Identification of mannose 6-phosphate in two asparagine-linked sugar chains of recombinant transforming growth factor-beta 1 precursor

Recombinant transforming growth factor-beta 1 (TGF-beta 1) precursor produced and secreted by a clone of Chinese hamster ovary cells was found to be glycosylated and phosphorylated. Treatment of 32P-labeled precursor protein with N-glycanase indicated that phosphate was incorporated into asparagine-linked complex carbohydrate moieties. Fractionation of 32P-labeled glycopeptides followed by amino acid sequence analysis indicated that greater than 95% of the label was incorporated into two out of three glycosylation sites at Asn-82 and Asn-136 of the TGF-beta 1 precursor. Two-dimensional electrophoretic analysis of acid hydrolyzed precursor protein and precursor protein-derived glycopeptides indicated that 32P was incorporated as mannose 6-phosphate. Binding studies with the purified receptor for mannose 6-phosphate indicated that the TGF-beta 1 precursor could bind to this receptor and the binding was specifically inhibited with mannose 6-phosphate.     

Site-directed mutagenesis of cysteine residues in the pro region of the transforming growth factor beta 1 precursor. Expression and characterization of mutant proteins

Three cysteine residues are located in the pro region of the transforming growth factor beta 1 (TGF-beta 1) precursor at amino acid positions 33, 223, and 225. Previous studies (Gentry, L. E., Lioubin, M. N., Purchio, A. F., and Marquardt, H. (1988) Mol. Cell. Biol. 8, 4162-4168) with purified recombinant TGF-beta 1 (rTGF-beta 1) precursor produced by Chinese hamster ovary (CHO) cells revealed that Cys-33 can form a disulfide bond with at least 1 cysteine residue in mature TGF-beta 1, contributing to the formation of a 90-110-kDa protein. We now show that Cys-223 and Cys-225 form interchain disulfide bonds. Site-directed mutagenesis was used to change these Cys codons to Ser codons, and mutant constructs were transfected into COS cells. Analysis of recombinant proteins by immunoblotting showed that by substituting Cys-33 the 90-110-kDa protein is not formed, and thus, more mature dimer (24 kDa) is obtained, corresponding to a 3- to 5-fold increase in biological activity. Substitution of Cys-223 and/or Cys-225 resulted in near wild-type levels of mature TGF-beta 1. Furthermore, cells transfected with plasmid coding for Ser at positions 223 and 225 expressed only monomeric precursor proteins and released bioactive TGF-beta 1 that did not require acid activation, suggesting that dimerization of the precursor pro region may be necessary for latency.     

A role of the latent TGF-beta 1-binding protein in the assembly and secretion of TGF-beta 1.

Transforming growth factor-beta 1 (TGF-beta 1) is synthesized as latent complexes with high molecular weights. The large latent complex of TGF-beta 1 in platelets is composed of three components, i.e. the mature TGF-beta 1, which is non-covalently associated with a disulphide-bonded complex of the N-terminal remnant of the TGF-beta 1 precursor (TGF-beta 1-latency associated peptide) and the latent TGF-beta 1 binding protein (LTBP). The TGF-beta 1-latency associated peptide is sufficient for the latency of TGF-beta 1, whereas the functions of LTBP remain to be elucidated. In a human erythroleukemia cell line, HEL, the production of the latent form of TGF-beta 1 was induced more than 100-fold by phorbol 12-myristate 13-acetate. Analysis by Northern blotting revealed that both the TGF-beta 1 precursor and LTBP were induced in a coordinated fashion. Analysis by immunoprecipitation using antibodies against LTBP and the TGF-beta 1 precursor dimer revealed that LTBP has a molecular size of 205 kd under reducing conditions in this cell type, i.e. similar to that from cells transfected with cDNA for LTBP, but larger than the platelet form (125-160 kd). Limited tryptic digestion of LTBP in HEL cells and analysis by SDS-PAGE showed protein bands of similar sizes to those of platelet LTBP, suggesting that the difference in molecular sizes of LTBP involves cell-specific processing. The biosynthesis of the latent TGF-beta 1 was studied by pulse-chase analysis. LTBP became covalently associated with the TGF-beta 1 precursor within 15 min after synthesis in this cell line. Secretion of the large latent TGF-beta 1 complex was observed as early as 30 min after the synthesis of LTBP; at the same time, a free form of LTBP not bound to the TGF-beta 1 precursor was seen. In contrast, the TGF-beta 1 precursor remained inside the cells in an unprocessed form for a longer time period and the TGF-beta 1 precursor dimer without LTBP was secreted only very slowly. Furthermore, the results of partial tryptic digestion of this molecule suggested that it contained improper disulphide bonding. These results suggest that LTBP plays a critical role in the assembly and secretion of the latent TGF-beta 1.     

