Disulfide bond formation during the folding of influenza virus hemagglutinin.

To study the importance of individual sulfhydryl residues during the folding and assembly in vivo of influenza virus hemagglutinin (HA), we have constructed and expressed a series of mutant HA proteins in which cysteines involved in three disulfide bonds have been substituted by serine residues. Investigations of the structure and intracellular transport of the mutant proteins indicate that (a) cysteine residues in the ectodomain are essential both for efficient folding of HA and for stabilization of the folded molecule; (b) cysteine residues in the globular portion of the ectodomain are likely to form native disulfide bonds rapidly and directly, without involvement of intermediate, nonnative linkages; and (c) cysteine residues in the stalk portion of the ectodomain also appear not to form intermediate disulfide bonds, even though they have the opportunity to do so, being separated from their correct partners by hundreds of amino acids including two or more other sulfhydryl residues. We propose a role for the cellular protein BiP in shielding the cysteine residues of the stalk domain during the folding process, thus preventing them from forming intermediate, nonnative disulfide bonds.

Converting tissue plasminogen activator to a zymogen: a regulatory triad of Asp-His-Ser.

Unlike most serine proteases of the chymotrypsin family, tissue-type plasminogen activator (tPA) is secreted from cells as an active, single-chain enzyme with a catalytic efficiency only slightly lower than that of the proteolytically cleaved form. A zymogenic mutant of tPA has been engineered that displays a reduction in catalytic efficiency by a factor of 141 in the single-chain form while retaining full activity in the cleaved form. The residues introduced in the mutant, serine 292 and histidine 305, are proposed to form a hydrogen-bonded network with aspartate 477, similar to the aspartate 194-histidine 40-serine 32 network found to stabilize the zymogen chymotrypsinogen.

Correction of a defect in mammalian GPI anchor biosynthesis by a transfected yeast gene.

Glycosylphosphatidylinositol (GPI) serves as a membrane anchor for a large number of eukaryotic proteins. A genetic approach was used to investigate the biosynthesis of GPI anchor precursors in mammalian cells. T cell hybridoma mutants that cannot synthesize dolichol-phosphate-mannose (Dol-P-Man) also do not express on their surface GPI-anchored proteins such as Thy-1 and Ly-6A. These mutants cannot form mannose-containing GPI precursors. Transfection with the yeast Dol-P-Man synthase gene rescues the synthesis of both Dol-P-Man and mannose-containing GPI precursors, as well as the surface expression of Thy-1 and Ly-6A, suggesting that Dol-P-Man is the donor of at least one mannose residue in the GPI core.

The Saccharomyces cerevisiae DPM1 gene encoding dolichol-phosphate-mannose synthase is able to complement a glycosylation-defective mammalian cell line.

The Saccharomyces cerevisiae DPM1 gene product, dolichol-phosphate-mannose (Dol-P-Man) synthase, is involved in the coupled processes of synthesis and membrane translocation of Dol-P-Man. Dol-P-Man is the lipid-linked sugar donor of the last four mannose residues that are added to the core oligosaccharide transferred to protein during N-linked glycosylation in the endoplasmic reticulum. We present evidence that the S. cerevisiae gene DPM1, when stably transfected into a mutant Chinese hamster ovary cell line, B4-2-1, is able to correct the glycosylation defect of the cells. Evidence for complementation includes (i) fluorescence-activated cell sorter analysis of differential lectin binding to cell surface glycoproteins, (ii) restoration of Dol-P-Man synthase enzymatic activity in crude cell lysates, (iii) isolation and high-performance liquid chromatography fractionation of the lipid-linked oligosaccharides synthesized in the transfected and control cell lines, and (iv) the restoration of endoglycosidase H sensitivity to the oligosaccharides transferred to a specific glycoprotein synthesized in the DPM1 CHO transfectants. Indirect immunofluorescence with a primary antibody directed against the DPM1 protein shows a reticular staining pattern of protein localization in transfected hamster and monkey cell lines.

Ratio of male to female births in the offspring of BRCA1 and BRCA2 carriers.

A recent report based on 68 families, including 17 with mutations in BRCA1, suggested that there was an excess of female offspring born to BRCA1 mutation carriers. We have examined the gender ratio among offspring of 511 mutation carriers from 116 BRCA1 families, 77 and 39 from Australia and the United States, respectively. We found no evidence for a significant deviation from the expected proportion of female offspring in the Australian pedigrees, but there was an excess of female offspring in pedigrees from the USA. Ascertainment bias probably explains this bias, rather than a link with X-chromosome inactivation as previously suggested, because the families from the USA were ascertained for the purposes of linkage studies whereas those from Australia were ascertained through Familial Cancer Clinics to which they had been referred for clinical genetic counseling and mutation testing.

