Identification and analysis of discrete functional domains in the pro region of pre-pro-transforming growth factor beta 1

A series of site-specific insertion and deletion mutants was prepared in the pro domain of transforming growth factor beta 1 (TGF beta 1) encoded by simian TGF beta 1 cDNA. These mutants were transiently expressed in COS-1 cells and the ability of each to be properly processed, folded correctly, and secreted was determined by immunoblot analysis of cells and culture supernatants. Insertions in regions corresponding to amino acid residues 50, 154, and 170 blocked secretion; culture supernatants from COS-1 cells showed no immunologically reactive proteins, whereas intact cells contained high levels of the mutant polypeptides. Insertions in the middle portion of the pro domain at residues 81, 85, and 144 affected disulfide maturation of the mature TGF beta 1. An insertion at residue 110, on the other hand, appeared to destabilize the mature TGF beta 1 polypeptide, resulting in degraded growth factor. Relatively small (10 amino acids) to large (125 amino acids) deletion mutations in the pro domain of TGF beta 1, when expressed as the full-length pre-pro-TGF beta 1, appeared to block secretion. By contrast, if the pro domain (designated beta 1-latency-associated peptide [beta 1-LAP]) was expressed independently, deletion mutants in the region 40-110 were readily secreted by the COS-1 cells, whereas deletions in residues 110-210 either destabilized the structure of the protein or blocked its intracellular transport. Cross-linking assays employing radioiodinated TGF beta 1 and biological assays indicate that residues 50-85 of beta 1-LAP are required for association with mature TGF beta 1.     

Assembly of the mammalian muscle acetylcholine receptor in transfected COS cells

We have investigated the mechanisms of assembly and transport to the cell surface of the mouse muscle nicotinic acetylcholine receptor (AChR) in transiently transfected COS cells. In cells transfected with all four subunit cDNAs, AChR was expressed on the surface with properties resembling those seen in mouse muscle cells (Gu, Y., A. F. Franco, Jr., P.D. Gardner, J. B. Lansman, J. R. Forsayeth, and Z. W. Hall. 1990. Neuron. 5:147-157). When incomplete combinations of AChR subunits were expressed, surface binding of 125I-alpha-bungarotoxin was not detected except in the case of alpha beta gamma which expressed less than 15% of that seen with all four subunits. Immunoprecipitation and sucrose gradient sedimentation experiments showed that in cells expressing pairs of subunits, alpha delta and alpha gamma heterodimers were formed, but alpha beta was not. When three subunits were expressed, alpha delta beta and alpha gamma beta complexes were formed. Variation of the ratios of the four subunit cDNAs used in the transfection mixture showed that surface AChR expression was decreased by high concentrations of delta or gamma cDNAs in a mutually competitive manner. High expression of delta or gamma subunits also each inhibited formation of a heterodimer with alpha and the other subunit. These results are consistent with a defined pathway for AChR assembly in which alpha delta and alpha gamma heterodimers are formed first, followed by association with the beta subunit and with each other to form the complete AChR.     

Localization of dystrophin relative to acetylcholine receptor domains in electric tissue and adult and cultured skeletal muscle

Two high-affinity mAbs were prepared against Torpedo dystrophin, an electric organ protein that is closely similar to human dystrophin, the gene product of the Duchenne muscular dystrophy locus. The antibodies were used to localize dystrophin relative to acetylcholine receptors (AChR) in electric organ and in skeletal muscle, and to show identity between Torpedo dystrophin and the previously described 270/300-kD Torpedo postsynaptic protein. Dystrophin was found in both AChR-rich and AChR-poor regions of the innervated face of the electroplaque. Immunogold experiments showed that AChR and dystrophin were closely intermingled in the AChR domains. In contrast, dystrophin appeared to be absent from many or all AChR-rich domains of the rat neuromuscular junction and of AChR clusters in cultured muscle (Xenopus laevis). It was present, however, in the immediately surrounding membrane (deep regions of the junctional folds, membrane domains interdigitating with and surrounding AChR domains within clusters). These results suggest that dystrophin may have a role in organization of AChR in electric tissue. Dystrophin is not, however, an obligatory component of AChR domains in muscle and, at the neuromuscular junction, its roles may be more related to organization of the junctional folds.     

The NSR1 gene encodes a protein that specifically binds nuclear localization sequences and has two RNA recognition motifs

We previously identified a protein (p67) in the yeast, Saccharomyces cerevisiae, that specifically recognizes nuclear localization sequences. We report here the partial purification of p67, and the isolation, sequencing, and disruption of the gene (NSR1) encoding this protein. p67 was purified using an affinity column conjugated with a peptide containing the histone H2B nuclear localization sequence from yeast. Using antibodies against p67 we have cloned the gene for this protein. The protein encoded by the NSR1 gene recognizes the wild-type H2B nuclear localization sequence, but does not recognize a mutant H2B sequence that is incompetent for nuclear localization in vivo. Interestingly, the NSR1 protein has two RNA recognition motifs, as well as an acidic NH2 terminus containing a series of serine clusters, and a basic COOH terminus containing arg-gly repeats. We have confirmed the nuclear localization of p67 by immunofluorescence and found that a restricted portion of the nucleus is highlighted. We have also shown that NSR1 (p67) is required for normal cell growth.     

