three different active sites: (1) an N-terminal region involved in the inhibition of fibroblast spreading, (2) a stretch encoded by the Nogo-A-specific exon that restricts neurite outgrowth and cell spreading and induces growth cone collapse, and (3) a C-terminal region (Nogo-66) with growth cone collapsing function. We show that Nogo-A-specific active fragments bind to the cell surface of responsive cells and to rat brain cortical membranes, suggesting the existence of specific binding partners or receptors. Several antibodies against different epitopes on the Nogo-A-specific part of the protein as well as antisera against the 66 aa loop in the C-terminus stain the cell surface of living cultured oligodendrocytes. Nogo-A is also labeled by nonmembrane-permeable biotin derivatives applied to living oligodendrocyte cultures. Immunofluorescent staining of intracellular, endoplasmic reticulum-associated Nogo-A in cells after selective permeabilization of the plasma membrane reveals that the epitopes of Nogo-A, shown to be accessible at the cell surface, are exposed to the cytoplasm. This suggests that Nogo-A could have a second membrane topology. The two proposed topological variants may have different intracellular as well as extracellular functions.

DNA microarrays are powerful tools for the analysis of gene expression on a genomic scale. The importance of individual regulatory events for the process under study can however not be deduced unequivocally without additional experiments. We devised a strategy to identify central regulators of cancer drug responses by combining the results of microarray experiments with efficient methods for phenotypic testing of candidate genes. We exposed murine FL5.12 pro-B cells to cisplatin, camptothecin, methotrexate or paclitaxel, respectively and analysed the patterns of gene expression with cDNA microarrays. Drug-specific regulatory events as well as intersections between different apoptotic pathways, including previously studied responses to staurosporine and interleukin-3 (IL-3) deprivation, were identified. Genes shared by at least three pathways were chosen for further analysis. Ectopic expression of three such genes, TEAP, GP49B, and Lipin1 was found to have an anti-proliferative effect on pro-B cells. Interestingly, we identified hemoglobin alpha as a strong pro-apoptotic regulator. While hemoglobin-expressing cells were growing normally in the presence of IL-3, they displayed accelerated apoptosis with similar kinetics as Bax overexpressing cells upon IL-3 removal. The pro-apoptotic effect of hemoglobin was suppressed by Bcl-2 and was characterized by enhanced stimulation of caspase activity.

A new cDNA encoding a protein of 362 amino acids designated SH3GLB1, for SH3 domain GRB2-like endophilin B1, was identified in a yeast two-hybrid screen devoted to the identification of new partners interacting with the apoptosis inducer Bax. SH3GLB1 shows strong similarities to the SH3 domain-containing proteins of the endophilin family and presumably represents the human homologue of the potential Caenorhabditis elegans SH3 containing-protein identified by systematic translation of the C. elegans genome (GenBank Accession No. U46675). Reversing prey to bait in the yeast screen, a second protein, SH3GLB2, of 395 amino acids showing 65% identity to SH3GLB1 was identified as an interacting partner of SH3GLB1. The discovery of SH3GLB1 itself in the screening with SH3GLB1 as a bait and further mapping experiments demonstrated that a core coiled-coil-type region is required for the formation of SH3GLB homo- and/or heterodimers, whereas the SH3 domain is not involved in these interactions. Interestingly, the similarities with the endophilin proteins cover the entire sequence of the SH3GLB family, suggesting a common fold and presumably a common mode of action. Furthermore, SH3GLB members colocalize to the cytoplasmic compartment of the cell together with Bax and are excluded from the nucleus. SH3GLB1 and SH3GLB2 do not significantly influence the onset and time course of Bax-mediated apoptosis in HeLa or 293T cells.

Uncoupling proteins (UCPs) are members of the superfamily of the mitochondrial anion carrier proteins (MATP). Localized in the inner membrane of the organelle, they are postulated to be regulators of mitochondrial uncoupling. UCP2 and 3 may play an important role in the regulation of thermogenesis and, thus, on the resting metabolic rate in humans. To identify interacting proteins that may be involved in the regulation of the activity of UCPs, the yeast two-hybrid system was applied. Segments of hUCP2 containing the hydrophilic loops facing the intermembrane space, or combinations of these, were used to screen an adipocyte activation domain (AD) fusion library. The 14.3.3 protein isoforms θ, β, ζ were identified as possible interacting partners of hUCP2. Screening of a human skeletal muscle AD fusion library, on the other hand, yielded several clones all of them encoding the γ isoform of the 14.3.3 family. Mapping experiments further revealed that all these 14.3.3 proteins interact specifically with the C-terminal intermembrane space domain of both hUCP2 and hUCP3 whereas no interactions could be detected with the C-terminal part of hUCP1. Direct interaction between UCP3 and 14.3.3 θ could be demonstrated after in vitro translation by coimmunoprecipitation. When coexpressed in a heterologous yeast system, 14.3.3 proteins potentiated the inhibitory effect of UCP3 overexpression on cell growth. These findings suggest that 14.3.3 proteins could be involved in the targeting of UCPs to the mitochondria.