Characterization of intraocular tumors arising in transgenic mice.

PURPOSE: To characterize intraocular tumors that arise by in situ transformation in the choroid-retinal pigment epithelium (RPE) in transgenic mice bearing the SV40 oncogene under the control of the mouse tyrosinase promoter. METHODS: Tumors from TySV40 transgenic mice were characterized in vivo and in vitro by immunohistology, compound microscopy, and electron microscopy. Tumor cell lines were established and characterized for growth and metastatic potential in the eyes of nude mice. RESULTS: On light microscopy, ocular tumors were predominantly epithelioid, although occasional clusters of spindle cells were also present. Transmission electron microscopy revealed the presence of numerous basal infoldings and abundant multilaminated basement membranes on the ocular tumors. Tumors stained with antibodies to melanoma-associated antigens, gangliosides GD2 and GD3, and the SV40 T antigen. Radiolabeled transgenic tumor cells preferentially localized in the liver after intravenous injection in normal mice. Intracamerally transplanted transgenic tumors metastasized from the eyes to the livers of nude mice. CONCLUSIONS: In TySV40 transgenic mice, intraocular tumors develop that arise at the choroid-RPE interface, and they display morphologic and ultrastructural features consistent with RPE carcinomas. However, the transgenic tumors express melanoma-associated antigens and a propensity to metastasize to the liver, two features characteristic of uveal melanomas. The TySV40 transgenic murine tumors represent potentially useful tools for investigations into the biology and metastasis of intraocular neoplasms.

Inhibitor-resistant tissue-type plasminogen activator: an improved thrombolytic agent in vitro.

Platelet-rich clots are inefficiently lysed by current fibrinolytic agents. Platelets contain a great deal of plasminogen activator inhibitor 1 (PAI-1), the principal endogenous inhibitor of tissue-type plasminogen activator (t-PA). We have tested whether PAI-1 resistant t-PAs would be more effective thrombolytic agents in an in vitro model of platelet-rich clots. Clots were formed with recalcified human plasma without or with the addition of platelets. The lysis of these clots was followed by the release of incorporated 125I-fibrinogen. Mutant and wild-type t-PA were almost equally effective against clots lacking platelets but the mutant was twice as effective at lysing platelet-rich clots. A mechanism for this effect is suggested by the demonstration that a complex between wild-type t-PA and extruded platelet contents resembles that between purified t-PA and PAI-1 and that the PAI-1 resistant t-PA does not interefer with formation of this adduct. Because of its enhanced ability to lyse platelet-rich clots in vitro, further in vivo work may find that PAI-1 resistant t-PA is a more efficacious therapeutic agent than wild-type t-PA in situations where platelets contribute to the failure of thrombolysis.

Expression and regulation of endothelin precursor mRNA in avascular human amnion.

Posttranslational processing of preproendothelin in endothelial cells gives rise to endothelin, a 21 amino acid polypeptide that is a potent vasoconstrictor. Endothelin production is believed to be mediated principally by transcriptional mechanisms. Previously, preproendothelin mRNA expression has been detected only in vascular endothelial tissue and cells. In this study, we found that preproendothelin mRNA is expressed in an avascular human tissue, namely, amnion, an extraembryonic fetal membrane. Preproendothelin mRNA was not detected in avascular chorion laeve tissue (also an extraembryonic fetal membrane), in the highly vascularized fetal trophoblast, or in maternal uterine tissues. Furthermore, we found that preproendothelin gene expression is retained in human amnion cells maintained in primary monolayer culture. Using the amnion cells in primary monolayer culture to investigate the regulation of preproendothelin mRNA expression, we found that epidermal growth factor (EGF) and interleukin-1 (IL-1) act to stimulate preproendothelin mRNA levels; in addition, the induction of preproendothelin mRNA by either of these agents is enhanced upon simultaneous treatment with cycloheximide. These findings are indicative that preproendothelin gene expression in amnion is regulated positively by EGF and IL-1 and that inhibition of protein synthesis leads to superinduction of preproendothelin mRNA. In human umbilical cord endothelial cells, neither IL-1 nor EGF stimulate preproendothelin mRNA expression but inhibition of protein synthesis does lead to increased levels of preproendothelin mRNA. The amnion, therefore, provides a useful system for expansion of our understanding of the tissue specific expression and regulation of preproendothelin mRNA.