Identification of a novel transforming growth factor-beta (TGF-beta 5) mRNA in Xenopus laevis

A novel transforming growth factor-beta (TGF-beta) mRNA of about 3.0 kilobases, which encodes a putative protein of 382 amino acids, has been identified in amphibians by cDNA cloning. This mRNA, which we designate as TGF-beta 5, is developmentally regulated and highly expressed beginning at early neurula (stage 14) and in many adult tissues in Xenopus laevis. Following the first methionine, the putative precursor protein has a hydrophobic region, approximately 22 amino acids long, which probably represents a signal sequence, similar to that found in TGF-beta s 1-3. The precursor also has potential sites for glycosylation, integrin binding (RGD), and a tetrabasic amino acid (RKKR) site for potential cleavage of the precursor peptide to a biologically active protein. The putative mature protein consists of 112 amino acids with 9 cysteines and has 76, 66, 69, and 72% identity to TGF-beta s 1-4, respectively.     

Molecular events in the processing of recombinant type 1 pre-pro-transforming growth factor beta to the mature polypeptide.

Recently, the simian type 1 transforming growth factor beta (TGF-beta 1) cDNA was expressed at high levels in Chinese hamster ovary (CHO) cells by dihydrofolate reductase-induced gene amplification (L.E. Gentry, N.R. Webb, G.J. Lim, A.M. Brunner, J.E. Ranchalis, D.R. Twardzik, M.N. Lioubin, H. Marquardt, and A.F. Purchio, Mol. Cell. Biol. 7:3418-3427, 1987). We have now purified and characterized the recombinant proteins released by these cells. Analyses of the precursor proteins by amino acid sequencing identified potentially important proteolytic processing sites. Signal peptide cleavage occurs at the Gly-29-Leu-30 peptide bond of pre-pro-TGF-beta 1, yielding pro-TGF-beta 1 (30 to 390). In addition, proteolytic processing of the precursor to yield mature TGF-beta 1 occurs at the dibasic cleavage site immediately preceding Ala-279, indicating that CHO cells possess the appropriate processing enzyme. Greater than 95% of the biological activity detected in the conditioned medium of the CHO transfectant was due to mature, properly processed growth factor. Highly purified recombinant TGF-beta 1 had the same specific biological activity as natural TGF-beta 1. The concentration of TGF-beta 1 required for half-maximal inhibition of Mv1Lu mink lung epithelial cell growth was approximately 1 to 2 pM. Purified precursor inhibited mink lung cell proliferation at 50 to 60 pM concentrations. The purified precursor preparation was shown to consist of pro-TGF-beta 1 (30 to 390), the pro region of the precursor (30 to 278), and mature TGF-beta 1 (279 to 390) interlinked by at least one disulfide bond with the pro portion of the precursor. These recombinant forms of TGF-beta1 should prove useful for further structural and functional studies.     

cDNA cloning of porcine transforming growth factor-beta 1 mRNAs. Evidence for alternate splicing and polyadenylation

Most eukaryotic cells encode principally a 2.5-kilobase (kb) transforming growth factor (TGF)-beta 1 mRNA. However, we have found two major TGF-beta 1 RNA species, 3.5 and 2.5 kb long, in porcine tissues. The 3.5-kb species has a longer 3'-untranslated sequence generated by the selection of an alternate polyadenylation site. There is a 117-nucleotide sequence within this unique 3' region, which is similar to the PRE-1 repetitive sequence of unknown function, reported earlier in the porcine genome. We have also cloned and characterized an alternately spliced mRNA species specific for the TGF-beta 1 gene, in which exons IV and V of the corresponding human TGF-beta 1 gene are deleted. The nucleotide sequence of this cDNA clone predicts a putative precursor protein of 256 amino acids; the N-terminal 211 amino acids of this putative protein are identical to the TGF-beta 1 precursor protein (exons I, II, and III of the human TGF-beta 1 gene), but the C-terminal 45 amino acids are distinct, due to a frameshift in the translation of exons VI and VII. In addition we provide data for the existence of other mRNA species generated in a tissue-specific manner either by alternate splicing or by heterogeneous 5' leader sequences.     