BFL-1 is the smallest member of the BCL-2 family and has been shown to retard apoptosis in various cell lines. However, the structural basis for its function remains unclear. Molecular modeling showed that BFL-1 could have a similar core structure as BCL-xL, consisting of seven alpha helices, although both proteins share only the conserved BCL-2 homology domains (BH1 and BH2 domains), but otherwise have very limited sequence homology, particularly in the N-terminal region. We demonstrated in the yeast two-hybrid system that BFL-1 interacts strongly with human BAX but is not able to form homodimers nor to interact with human BCL-2 or BCL-xL. Overexpression experiments in REF52 rat fibroblasts showed that BFL-1 conferred increased resistance to apoptosis induced by serum deprivation. BFL-1 had also the ability to neutralize BAX lethality in yeast. BAX requires the BH3 domain for interaction with BFL-1. However, the minimal region of BFL-1 for the interaction with BAX in coimmunoprecipitation experiments was not sufficient to protect cells from apoptosis. Further examination of BFL-1 and several other anti-apoptotic proteins suggests a more general type of structure based on structural motifs, i.e. a hydrophobic pocket for the binding of proapoptotic proteins, rather than extended sequence homologies.

When the extracellular domain of rat low-affinity nerve growth factor receptor (NGFRe) was synthesized in Saccharomyces cerevisiae with the signal peptide of invertase, NGFRe was translocated to the endoplasmic reticulum (ER) and retained there. However, when NGFRe was fused to the C-terminus of the hsp150 delta-carrier, the hsp150 delta-NGFRe fusion protein was efficiently secreted to the growth medium with no apparent retention in the ER. The NGFRe portion was disulphide-bonded and its single N-glycosylation site was occupied. The hsp150 delta-carrier is an N-terminal signal peptide-containing fragment of a yeast secretory glycoprotein. Hsp150 delta-NGFRe, harvested from the culture medium, inhibited the cross-linking of [125I]NGF to authentic NGFR on the surface of human melanoma cells. Moreover, [125I]NGF could be chemically cross-linked to secretory hsp150 delta-NGFRe, suggesting that the NGFRe portion had adopted a ligand-binding conformation. However, inhibition of the cross-linking by unlabelled NGF was less effective than in the case of the authentic receptor. The hsp150 delta-carrier may have potential in the production of mammalian proteins, which require elaborate folding and disulphide formation in the ER.

We have used four glycoproteins as markers to study how disulfide bond formation and protein folding effect the intracellular transport of proteins in yeast. Under normal conditions, the vacuolar enzyme carboxypeptidase Y (CPY) and the secretory stress-protein hsp150 acquired disulfide bonds in the endoplasmic reticulum (ER). Treatment of living cells with the reducing agent dithiothreitol (DTT) prevented disulfide formation of newly synthesized CPY and hsp150, resulting in retention of the proteins in the ER. When DTT was removed, the sulfhydryls were reoxidized, and the transport of the proteins to their correct destinations was resumed. Even mature CPY, located in the vacuole, could be reduced with DTT, and reoxidized after removal of the drug. DTT treatment blocked intracellular transport of hsp150 only when present during the synthesis and translocation of the protein. Reduction of folded hsp150, accumulated in the ER due to a sec block prior to DTT treatment, did not inhibit its secretion. The Kar2p/BiP protein, a component of the ER lumen, was found to be associated with fully translocated reduced hsp150, but not with native hsp150, suggesting that Kar2p/BiP may be involved in the putative retention mechanism. The cysteine-free pro-alpha-factor, and invertase which was shown to have free sulfhydryls, were secreted and modified similarly in the presence and absence of DTT, showing that the secretory pathway of yeast functioned under reducing conditions.