Tissue-type plasminogen activator-induced invasion and metastasis of murine melanomas.

The role of tissue-type plasminogen activator (tPA) in the 'spontaneous' as well as 'experimental' metastasis of ocular melanomas in mice was evaluated by transfecting the D5.1G4 murine melanoma cell line that possesses low metastatic activity and low tPA activity with a full length cDNA encoding human tPA. For comparison, a highly metastatic melanoma cell line (Queen's) that constitutively expresses high tPA production, was transfected with a cDNA coding for human plasminogen activator inhibitor type 1 (PAI-1). Unlike non-transfected controls, transfected D5.1G4 melanoma cells expressed high levels of tPA and produced extensive pulmonary metastases following intravenous injection. By contrast, PAI-1 transfected Queen's melanoma cells expressed low tPA activity and displayed significantly reduced metastatic potential compared with nontransfected controls. Moreover, PAI-1 transfected Queen's melanoma cells did not metastasize from the eye while nontransfected parental cells produced extensive spontaneous metastases. Expression of tPA activity in transfected and nontransfected cell lines was completely blocked by an anti-tPA antibody. This antibody significantly inhibited the organ localization and frequency of lung metastases of both Queen's and tPA-transfected D5.1G4 melanomas. This study demonstrates that tPA is involved in the metastasis of murine intraocular melanomas.

Rejection of intraocular tumors from transgenic mice by tumor-infiltrating lymphocytes.

The present study examined the role of tumor infiltrating lymphocytes (TIL) in the rejection of intraocular tumors from SV40 transgenic mice. Tumor cells from an intraocular tumor arising in an SV40 transgenic FVB/N mouse were transplanted into the eyes of syngeneic FVB/N mice and the TIL isolated. TIL were assessed for direct cytolytic activity in vitro. TIL were also transferred passively to immunosuppressed FVB/N mice to determine if they could mediate intraocular tumor rejection. The role of CD4+ and CD8+ T cells in intraocular tumor rejection was evaluated by depleting the respective cell populations in FVB/N hosts prior to intraocular tumor challenge. The results showed that intraocular tumors undergoing rejection in immunocompetent syngeneic hosts became infiltrated with T cells, with the CD8+ subset predominating at the time of rejection. By contrast, athymic nude mice did not reject the intraocular tumors nor did the tumors become infiltrated with TIL. TIL displayed direct, tumor-specific cytolytic activity immediately after isolation from the tumor-containing eyes. FVB/N hosts depleted of CD4+ T cells were unable to reject their intraocular tumors. In vivo depletion of CD8+ T cells delayed, but did not prevent tumor rejection. Adoptively transferred TIL mediated swift rejection of intraocular tumors in immunoincompetent recipients. Recipients of TIL, but not recipients of normal spleen cells, acquired significant tumor-specific CTL activity that was demonstrable in vitro. The results strongly suggest, but do not prove, that TIL mediate rejection of intraocular tumors from transgenic mice by direct cytolysis. Although CD4+ T cells are necessary for tumor rejection and are capable of direct cytolysis, the predominant effector cells are CD8+ CTL.

Common and divergent peptide binding specificities of hsp70 molecular chaperones.

We have studied the binding of synthetic peptides to three hsp70 molecular chaperones, DnaK, BiP, and hsc70, as a model for the interaction of hsp70 proteins with unfolded regions of target polypeptides. We measured the ability of 53 peptides to inhibit the formation of complexes between the hsp70 proteins and denatured lactalbumin. Peptides that bound with highest affinity to all three hsp70 proteins contained stretches of at least 7 residues that included large hydrophobic and basic amino acids, but few or no acidic residues. Amino acid substitutions within one heptameric peptide showed that an important feature for its binding to all three chaperones was a large hydrophobic residue in position 4, while specificity differences between the chaperones were revealed by substitutions at positions 2 and 6. Such specificity differences were frequently observed with other peptides, the most extreme example being a peptide rich in basic residues that bound with high affinity to DnaK, intermediate affinity to hsc70, and negligible affinity to BiP. Substitution of a lysine residue at position 2 in this peptide by tyrosine abolished the specificity difference by increasing the affinities of the DnaK and hsc70 proteins 5- and 20-fold, respectively, and that of BiP by greater than 2 orders of magnitude. Thus, hsp70 proteins can exhibit common or exclusive binding specificities, depending on the peptide sequence.

Molecular cloning and analysis of functional cDNA and genomic clones encoding bovine cellular retinoic acid-binding protein.