Retention of the transforming growth factor-beta 1 precursor in the Golgi complex in a latent endoglycosidase H-sensitive form.

Transforming growth factor-beta 1 (TGF-beta 1) is synthesized as a latent high molecular weight complex in a human erythroleukemia cell line, HEL, treated with phorbol 12-myristate 13-acetate. The complex is comprised of three components: mature TGF-beta 1, the TGF-beta 1 latency-associated peptide (beta 1-LAP), and the latent TGF-beta 1-binding protein (LTBP). LTBP plays an important role in the assembly and secretion of the latent TGF-beta 1 complex; if the TGF-beta 1 precursor fails to bind to LTBP, much of it remains inside the cells and may contain anomalous disulfide bond(s) between beta 1-LAP and the mature TGF-beta 1 molecule (Miyazono, K., Olofsson, A., Colosetti, P., and Heldin, C.-H. (1991) EMBO J. 10, 1091-1101). In the present work, we have investigated the subcellular localization and properties of the TGF-beta 1 precursor retained intracellularly. When the HEL cells were metabolically labeled and chased for up to 72 h, a considerable part of the TGF-beta 1 precursor was still observed intracellularly in an unprocessed form. The secreted form of the TGF-beta 1 precursor was resistant to endoglycosidase H, whereas the intracellular form of the TGF-beta 1 precursor was sensitive to endoglycosidase H, regardless of the presence or absence of swainsonine, an inhibitor of mannosidase II. Indirect immunofluorescence microscopy revealed that the TGF-beta 1 precursor co-localized with mannosidase II, a marker for the Golgi complex, but not with protein disulfide isomerase, a marker for the endoplasmic reticulum. The intracellular TGF-beta 1 precursor was prepared from phorbol 12-myristate 13-acetate-treated HEL cells and tested for TGF-beta 1 bioactivity. Half-maximal inhibition of the DNA synthesis in mink lung epithelial cells, Mv1Lu, was observed at 80 pM of the acid-treated TGF-beta 1 precursor, whereas nontreated material showed minimal growth inhibitory activity. Taken together, these results indicate that the TGF-beta 1 precursor is retained inside the cells in the Golgi complex, mainly in a latent, immature form.     

Proteolytic processing of mullerian inhibiting substance produces a transforming growth factor-beta-like fragment

Mullerian inhibiting substance (MIS) is a differentiation factor that causes the Mullerian duct to regress during the development of the male reproductive tract. The active form is a disulfide-linked dimer consisting of two identical 70-kDa subunits. Recently, the amino acid sequence for MIS was deduced from its gene sequence and revealed that the carboxyl-terminal region shares homology with transforming growth factor (TGF)-beta. Since TGF-beta is produced as a large latent precursor that requires proteolytic activation for activity, we sought to determine if MIS might undergo a similar processing event. Here we demonstrate that typically 5 to 20% of the protein in MIS preparations is cleaved at a site 109 amino acids from the carboxyl terminus. Concurrent cleavages from both chains of the MIS dimer produces a 25-kDa TGF-beta-like fragment and a high molecular mass complex derived from the amino terminus of the protein. Although the two fragments are noncovalently linked, they remain tightly associated after cleavage, and thus are structurally organized like TGF-beta within its precursor. The same cleavage products also can be generated by limited proteolysis with plasmin, which provides a simple method for converting the entire preparation into the cleaved form. The plasmin-digested MIS is fully active in the organ culture assay.     