Summary When the genes coding for the outer membrane (OM) proteins OmpA and OmpF of Escherichia coli are fused to a signal sequence of a bacillar exoenzyme and expressed in Bacillus subtilis they remain cell-bound and the signal sequence is not cleaved. To identify the step of arrest in the export of these proteins we studied their accessibility to protease applied to intact protoplasts; they remained resistant indicating fully intracellular localization. Both proteins appeared associated with the cell membranes in sedimentation and flotation centrifugation experiments. However, OmpA and OmpF proteins synthesized in B. subtilis without a signal sequence were similarly associated with membranes in centrifugation experiments whereas electron microscopy showed the presence of intracytoplasmic inclusion bodies not obviously attached to the cytoplasmic membrane. We conclude that OmpA and OmpF proteins even when provided with a functional signal sequence do not enter the export pathway in B. subtilis, probably owing to lack of a specific export component in B. subtilis.

The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin inhibitor (mTOR) pathway is often constitutively activated in human tumor cells, providing unique opportunities for anticancer therapeutic intervention. NVP-BEZ235 is an imidazo[4,5-c]quinoline derivative that inhibits PI3K and mTOR kinase activity by binding to the ATP-binding cleft of these enzymes. In cellular settings using human tumor cell lines, this molecule is able to effectively and specifically block the dysfunctional activation of the PI3K pathway, inducing G1 arrest. The cellular activity of NVP-BEZ235 translates well in in vivo models of human cancer. Thus, the compound was well tolerated, displayed disease stasis when administered orally, and enhanced the efficacy of other anticancer agents when used in in vivo combination studies. Ex vivo pharmacokinetic/pharmacodynamic analyses of tumor tissues showed a time-dependent correlation between compound concentration and PI3K/Akt pathway inhibition. Collectively, the preclinical data show that NVP-BEZ235 is a potent dual PI3K/mTOR modulator with favorable pharmaceutical properties. NVP-BEZ235 is currently in phase I clinical trials. [Mol Cancer Ther 2008;7(7):1–13 [Mol Cancer Ther 2008;7(7):1851–13]

Nogo-A, a membrane protein enriched in myelin of the adult CNS, inhibits neurite growth and regeneration; neutralizing antibodies or receptor blockers enhance regeneration and plasticity in the injured adult CNS and lead to improved functional outcome. Here we show that Nogo-A-specific knock-outs in backcrossed 129X1/SvJ and C57BL/6 mice display enhanced regeneration of the corticospinal tract after injury. Surprisingly, 129X1/SvJ Nogo-A knock-out mice had two to four times more regenerating fibers than C57BL/6 Nogo-A knock-out mice. Wild-type newborn 129X1/SvJ dorsal root gangliain vitrogrew a much higher number of processes in 3 d than C57BL/6 ganglia, confirming the stronger endogenous neurite growth potential of the 129X1/SvJ strain. cDNA microarrays of the intact and lesioned spinal cord of wild-type as well as Nogo-A knock-out animals showed a number of genes to be differentially expressed in the two mouse strains; many of them belong to functional categories associated with neurite growth, synapse formation, and inflammation/immune responses. These results show that neurite regenerationin vivo, under the permissive condition of Nogo-A deletion, and neurite outgrowthin vitrodiffer significantly in two widely used mouse strains and that Nogo-A is an important endogenous inhibitor of axonal regeneration in the adult spinal cord.

Recently we showed that alpha globin is a novel pro-apoptotic factor in programmed cell death in the pro-B cell line, FL5.12. Alpha globin was also upregulated in various other cell lines after different apoptotic stimuli. Under withdrawal of IL-3, overexpression of alpha globin accelerated apoptosis in FL5.12. Here, we have studied how transcription of alpha globin is placed in the broader context of apoptosis. We used Affymetrix chip technology and RT QPCR to compare expression patterns of FL5.12 cells growing with or without IL-3 to search for transcription factors which were concomitantly upregulated with alpha globin. The erythroid-specific transcription factor GATA-2 was the earliest and most prominently upregulated candidate. GATA-1 was expressed at low levels and was weakly induced while GATA-3 was completely absent. To evaluate the influence of GATA-2 on alpha globin expression and cell viability we overexpressed GATA-2 in FL5.12 cells. Interestingly, high expression of GATA-2 resulted in cell death and elevated alpha globin levels in FL5.12 cells. Transduction of antisense GATA-2 prevented both increase of GATA-2 and alpha globin under apoptotic conditions and delayed cell death. We suggest a role of GATA-2 in apoptosis besides its function in maintenance and proliferation of immature hematopoietic progenitors.