A recombinant cDNA clone, pCRABP-HS1, encoding cellular retinoic acid-binding protein was isolated from a bovine adrenal cDNA library. COS-7 cells transfected with pCRABP-HS1 produced a biologically active retinoic acid-binding protein molecule of the expected molecular mass (15.5 kDa). RNA blot hybridization analysis using pCRABP-HS1 as a probe revealed a single 1050-nucleotide mRNA species in bovine adrenal, uterus, and testis, tissues that contain the highest levels of retinoic acid-binding activity. No hybridization was detected in RNA extracted from ovary, spleen, kidney, or liver, which contain relatively low levels of cellular retinoic acid-binding protein activity. Analysis of genomic clones isolated from an EcoRI bovine genomic library demonstrated that the bovine cellular retinoic acid-binding protein gene is composed of four exons and three introns. Two putative promoter sequences were identified in the cloned 5' sequence of the gene.

Expression of early genes of origin-defective mutants of simian virus 40.

The nucleotide sequences of eight origin-defective mutants of simian virus 40 have been determined. All of the mutants have suffered deletions, which range in size from 4 to 241 nucleotides. Some of the mutants induce the synthesis of tumor (T) antigen, others do not. Viral mRNA extracted from rat cells transformed by two of the T-antigen-positive mutants has been analyzed by the S1 nuclease technique of Berk and Sharp. Irrespective of the size or the location of the deletions, the 5' ends of viral mRNAs are located approximately the same distance from the A+T-rich region (A-T-T-T-A-T) rather than at a specific site in the viral genome.

Peptide-dependent stimulation of the ATPase activity of the molecular chaperone BiP is the result of conversion of oligomers to active monomers.

The molecular chaperone BiP purified from bovine liver (bBiP) exhibits a low basal level of ATPase activity that can be stimulated 3-6-fold by synthetic peptides (Flynn, G. C., Chappell, T. G., and Rothman, J. E. (1989) Science 245, 385-390). By contrast, recombinant murine BiP (rBiP) purified to homogeneity following expression in Escherichia coli exhibits a higher basal level of ATPase activity and is much less stimulated by synthetic peptides. Nondenaturing gel electrophoresis showed that rBiP is predominantly monomeric, while bBiP exists in multiple forms probably corresponding to differentially modified monomeric, dimeric, and higher oligomeric species. Some, but not all, synthetic peptides cause conversion of the oligomeric and modified species of bBiP to a monomeric form. We propose that the peptide-dependent ATPase stimulation observed for BiP reflects the conversion of inactive oligomeric and/or modified species into active monomers.

Interleukin-1 promotes hyperglycemia and insulitis in mice normally resistant to streptozotocin-induced diabetes.

By administering physiological doses of interleukin-1 (IL-1) concurrently with multiple low doses of the beta cell toxin streptozotocin (MSZ), we observed an augmentation of diabetes by IL-1 in four different strains of mice. Augmentation of hyperglycemia by IL-1 was most prominent in the two MSZ-resistant mouse strains Balb/cJ and A/J. Furthermore, concurrent treatment with IL-1 and MSZ rendered these MSZ-resistant mice susceptible to the development of significant insulitis when compared to mice treated with MSZ alone. Development of insulitis was dependent upon the dose of IL-1; it was only observed at an IL-1 dose of 250 ng/kg body weight. Analysis of the leukocytic infiltrate in the islets of mice after treatment with 250 ng/kg IL-1 plus MSZ revealed the presence of L3T4+ and Lyt-2+ T lymphocytes. Administration of MSZ alone or IL-1 alone did not produce diabetes in the MSZ-resistant mice, indicating that neither of these agents was toxic to the beta cells by itself. We conclude that IL-1 synergizes with MSZ to augment diabetes in mice that are normally resistant to the diabetogenic effects of MSZ.

Expression of a full-length cDNA coding for human intestinal lactase-phlorizin hydrolase reveals an uncleaved, enzymatically active, and transport-competent protein.