The influence of particle size and multiple apoprotein E-receptor interactions on the endocytic targeting of beta-VLDL in mouse peritoneal macrophages

Low density lipoprotein (LDL) and beta-very low density lipoprotein (beta-VLDL) are internalized by the same receptor in mouse peritoneal macrophages and yet their endocytic patterns differ; beta-VLDL is targeted to both widely distributed and perinuclear vesicles, whereas LDL is targeted almost entirely to perinuclear lysosomes. This endocytic divergence may have important metabolic consequences since beta-VLDL is catabolized slower than LDL and is a more potent stimulator of acyl-CoA/cholesterol acyl transferase (ACAT) than LDL. The goal of this study was to explore the determinants of beta-VLDL responsible for its pattern of endocytic targeting. Fluorescence microscopy experiments revealed that large, intestinally derived, apoprotein (Apo) E-rich beta-VLDL was targeted mostly to widely distributed vesicles, whereas small, hepatically derived beta-VLDL was targeted more centrally (like LDL). Furthermore, the large beta-VLDL had a higher ACAT-stimulatory potential than the smaller beta-VLDL. The basis for these differences was not due to fundamental differences in the means of uptake; both large and small beta-VLDL were internalized by receptor-mediated endocytosis (i.e., not phagocytosis) involving the interaction of Apo E of the beta-VLDL with the macrophage LDL receptor. However, large beta-VLDL was much more resistant to acid-mediated release from LDL receptors than small beta-VLDL. Furthermore, partial neutralization of the multiple Apo Es on these particles by immunotitration resulted in a more perinuclear endocytic pattern, a lower ACAT-stimulatory potential, and an increased sensitivity to acid-mediated receptor release. These data are consistent with the hypothesis that the interaction of the multivalent Apo Es of large beta-VLDL with multiple macrophage LDL receptors leads to a diminished or retarded release of the beta-VLDL from its receptor in the acidic sorting endosome which, in turn, may lead to the widely distributed endocytic pattern of large beta-VLDL. These findings may represent a physiologically relevant example of a previously described laboratory phenomenon whereby receptor cross-linking by multivalent ligands leads to a change in receptor targeting.     

Transforming growth factor type beta 1 (TGF-beta 1) down-regulates interleukin-2 production and up-regulates interleukin-2 receptor expression in a thymoma cell line

Transforming growth factor type beta 1 (TGB-beta 1) belongs to a family of polypeptides with regulatory effects on growth and differentiation of a variety of cell types. TGB-beta 1 plays an important role in regulation of immune response by acting as a negative control signal for T cell proliferation through still unknown mechanisms. In this study we have analysed the effects of TGB-beta 1 on EL 4-6.1, a variant of the murine EL 4 thymoma, which can be induced by phorbol 12-myristate 13-acetate (PMA) and/or interleukin 1 (IL-1) to secrete interleukin 2 (IL-2) and express IL-2 receptors (IL-2R). Using this defined model system, we show that TGB-beta 1 simultaneously down-regulates IL-2 expression and up-regulates the number of both high and low affinity IL-2R. These changes correlate with changes at the mRNA level, suggesting an effect at the pre-translational level. The specificity of both TGF-beta 1 effects was demonstrated using a neutralizing antiserum to TGF-beta 1. Our data also suggest that TGF-beta 1 does not interfere with early activation signals of PMA and/or IL-1. This model might be useful for elucidating the complex role of TGF-beta 1 in the regulation of T cell responses.     

Evidence of synergy between Thy-1 and CD3/TCR complex in signal delivery to murine thymocytes for cell death.

The potential role of Thy-1 in CD3/TCR complex-mediated signal delivery to murine thymocytes was studied. Ag-mimicking cross-linked anti-CD3 mAb stimulated suspension of thymocytes from adult (6 to 8 wk old) mice for a brisk free cytoplasmic calcium ion ([Ca2+]i) rise, low level of inositol phosphate production, and marginal increase in tyrosine-specific phosphorylation of 110/120-kDa and 40-kDa cellular proteins. Weak but sustained [Ca2+]i rise, low inositol phosphate production, and weak protein tyrosine phosphorylation were also induced by the cross-linked anti-Thy-1 mAb that mimicked the putative natural ligand. The signal delivered via either of these two pathways was however insufficient for definitively promoting cell death and DNA fragmentation in the adult thymocytes. Here we demonstrated that anti-Thy-1 mAb synergized with anti-CD3 mAb for inducing a long-lasting prominent [Ca2+]i rise, definite inositol 1,4,5-triphosphate and inositol 1,3,4,5-tetrakiphosphate production, and extensive tyrosine-specific phosphorylation of 110/120-, 92-, 75-, and 40-kDa proteins, which resulted in marked promotion of cell death and DNA fragmentation in the adult thymocytes. This unique anti-Thy-1 antibody activity was confirmed to be directed to glycosylphosphatidylinositol-anchored Thy-1, and was distinguished from the known anti-L3T4 activity that augmented the CD3-mediated signal transduction in a different manner. The synergistic actions of anti-CD3 and anti-Thy-1 mAb obligatorily required the cross-linking of the two mAb together. The anti-CD3 and anti-Thy-1 mAb cross-linked together acted on immature thymocytes from newborn (less than 24 h after birth) mice for rather more extensive promotion of protein tyrosine phosphorylation and cell death. In addition, they affected peripheral T lymphocytes for accelerating protein tyrosine phosphorylation but not cell death. These results suggest a novel function of glycosylphosphatidylinositol-anchored Thy-1 as a possible unique intrathymic intensifier of the CD3/TCR complex-delivered signal for negative thymocyte selection.     