To investigate the role of the myelin-associated protein Nogo-A on axon sprouting and regeneration in the adult central nervous system (CNS), we generated Nogo-A-deficient mice. Nogo-A knockout (KO) mice were viable, fertile, and not obviously afflicted by major developmental or neurological disturbances. The shorter splice form Nogo-B was strongly upregulated in the CNS. The inhibitory effect of spinal cord extract for growing neurites was decreased in the KO mice. Two weeks following adult dorsal hemisection of the thoracic spinal cord, Nogo-A KO mice displayed more corticospinal tract (CST) fibers growing toward and into the lesion compared to their wild-type littermates. CST fibers caudal to the lesion—regenerating and/or sprouting from spared intact fibers—were also found to be more frequent in Nogo-A-deficient animals.

Înjured axons in mammalian peripheral nerves often regenerate successfully over long distances, in contrast to axons in the brain and spinal cord (CNS). Neurite growth-inhibitory proteins, including the recently cloned membrane protein Nogo-A, are enriched in the CNS, in particular in myelin. Nogo-A is not detectable in peripheral nerve myelin. Using regulated transgenic expression of Nogo-A in peripheral nerve Schwann cells, we show that axonal regeneration and functional recovery are impaired after a sciatic nerve crush. Nogo-A thus overrides the growth-permissive and -promoting effects of the lesioned peripheral nerve, demonstrating its in vivo potency as an inhibitor of axonal regeneration.

bax is an apoptosis-inducing member of the bcl-2 multigene family. We have studied interactions of human Bax with itself, and with the apoptosis-preventing members Bcl-2 and Bcl-xL using a yeast two-hybrid system. Exhaustive Bax truncations were constructed and their interactions with full-length family members studied. Bax interacted similarly with itself as with the apoptosis-suppressing family members Bcl-2 and Bcl-xL in quantitative two-hybrid studies. A region of 41 amino acids covering the recently discovered BH3 domain of Bax was found to be necessary and sufficient for all interactions of Bax. Bax truncations containing BH3, but lacking BH1 and BH2 homology domains, interacted with the other family members markedly more strongly than full-length Bax, which may reflect conformational changes required for the interactions of full-length Bax. The minimum requirements for Bax homodimerization were found to be the BH3 domain from one Bax molecule and a region covering BH3 plus BH1 from another. We also studied the apoptosis-inducing activity of the Bax truncations upon microinjection of expression plasmids into rat fibroblasts. The BH3 region was required for the apoptosis-inducing activity of Bax, whereas BH1, BH2 and the N-terminus of Bax were dispensable.

Escherichia coli beta-lactamase was secreted into the culture medium of Saccharomyces cerevisiae in biologically active form, when fused to the C-terminus of the hsp150 delta-carrier. The hsp150 delta-carrier is an N-terminal fragment of the yeast hsp150 protein, having a signal peptide and consisting mostly of a 19 amino acid peptide repeated 11 times in tandem. Here we expressed the hsp150 delta-carrier fragment alone in S. cerevisiae. Apparently due to a positional effect of the gene insertion, large amounts of the hsp150 delta-carrier were synthesized. About half of the de novo synthesized carrier molecules were secreted into the culture medium, the rest remaining mostly in the pre-Golgi compartment. The extensively O-glycosylated carrier fragment was purified from the culture medium under non-denaturing conditions. Circular dichroism spectroscopy showed that it had no regular secondary structure. Nuclear magnetic resonance spectroscopy showed that a non-glycosylated synthetic peptide, the consensus sequence of the repetitive 19 amino acid peptide, also lacked secondary structure. The unstructured carrier polypeptide may facilitate proper folding and secretion of heterologous proteins attached to it.

We have studied the relationship between folding and secretion competence of hsp150-beta lactamase fusion proteins in Saccharomyces cerevisiae. hsp150 is a secretory protein of yeast, and beta-lactamase was chosen, since its folding can be monitored by assaying its enzymatic activity. The hsp150 pre-pro-protein consists of a signal peptide, subunit I, a repetitive region, and a unique C terminus. Fusion of beta-lactamase to the C terminus of hsp150 produced Cla-bla protein, which was secretion-competent but inactive. The Pst-bla protein, where beta-lactamase was fused to subunit I, was also inactive and mostly secreted, but part of it remained in the pre-Golgi compartment. When beta-lactamase was fused to the C-terminus of the repetitive region, the fusion protein (Kpn-bla) was translocated to the endoplasmic reticulum, acquired disulfide bonds, and adopted an enzymatically active conformation. Kpn-bla was secreted to the medium without decrease of specific activity or retention in the cell. Folding of Kpn-bla to an active and transport-competent form required co-translational disulfide formation, since treatment of cells with dithiothreitol resulted in endoplasmic reticulum-retained inactive Kpn-bla. When dithiothreitol was removed, Kpn-bla resumed transport competence but remained inactive. Reduction of prefolded Kpn-bla did not inhibit enzymatic activity or transport. The repetitive hsp150 carrier may have use in heterologous protein production by conferring secretion competence to foreign proteins in yeast.