Lactase-phlorizin hydrolase (LPH) (EC 3.2.1.23/62) is a major intestinal microvillar membrane glycoprotein that digests lactose, the main carbohydrate of milk. To investigate structure/function relationships of LPH and to assess the impact of intracellular processing on the function of LPH and on its transport to the cell surface, we have expressed a full-length cDNA encoding LPH in mammalian COS-1 cells. Analysis of the expressed protein by immunoprecipitation with monoclonal anti-LPH antibodies and treatments with endo-beta-N-acetylglucosaminidase H and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two polypeptides with apparent molecular masses of 215 and 230 kDa, representing the mannose-rich (pro-LPHh) and complex (pro-LPHc) glycosylated forms of the precursor. By contrast to pro-LPH in human enterocytes, the expressed pro-LPH in COS-1 cells does not undergo intracellular proteolytic cleavage to generate a form similar to the mature enzyme of the brush-border membrane. Intracellular cleavage, however, is not essential for the molecule to acquire its enzymatic activity since pro-LPH in COS-1 cells is enzymatically as active as LPH isolated from intestinal brush-border membranes. Indirect immunofluorescent staining of transfected cells demonstrated that pro-LPH is expressed at the cell surface. This was further corroborated by the sensitivity of the complex glycosylated form (pro-LPHc) to trypsin in the medium. Our results provide the first conclusive evidence that pro-LPH is an enzymatically active molecule and that the intracellular proteolysis of pro-LPH is not essential for the generation of transport-competent forms of LPH.

Complexes of tissue-type plasminogen activator and its serpin inhibitor plasminogen-activator inhibitor type 1 are internalized by means of the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor.

Tissue-type plasminogen activator and urokinase are serine proteases secreted by many cell types that participate in biological processes, such as tissue restructuring, cell migration, and tumor metastasis. Clinically, these proteases are used to dissolve coronary fibrin clots that are the proximal causes of acute myocardial infarction. In vivo, the activity of these enzymes is controlled by plasminogen-activator inhibitors, members of the serpin family of protease inhibitors. This study shows that tissue-type plasminogen activator-inhibitor complexes bind in solution to low density lipoprotein receptor-related protein (LRP), a large heterodimeric ubiquitous membrane receptor. In cultured cells, endocytosis and degradation of these complexes is reduced by polyclonal antibodies directed against LRP and inhibited by a M(r) 39,000 protein that binds to LRP and inhibits its interaction with previously known ligands, including apolipoprotein E and alpha 2-macroglobulin. We propose a role for LRP in the clearance of plasminogen activator-inhibitor complexes that is analogous to its function in the endocytosis of alpha 2-macroglobulin-protease complexes.

Glycosylation requirements for intracellular transport and function of the hemagglutinin of influenza virus.

The contribution of each of the seven asparagine-linked oligosaccharide side chains on the hemagglutinin of the A/Aichi/68 (X31) strain of influenza virus was assessed with respect to its effect on the folding, intracellular transport, and biological activities of the molecule. Twenty mutant influenza virus hemagglutinins were constructed and expressed, each of which had one or more of the seven glycosylation sites removed. Investigations of these mutant hemagglutinins indicated that (i) no individual oligosaccharide side chain is necessary or sufficient for the folding, intracellular transport, or function of the molecule, (ii) at least five oligosaccharide side chains are required for the X31 hemagglutinin molecule to move along the exocytic pathway to the plasma membrane, and (iii) mutant hemagglutinins having less than five oligosaccharide side chains form intracellular aggregates and are retained in the endoplasmic reticulum.

A 22 bp cis-acting element is necessary and sufficient for the induction of the yeast KAR2 (BiP) gene by unfolded proteins.

The KAR2 gene of Saccharomyces cerevisiae codes for an essential chaperone protein (BiP) that is localized in the lumen of the endoplasmic reticulum (ER). The high basal rate of transcription of KAR2 is increased transiently by heat shock: prolonged induction occurs when unfolded proteins accumulate in the ER. Three cis-acting elements in the KAR2 promoter control expression of KAR2: (i) a GC-rich region that contributes to the high level of constitutive expression, (ii) a functional heat shock element (HSE) and (iii) an element (UPR) that is involved in the induction of BiP mRNA by unfolded proteins. By analyzing internal deletion mutants of the KAR2 promoter, we demonstrate here that these three elements regulate transcription of KAR2 independently. Furthermore, the 22 bp UPR element causes a heterologous (CYC1) promoter to respond to the presence of unfolded proteins in the ER. Extracts of both stressed and unstressed yeast cells contain proteins that bind specifically to synthetic HSE and UPR elements and retard their migration through gels. Binding proteins specific for the UPR element can be fractionated by ammonium sulfate precipitation. Two of the proteins UPRF-1 and UPRF-2 (which is apparently a proteolytic degradation product of UPRF-1) bind inefficiently to mutant versions of the UPR that are unable to confer responsiveness to unfolded proteins to the (CYC1) promoter. UPRF-1 therefore displays the properties expected of a transcription factor that is involved in the sustained response of the KAR2 promoter to unfolded proteins in the ER. These experiments show that yeast cells can activate a transcription factor that stimulates expression of a nuclear gene in response to the accumulation of unfolded proteins in another cellular compartment.