Transient production and secretion of human transforming growth factor TGF-beta 2

Transient transfection of simian COS cells with a recombinant plasmid encoding the human transforming growth factor TGF-beta 2 precursor protein results in the production of a latent, biologically inactive protein. Upon acidification, recombinant TGF-beta 2 exhibits full biological activity, including inhibition of mink lung epithelial cell growth, stimulation of anchorage-independent growth of murine embryonic fibroblasts, and competition for TGF-beta receptor binding. Further analysis of conditioned media with antiserum to either a pro- [amino acid (aa) residues 1-220] or mature [aa 297-414] peptide of the TGF-beta 2 precursor suggests that TGF-beta 2, similar to TGF-beta 1 production in Chinese hamster ovary cells [Gentry et al., Mol. Cell. Biol. 7 (1987) 3418-3427], is initially synthesized as a larger precursor protein which is proteolytically cleaved to yield the mature 112-aa transforming growth factor.     

The autocrine production of transforming growth factor-beta 1 during lymphocyte activation. A study with a monoclonal antibody-based ELISA

We describe the production and characterization of three mAb to transforming growth factor-beta (TGF-beta) and the use of two of them for the development of a TGF-beta 1-specific ELISA and for the study of the regulation of immune function in vitro. All three mAb bound recombinant human TGF-beta 1 (rHuTGF-beta 1) with high affinity and recognized the dimer form of this molecule in immunoblots. mAb 2G7 immunoprecipitated rHuTGF-beta 1, TGF-beta 2, and rHuTGF-beta 3 and neutralized the growth inhibitory activity of all three molecules in vitro on mink lung epithelial-like cells, Mv1Lu, indicating a shared neutralization epitope. mAb 4A11 neutralized and immunoprecipitated only rHuTGF-beta 1, and mAb 12H5 immunoprecipitated rHuTGF-beta 1 but had no effect on the bioactivity of either rHuTGF-beta 1, TGF-beta 2, or rHuTGF-beta 3. These results suggest that a second neutralization epitope may be unique to TGF-beta 1. The ELISA was developed with mAb 4A11 and 12H5, with a range of 0.63 to 40 ng/ml, i.e., a sensitivity of 0.63 ng/ml or 63 pg/sample. The assay is accurate, precise, and specific for the active but not the inactive or latent TGF-beta 1 complex and fails to react with TGF-beta 2, rHuTGF-beta 3, inhibin A, and activin A. Supernatants obtained from serum-free cultures of human PBMC from multiple donors contained significant quantities of TGF-beta 1 (3 to 15 ng/ml), which was detected in the ELISA only after pH 2 treatment to convert latent TGF-beta to the active form. Treatment of the PBMC with either recombinant human IL-2 (rHuIL-2) or PHA-P/PMA enhanced the production of latent TGF-beta 1. mAb 4A11 and 2G7, but not mAb 12H5 enhanced both the proliferative response of PBMC to rHuIL-2/rHuTNF-alpha and PHA-P and the development of the rHuIL-2/rHuTNF-alpha treated PBMC into LAK cells with cytotoxic activity against COLO target cells. These findings suggest that although PBMC secrete latent TGF-beta 1, mechanisms that convert the latent TGF-beta complex into an active form exist in vitro and that the endogenously produced TGF-beta can regulate immune functions in an autocrine fashion.     

Transforming growth factor-beta 1: histochemical localization with antibodies to different epitopes

We have localized transforming growth factor-beta (TGF-beta) in many cells and tissues with immunohistochemical methods, using two polyclonal antisera raised to different synthetic preparations of a peptide corresponding to the amino-terminal 30 amino acids of TGF-beta 1. These two antibodies give distinct staining patterns; the staining by anti-CC(1-30) is intracellular. This differential staining pattern is consistently observed in several systems, including cultured tumor cells; mouse embryonic, neonatal, and adult tissues; bovine fibropapillomas; and human colon carcinomas. The extracellular staining by anti-CC(1-30) partially resembles that seen with an antibody to fibronectin, suggesting that extracellular TGF-beta may be bound to matrix proteins. The intracellular staining by anti-LC(1-30) is similar to that seen with two other antibodies raised to peptides corresponding to either amino acids 266-278 of the TGF-beta 1 precursor sequence or to amino acids 50-75 of mature TGF-beta 1, suggesting that anti-LC(1-30) stains sites of TGF-beta synthesis. Results from RIA and ELISAs indicate that anti-LC(1-30) and anti-CC(1-30) recognize different epitopes of this peptide and of TGF-beta 1 itself.     