We have cloned and characterized the HSP150 gene of Saccharomyces cerevisiae, which encodes a glycoprotein (hsp150) that is secreted into the growth medium. Unexpectedly, the HSP150 gene was found to be regulated by heat shock and nitrogen starvation. Shifting the cells from 24 degrees C to 37 degrees C resulted in an abrupt increase in the steady-state level of the HSP150 mRNA, and de novo synthesized hsp150 protein. Returning the cells to 24 degrees C caused a rapid decrease in mRNA and protein synthesis to basal levels. The HSP150 5'-flanking region contains several heat shock element-like sequences (HSE). To study the function of these sequences, a strain bearing a disrupted copy of the HSP150 gene was transformed with plasmids in which the coding region of HSP150, or a HSP150-lacZ fusion gene, was preceded by 5' deletion derivatives of the HSP150 promoter. Site-directed mutagenesis of one HSE-like element, located between the TATA box and transcription initiation sites, abolished heat activation of transcription. In addition to heat shock, the HSP150 gene is regulated by the availability of nutrients in the growth medium. The HSP150 mRNA level was increased by nitrogen limitation at 24 degrees C, even when under the control of a HSP150 promoter region of 137 bp carrying the mutagenized HSE.

Several heat shock proteins serve as molecular chaperons, assisting in protein maturation by reducing malfoding, mostly in the cytoplasm, nucleus or mitochondria. Some of them, like the BiP protein, which belongs to the 70 kD heat shock proteins, are resident ER-proteins. We have cloned and characterized the first gene, the HSP150 gene of S. cerevisiae which codes for a heat shock protein which is secreted to the exterior of the cell. The function of the protein is as yet unknown, however, it is conserved in divergent yeasts (Russo et al., 1992).

The secretion of the outer membrane proteins OmpA and OmpF of Escherichia coli has previously been found to be blocked at an early intracellular step, when these proteins were fused to a bacillar signal sequence and expressed in Bacillus subtilis. We have now fused these proteins to long secretable polypeptides, the amino-terminal portions of alpha-amylase or beta-lactamase. In spite of this, no secretion of the fusion proteins was detected in B. subtilis. With the exception of a small fraction of the beta-lactamase fusion, the proteins were cell-bound with uncleaved signal sequences. Protease accessibility indicated that the fusion proteins were not even partially exposed on the outer surface of the cytoplasmic membrane. Thus there was no change of the location compared to the OmpA or OmpF fused to the signal sequence only. We conclude that, like OmpA and OmpF, the fusion proteins fold into an export-incompatible conformation in B. subtilis before the start of translocation, which we postulate to be a late post-translational event.

SUMMARY: We have inserted a C-terminally truncated gene of the major outer membrane protein OmpA of Escherichia coli downstream from the promoter and signal sequence of the secretory α-amylase of Bacillus amyloliquefaciens in a secretion vector of Bacillus subtilis. B. subtilis transformed with the hybrid plasmid synthesized a protein that was immunologically identified as OmpA. All the protein was present in the particulate fraction. The size of the protein compared to the peptide synthesized in vitro from the same template indicated that the α-amylase derived signal peptide was not removed; this was verified by N-terminal amino acid sequence determination. The lack of cleavage suggests that there was little or no translocation of OmpA protein across the cytoplasmic membrane. This is an unexpected difference compared with periplasmic proteins, which were both secreted and processed when fused to the same signal peptide. A requirement of a specific component for the export of outer membrane proteins is suggested.

Here we studied the glycosylation of a mammalian protein, the ectodomain of rat nerve growth factor receptor (NGFRe), in Saccharomyces cerevisiae. NGFRe is secreted to the culture medium of S. cerevisiae if it is fused to a polypeptide (hsp 150 delta) carrier. The hsp 150 delta-carrier has 95 serine and threonine residues, which were extensively O-glycosylated. In spite of 41 potential sites, NGFRe lacked O-glycans, whether fused to the carrier or not. Distortion of the conformation of NGFRe by inhibition of disulfide formation did not promote O-glycosylation, whereas N-glycosylation was enhanced. Thus, the serine and threonine residues of the hsp 150 delta-NGFRe fusion protein were highly selectively O-glycosylated.