Delineation of type-specific regions on the envelope glycoproteins of human T cell leukemia viruses.

Two different approaches were used to map the type-specific regions on human T cell leukemia virus (HTLV) envelope glycoproteins. 1) Antibody reactivities of polymerase chain reaction-confirmed HTLV-I or HTLV-II carriers' sera were analyzed by Western blot assay with seven recombinant proteins containing different regions of HTLV-I or HTLV-II envelope proteins. 2) Rabbit antibodies elicited by nine HTLV-I Env synthetic peptides were used to react with the native HTLV envelope glycoproteins in an antibody-dependent cellular cytotoxicity (ADCC) assay. The results of the Western blot analysis showed that RP-B2, which contains amino acid residues 166 to 213 from HTLV-II exterior glycoprotein, was specifically reactive with 90.6% (48 of 53) of the HTLV-II carriers' sera but not with any of the HTLV-I carriers' serum (0 of 71). In contrast, RP-B, which contains amino acid residues 166 to 229 from HTLV-I exterior glycoprotein, was reactive with 85.1% (114 of 134) of the HTLV-I carriers' sera but not with any HTLV-II carrier serum (0 of 62). Furthermore, anti-HTLV-I Env synthetic peptide antibody-mediated ADCC identified several distinguishing HTLV-I ADCC epitopes in the middle region (amino acid residues 177 to 257) of the HTLV-I exterior glycoprotein. Therefore, HTLV type-specific epitopes reside mainly in a 69-amino acid sequence bounded by two cysteine residues (amino acids 157 and 225 for HTLV-I and 153 and 221 for HTLV-II), in the middle region of the exterior envelope glycoproteins.     

IFN-alpha induces autocrine production of IL-6 in myeloma cell lines.

IL-6 is a major tumor growth factor in human multiple myeloma. Myeloma cell lines, which have the same phenotypic characteristics and Ig gene rearrangements as the original fresh myeloma cells and whose growth is strictly dependent on exogenous IL-6 similar to fresh myeloma cells, have been reproducibly established. We show here that IFN-alpha stimulated the growth of five of six of these human myeloma cell lines by inducing an autocrine production of IL-6 in myeloma cells. Indeed, IFN-alpha induced IL-6 mRNA accumulation and IL-6 production in myeloma cells and the IFN-alpha-induced growth of these cells was inhibited by anti-IL-6 mAb. Moreover, IFN-alpha made possible the rapid emergence of autonomously growing myeloma cell sublines, which produced IL-6 as an autocrine growth factor. As IFN-alpha has a potential therapeutical interest for multiple myeloma, the present study opens up new directions for studying its effects on the myeloma clone in vivo.     

Binding and complement activation by C-reactive protein via the collagen-like region of C1q and inhibition of these reactions by monoclonal antibodies to C-reactive protein and C1q

Ligand-complexed C-reactive protein (CRP), like aggregated or complexed IgG, can react with C1q and activate the classical C pathway. Whereas IgG is known to bind to the globular region and not to the collagen-like region (CLR) of C1q, the site of interaction of C1q with CRP has not been defined. CRP-trimers were prepared by cross-linking and found to bind to C1q and to activate the C system. Heat-aggregated IgG (Agg-IgG) did not block the binding of CRP-trimers to C1q, nor did CRP-trimers block binding of Agg-IgG to C1q, suggesting that CRP and IgG bind at different sites. ELISA and Western blot analysis showed that CRP-trimers bound to the CLR, whereas Agg-IgG bound only to the globular region; similarly, anti-CLR mAb inhibited binding of CRP-trimers to C1q whereas anti-globular region mAb did not. Reactivity with CRP-trimers as well as with Agg-IgG was retained after reduction/alkylation and SDS treatment of C1q. A group of 22 anti-CRP mAb directed against at least six distinct native-CRP epitopes and eight distinct neo-CRP epitopes was tested for ability to inhibit the CRP-CLR interaction; one mAb, anti-native CRP mAb 8D8, with strong inhibitory activity was identified. Fab' of 8D8 blocked binding of CRP-trimers to intact C1q as well as CLR, and also inhibited CRP (CRP-trimers and CRP-protamine complexes) induced C activation, but had no effect on C1q binding or C activation by Agg-IgG. These results indicate that a conformation-determined region on CRP binds to a sequence-determined region on the CLR of C1q in an interaction which leads to C activation. Anti-CRP and anti-C1q mAb that specifically inhibit this interaction are described.