Decreased transforming growth factor-beta response and binding in insulin-independent teratoma-derived cell lines with increased tumorigenic properties.

The mouse C3H teratoma-derived cell line 1246 is an adipogenic cell line which stringently requires insulin to proliferate and differentiate in defined medium. From this cell line an insulin-independent cell line called 1246-3A was isolated. It was found that, in contrast to 1246 cells, 1246-3A cells had lost the ability to differentiate and became tumorigenic when injected at a density of 10(6) cells/mouse into syngeneic host C3H mice. In addition, they produce in their culture medium transforming growth factor alpha- and beta-like polypeptides which stimulate their proliferation. Highly tumorigenic insulin-independent cell lines able to give rise to tumor when injected at a density of 10(4) cells/mouse were isolated by using an in vitro-in vivo shuttle technique. The highly tumorigenic cell lines have lost the response to TGF-beta 1. The binding of TGF-beta 1 to the nontumorigenic parent cell line or to cells displaying increased tumorigenic properties was investigated. The data presented here indicate that the increased tumorigenicity is accompanied by a progressive decrease of specific binding of TGF-beta 1 to the cells. However, the decreased number of cell surface TGF-beta 1 binding sites in the highly tumorigenic cells did not correlate with an increase in the secretion of TGF-beta protein by the tumorigenic cells, as all of TGF-beta produced by the cells was in a latent form. Affinity cross-linking experiments indicated that the 1246 cell line displayed several TGF-beta cross-linked molecular species (MW 140, 92, and 70 kDa). Increase of tumorigenicity was accompanied by a marked decrease in the intensity of the cross-linked bands, particularly of the 92 and 70 kDa species.     

Transforming growth factor-beta inhibition of interleukin-1 activity involves down-regulation of interleukin-1 receptors on chondrocytes.

Interleukin-1 (IL-1) plays an important role in cartilage destruction associated with inflammatory and degenerative arthritis because of its ability to induce matrix degrading enzymes. Previously, we have shown that the IL-1-induced chondrocyte protease activity was inhibited by transforming growth factor-beta (TGF-beta). In this paper, we show that TGF-beta inhibits the IL-1-induced synthesis of collagenase and stromelysin by reducing the steady-state mRNA levels in rabbit articular chondrocytes. We further demonstrate that TGF-beta-treated chondrocytes show reduced 125I-IL-1 binding that returns to a normal level when TGF-beta is removed from the culture medium. The inhibitory effect of TGF-beta is observed for both naturally occurring as well as fibroblast growth factor (FGF)-inducible binding sites (receptors). Scatchard analysis of receptor-ligand interactions demonstrate that the reduced binding is due to a reduction in the number of receptors for IL-1 and is not due to changes in affinity. Affinity cross-linking studies suggest that control chondrocytes contain two major cross-linked bands of Mr = 116 and 80 kDa and a minor band of Mr = 100 kDa. FGF-treated cells show enhanced levels of all the bands, plus an additional 200-kDa band. TGF-beta treatment of chondrocytes results in the reduction of all of these bands in both control as well as FGF-induced cells. These observations suggest that the ability of TGF-beta to down-regulate the IL-1 receptor may be a mechanism by which it exerts its effects in antagonizing the IL-1 activity on chondrocytes.     

The murine transforming growth factor-beta precursor

Transforming growth factor-beta (TGF-beta) is a homodimeric polypeptide which can act, often in cooperation with other growth factors, as a mitogenic factor for a variety of cells. TGF-beta can also exert growth inhibitory activity on many other cell lines. We have isolated cDNAs coding for the murine TGF-beta cDNA precursor. The deduced amino acid sequence localizes the 112-amino acid long TGF-beta monomer to the C terminus of the precursor. Two areas of the precursor exhibit a marked degree of homology to the human counterpart. One of these regions comprises the mature TGF-beta monomer, while the other corresponds to the NH2 terminus of the precursor and suggests an important biological function for this area. Northern hybridization results identify a major 2.5-kilobase TGF-beta mRNA and several minor TGF-beta mRNA species.     

Transforming growth factor beta (TGF-beta) type V receptor has a TGF-beta-stimulated serine/threonine-specific autophosphorylation activity.

The transforming growth factor beta (TGF-beta) type V receptor, a newly identified high molecular weight TGF-beta receptor (M(r) approximately 400,000) has been purified from bovine liver plasma membranes (O'Grady, P., Kuo, M.-D., Baldassare, J. J., Huang, S. S., and Huang, J. S. (1991) J. Biol. Chem. 266, 8583-8589). The purified TGF-beta type V receptor underwent autophosphorylation at serine residues when incubated with [gamma-32P]ATP in the presence of 0.1% beta-mercaptoethanol and 2.5 mM MnCl2. This phosphorylation was stimulated by preincubation with TGF-beta. The preferred exogenous substrate for the Ser/Thr-specific phosphorylation activity of the type V receptor was found to be bovine casein. The TGF-beta type V receptor could be affinity-labeled with 5'-p-[adenine-8-14C]fluorosulfonylbenzoyl adenosine. Polylysine appeared to stimulate the autophosphorylation of the TGF-beta type receptor in the presence of [gamma-32P]ATP and the incorporation of 5'-p-[adenine-8-14C]fluorosulfonylbenzoyl adenosine into the TGF-beta type V receptor. The amino acid sequence analysis of the peptide fragments produced by cyanogen bromide cleavage of the purified TGF-beta type V receptor revealed that a peptide, namely CNBr-19, contained an amino acid sequence which shows homology to the putative ATP binding site of the receptors for activin, the Caenorhabditis elegans daf-1 gene product, and TGF-beta type II receptor (Lin, H. Y., Wang, Y.-F., Ng-Eaton, E., Weinberg, R. A., and Lodish, H. F. (1992) Cell 68, 775-785). These results suggest that the TGF-beta type V receptor is a Ser/Thr-specific protein kinase and belongs to the new class of membrane receptors associated with a Ser/Thr-specific protein kinase activity.     

A new type of transforming growth factor-beta, TGF-beta 3.

A new type of TGF-beta, TGF-beta 3, has been identified by cDNA characterization. The amino acid sequence of mature TGF-beta 3 and its precursor has been derived from porcine and human cDNA sequences. The human TGF-beta 3 gene is spread over seven exons as in the case of the TGF-beta 1 gene. Comparison with TGF-beta 1 and -beta 2 indicates a strong conservation of the mature sequences, but a relaxed homology in the precursor segments. TGF-beta 3 mRNA is mainly expressed in cell lines from mesenchymal origin, suggesting a biological role different from the other TGFs-beta.     

epr, which encodes glycylglycine endopeptidase resistance, is homologous to femAB and affects serine content of peptidoglycan cross bridges in Staphylococcus capitis and Staphylococcus aureus.

Staphylococcus capitis EPK1 produces a glycylglycine endopeptidase, ALE-1 (M. Sugai, T. Fujiwara, T. Akiyama, M. Ohara, H. Komatsuzawa, S. Inoue, and H. Suginaka, J. Bacteriol. 179:1193-1202, 1997), which hydrolyzes interpeptide pentaglycine chains of cell wall peptidoglycan of S. aureus. Characterizations of the enzyme activity and cloning of ale-1 revealed that ALE-1 is very similar to prolysostaphin produced by S. simulans bv. staphylolyticus. Strain EPK1 is resistant to lysis by ALE-1 and by lysostaphin. A gene that renders the cells resistant to glycylglycine endopeptidase (epr) was found 322 bp upstream of and in the opposite orientation to ale-1. The deduced amino acid sequence of epr showed similarities to FemA and FemB, which have been characterized as factors essential for methicillin resistance of S. aureus. Inactivation of either femA or femB causes decreased resistance to methicillin, increased resistance to lysostaphin, and decreased glycine content in the interpeptide chains of peptidoglycan. Therefore, femAB is suggested to be involved in the addition of glycine to pentapeptide peptidoglycan precursor. S. aureus with epr on a multicopy plasmid had phenotypes similar to those of femAB mutants except that it did not alter resistance level to methicillin. These results suggest that epr and femAB belong to the protein family involved in adding amino acids to the pentapeptide peptidoglycan precursor and that epr is involved in the addition of serine to the pentapeptide.     

Platelet factor 4 selectively inhibits binding of TGF-beta 1 to the type I TGF-beta 1 receptor.

A low molecular weight inhibitor of TGF-beta 1 binding was detected in partially purified human platelet extracts by using Hep 3B hepatoma cells in the binding assays. The inhibitory protein was purified to homogeneity and was identified as platelet factor 4 on the basis of its amino acid sequence. TGF-beta 1 binding to Hep 3B cells was almost completely inhibited by 100 nM concentrations of platelet factor 4, but TGF-beta 1 binding to NRK 49F fibroblasts was inhibited only slightly. Affinity cross-linking experiments revealed that these differences in the inhibition of TGF-beta 1 binding by platelet factor 4 were due to differences in the complements of TGF-beta 1 binding proteins present on these two cell types. In Hep 3B cells the majority of bound TGF-beta 1 was cross-linked to a complex which had an apparent molecular weight of 70 kDa. TGF-beta 1 binding to this protein was the most sensitive to inhibition by platelet factor 4. Based on its size and TGF-beta 1 binding properties, we believe this protein is the type I TGF-beta 1 receptor. Hep 3B cells also had a high-affinity TGF-beta 1 binding protein which appeared as an 80 kDa complex, and which we believe to be the type II TGF-beta 1 receptor. TGF-beta 1 binding to this protein was not inhibited by platelet factor 4. TGF-beta 1 was also cross-linked to complexes of higher molecular weights in Hep 3B cells, but it was not clear whether any of them represented the type III TGF-beta 1 receptor. In NRK 49F cells, the majority of bound TGF-beta 1 was cross-linked to a high molecular weight complex which probably represented the type III TGF-beta 1 receptor. NRK 49F cells also had type I TGF-beta 1 receptors and platelet factor 4 inhibited binding to these receptors in the NRK cells. Since the type I receptor contributed only a small percentage of total TGF-beta 1 binding, however, the overall effects of platelet factor 4 on TGF-beta 1 binding to NRK 49F cells were negligible. We were unable to demonstrate specific or saturable binding of platelet factor 4 to Hep 3B cells using either direct binding or affinity cross-linking assays. Thus, it is not clear whether platelet factor 4 inhibits TGF-beta 1 binding by competition for binding to the type I receptor. Modest concentrations of TGF-beta 1 reduced the adherence of Hep 3B cells to tissue culture dishes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Transforming growth factor beta 1: importance of glycosylation and acidic proteases for processing and secretion

The role of glycosylation of the transforming growth factor-beta 1 (TGF-beta 1) precursor was investigated by treating a transfected Chinese hamster ovary (CHO) cell line expressing high levels of recombinant TGF-beta 1 (TGF-beta 3-2000 cells) with a series of glycosylational inhibitors. Tunicamycin, a nucleoside antibiotic which prevents the formation of the dolichol intermediate necessary for oligosaccharide addition of the nascent polypeptide chain, appeared to block secretory exit and led to an increase in the cellular associated, nonglycosylated pro-TGF-beta 1 form. 1-Deoxymannojirimycin and swainsonine, inhibitors of the mannosidases I and II, respectively, blocked complete glycoprotein processing of the TGF-beta 1 precursor as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by sensitivity to glycosidases. However, the abnormal TGF-beta 1 polypeptides containing the altered carbohydrate side chains were secreted readily by the CHO cells. In contrast, inhibitors of the glucosidases at the first step in glycoprotein remodeling, 1-deoxynojirimycin and castanospermine, markedly inhibited secretion of the TGF-beta 1 polypeptides from transfected CHO cells. In all cases, these inhibitors did not appear to affect proteolytic processing of the TGF-beta 1 polypeptides. Furthermore, inhibitor treatment did not affect mannose-6-phosphorylation of the TGF-beta 1 polypeptides. These results suggest that glycosylation and early stage remodeling of oligosaccharide side chains are necessary for secretion of TGF-beta 1. Treatment of the transfected CHO cells with weak bases (NH4Cl and chloroquine), or a monovalent ionophore (monensin), prevented proteolytic processing of the TGF-beta 1 precursor indicating that cleavage occurs by proteases in an acidic cellular compartment.